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Chap.

1 Scope and History of Microbiology

Why Study Microbiology? 1. Impact on Human Health 2. Balance of Nature - food source, play a role in decomposition, help other animals digest grass (cattle, sheep, termites). 3. Environmental provide safe drinking water; development of biodegradable products; use bacteria to clean up oil spills, etc. called bioremediation. 4. Industrial foodstuffs (beer, wine, cheese, bread), antibiotics, insulin, genetic engineering 5. Agricultural - research has led to healthier livestock and disease-free crops.

I.

Microbiology defined - The study of microbiology is the study of microorganisms, which are organisms that are invisible to the naked eye.

II. Classification of Microorganisms The 5 major groups of microorganisms: bacteria, algae, fungi, protozoa, and viruses. We will also study some other smaller groups such as prions and viroids. The one property that links these groups together is their very small size!
2 types of cells (viruses, prions and viroids are acellular without a cell): 1. Prokaryotic ("before nucleus") these guys are cells, but they have no internal membrane bound structures (no membrane-bound nucleus or membrane-bound organelles); includesonly the bacteria. (membrane bound nucleus and membrane-bound organelles); includes organisms such as protozoans, fungi, algae, animals, plants.

2. Eukaryotic ("true nucleus") do have internal membrane bound structures

A. Bacteria (singular - bacterium) (study of bacteria bacteriology) 1. prokaryotic 2. unicellular 3. size: 1/1000 the volume of a typical eukaryotic cell 4. 2 groups (discovered in 1970's) - we'll discuss more later a. Archaeobacteria - ancient bacteria b. Eubacteria - true bacteria

5. some shapes: bacillus (rod), coccus (spherical), spirillum (spiral), vibrio (curved rod) 6. motile or nonmotile 7. how do they obtain their energy? a. photosynthetic autotrophs - use energy from the sun to produce their own carbohydrates for energy. b. chemosynthetic autotrophs - process inorganic molecules for energy (ex. sulfur or iron). c. heterotrophs - depend on outside sources of organic molecules (ex. carbohydrates or sugars) for energy 8. temperature extremes: -20oC to 110oC (thats really cold & really hot! freezing is OoC and boiling is 100oC) 9. examples of diseases? B. Algae (singular - alga) - not a focus in this course. 1. eukaryotic 2. unicellular or multicellular 3. size: some microscopic, some macroscopic (ex. kelp) 4. motile or nonmotile 5. how do they obtain their energy? photosynthetic autotrophs 6. disease causing? no C. Fungi (singular - fungus) (study of fungi mycology) 1. eukaryotic 2. unicellular or multicellular (yeasts are unicellular, molds are multicellular) 3. nonmotile 4. how do they obtain their energy? a. heterotrophs b. Why are they ecologically important? Scavengers; they live off dead matter and thus, decompose it. 5. examples of diseases (called mycoses)?

examples of nonpathogenic fungi? D. Protozoa ("first animals") 1. eukaryotic 2. unicellular 3. motile or nonmotile 4. how do they obtain their energy? Heterotrophs 5. disease causing 2 examples: malaria & giardiasis (one of the dont drink the water diseases) E. Viruses - (study of viruses virology) 1. acellular, so not considered prokaryotic or eukaryotic; obligate intracellular parasites; when they are outside of a host cell, there is no evidence that these guys are alive. 2. basic structure of a virus - a piece of nucleic acid (RNA or DNA) enclosed by a protein coat (capsid); possess no nucleus, organelles, cell membrane, or cytoplasm. 3. size - 1/10 to 1/1000 the size of an ordinary bacterial cell. 4. nonmotile 5. examples of diseases?
Important Note: We will consider a sixth group, the helminths (worms), in our study of microbiology. While most of the adult stages of these worms are macroscopic, many of them go through a microscopic stage in their life cycles (egg & larval stages). Some examples of helminths are tapeworms, hookworms, pinworms, heartworms, and Chinese liver flukes. More to come later!!

III. A Brief History of Microbiology C. Leeuwenhoeck (lived 1632-1723) 1. What discovery is he credited with? First person to use microscopes to observe microbes; as a hobby he made small handheld microscopes; he called microorganisms animalcules.

C. Hooke 1. What discovery is he credited with? He first described cellulae (small rooms) in cork in 1665. His discovery led to the formulation of the cell theory, which states that cells are the basic organizational unit of all living things. C. Redi and Spontaneous Generation 1. What is this theory? Living organisms arise from nonliving things (ex. maggots come from rotting meat) 2. Who disproved this theory and how? In the late 1600s Francisco Redi showed that maggots developed only in meat that flies could reach to lay eggs on. 3. Many insisted that he only disproved spontaneous generation for macroorganisms; maybe microbes were an exception. D. Needham vs. Spallanzani - still trying to prove or disprove the theory of spontaneous generation. 1. What was Needham's hypothesis, experiment, & conclusions? Everyone knew boiling killed microbes; so, he would boil chicken broth, put it in a flask, & seal it; if microbes grew, then it could only be because of spontaneous generation; they did grow. [We now know that microbes grew because the flask was not sterilized before he poured in the broth!] 2. What was Spallanzani's hypothesis, experiment, & conclusions? He was not convinced by Needham's experiment. He put broth in a flask, sealed it (creating a vacuum), & then boiled it. There were no microbes in the cooled broth! Critics said he didn't disprove spontaneous generation - they said he just proved that spontaneous generation required air.

E. Pasteur's Epic Experiments (1859) 1. What was his experimental method? To offset the argument that air was necessary for spontaneous generation, Pasteur allowed the free passage of air, but prevented the entry of microbes. He boiled meat broth in a flask & then drew out & curved the neck of the flask in a flame. No microbes developed in the flask. When he tilted the flask so some broth flowed into the curved neck & then tilted it back so the broth was returned to the base of the flask, microbes grew. Gravity had caused the microbes that had entered the flask in air & dust to settle at the low point of the neck, never reaching the broth in the base until the broth washed them in. 2. Pasteur's success was partly due to good luck. He used meat, which contains few bacterial endospores (endospores are resistant to heat; many experiments done prior to Pasteur's used vegetable broths - plants contains many endospore-forming bacteria.) 3. What 3 things did Pasteur's experiments prove? a. No living things arise by spontaneous generation. b. Microbes are everywhere - even in the air and dust c. The growth of microbes causes dead plant & animal tissue to decompose & food to spoil (this led him to develop the technique of pasteurization - he developed it to keep wine from spoiling). 4. Pasteur also contributed to the development of vaccines. F. The Germ Theory of Disease 1. What is the germ theory of disease? Microbes (germs) cause disease and specific microbes cause specific diseases.

2. Who proved this theory? Robert Koch in the late 1870's. 3. What disease was he studying? anthrax - disease of cattle/sheep; also in humans 4. What was his experimental method? He observed that the same microbes were present in all blood samples of infected animals. He isolated and cultivated these microbes (now known to be Bacillus anthracis ). He then injected a healthy animal with the cultured bacteria & that animal became infected with anthrax & its blood sample showed the same microbes as the originally infected animals. 5. What did his experiments prove? Particular microbes cause particular diseases. 6. What are Koch's 4 Postulates? 1.) The causative agent must be present in every individual with the disease. 2.) The causative agent must be isolated & grown in pure culture (how did he invent pure cultures?; with Frau Hesse's help, he developed the agar plate method(see p. 13). 3.) The pure culture must cause the disease when inoculated into an experimental animal. 4.) The causative agent must be reisolated from the experimental animal & reidentified in pure culture. G. What are Some Ways that We Can Control Infectious Diseases? 1. Immunity - stimulating the body's own ability to combat infection; from ancient times it was a recognized fact that people who suffered from certain diseases never got them again; infection could produce immunity.

a. Immunization defined: produce immunity by providing exposure to altered organisms that do not cause disease. b. Jenner & Smallpox - observed that dairymaids that contracted a mild infection of cowpox seemed to be immune to smallpox. He inoculated a boy with fluid from a cow pox blister and he contracted cowpox; he then inoculated him with fluid from a smallpox blister; the boy did not contract smallpox; the term vaccination came from vacca for cow. c. The first vaccines: 1.) Pasteur's discovery? attenuated bacteria can produce immunity 2.) Attenuated defined - weakened virus or bacteria that is unable to cause the disease (it was later discovered that killed microbes can also produce immunity) 3.) What vaccines did Pasteur develop? anthrax, rabies 2. Public Hygiene a. Improving sewage disposal. b. Assuring a clean public water supply. c. Food preservation & inspection. ex. Pasteurization - kills most microbes by exposing to heat. d. Improving personal hygiene. Semmelweiss & childbed fever e. Developing antiseptic techniques. Lister & carbolic acid he developed the first aseptic techniques. 3. Chemotherapy

a. Who is the father of chemotherapy? Paul Ehrlich he discovered a drug treatment for syphilis; he developed the guiding principle of chemotherapy, which is selective toxicity (the drug must be toxic to the infecting microbe, but relatively harmless to the hosts cells). b. What was the first major class of drugs to come into widespread clinical use? sulfa drugs c. Who discovered the first antibiotic? Flemming discovered (penicillin); antibiotics are antibacterial compounds produced by fungi and bacteria.

CHAPTER 2 INTRODUCTORY CHEMISTRY & BIOCHEMISTRY

INTRODUCTORY CHEMISTRY
Why is it necessary to study chemistry? Living things are made of matter (anything that occupies space & has mass) & matter follows the laws of chemistry. Even the characteristics we consider to be unique to living things are the result of chemical reactions (ex. movement as a result of muscle contractions).

I. THE ATOM A. Atom - smallest unit of matter unique to a particular element. B. Element - A substance made up of only one kind of atom - ex. carbon contains only carbon atoms. Elements can't be broken

down into other substances by ordinary chemical means. Each element displays unique properties (ex. some are gases, some are solids, some are metals, etc.). About 92 elements occur naturally (there are also some man-made elements). Some of the elements important to our study of living systems are carbon, oxygen, hydrogen, nitrogen, sodium, chlorine, phosphorus, and potassium. You may also be familiar with the elements lead, iron, iodine, gold, silver, and copper, nickel, and platinum. C. Some Important Things to Know about Atoms & Elements: 1. An atom consists of 2 basic parts: a. nucleus - the nucleus contains protons & neutrons: 1.) protons - positively charged; all atoms have protons; protons give the nucleus a positive charge. 2.) neutrons - neutral in charge; fxn.: "stabilizers"; all atoms except hydrogen have one or more neutrons. b. electrons negatively charged; occupy orbit energy levels or shells around the nucleus; attracted to the positive charge of the nucleus; in an atom, the number of electrons always equals the number of protons, so the atom, as a whole, has no charge; electrons determine the chemical properties of elements (ex. whether they are a liquid, solid, or gas, etc.). 2. atomic number = number of protons (or number of electrons); in an atom, the number of protons always equals the number of electrons; this number differs for each element.

3. atomic mass number = number of protons + number of neutrons; the number of electrons is not included in the mass number due to their insignificant mass. 4. The 6 elements important for building organic molecules like carbohydrates, lipids, proteins, and nucleotides are: carbon, hydrogen, oxygen, sulfur, nitrogen, & phosphorus. Note: Be able to determine the number of electrons, protons, & neutrons in an atom, given the atomic number & atomic mass. Ex. Sodium (Na) has an atomic number of 11 & an atomic mass of 23. A Sodium atom has 11 protons, 11 electrons, & 12 neutrons.

II.

CHEMICAL REACTIONS

A. More about Electrons 1. Electrons orbit around the nucleus at different energy levels or shells the 1st shell (k shell) can hold no more than 2 electrons; the next shell (l shell) can hold 8 electrons, 2 electrons in each of 4 orbitals; the next shell (m shell) also holds 8 electrons. These will be the only shells that we will deal with in this class. 2. An atom is the most stable when all of its shells are completely filled (the k shell fills first, then the l shell, then the m shell, & so on). 3. The atoms of many elements have partially filled outer shells, therefore they are not very stable; these atoms tend to react with other atoms to completely fill their outer shells &, in doing

so, they form chemical bonds; it is important to remember that it's the electrons of an atom that participate in the chemical bonds that form between atoms. Molecules are formed when 2 or more atoms are joined together by interactions between the electrons of their outer electron shells.

B. Chemical Bonds 1. Ionic bonds a. definition - in ionic bonds electrons are transferred to other atoms to completely fill outer shells; atoms are electrically neutral, but when they gain or lose electrons in combining with other atoms, they are called ions (charged atoms) & they take on a positive or negative charge; in other words, the transfer of electrons upsets the balance of protons & electrons in an atom; atoms that lose electrons are positively charged, atoms that gain electrons are negatively charged; ionic bonds involve the attractions between these oppositely charged ions. So, before you can have an ionic bond, you have got to have oppositely charged ions, & to create ions you have got to transfer electrons. b. example: NaCl (sodium chloride- table salt); Na (at. # 11) has one electron in its outer m shell - it needs 7 electrons to fill this shell - it is easier for Na just to give this electron away, & eliminate the m shell entirely. Cl (at. # 17) has 7 electrons in its outer m shell - it only needs 1 electron to fill its outer shell. Therefore, when Na & Cl

atoms react, Na gives up its outer electron to Cl. Because Na gives up an electron, it now has 11 protons & 10 electrons, resulting in a positively charged atom. Cl now has 17 protons & 18 electrons, resulting in a negatively charged atom. Na+ & Cl- ions are attracted to each other because of their opposite charges & an ionic bond is formed. The attraction is the ionic bond! Only the electron # changes when ions are formed!

2. Covalent bonds - more common in the human body & are more stable. a. definition - Electrons are not transferred, but are shared. The shared electrons spend part of their time around the nucleus of one atom & part of their time around the other. Each pair of electrons shared equals one covalent bond (if 2 pair of electrons are shared between 2 atoms, a double covalent bond is formed, a triple covalent bond occurs when 3 pr. of electrons are shared). We only discuss single covalent bonds. b. example: methane (CH4); a carbon atom can form four covalent bonds - it has 4 electrons in its outer shell, therefore it needs 4 more electrons to fill its outer shell; hydrogen has one electron in its outer shell, therefore it needs one electron to fill its outer shell. Rather than give 4 electrons away or accept 4 electrons, carbon shares its 4 electrons in its outer shell with 4 Hydrogen atoms.

c. polar vs. nonpolar - covalent bonds can be polar or nonpolar; if both atoms exert the same pull on the shared electrons (equal sharing), the bond is nonpolar (example: methane); if there is unequal sharing of electrons, the bond is polar; in molecules with polar covalent bonds, there is an atom that has a much larger nucleus (more protons) than the other atoms in the molecule; the atom with the most protons is more attractive to the shared electrons, so the electrons spend most of their time around this atom's nucleus; all of these electrons spending most of their time around a particular nucleus gives this part of the molecule a partial negative charge; the other atom(s) in the molecule acquire a partial positive charge, because the shared electrons are not spending much time around them. Shared electrons in polar covalent bonds are not spending all of their time around a particular nucleus - if this were the case then we would be talking about electrons being transferred (as in ionic bonds). 4. Hydrogen bonds - These bonds result from polar covalent bonds; they form between molecules & occur between the slightly negative atom of one molecule & the slightly positive atom of another molecule. These bonds can occur between hydrogen & oxygen & between hydrogen & nitrogen. Hydrogen bonds are weaker than ionic & covalent bonds, because the charges on the molecules are "partial" or weak charges.

C. Chemical Reactions - A chemical reaction when atoms or molecules (called reactants) collide and are transformed into

different combinations of the same atoms or molecules (called products). In this process, chemical bonds break and new ones form. In living systems special proteins called enzymes catalyze these chemical reactions (they make them go). We'll talk more about enzymes later. III. WATER A. Structure:

1. A single water molecule is made up of one oxygen atom & two hydrogen atoms (H2O).

2. Oxygen has 8 protons, while each hydrogen has only one proton. Oxygen forms a covalent bond with each hydrogen so that the outer shells of each atom are complete.

3. Because the oxygen atom has more protons (positives) than the hydrogen nuclei, the shared electrons have a greater attraction to the oxygen nucleus & spend more time around it than they do around the hydrogen nuclei (unequal sharing of electrons). The oxygen atom therefore has 10 electrons (8 of its own & 1 from each hydrogen) spending most of their time around its nucleus of 8 protons - this gives the oxygen end of the water molecule a partially negative charge. Since the hydrogen electrons are spending most of their time around the oxygen atom, the hydrogen atoms, which have one proton each, take on a partially positive charge. This results in a polar molecule (a molecule that

has partially positive & negative regions). Their polarity allows water molecules to interact with one another, forming hydrogen bonds. The same type of interaction is possible between water & many other polar substances. Polar substances are hydrophilic (water-loving) & nonpolar substances are hydrophobic (water-fearing).

B. PROPERTIES OF WATER - Hydrophilic & hydrophobic interactions underlie several properties of water that are biologically important.

1. Temperature-Stabilizing Effects:

Note: The temperature of a substance is a measure of how fast its molecules are moving; the higher the temperature of a substance, the faster its molecules are moving.

a.) heating water - It takes considerable heat to raise the temperature of water because the hydrogen bonds between the water molecules restrict the movement of the molecules; in order for the temperature of water to rise, a number of H bonds must be broken - this takes a lot of energy. This resistance to temperature change helps living cells to maintain a relatively constant temperature; this is important because biochemical reactions take place within a narrow temperature range (this has to do with the action of enzymes). This resistance to temperature change also helps organisms that live in aquatic or marine environments.

2. Water As a Solvent - the polarity of water is also responsible for water's capacity as a solvent (something that dissolves something else); water is an excellent solvent for ions & other polar molecules (solutes) in cells.

C. ACIDS AND BASES When molecules of inorganic acids, bases, or salts dissolve in water of body cells, they undergo ionization or dissociation (they break apart into their individual ions).

1. Acids & Bases Generally Defined

Acid - Defined as a solute that releases H+ ions in a solution [ex. HCl - hydrochloric acid dissociates into H+ ions & Cl- ions]

Base - Defined as a solute that removes H+ ions from a solution; many release OH- ions in this process. [ex. Mg(OH)2 - magnesium hydroxide dissociates into OH- ions & Mg++ ions].

2. pH Scale - Fluids are assigned a pH value (0 -14), which refers to the hydrogen ion concentration present in the fluid. The hydrogen ion concentration is abbreviated as [H+].

a. acid - pH below 7.0; base - pH above 7.0; neutral - pH = 7.0 b. pH = - log [ H+ ] (formula for calculating pH) c. It is a common misconception to think that as the [H+] increases, the pH also increases! The rule is: As [H+] increases, pH decreases! This can be seen from the following example: solution A: [H +] = 1 x 10-2 or 0.01 x 10-3] = 2 solution B: [H+ ] = 1 x 10-8 or 0.00000001 pH = -log[1 x 10-4] = 8 pH = -log[1

(A quick way to find the pH of these solutions is to look at the exponent or count the number of decimal places in the [H+])

Solution A is more acidic than Solution B - Solution has a higher [H+] than Solution B (0.001 > 0.0001); therefore, Solution A has a lower pH than B.

When you think about a pH value, think that this number is really the number of decimal places in the hydrogen ion concentration. The larger the number, the more decimal places there are, indicating a smaller hydrogen ion concentration.

3. Buffers - help maintain a constant pH by removing or adding H+ ions; the pH inside living systems is generally between 7.357.45 (exception: the hydrochloric acid in the digestive system makes the pH here 2-3); this pH range is important, as many biochemical reactions take place only within this range; buffers can combine with hydrogen ions &/or release them, & so help stabilize the pH.

BIOCHEMISTRY
4 Groups of Organic Compounds Important in Living Organisms: Carbohydrates Lipids Proteins Nucleotides

Organic compound defined A compound containing carbon (with exception of carbon dioxide); found in all living things.

THE CENTRAL ROLE OF CARBON

A. The Carbon Backbone - The processes of life are primarily the result of the chemistry compounds of carbon. Because of carbon's tendency to form four covalent bonds in four different directions, carbon can form an unbelievably large number of different compounds of high complexity; organic compounds derive their basic shapes from the carbon atoms; this shape helps determine the compound's function in living systems.

B. Functional groups - The structure & behavior of organic compounds also depends on the properties of their functional groups; functional groups are groups of atoms (ex. hydrogen, oxygen, nitrogen, phosphorus, sulfur) attached to the carbon backbone.

I. CARBOHYDRATES:

A. Structure: generally made up of only three elements: carbon, hydrogen, & oxygen

B. Three Principle Classes of Carbohydrates: 1. Monosaccharide a. Structure - composed of single sugar molecule; the atoms in a sugar molecule can form a straight chain or a ring (rings are more common in the body).

b. Examples - Glucose, Ribose, Fructose, Galactose d. Functions - monosaccharides are important energy molecules in living things; glucose is the primary energy source for humans & many other animals; also important as building blocks of larger sugars.

2. Oligosaccharides - composed of short chains of monosaccharides; Examples: a. Sucrose (table sugar) is a disaccharide composed of glucose & fructose; sucrose is the form in which sugars are transported in plants. b. Lactose (milk sugar) is a disaccharide composed of glucose & galactose.

Sucrose = Glucose

Fructose

2.

Polysaccharides - straight or branched chains of many monosaccharide units

a. Storage Polysaccharides 1.) Starch - sugar storage in plants. 2.) Glycogen animal starch; principle storage form for glucose in higher animals; this energy storage is short term; lipids are used for long term energy storage.

b. Structural Polysaccharides 1.) Cellulose Principal component of the plant cell wall; also found in the cell walls of algae and fungi. Monosaccharides are bonded together in such a way that the molecule resists breakdown by multicellular organisms. We dont have the digestive enzymes to break the bonds; however, some microorganisms do have these enzymes; this is why microbes are so important in the gut of a termite, cow, etc.) 2.) Chitin contains nitrogen; forms the cell wall of some fungi (its the same stuff insect exoskeletons are made of!) II. PROTEINS

A. Protein Structure:

1. Proteins are composed of subunits called amino acids (there are 20 different amino acids that make up proteins). Amino acids contain carbon, hydrogen, oxygen, and nitrogen. Some also contain sulfur. Amino acids have a structure similar to the one below. The R stands for some other atom or group of atoms bonded to the central carbon atom in the molecule. The sequence of amino acids in a chain helps determine the structure &

shape of the protein & therefore the function of the protein; there are many possible combinations of amino acids that produce the many different kinds of proteins.

N----C----C

OH

2. Peptide bonds - linkage formed between one amino acid & another amino acid; the name of these bonds is why chains of amino acids are called polypeptides.

3. Producing the three dimensional structure of a protein: We have been discussing proteins as "chains of amino acids." However, the final structure of proteins is not a straight chain of amino acids. Proteins are very complex, threedimensional molecules, with numerous twists & folds. The amino acid chain of every kind of protein is folded in a very specific way [the chain will twist & fold itself based on the linkage of amino acids in a specific sequence & the environmental conditions (i.e., temperature & pH)]. There are several bonds & forces which give a protein its specific 3-D structure (i.e., hydrogen bonds, ionic bonds, etc.); these bonds link distant parts of the molecule, forming loops, twists, etc. Destruction of a protein's 3-D structure by extreme heat or pH is calleddenaturation (see "enzymes" on the next page for how this occurs). Analogy for protein structure: Think of a phone cord. Pull it straight (like a straight chain of a.a.), then let it twist, then roll the twisted cord

into a ball. (Every type of protein folds and twists in a very specific way.) B. Some Specific Functions of Proteins: 1. Structural Proteins: collagen in connective tissue, keratin in the skin, cytoskeleton in cells 2. Functional Proteins: a. membrane transport proteins transport substances across the cell membrane b. cell movement ex. flagellum c. enzymes as catalysts (enzymes speed up the rate of chemical reactions); all life processes are primarily the result of chemical reactions; molecules in living things require enzymes in order to react; without enzymes, chemical reactions in living things cant occur; see below for more information on enzymes. d. antibodies in the immune response

C. Enzymes a large, globular protein molecule that accelerates a specific chemical reaction. Virtually all chemical reactions that take place in cells involve enzymes!!! Most of a cells proteins are enzymes.

1. Why are enzymes needed? In order for particular molecules to react with one another, they must be in close proximity & must collide with sufficient force to overcome the mutual repulsion of their negatively charged electron clouds & to break existing chemical bonds within the molecules. The force with which they collide depends on their kinetic energy (energy of motion). Most chemical reactions require an initial input of energy to get started, which increases the kinetic energy of the molecules, enabling a greater number of them to collide with sufficient force. In the chemistry lab, we can supply this

energy with heat. In a cell, many different reactions are going on at the same time, therefore heat cannot be used as it would be nondiscriminatory (it would affect many reactions at the same time). Cells get around this problem by using enzymes, which serve as catalysts (they get the chemical reactions going). The enzymes form a temporary association with the molecules that are to react, bringing them close to one another & weakening the existing chemical bonds, making it easier for new ones to form.

2. Enzyme Structure & Function - Enzymes are large, complex, globular proteins consisting of one or more polypeptide chains. The molecules that enzymes acts on are known as the substrates. Enzymes are folded to form a groove or pocket (called an active site) on their surface into which the substrate fits & where the chemical reactions take place. See diagram below:

3. Effects of Temperature & pH on Enzyme Function 1. Temperature: As the temperature increases, so does the rate of enzyme catalyzed reactions, but only up to a certain point. At high temperatures, the enzymes are denatured (due to the vibration of molecules at high temperatures, the bonds that maintain the enzyme's structure are broken & the protein unfolds). If denaturation is severe, the damage to the enzyme is irreversible. 2. pH: The shape of the enzyme depends partly on attraction between positively & negatively charged amino acids. As the pH changes (acidic - more H+, basic - fewer H+), these charges change, changing the shape of the enzyme & its function. Remember: the optimum pH for most enzymes is 6-8. (exception: the stomach which has a pH of 2)

Note: All proteins can be denatured by heat and extreme pH.

III. PEPTIDOGLYCAN
This molecule has both protein and polysaccharide components and it forms the cell wall of eubacteria. It may be one or several layers thick. It is an extremely strong protective covering. Glycan strands in all eubacteria are made of alternating units of 2 modified sugars, N-acetylglucosamine (NAGA) and N-acetylmuramic acid (NAMA). (Its structure is similar to a chain link fence!) More later!

IV. LIPIDS
A. General Structure - all lipids are mostly nonpolar (hydrophobic) & are insoluble in polar solvents such as water; lipid structure varies greatly & is discussed below for each type. B. Some General Functions: 1. long term energy storage (example: glycerides); energy is stored in the chemical bonds; excess carbohydrates, proteins or fats are converted to triglycerides & are stored in adipose (fat) tissue. 2. structural (example: phospholipids make up the cell membrane of cells) C. Types of Lipids:

1. Lipids with Fatty Acids Glycerides & Phospholipids: a. Glycerides 1.) Structure: classified as mono-, di-,

& triglycerides, depending on the number of

fatty acids attached a single glycerol molecule; glycerol has 3 carbon atoms & 3 hydroxyl (OH) groups; fatty acids are long, nonpolar chains composed of hydrogen & 4 to 24 carbon atoms, with a carboxyl (COOH) group at one end.

a.) Saturated fatty acids - all carbons in the fatty acid tails are joined together by single carbon to carbon bonds & as many hydrogen atoms as possible are linked to the carbons (the carbons are said to be "saturated" with hydrogens); triglycerides with many saturated fatty acids are solid at room temperature; occur mostly in animal tissues, but also in a few plant products; examples: butter, lard, cocoa butter, palm oil, coconut oil; the liver produces cholesterol from some breakdown products of saturated fats. b.) Unsaturated fatty acids - one or more double bonds occur between carbon atoms in the fatty acid tails; this cuts down on the number of hydrogen atoms that can bond to the carbons; liquids at room temperature; the double bonds create a kink in the shape of the molecule prevent the fatty acids from packing close together & becoming solidified; unsaturated fatty acids are more common in plants; monounsaturated fatty acids are better for you that the polyunsaturated ones; the polys can produce compounds called trans fatty acids, which increase the risk of heart disease. A triglyceride molecule:
H H H H H H H H H H H

H--C---O---C--CC--C--C--CCCCC--CH O H H H H H H H H H H

saturated f.a.

H H H H

H H

H H

H--C---O---C--CC--C--C--C--CCCCC--H O H H H H H H H H H H

saturated f.a.

H H

H H

H H

HC---O---CCCC==CCCCCCC---H H O H H H H H H H H

unsaturated f.a.

2.) Functions of Glycerides a.) Energy - For most organisms and cellular microorganisms, sugars in excess of what can be stored as glycogen are converted into fats for more permanent storage; this is not the case in bacteria!

b. Phospholipids 1.) Structure - 2 fatty acids & 1 phosphate group are linked to a glycerol molecule; a small polar group is linked to the phosphate group; this results in a molecule with a dual nature - the molecule has a nonpolar, hydrophobic end & a polar, hydrophilic end.

2.) Function: Structural. The phosphate end of the molecule & its polar group are called the "head" of the molecule; the two fatty acids are called the "tails" of the molecule; the head is hydrophilic ("water-loving"), while the 2 fatty acid "tails" are hydrophobic ("water-fearing"). This arrangement forms the structural basis of cellular membranes & is called the phospholipid bilayer.

Phosphate Head (polar)

2 Fatty AcidTails (nonpolar)

V
2. Lipids without Fatty Acids: Steroids a. Structure - different from other lipids; they consist of 4 interlocking carbon rings with numerous hydrogens attached; while they have no fatty acids, they are still nonpolar & hydrophobic, so they are classified as lipids.

b. Some Examples: 1.) cholesterol - important component in eukaryotic cell membranes & serves as the starting material for the synthesis of other steroids. Not found in the cell membranes of bacteria with the exception of the Mycoplasms. V. NUCLEOTIDES

A. Structure: nucleotide = phosphate(s) + monosaccharide + a nitrogencontaining compound (called a base); its the bases that spell out the genetic message in DNA & RNA).

B. Functions:

1.

Nucleotides are the basic subunits of nucleic acids such as

a. DNA (deoxyribonucleic nucleic acid) - carrier of the genetic message - makes up chromosomes in the nucleus of the cell.

b. RNA (ribonucleic acid) - transcribes genetic message present in DNA & produces proteins from it.

2. Nucleotides also make up the adenosine phosphates (ex. ATP adenosine triphosphate used for energy molecule in the cell).

3. Nucleotides make up some coenzymes (ex. NAD & FAD); these molecules function as electron carriers in some biochemical reactions; they are called the cells reducing power. Well talk about this more in the metabolism chapter.

Chp. 3 Microscopy and Staining

I.

Principles

Microscopy is the technology of making very small things visible to the human eye. Most microbes are so small that they are measured in micrometers or nanometers. A typical bacterial cell is about 1 um while a virus is more in the range of 10-100 nm. Remember, Leeuwenhoek was probably the 1st to see microorganisms in the 1600s with his invention of his simple microscopes.

Resolution is the ability to see two objects as separate, discreet entities.kind of like the ability to see railroad tracks as being separate tracks.GOOD resolution is being able to distinguish the two tracks as separate..once the two tracks merge into one, the resolution is poor!!!

Refraction is the bending of light as it passes from one medium to another of different density. Immersion oil, which has the same index of refraction as glass, is used to replace air and to prevent refraction at a glass-air interface. An example would be when one looks at objects just below the surface of water in a pond or other body of water..the objects become refracted or distorted from the true image.

The total magnification of a light microscope is calculated by multiplying the magnifying power of the objective lens by the magnifying power of the ocular lens. Increased magnification is of no value unless good resolution can also be maintained.

Scanning (3X) x (10X) = 30X total Low power (10X) x (10X) = 100X total High dry (40X) x (10X) = 400X total

Oil immersion (100X) x (10X) = 1000X total

Most microscopes are designed so that when the microscopist increases or decreases the magnification by changing from one objective lens to another, the specimen will remain very nearly in focus. Such microscopes are said to be parfocal (par means equal).

Types of microscopes:

Compound Light*** (this is what we will useknow the parts and functions.we will spend more time on this in the lab.

Dark-Field-

Phase-ContrastFluorescence-

Transmission-

Scanning Electron-

II.

Techniques of Light Microscopy

A.

Preparation of specimens

Wet mounts are used to view living organisms. The hanging drop technique is a special type of wet mount, often used to determine whether organisms are motile.

Smears of appropriate thickness are allowed to air-dry completely and are then passed through an open flame. This process, called heat fixation, kills the organisms, causing them to adhere to the slide and more readily accept stains.

B.

Principles of Staining

A stain, or dye, is a molecule that can bind to a structure and give it color.

Most microbial stains are cationic (positively charged), or basic dyes, such as methylene blue, crystal violet, or safrannin. Some are anionic dyes (negatively charged), or acidic dyes, such as nigrosin or India ink.

MOST bacterial surfaces are negatively charged so they will attract the basic dyes. Simple stains use one dye and reveal basic cell shapes and arrangements. Differential stains use two or more dyes and

distinguish various properties or organisms. The Gram stain, spore stain, and acid-fast stain are examples.

Negative stains color the background around cells and their parts, which resist taking up the stain. (acidic dyes will stick to the glass slide since glass has a + charge).

Imagine a magnet when thinking of basic and acidic dyes..basic dyes (+) will attract to bacteria due to their (-) parts but will be repelled by the glass because of its (+) charge!! Acidic dyes, on the other hand, will attract to the (+) glass but be repelled by the (-) bacterial parts!!

We will also be covering these in great detail in the laboratory.

Chapter 4 Characteristics of Prokaryotic & Eukaryotic Cells


All cells have: 1. Cell or plasma membrane (separates the cell from the outer environment) 2. Genetic material (DNA) 3. Cytoplasm. I. TWO GENERAL TYPES OF CELLS:

A. Prokaryotic ("before nucleus") - a cell lacking a membrane-bound nucleus & membrane-bound organelles (ex. bacteria); these cells do have some organelles, but they are not membrane-bound; all prokaryotic cells have a cell wall, its primary component being peptidoglycan; prokaryotic cells are much smaller than eukaryotic cells (about 10 times smaller); their small size allows them to grow faster & multiply more rapidly than eukaryotic cells (they have a higher surface area to volume ratio than larger cells; thus, because they are small, they can easily meet their modest nutritional needs and grow rapidly). This group includes all bacteria.

B. Eukaryotic ("true nucleus") - a cell having a membrane-bound nucleus & membrane-bound organelles (little organs specialized structures that perform specific functions within the cell); evolved about 2 million years after the prokaryotes; cell walls are sometimes present, but they are composed of cellulose or chitin; organisms with eukaryotic cells include fungi, algae, protozoa, plants, & animals. It is important to know the differences between prokaryotic and eukaryotic cells; allows us to control disease-causing bacteria without harming our own cells. II. PROKARYOTIC CELL STRUCTURE A. Appendages 1. Pili - straight hairlike appendages; they are usually short; all gram negative bacteria have pili; function is to attach bacteria to other bacteria, other cells, or other surfaces (not for locomotion): a. sex pili allow one bacterial cell to adhere to another (cells can actually exchange genetic material through the pili - this is the closest bacteria get to sexual reproduction!); called conjugation. b. other types of pili attach bacteria to plant or animal cells to maintain themselves in a favorable environment; if pili have been lost (maybe due to a mutation) in disease-causing bacteria, the bacteria will not be able to establish an infection.

2. Flagella (singular flagellum) - long, thin structures that extend outward from the surface of the envelope; function is locomotion - bacteria with flagella are motile; flagella rotate to propel the bacterium. Bacteria can have 1, 2, or many flagella (ex. of a bacteria with many flagella Salmonella).

3. Axial Filaments - bundles of flagella which wrap around the cell body between the cell wall and the outer membrane; together they form a helical bulge that moves like a corkscrew as the entrapped flagella turn & propel the cell; found only in one type of bacteria called the spirochetes; this unique form of movement is well suited to the viscous environment (mud & mucous) where the bacteria is generally found. Ex. of bacteria with a.f. Treponema (causes syphilis) and Borrelia (causes Lyme disease).

B. Cell Envelope (layers from outside to inside) (BE ABLE TO DIAGRAM!)

1. Glycocalyx - found in most bacteria; slimy or gummy substance that becomes the outermost layer of the cell envelope; a thick glycocalyx is often called a capsule; a thin glycocalyx is often called a slime layer; functions: a. protection from drying out b. helps a cell adhere to a surface where conditions are favorable for growth c. provide protection against phagocytosis (engulfment & destruction by cells such as white blood cells) - a slippery glycocalyx makes it difficult for the phagocyte to grab hold of the bacterium. 2. Outer Membrane - primarily found in gram negative bacteria (ex. E. coli,

Salmonella, Shigella, Pseudomonas, Proteus, Neisseria gonorrhoeae); composed


of a bilayer membrane; the inner layer is composed of phospholipids; the outer layer is composed of lipopolysaccharides (LPSs), a compound that's not found in any other living organism!; part of the LPS is hydrophobic, part is hydrophilic; most molecules are transported across the outer membrane and into the cell through special proteins called porins; these porins create small pores or

channels in the outer membrane that allow molecules to diffuse in; function of the outer membrane is mainly protection - because of the outer membrane, gram negative bacteria are generally more resistant than gram positive bacteria to many toxic compounds, including antibiotics (antibiotics are too large to diffuse through the porins).

More about LPSs These compounds are endotoxins and are only released when the bacteria die and their cell walls are broken down. Endotoxins cause fever and dilate blood vessels (drop in blood pressure results). Killing the bacteria may increase the concentrations of this toxin!

3. The Cell Wall - The structure described below is found in all eubacteria except the mycoplasmas (these bacteria lack a cell wall); in archaeobacteria, the cell walls are composed of a different type of peptidoglycan or protein & some do not have cell walls. In gram negative bacteria, the cell wall lies just inside the periplasm; in gram positive bacteria, it lies just inside the glycocalyx, if one exists. a. Structure & Composition of Cell Wall in Eubacteria 1.) The chief component is peptidoglycan. 2.) Peptidoglycan is composed of long chains of polysaccharides (glycan) cross-linked by short proteins (peptides). 3.) When linked together these chains create the single rigid meshlike molecule that forms the bacterial cell wall (resembles a chain link fence!) 4.) A major difference between G(+) & G(-) bacterial cell walls: a.) G(-): peptidoglycan mesh is only one layer thick. b.) G(+): peptidoglycan wall is many layers thick.

b. Cell Wall Function In many cases, the cell wall is very porous and does not regulate the transport of substances into the cell. Two major functions of the cell wall are maintaining shape and withstanding turgor pressure. Both are discussed below.

1.) Cell Shape - one fxn. of the cell wall is to confer shape on the bacterium; most bacteria fall into one of these general groups. However, some bacteria have irregular shapes. Even bacteria of the same kind or within the same culture sometimes vary in size and shape (especially in aging cultures). a.) cocci (singular - coccus) - spherical b.) bacilli (singular - bacillus) - rod-shaped c.) spirilli (singular - spirillum) - spiral-shaped d.) vibrio - comma-shaped

In addition to these characteristic cell shapes, cells can also be found in distinctive groups of cells: pairs, chains, tetrads (cubes), grape-like clusters, etc.

2.) Withstanding Turgor pressure A cell's turgor pressure is the internal pressure from its contents. Ordinarily, a bacterium is in a hypotonic solution (a more dilute solution that has less solute and more water than the inside of the bacterium) and water tries to move from a high water concentration to a low water concentration; that is, water tries to move inside the bacterium (see tonicity under osmosis later in the handout). Without the cell wall, the water would continue to more inside the cell, and the cell would lyse or burst; the cell wall withstands turgor pressure, so that the cell does not lyse.

Action of some antibiotics (ex. penicillin) - Bacteria produce enzymes that reseal breaks in the peptidoglycan cell wall that occur during normal growth and division; penicillin binds to these enzymes, inactivating the enzymes so that the breaks cannot be resealed. The bacteria then lyse.

Lysozyme, an enzyme found in tears, digests (breaks down) peptidoglycan. c. Mycoplasmas - group of bacteria that lack a cell wall; they avoid lysis from turgor pressure by maintaining a nearly equal pressure between their cytoplasm and their external environment by actively pumping sodium ions out of the cell; additionally, their cell membranes are strengthened because they contain cholesterol, a lipid found in eukaryotic cell membranes.

4. Periplasm - used to be called a space, because of the way it looked in electron micrographs; found between the cell membrane and the peptidoglycan cell wall; therefore, only found in gram negative cells; composed of a gelatinous material containing proteins; one function of these proteins is that break down certain nutrients into smaller molecules that can pass through the cell membrane.

5. Plasma or Cell Membrane - membrane that encloses the cytoplasm of any cell; major function is to contain the cytoplasm and to transport and regulate what comes in and what goes out of the cell. Many prokaryotic cell membranes are similar to eukaryotic cell membranes. Its structure is referred to as the Fluid Mosaic Model, because the structure behaves more like a fluid than a solid. Contains: Membrane Lipids: (composed primarily of phospholipid molecules) a.) phospholipid bilayer (hydrophobic fatty acid tails & hydrophilic phosphate heads review chemistry handout on phospholipids)

Membrane Proteins: (proteins float in the fluid lipid bilayer) a.) Integral proteins - inserted in the bilayer; mainly involved in transport. 1.) carrier proteins - bind to specific substances & transport them across the cell membrane. 2.) channel proteins - proteins with a channel through which small, water soluble substances move across the cell membrane. b.) Peripheral proteins - usually attached to membrane surface; some are enzymes; some are involved in the electron transport chain and/or photosynthesis (well talk about these processes in the metabolism chapter); others are involved in the changes in cell shape that occur during cell division.

Note: Archaeobacteria Cell Membranes - there are different kinds of bonds in the phospholipid molecules that link the lipids (tails) to the glycerol molecule (head); these bonds are stronger and may help these bacteria survive extreme temperature and pH.

Cell Membrane Invaginations - the cell membrane sometimes invaginates or folds back on itself, forming structures that extend into the cytoplasm; since prokaryotic cells lack organelles, these invaginations provide increased surface area for peripheral proteins (enzymes) to catalyze chemical reactions.

C. Cytoplasm - matrix composed primarily of water (90%) & proteins. Contains the following:

1. Nucleoid - or nuclear region is a mass of DNA; well defined, although it is not surrounded by a membrane; most of a bacterium's DNA is arranged in a single

circular molecule called achromosome; some bacteria also contains smaller circular DNA molecules called plasmids (to be discussed later).

2. Ribosomes - site of protein synthesis; prokaryotic ribosomes are smaller than eukaryotic ribosomes. Antibiotics such as tetracycline, erythromycin, and streptomycin can specifically target bacterial ribosomes & not harm the host's eukaryotic ribosomes.

3. Endospores - extremely hardy, resting (non-growing) structures that some bacteria, principally G(+), produce through the process of sporulation when nutrients are exhausted; when favorable conditions return, endospores germinate to produce new vegetative cells, which grow & reproduce; they are able to withstand harsh environmental conditions because they contain so little water and high concentrations of calcium and dipicolinic acid; when favorable conditions return, the spore germinates into a new vegetative cell.

Some of endospore-producing bacteria are pathogenic to humans. Ex. Clostridium tetani causes tetanus (other species of this genus cause botulism and gas gangrene). Bacillus is another genus of bacteria that forms spores. We will learn how to stain bacteria so you may observe these spores. III. EUKARYOTIC CELL STRUCTURE A. Appendages 1. Cilia - short, hairlike, motile cellular extensions that occur on the surfaces of certain cells; ex. some protozoa (called Ciliates) use cilia for motility & feeding. 2. Flagella - in humans, the single, long, hairlike cellular extension that occurs in sperm cells; beat in waves (prokaryotic flagella rotate!); some protozoans use flagella for motility.

B. Cell Wall 1. Animal cells - no cell wall! 2. Plant cells - made of cellulose 3. Fungi - in most made of cellulose; some made of chitin (polysaccharide containing nitrogen - similar to exoskeletons of insects) and cellulose. 4. Algae - made of cellulose 5. Protozoans - no cell wall!

C. Glycocalyx - A glycocalyx may exist outside the plasma membrane; composed of carbohydrate chains from glycoproteins in cell membrane.

D. Plasma Membrane - already described; differences are between prokaryotes & eukaryotes: 1. proteins involved in electron transport chain and photosynthesis are not found in cell membrane, but are found in cytoplasmic organelles (mitochondria and chloroplast respectively), and 2. cell membrane contains cholesterol (in prokaryotes, only mycoplasmas have cholesterol in their cell membrane).

E. Cytoplasm

1. Cytoskeleton (not found in prokaryotes) a. structure - network of filamentous protein structures. b. functions - give the cell shape (support & rigidity); anchor the organelles; transport substances through the cell (cytoplasmic streaming), cytoplasmic streaming also enables some eukaryotes to move (formation of pseudopods); involved in cell division; involved in cell motility (flagella).

F. Nucleus 1. Structure in eukaryotic cells: a. nuclear envelope - double membrane with nuclear pores that surrounds the nucleus. b. chromosomes - genetic material composed of DNA & associated; chromosomes are linear. 2. Function: a. carrier of the hereditary information, which exerts a continuing influence over the ongoing activities of the cell through protein synthesis; "control center of the cell." b. isolates the DNA in eukaryotic cells.

G. Ribosomes (may be free in the cytoplasm or attached to rough endoplasmic reticulum & the nucleus) 1. Structure - not membrane-bound; made up of RNA & protein. 2. Function - sites of protein synthesis (where amino acids are assembled into polypeptides).

H. Membrane-bound Organelles - Eukaryotic cells have specializes membrane-bound organelles that carry out specific functions such as photosynthesis (chloroplasts), ATP production (mitochondria), lipid & protein synthesis (endoplasmic reticulum, golgi complex), cellular digestion (lysosomes), & transport (vesicles). We will not discuss these organelles in detail, since the focus of this class will be on prokaryotes. You will discuss these organelles in detail in Anatomy & Physiology I.

a. ENDOPLASMIC RETICULUM

1.) 2.)

Structure: interconnecting flattened sacs, tubes, & channels. Types & Functions: (both types support the cytoplasm & provide more surface area inside the cell for chemical reactions to take place) a.) rough E. R. - (ribosomes are attached to it) - function: initial modification of proteins; process: polypeptide chains are formed at the ribosome & some of them are transported into the r. e. r. for modification; the polypeptides are then packaged in transport vesicles or sacs (a piece of the e. r. pinches off around the polypeptide); these vesicles transport the polypeptides to the golgi complex for further modification into proteins. b.) smooth E. R. - (no ribosomes attached) - function: main site of lipid synthesis; lipids are then sent to the golgi body in transport vesicles for further modification & distribution.

b. GOLGI COMPLEX 1.) Structure - 4 to 8 flattened, membrane-bound sacs loosely stacked on top of one another surrounded by vesicles; looks like a stack of pancakes. 2.) Function - final modification of proteins & lipids. 3.) Process: transport vesicles from the r.e.r. or s.e.r. fuse with the golgi complex; proteins & lipids are processed in the golgi complex; the finished product is pinched off in a piece of golgi membrane (another vesicle) & is transported to the part of the cell where it is needed; the golgi complex processes, packages, & distributes the material the cell manufactures (the Wal-Mart distribution center).

c. VESICLES 1.) Structure - membrane-bound sacs that could be pinched off pieces of golgi complex, E.R., or cell membrane 2.) Function - transport material within the cell & into & out of the cell. 3.) Some specialized vesicles:

a.) Lysosomes - contain enzymes for breaking down proteins, lipids, etc. (digestion within the cell); they fuse with other vesicles (such as phagocytic vesicles) to degrade or digest their contents. b.) Peroxisomes contain enzymes (peroxisomes) that break down toxic hydrogen peroxide into water and oxygen (you see the oxygen bubbles when you apply hydrogen peroxide to tissue).

d. MITOCHONDRIA 1.) Structure - usually shown oval shaped; double membrane: smooth outer

membrane & a folded inner membrane (folds provide more surface area for chemical reactions to take place).
2.) Function - break down energy containing organic molecules (ex.

carbohydrates) & repackage the energy into smaller units (ATP) that can be used by the cells; called the "powerhouse" of the cell.

e. CYTOSKELETON 1.) Structure - network of filamentous protein structures called microtubules & microfilaments. 2.) Functions - give the cell shape (support), anchor the organelles, transport substances through the cell, involved in cell division.

f. CENTRIOLES 1.) Structure - paired cylindrical structures composed of protein filaments 2.) Function - during cell division, organize a microtubule network, called spindle fibers; spindle fibers are responsible for moving the chromosomes around in the cell during division.

IV. CELL MEMBRANE TRANSPORT

A. PASSIVE TYPES OF TRANSPORT ACROSS THE CELL MEMBRANE 1. Most passive transport processes depend on the process of DIFFUSION a. Definition - the net movement of particles from a greater concentration to a lower concentration (down a concentration gradient) to distribute the particles uniformly; it's apassive process - molecules move by their own kinetic energy - requires no energy expenditure by the cell (no ATP); molecules will diffuse freely until an equilibrium is reached (equal distribution on both sides)

b. Simple Diffusion through the Cell Membrane - The lipid interior of the cell membrane is a barrier to simple diffusion; most polar molecules (polar molecules get "stuck" in the nonpolar fatty acid tails). Small, nonpolar, lipid soluble molecules like fats, carbon dioxide, oxygen, & alcohol move easily through the cell membrane by simple diffusion. Polar & charged molecules can diffuse through the membrane if they are small enough to pass through pores in channel proteins.

c. Osmosis - a special case of diffusion; the movement of water across a semipermeable membrane - water moves from a high water concentration to a low water concentration (or from a low solute concentration to a high solute concentration); water moves across cellular membranes through pores in channel proteins or through momentary openings in the membrane. Tonicity: (describes the relative concentrations of solute in two fluids, such as the fluid inside & outside a cell); 3 cases: 1.) isotonic solutions ("iso" = same) - two or more solutions that have equal concentrations of solute.

2.) hypotonic solution ("hypo" = less) - one solution has less solute (more water) than the other; a cell that is in a hypotonic environment will lyse (burst); ex. placing a cell in distilled water would cause the cell to lyse - water would move into the cell to where the water concentration is lower. 3.) hypertonic solution ("hyper" = more) - one solution crenate (shrink), because the water

has more solute (less water) than the other; a cell that is in a hypertonic environment will in the cell moves out of the cell to an area of lower water concentration; ex. placing a cell in water with a high salt or sugar concentration would cause the cell to crenate water would move out of the cell to where the water concentration is lower.

Note: The above examples describe the environment that the cell is in (i. e., the solution is hypotonic or hypertonic to the cell). You can also talk about the cell in relation to its environment (i. e., the cell is hypertonic or hypotonic to its environment). You have to make this distinction!! The cells in our bodies try to maintain the isotonic condition so that they are not in danger of lysing or crenating.

d. Facilitated Diffusion - Again, only small, nonpolar molecules readily diffuse across the cell membrane. Polar & charged molecules get "stuck" in the fatty acid part of the lipid bilayer. Small, polar molecules, like water, and some ions can diffuse through channel proteins. Most biologically important molecules, however, are polar & are much larger than water (ex. glucose) and cannot fit through channel proteins. Special selective carrier proteins are located in the membrane to transport molecules like glucose. In facilitated diffusion, carrier proteins move molecules from a high concentration to a low concentration like in simple diffusion; it is believed that changes in the shape of the carrier protein allow it to envelop and then release the transported substance. Note: Few prokaryotes transport in this way; but may compounds, including most sugars, enter most eukaryotic cells in this way.

B. ACTIVE TYPES OF TRANSPORT ACROSS THE CELL MEMBRANE These processes use energy (ATP)!!!

1. Active Transport - Carrier proteins move molecules move from low concentration to high concentration (against the concentration gradient). Example: a. In prokaryotes - most nutrients are transported in this way because many prokaryotes live in low nutrient environments; group translocation is a form of active transport that occurs in some prokaryotes with certain molecules; in group translocation, a molecule is transported into the cell and at the same time chemically changed in to a slightly different molecule; this occur so that the molecule cannot leave the cell.

2. Vesicle Mediated Transport by Eukaryotes - We will concentrate on the type of vesicle mediated transport called endocytosis, since this is how white blood cells eat bacteria, etc.

a. Endocytosis - substances are imported into the cell; vesicles (sacs) are formed from the cell membrane, sometimes in response to the triggering of a receptor membrane protein (called receptor-mediated endocytosis); the cell membrane envelopes the substance to be imported & pinches off to form a vesicle that moves into the cytoplasm; endocytic vesicles can then fuse with enzymecontaining vesicles called lysosomes to digest their contents.

When solid material is imported into the cell, this type of endocytosis is specifically called phagocytosis ("cell eating"); ex. a white blood cell engulfing a bacteria.

Chapter 5....associated with the below chapters with respect to diseases Chapter 18-23 - Eukaryotic Organisms [Fungi, Protozoans, Helminths, & Arthropod Vectors]

Features that distinguish protozoal & helminthic infections : 1.) More important in tropical countries than in countries like the U. S. However, parasitic diseases are becoming more prevalent in the U. S. as more infected people move here; also because people with immune deficiencies such as AIDS are more susceptible to certain parasites. 2.) How the immune system responds to these parasites is a mystery. An immune response is activated, but the immune system is seldom able to rid the body of them. 3.) They have more complex life cycles, with multiple hosts involved.

I. FUNGI
A. Some General Characteristics: eukaryotic cells nonmotile heterotrophic (use organic compounds a carbon source; they cant make their own sugars; no photosynthesis) prefer more acidic conditions than bacteria can tolerate higher osmotic pressure and lower moisture than bacteria larger than bacteria and have more cellular and morphologic detail cannot tolerate the high temps. that bacteria can (fungal spores arent as resistant as bacterial spores)

most are aerobic; some are facultative anaerobes (ex. yeasts) & some are anaerobes

important in ecosystems as decomposers (called saprophytes - they obtain nutrients by decomposing dead & decaying matter); some are parasites, causing disease (mycosis; mycoses is plural); some produce toxins that cause disease (mycotoxicosis; mycotoxicoses is plural).

major cause of plant diseases the study of fungi is mycology

B. General Morphology: most, with exception of unicellular species, have a vegetative structure called a mycelium (a multinucleate mass of cytoplasm enclosed within a system of rigid, branched, tube-like filaments called hyphae). hyphae can be coenocytic (undivided network of branching tubes) or have septa (cross walls). cells walls are composed of cellulose, chitin (contains nitrogen - also found in the exoskeletons of insects, crayfish, etc.), or a combination of the two. specific morphology will be discussed later for each group of fungi

C. Reproduction - Fungi are classified by how they reproduce (sexually or asexually). [functions of spores include dissemination and reproduction]

1. Asexual Reproduction - Occurs by elongation of hyphae, budding, or asexual spore production.

Asexual spores are specialized cells that are dispersed & germinate in a favorable environment to produce a new fungus; they are products of a type of

cell division called mitosis(one cell divides to form 2 daughter cells that are identical to one another and to the original parent cell). Types: sporangiospores , conidiospores.

2. Sexual Reproduction - Occurs by producing sexual spores, which form following sexual fusion of gametes (similar to sperm & eggs). Types: zygospores, ascospores, & basidiospores.

D. 2 General Groups of Fungi - Yeasts vs. Molds


[These are descriptive terms, not taxonomic! These organisms belong to many groups of fungi.]

1. Yeasts - characteristics: nonfilamentous, unicellular reproduce asexually by budding reproduce sexually by producing various kinds of spores aerobic or facultative anaerobes used to prepare bread, wine, beer, etc. (fermentation of carbohydrates produces ethanol & carbon dioxide) ex. Saccharomyces cerevisiae (cervesa means beer in Spanish) some are pathogenic; ex. Candida albicans (causes yeast infections, thrush; see below)

2. Molds - characteristics: filamentous, multicellular

have a vegetative structure called a mycelium (a multinucleate mass of cytoplasm enclosed within a system of rigid, branched, tube-like filaments called hyphae).

hyphae can be coenocytic (undivided network of branching tubes) or have septa (cross walls).

also possess reproductive hyphae which produce different kinds of spores (discussed above and below)

see below for examples.

E. Classification of Some of the Lower Fungi:

1. Zygomycetes:

a. Characteristics: coenocytic hyphae, produce sporangiospores (asexual spores) & zygospores (sexual spores). b. Ex. Rhizopus nigricans - black mold that develops on stale bread; the tiny black dots on the mold are the sporangia, which hold the sporangiospores; sporangia look like tiny mushroom caps. c. can be opportunistic; some are pathogenic in the immnocompromised

F. Classification of Some of the Higher Fungi:

1. Ascomycetes (Sac Fungi)

a. Characteristics: includes molds with septate hyphae and some yeasts; ascospores (sexual spores) develop within sacs called asci (sing. ascus); also produce conidiospores(asexual spores).

b. Examples: 1.) Saccharomyces cerevisiae - yeast is used to make beer, bread, wine; cervesa means beer in Spanish. 2.) Trichophyton - causes athlete's foot (tinea pedis); ringworm of the feet; other species infect different parts of the body (dandruff, nail fungus, jock itch) 3.) Penicillium spp. - conidiospores form long chains on branching conidiophores, creating a brush-like structure that looks like a broom (penicillus means brush"); some species produce the antibiotic penicillin. 4.) Aspergillus spp. - form long chains on a globelike conidiophore; cause aspergillosis, a pulmonary disease of animals & humans; infection is often secondary to tuberculosis, immunodeficiency, & steroid therapy. 5.) Histoplasma capsulatum - causes Mississippi Valley fever (histoplasmosis); can get from bird droppings and bat guano; endemic disease in this area; pulmonary disease. 6.) Candida albicans - part of our natural flora; opportunistic; becomes a problem when defenses are weakened or balance of microbes is upset (ex. from antibiotic treatment); cause of vaginal & intestinal yeast infections & thrush in the mouth ("cottage cheese patches") - called candidiasis. 2. Basidiomycetes (Club Fungi) a. Characteristics: many form basidiocarps (mushrooms, puffballs, or shelflike bodies on trees); some are molds, a few are yeasts; produce conidiospores; also producebasidiospores (sexual spores); basidiospores form on the "gills" of mushroom basidiocarps.

b. Examples:
1.) Amanita - poisonous mushroom; toxin causes a mycotoxicosis

2.) Cryptococcus - yeast cells surrounded by a capsule; causes fatal meningitis (cryptococcosis); transmission inhalation of contaminated dust; found in 8% of AIDS patients.

3. Deuteromycetes (Imperfect Fungi) a. Characteristics: called the imperfect fungi because no sexual stage has been observed; we put them in this group until a sexual stage is observed; these fungi grow as yeasts or molds; identify on basis of shape & arrangement of their conidiospores (asexual spores); some species are pathogenic; many of these fungi have recently been placed in other phyla.

G. Dimorphic Fungi - Some fungi switch between a single-celled yeast phase of growth & a mycelial phase (called dimorphism); discovered by Pasteur; some species will switch if oxygen supply decreases. Pathogenic dimorphic fungi are mycelial outside of the host & single-celled inside the host. With pathogenic species, it is usually high body temperature that causes the switch. Candida changes in response to the higher nutrient concentrations found in the body. The problem with dimorphism is that single cells are more readily spread in bloodstream, leading to systemic infections.

H. Mycoses (Fungal Diseases) Humans usually acquire fungal disease from nature; they are not highly contagious. mycotoxicosis vs. opportunistic mycoses: See above for diseases Some produce toxins that are hallucinogenic; ex. muscarin - produced by a mushroom Some produce toxins that are highly poisonous; ex. 1.) Claviceps (rye mold) - produces ergot; causes death to anyone eating bread made from contaminated rye; LSD is made form fruiting structures (causes hallucinations)

2.) Aspergillus produces aflatoxin; which grows in many plant materials; low levels of toxin can be carcinogenic. 3.) Amanita - poisonous mushroom

I. Antibiotics: Penicillins produced by Penicillium ; Cephalosporins produced by Cephalosporium.

II. PROTOZOA
A. General Characteristics: Unicellular eukaryotes. The protistan lineages continue into the kingdoms of plants, fungi, and animals. Limited to a moist environment because they lack a cell wall Heterotrophs Most reproduce asexually by fission (one cell divides to form 2 identical daughter cells & budding; some (ex. Plasmodium that causes malaria) under go schizogony (multiple fission). Sexual reproduction occurs by conjugation, the fusion of vegetative cells, or by the fusion of specialized gametes called gametocytes. Some have complex life cycles, requiring multiple hosts and changing their morphology (ex. Plasmodium uses the mosquito as an intermediate host) Trophozoite - active, motile, feeding stage of protozoans; parasitic stage that causes the disease in the host. Cyst - resistant, inactive stage; how diseases are usually transmitted by the fecaloral route; usually more useful than trophozoites for lab identification.

B. Classification: [based on mode of locomotion or motility]

1. Mastigophora or Zoomastigophora (move by means of flagella) a. Trypanosoma gambiense - infects the blood and tissue fluids; causes African sleeping sickness (it leads to the loss of consciousness and death when it invades the CNS); can also infect cattle; vector is the tsetse fly. b. Giardia lamblia - body has the appearance of a human face (4 eyes are nuclei); have 2-6 flagella; form cysts; causes a waterborne dysentery (traveler's diarrhea); one of the dont drink the water diseases; firs sigh is usually an explosive, foul-smelling watery diarrhea followed by copious amounts of campers are a high-risk group because of asylvatic cycle (parasite is found in mountain streams contaminated with human feces or animal feces, especially beavers). c. Trichomonas vaginalis - causes vulvovaginitis; numerous flagella

2. Sarcodina (move by means of pseudopodia or "false feet" - temporary extensions of the cell body caused by protein filaments of the cytoskeleton pushing on the cell membrane); feed on algae, bacteria, and other protozoans by phagocytosis. a. Amoeba proteus - freshwater; not pathogenic b. Entamoeba histolytica - causes amoebic dysentery; usually acquired by consuming fecally contaminated water or food; flies and cockroaches can also be mechanical vectors; produce cysts; first protozoan to be shown to be a pathogen (1875); one of the dont drink the water diseases; trophozoites may invade the intestinal mucosa where they can cause ulceration and escape into the blood vessels; they may allow bacteria in fecal material to enter the body cavity and cause peritonitis. c. Naegleria fowleri - causes amoebic meningioencephalitis; usually seen in swimmers.

d. Acanthamoeba polyphaga accumulates on the water surface of contaminated hot tubs when tubs are covered; cause ulceration of the eyes and skin; can invade the central nervous system and cause meningioencephalitis.

3.

Ciliophora (move by means of cilia) a. Paramecium caudatum - freshwater; not pathogenic b. Balantidium coli - only ciliophoran that causes disease; produces cysts; causes diarrhea of large intestine; rare except in the Philippines; symptoms are similar to those of amoebic dysentery.

4.

Apicomplexa or Sporozoa or Haemosporina - Basically nonmotile. All have an infectious, sporelike stage (sporozoite) that is often transmitted to new hosts by an insect vector. All are parasitic (obligate parasites - cannot live apart from the host). Some have elaborate life cycles, changing body form (trophozoite, sporozoite, merozoite); life cycle includes schizogony (multiple fission). Examples a. Plasmodium vivax - causes malaria; vector is the mosquito; kills 1-3.5 million people each year; malaria = bad air; used to infect people with malaria to stop the progression of syphilis (fevers would kill the bacteria). b. Toxoplasma gondii - causes toxoplasmosis; humans acquire the disease by consuming cysts in the meat of infected animals or ingesting material contaminated by cat feces containing the parasite (can get it from cleaning the litter box - doctors warn pregnant women not to do this). c. Cryptosporidium - form cysts; cause enteritis & diarrhea; can occur in water supplies; can also be transmitted by fecal-oral transmission from kittens/puppies; resistant to chlorine (it can survive full-strength Chlorox!); threat only AIDS patients and those immunocompromised; no effective treatment found.

d. Pneumocystis carinii - may be a fungus!!; causes pneumocystis pneumonia; spread in respiratory droplets; common in AIDS patients.

III. HELMINTHS Flatworms & Roundworms

General Characteristics: Animals Cephalization - concentration of sensory receptors toward the anterior end. Organ/system level or organization/ Sexual reproduction. Most flatworms are monoecious (male & female reproductive organs in same animal). Roundworms are dioceious (separate sexes).

A. Platyhelminthes (Flatworms = Trematodes + Cestodes) - most are free-living; marine and freshwater; predators, scavengers, or parasitic; some have regenerative capabilities.

1. Trematoda (Flukes) - all parasitic of vertebrates; have complex life cycles that
include sexual and asexual phases; they require at least 2 kinds of organisms to complete the cycle - they reach sexual maturity in a primary or definitive host (always a vertebrate), their larval stages develop or become encysted in an intermediate host (usually an invertebrate). a. Clonorchis sinensis (Chinese or Human Liver Fluke) Adults live in bile ducts (in the liver) of humans (definitive host) Intermediate hosts: snail (first) and fish (second) Life cycle: a snail ingests the eggs; the eggs hatch & release a larval stage which goes through several transformation before finally forming a tadpole-like cercariae; the cercariae bore through the flesh of the snail,

& escape into the water; they swim until they find the appropriate species of fish; they encyst in the muscle tissues of the fish (forming metacercariae); the adult flukes develop in livers of humans who eat raw, infected fish; eggs of the parasite are excreted in the feces; when human feces end up in ponds, etc., snails ingest the eggs & the cycle repeats itself.]

b. Schistosoma mansoni (Schistosomes or Blood Flukes) - adults live in circulatory system; spiny eggs break through the blood vessel wall and through the gut wall to be expelled in feces; eggs hatch into cercaria in water; cercaria then penetrate skin when a person is bathing or swimming; cause spleen and liver enlargement, dysentery, and cirrhosis of the liver

2. Cestoda (Tapeworms) - intestinal parasites of vertebrates; no digestive system like


in trematodes & nematodes; they absorb nutrients through their tegument! Morphology: scolex (head) with suckers and/or hooks (for attachment), proglottids (body units - each one has male and female reproductive organs): immature proglottids (closest to the scolex), mature proglottids (next closest to the scolex), and gravid proglottids (furthest from the scolex - in these proglottids, the uterus is filled with eggs).

General life cycle of tapeworms: the gravid proglottids break off and are passed in the definitive host's feces; larval forms hatch when the eggs are ingested by the intermediate host; larvae then encyst in the intermediate host (called a cysticercus or bladder worm); adult worms usually develop in the definitive host when raw or poorly cooked infected meat is eaten. Examples:

a. Taenia solium (pork tapeworm) reaches a length of 2-7 meters; primary host: humans, etc.; intermediate host: swine

Humans can be infected with the adults by consuming rare pork containing cysticerci larvae; larvae then develop into adults in digestive tract of the human. Humans can also be infected with larval forms when they accidentally ingest eggs (they get them from other infected humans who contaminate food, etc. with the eggs when they dont use proper hygiene after going to the bathroom). In this case every organ in the body may harbor cysticerci. When a cysticercus dies, it releases toxins and usually causes a severe allergic reaction, which is sometimes fatal.

b. Taenia saginata (beef tapeworm) reaches a length of 5-25 meters; primary host: humans, etc.; intermediate host: cattle, sheep, etc.; life cycle similar to that of beef.

T. solium above; beef riddles with encysted larvae is called measly

c. Echinococcus granulosus (dog tapeworm); small - only 3 proglottids long; typical life cycle: dogs are infected by adults when they eat raw butchered livestock containing larvae (ex. raw bones, etc.) eggs are passed in feces of dog; livestock eat vegetation with eggs when grazing larvae hatch and encyst in the muscle tissue of livestock

Humans can get hydatid cysts (larvae) from ingesting the eggs (the eggs are passed in feces of dog, dog licks himself, then dog licks your face). These cysts develop in the liver, lungs, and brain. Each fluid-filledcyst, containing many larvae, can reach the size of a grapefruit.

d. Dipylidium caninum (dog & cat tapeworm) - often seen in children; flea is the intermediate host - it eats the eggs on an animal; larvae develop in flea; if a dog,

cat, or human ingests the flea, the adult will develop. Note: Larvae are not transmitted through the bite of the flea!!

e. Hymenolepis nana (dwarf tapeworm) - most common tapeworm of humans in the world; intermediate host is a grain beetle; humans can ingest the eggs in cereals and other foods that contain parts of the insects; intermediate host is optional (meaning that if you ingest the eggs of this worm, you get an adult infection).

B. Nematoda (Nematodes)

General characteristics: Nematodes are everywhere!!!! They are freeliving in soil, fresh & salt water, & are parasitic in plants and animals. Dioecious (separate sexes). Possess a nonliving cuticle, which is secreted by the epidermis and is resistant to the digestive enzymes of the hosts. More highly developed than flatworms. Adults do not latch onto the host like the tapeworms.

1. Ascaris lumbricoides (intestinal roundworm of pigs, horses, humans) largest intestinal nematode in humans. disease is called ascariasis Eggs can remain viable even in preservative! Females are longer; males are shorter & have a hooked posterior end.

2. Enterobius vermicularis (pinworm)

parasitizes large intestine of humans (especially children) at night the females migrate to the perianal region to lay eggs their presence there causes itching hosts become infected by accidentally ingesting the eggs.

3. Ancylostoma caninum & Necator americanus (hook worms) larvae can penetrate the skin of a barefoot person once inside, the larvae travels through the bloodstream to the lungs, moves up the trachea, is coughed up & swallowed; it matures in the small intestine. Ancyclostoma has fangs; Necator has cutting plates.

4. Trichinella spiralis (pork roundworm) humans usually become infected by eating insufficiently cooked pork larvae are encysted in the muscle tissues of the pig causes trichinosis; larval migration can cause death.

5. Wucheria bancrofti adults live in lymph nodes where obstruction of lymph vessels leads to grotesque enlargement of these nodes & to a condition called elephantiasis a mosquito is the intermediate host.

6. Dirofilaria immitis - causes heartworm disease in dogs; a few cases in humans & cats; mosquito is intermediate host.

7. Trichuris trichiura - (whipworm) - adults partially embed in the mucosa of the large intestine; each adult produces 1,000-7,000 eggs/day!

Chap. 7 - Microbial Growth

The term microbial growth refers to the growth of a population (or an increase in the number of cells), not to an increase in the size of the individual cell. Cell division leads to the growth of cells in the population. Two Types of Asexual Reproduction in Microbes: 1.) Binary Fission - Bacterial reproduction occurs through fission, a primitive form of cell division that does not employ a spindle fiber apparatus. [A spindle fiber apparatus made of protein filaments is responsible for moving the chromosomes around during cell division (mitosis & meiosis) in most eukaryotic cells. Bacteria do not have these structures.] The bacterial cell doubles in size and replicates its chromosome. Following DNA replication, the two chromosomes attach to separate sites on the plasma membrane, and the cell wall is laid down between them, producing two daughter cells.

2.) Budding - A few bacteria and some eukaryotes (including yeasts) may also replicate by budding,
forming a bubble-like growth that enlarges and separates from the parent cell.

I. Microbial Growth
A. Phases of Growth - A microbial lab culture typically passes through 4 distinct, sequential phases of growth that form the standard bacterial growth curve: (Not all growth phases occur in all cultures). See graph; be able to draw & label.

1. Lag Phase - In the lag phase, the number of cells doesn't increase. However, considerable metabolic activity is occurring as the cells prepare to grow. (This phase may not occur, if the cells used to inoculate a new culture are in the log phase & provided conditions are the same). 2. Log Phase (logarithmic or exponential phase) - cell numbers increase exponentially; during each generation time, the number of cells in the population increases by a factor of two). The number of microbes in an

exponentially increasing population increases slowly at first, then extremely rapidly. Organisms in a tube of culture medium can maintain log growth for only a limited time, as nutrients are used up, metabolic wastes accumulate, microobes suffer from oxygen depletion. 3. Stationary Phase - The number of cells doesn't increase, but changes in cells occur: cell become smaller and synthesize components to help them survive longer periods without growing (some may even produce endospores); the signal to enter this phase may have to do with overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.). 4. Death Phase - In this phase, cells begin to die out. Death occurs exponentially, but at a low rate. Death occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily die during this phase!

B.

Continuous Culture of Microbes

In the lab, cultures usually pass through all growth phases - not in nature. In nature, nutrients continuously enter the cell's environment at low concentrations, and populations grow continually at a low but steady rate. The growth rate is set by the concentration of the scarcest or limiting nutrient, not by the accumulation of metabolic byproducts - in nature there is always some other microbe that can use these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the media.

II. Measuring Numbers of Microbes


A. Indirect Measurements (measure a property of the mass of cells and then ESTIMATE the number of microbes)

1. Turbidity Can hold tube up to the light and look for cloudiness as evidence of growth (difficult to detect slight growth). A spectrophotometer can measure how much light a solution of microbial cell transmits; the greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the less light that will be transmitted. Disadvantages: Not sensitive in terms of numbers of bacterial cells & not useful for detecting minor contamination. 2. Metabolic Activity - 3 ways: a. The rate of formation of metabolic products, such as gases or acids, that a culture produces. b. The rate of utilization of a substrate, such as oxygen or glucose. c. The rate of reduction of certain dyes. Ex. methylene blue becomes colorless when reduced.

B. Direct Measurements - Give more accurate measurements of numbers of microbes. 1. Direct Counts - Coulter Counter - electronic counter; rapid & accurate only if bacterial cells are the only particles present in the solution. [gives a total count - live & dead cells]. 3. Plate Count Bacterial colonies are viewed through the magnifying glass against a colony-counting grid; called a Quebec colony counter (we have this in the lab). [gives a viable count] 4. Filtration - A known volume of liquid or air is drawn through a membrane filter by vacuum. The pores in the filter are too small for microbial cells to pass through. Then the filter is placed on an appropriate solid medium and incubated. The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered. This technique is great for concentrating a sample, ex. a swimming pool, where small populations may go undetected using some other methods. [gives a viable count]

III. Growth Factors - Microbes can exist in a great many environments because
they are small, easily dispersed, need only small quantities of nutrients, are diverse in their nutritional requirements.

A. Physical Factors 1. pH bacteria can classified as: a. acidophiles (acid-loving) grow best at a pH of 1 to 5.4; Ex. Lactobacilllus (ferments milk) b. neutrophiles exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in this category. c. alkaliphiles (base loving) exist from pH to 7.0 to 11.5; ex. Vibrio

cholerae (causes cholera)

2. Temperature bacteria can be classified as: a. psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC. b. mesophiles - grow best between 25oC and 40 C; human body temp is 37oC. c. thermophiles (heat-loving) 50oC to 60oC; found in compost heaps and in boiling hot springs.

3. Moisture only the spores of sport-forming bacteria can exist in a dormant state in a dry environment.

4. Hydrostatic pressure pressure exerted by standing water (ex. lakes, oceans, etc.); some bacteria can only survive in high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the enzymes lose their shape and denature and the cell dies.

5. Tonicity (hypotonic, hypertonic, isotonic) The use of salt as a preservative in curing meats and the use of sugar in making jellies is based on the fact that a hypertonic environment kills or inhibits microbial growth. Halophiles (salt lovers) inhabit the oceans.

6. Radiation UV rays and gamma rays can cause mutations in DNA and even kill microorganisms. Some bacteria have enzyme systems that can repair some mutations. B. Oxygen Requirements 1. strict or obligate anaerobes oxygen kills the bacteria; ex. Clostridium

tetani
2. strict or obligate aerobes lack of oxygen kills the bacteria; ex. Pserdomonas 3. facultative anaerobes can shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. E. coli, Staphylococcus 4. aerotolerant the bacteria dont use oxygen, but oxygen doesnt harm them; ex. Lactobacillus 5. microaerophiles like low oxygen concentrations and higher carbon dioxide concentrations; ex. Campylobacter

C. Nutritional (Biochemical) Factors Nutrients needed by microorganisms include: Carbon carbon containing compounds are needed as an energy source (ex. glucose) and for building blocks. Nitrogen - needed for amino acids and nucleotides; some can synthesize all 20 amino acids; others have to have some provided in their medium. Sulfur needed for amino acids, coenzymes,

Phosphorus needed for ATP, phospholipids, and nucleotides Vitamins a vitamin is an organic substance that an organism requires in small amounts and that is typically used as a coenzyme; many bacteria make their own, but some are required in the medium; microbes living in the human intestine manufacture vitamin K, needed for blood clotting, and some of the B vitamins, thus benefiting their host. Certain trace elements ex. copper, iron, zinc, sodium, chloride, potassium, calcium, etc.; often serve as cofactors in enzymatic reactions.

III.

CULTURING BACTERIA

A. Methods of Obtaining Pure Cultures (a culture that contains only 1 species of organism) 1. The Streak Plate Method Bacteria are picked up on a sterile wire loop, and the wire is moved lightly along the agar surface, depositing streaks of bacteria on the surface. The loop is flamed and a few bacteria are picked up from the region already deposited and streaked onto a new region. Fewer and fewer bacteria are deposited as the streaking continues, and the loop is flamed after each streaking. Individual organisms (individual cells) are deposited in the region streaked last. After the plate is incubated at a suitable growth temperature for the organism, small colonies (each derived from a single bacterial cell) appear. The loop is used to pick up a portion of an isolated colony and transfer it to another medium for study. The use of aseptic technique assures that the new medium will contain organisms of a single species. Well do this in lab.

IV.

CULTURE MEDIA

A. Types of Media

1. Synthetic medium prepared in the lab from materials of precise or reasonably well-defined composition. 2. Complex medium contains certain reasonably familiar materials but varies slightly in chemical composition from batch to batch (contains extracts from beef, yeasts, blood); ex. nutrient agar, nutrient broth

B. Selective & Differential Media (we will learn about these in detail in lab!)
1. Selective one that encourages the growth of some bacteria but suppresses the growth of others. 2. Differential has an ingredient that causes an observable change in the medium when a particular biochemical reaction occurs (ex. a color or pH change).

C. Controlling Oxygen Content of Media


1. Candle jars the inoculated tube or plate is placed in a jar; a candle is lit before the jar is sealed; the burning candle uses the oxygen in the jar and adds carbon dioxide to it; when the carbon dioxide extinguishes the flame, condition are optimum for the growth of microorganisms that require small amounts of carbon dioxide (ex. Neisseria gonorrhoeae) 2. Thioglycollate medium oxygen-binding agent added to the medium to prevent oxygen from exerting toxic effects on anaerobes; media is usually dispensed in sealed screw-cap tubes. 3. Anaerobic Chamber (Brewer Jar) A catalyst is added to a reservoir in the lid of the jar. Water is added to the gas-pak. Water is converted into hydrogen gas and carbon dioxide. The hydrogen gas can then bind with any oxygen in the jar to form water. A methylene blue test strip is included in the jar to ensure that anaerobic conditions are reached. When oxidized (oxygen is present) the strip is blue; when reduced (no oxygen), the strip is clear.

H2O

----------->

CO2

H2

H2

O2

----------->

H2O

Chapter 8 - Metabolism of Microbes

I. A Model for Metabolism (using E. coli as an example) What is metabolism? All the biochemical reactions that take place in a cell.

The Model: Metabolism leading to the synthesis of a new microbial cell has 3 requirements: 1. Raw Materials - nutrients composed of carbon (carbohydrates, proteins, etc.) 2. Driving Force a. energy - (ATP - adenosine triphosphate) - Some chemical produce ATP; some chemical reactions use ATP. b. reducing power - Many biochemical reactions involve oxidation (removal of electrons from a compound) & reduction (addition of electrons to a compound; E. coli stores electrons in coenzymes called NAD (nicotinamide adenine dinucleotide) & NADP (nicotinamide adenine dinucleotide phosphate). These compounds capture electrons in the form of hydrogen atoms from compounds that are being oxidized, thus forming NADH & NADPH). NAD & NADP stores the cell's reducing power. Bacteria will then use this reducing power to build its cellular components (it will reduce other compounds in this process). Hint: O.I.L. = oxidation is loss; R.I.G. = reduction is gain

3. A Plan - contained in the cell's DNA; this is the genetic code.

II.

Metabolism: An Overview
1. Entry Mechanisms - Raw materials from the environment are transported into the cells by various mechanisms. 2. Catabolic Reactions - reactions in which raw materials are broken down into smaller molecules (precursor molecules); there are 12 precursor molecules that are required to synthesize building blocks (amino acids, monosaccharides, nucleotides, fatty acids) that will build a new cell. 3. Anabolic Reactions - reactions that build up larger molecules from smaller ones: a.) biosynthesis - 12 precursor molecules are put together to produce building blocks. b.) polymerization - building blocks are joined together to form macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan); ex. amino acids are put together to form a protein, nucleotides are put together to form DNA. c.) assembly - macromolecules are assembled into biological structures; ex. peptidoglycan forms a cell wall.

A. Flow of Materials - Sequential Steps:

raw materials precursor molecules buliding blocks macromolecules B. Driving Force - Where is ATP & Reducing Power Produced & Used in the Above Reactions? 1. Entry Mechanisms - Many materials that move into the cell are moving from low to high concentration; this requires ATP; remember the bacterial is usually hypertonic to its environment. 2. Catabolic Reactions - In general, the catabolic reactions transform raw materials into precursor molecules, reducing power, & ATP.

3. Anabolic Reactions - In general, these reactions use precursor molecules, ATP, & reducing power.

III. Aerobic Metabolism (We'll use E. coli as the example)

(Aerobic means that this type of metabolism requires oxygen)

A. Catabolic Reactions - Supply the cell with the 12 precursor molecules, reducing power (NADH & NADPH), & ATP.

1. Precursor Molecules - A minimum of 3 different pathways are required to produce all 12 precursor molecules: (Well look at these later in this handout) a. glycolysis - produces 6 b. tricarboxylic acid (TCA) cycle - produces 4 c. pentose phosphate pathway - produces 2 (we wont discuss this one)

2. Reducing Power - Many steps in a catabolic pathway involve oxidation (loss of electrons)/reduction (gain of electrons) reactions. The electrons (along with hydrogens) are usually transferred from the molecule being oxidized to the coenzymes NAD or NADP, to form NADH & NADPH. NADH & NADPH are used to reduce other molecules or are used to generate new ATP molecules.

3.

ATP a. Stored Energy - The principal compound that stores chemical energy in all cells is ATP (adenosine triphosphate). The energy-storage capabilities of ATP depend on the 2 bonds that join the 3 phosphate groups in the ATP molecule. These bonds are among the most highly reactive bonds

found in biochemicals. They are called high-energy bonds. The phosphate groups that are joined in ATP by high-energy bonds are readily donated to other compounds. These compounds that receive a phosphate group from ATP are termedphosphorylated compounds. After receiving a high-energy phosphate, phosphorylated

compounds can then participate in chemical reactions that would not occur if the compounds were unphosphorylated. This is why we say that the energy "stored" in the bonds of ATP is used to drive other metabolic reactions.

b. ATP Formation from ADP

[most of the ATP is made by

chemiosmosis]

1.) Chemiosmosis & the Electron Transport System- The electron transport system is made up of a series or chain of compounds. Some of these compounds are hydrogen-carriers and some are electron-carriers. NADPH/NADH transfers hydrogen atoms to a hydrogen-carrier in the chain. This hydrogen-carrier then passes hydrogen electrons to an electron-carrier in the chain; the hydrogen ions are pumped out of the cell (or out of the inner mitochondrial compartment, if you're talking about eukaryotes). Each compound in the chain will then alternately accept & then release hydrogen electrons; as electrons are passed along the chain they drop to lower and lower energy levels. At the end of the chain, electrons are accepted by oxygen to form water. (The final electron acceptor is oxygen!! This is why this process is calledaerobic respiration!).

ATPase is an enzyme located in the cell membrane of prokaryotes; in eukaryotes it's located in the inner mitochondrial membrane. In E. coli, the pumping of hydrogen ions (H+) out of the cell creates a H+ concentration gradient & an electrical gradient across the cell membrane (there are more H+ outside the cell than inside). H+ then flows back into the cell (H+ ions want to move from a high concentration to a low concentration). The ions move

through the membrane-bound ATPase, converting ADP to ATP. This process is called chemiosmosis.

4. Catabolic Pathways (breaking down) - Chemical reaction sequences that transform raw materials into the 12 precursor molecules, store energy in the form of ATP, & store reducing power in the form of NADPH/NADH. Central metabolism (described below) begins with the monosaccharide glucose. Most organisms have other catabolic pathways, which use substrates other than glucose (ex. humans can use protein & lipids, if glucose concentrations are low). These pathways all eventually feed into central metabolism at various points. a. Glycolysis (= "glucose splitting") - This pathway begins with glucose (a 6 carbon sugar) and ends with 2 pyruvic acids or pyruvates (3 carbon molecules). The initial reactions in glycolysis require ATP. Later reactions in glycolysis produce a small amount of ATP. A small amount of NADH is also produced, which will result in the production of more ATP in the electron transport system. The main function of glycolysis is to split glucose.

b. Kreb's Cycle - Some of the pyruvate formed by glycolysis is used in biosynthesis, the rest is oxidized to another precursor molecule, acetyl CoA (coenzyme A). Acetyl-CoA then enters the Kreb's Cycle by combining with a precursor molecule oxaloacetate to form citrate. In a series of 6 reactions, carbon dioxide is released & oxaloacetate is regenerated (this is where most of the carbon dioxide you exhale comes from!). In this cycle, only a small amount of ATP is formed (called substrate level phosphorylation). However, considerable reducing power is stored in the form of NADH, NADPH, & FADH2 (flavine adenine dinucleotide), which are used to produce ATP in the electron transport system.

c. Pentose Phosphate Pathway - we wont discuss.

5. Anabolic Pathways (building up) - use precursor molecules, ATP, & reducing power produced in above catabolic reactions.

a. Biosynthesis - E. coli converts precursor molecules produced in catabolism into building blocks (amino acids, monosaccharides, nucleotides, fatty acids) of macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan). Organisms that can't make a given building block grow only if that molecule is provided in the medium (or diet). Ex. E. coli can make all 20 amino acids. Humans are unable to make 9 of the 20, so these nutrients must come from our diets.

b. Polymerization - In these reactions, building blocks produced in biosynthesis are joined to form macromolecules. The major cellular polymerization reactions are DNA replication, RNA synthesis, protein synthesis, & polysaccharide & peptidoglycan synthesis. For most macromolecules, building blocks must be joined in a specific order. For ex., amino acids must be arranged in the proper order to produce the right proteins. If you change the amino acid sequence, you change the proteins structure and therefore change its function. Some polymerization is determined directly by the organism's DNA (ex. DNA replication, RNA synthesis, protein synthesis). Other reactions are indirectly determined by DNA (ex. polysaccharide & peptidoglycan synthesis). In the latter, building blocks are ordered by the enzymes that catalyze the polymerization reactions. Because enzymes are proteins & protein structure is determined by DNA, the reactions the enzymes catalyze are indirectly determined by DNA.

c. Assembly - Assembly of macromolecules into cellular structures (ex. flagella, ribosomes, cell wall) may occur spontaneously (self-assembly) or as a result of reactions catalyzed by enzymes.

IV. Anaerobic Metabolism

Anaerobic metabolism allows cells to grow in the absence of oxygen. Strict anaerobes are capable of only anaerobic metabolism. Facultative anaerobes are capable of both aerobic & anaerobic metabolism. (ex. E. coli)

A. Anaerobic Respiration - Involves an electron transport chain, but uses a compound other than oxygen as the final electron acceptor, allowing the cell to generate ATP by chemiosmosis. Compounds that can be used as final electron acceptors include sulfate & nitrate. 1. Nitrate users: These organisms, including E. coli, play a role in the nitrogen cycle (removing nitrogen from terrestrial & aquatic environments & returning it to the atmosphere). Some microbes reduce nitrate to nitrite. Some microbes reduce nitrite further to nitrogen gas. Well see this in lab! 2. Sulfate users: These organisms, called sulfur reducers, play a role in the sulfur cycle. Sulfate is reduced to hydrogen sulfide gas. Sulfate reducers typically grow in marine & river mud flats, giving these environments a rotten-egg odor & turning the mud black. Well see this in lab!

B. Fermentation - This type of anaerobic metabolism uses no electron transport chain.

Some differences between fermentation & respiration (aerobic or anaerobic): 1. Fermentation generates fewer ATP per molecule of substrate. (ex. E. coli can produce 38 ATP/molecule of glucose by aerobic respiration, but only 3 ATP by fermentation.)

2. Because many molecules of substrate must be metabolized to supply a cell's ATP requirements, the substrate must be in abundance in order for the microbe to grow. 3. Sugars are usually the only substrate that can be used in fermentation. 1. Lactic Acid Fermentation - This type of fermentation is carried out by lactic acid bacteria, the microbes that cause milk to sour (used to produce yogurt & buttermilk). Muscle tissue of animals also carries out lactic acid fermentation, when deprived of oxygen (during strenuous exercise - this is what makes your muscles sore). Steps: 1. Glycolysis - Glucose is metabolized to produce 2 molecules of pyruvic acid (a little ATP & NADH is also produced). 2. Pyruvic Acid Oxidation - pyruvic acid is oxidized to lactic acid using NADH, thus using up the reducing power stored in glycolysis.

Parts of the Kreb's cycle & Pentose Phosphate pathway are used to help generate the 12 precursor metabolites; they cannot all be formed in fermentation.

2. Alcoholic Fermentation This type of fermentation is typical of yeast, a type of fungi. In this pathway, pyruvic acid is converted to carbon dioxide and ethanol. glucose pyruvic acid carbon dioxide + ethanol

V. Nutritional Classes of Microorganisms Microbes are classified according to nutritional class, which depends on 2 factors: 1.) How it generates ATP & reducing power: chemo- (from oxidation of inorganic compounds like sulfur nitrite, ammonia, iron) or photo- (sun)

2.) The source of carbon atoms it uses to make precursor metabolites: auto(obtain carbon from carbon dioxide) or hetero- (obtain carbon from organic compounds like carbs, proteins, lipids, etc.)

Can have combinations of all of these: chemoautotrophs, chemoheterotrophs, photoautotrophs, photoheterotrophs.

Chap. 9 & 10 Microbial Genetics (DNA Replication & Protein Synthesis), Recombinant DNA, Genetic Engineering

I. THE BASIS OF HEREDITY All information necessary for life is stored in an organisms chromosomes, which are made up of DNA (exception: some viruses only have RNA). A chromosome is circular (prokaryotes) or linear (eukaryotes). Remember the organic molecules stuff we covered at the beginning of the semester? Nucleic acids (DNA & RNA) are made up of building blocks called nucleotides (more on structure below). In DNA, nucleotides are arranged in a twisted double chain called a helix. The particular nucleotide sequence spells out the genetic code that provides information for the synthesis of new DNA (DNA replication necessary for cell division) and for the synthesis of proteins. A typical prokaryotic cell contains a single circular chromosome. Bacteria may also have a small, circular piece of extrachromosomal DNA called a plasmid. Human body cells, examples of eukaryotic cells, have 46 linear chromosomes. A gene, the basic unit of heredity, is a liner sequence of DNA nucleotides that form a functional unit of the chromosome or plasmid. All information for the structure and function of an organism is coded in its genes. The information in specific gene is not always the same; different versions of the same gene are called alleles. Using humans as an example, the hair color gene is always found at the same location on a chromosome, but the different versions or alleles that can exist for hair color are blond, brunette, red, etc. Because prokaryotes only have one chromosome, so they generally only have one allele for a particular gene. Many eukaryotes have 2 sets of chromosomes and thus 2 alleles of each gene, which may be the same or different. For example in humans, we have 46 chromosomes or 23 pair. In each pair, you get one chromosome from your mom and one for

your dad. You may have a blonde hair allele from your mom and a dark hair allele from your dad (so you get dark hair since the dark hair allele is dominant).

II. DNA STRUCTURE - THE WATSON-CRICK MODEL


deoxyribonucleic acid]

[DNA =

In the Watson-Crick model, the DNA molecule is a double-stranded helix, shaped like a twisted "ladder." Remember that nucleic acids (DNA & RNA) are made up of building blocks callednucleotides. Each nucleotide is made up of a sugar, a phosphate, and a nitrogenous base. When we put these nucleotides together to build a DNA ladder, the sides of the ladder are composed of alternating phosphate groups & sugar molecules. The rungs of the ladder are made up of paired nitrogenous bases joined in the middle by hydrogen bonds. The nitrogenous bases areadenine, thymine, guanine, & cytosine; adenine always pairs with thymine (A-T or T-A) & guanine always pairs with cytosine (G-C or C-G) [This is called complementary base pairing]. These 4 bases spell out the genetic message or code! DNA enters into 2 kinds of reactions: 1.) Replication - replicates the DNA before cell division, so that each new daughter cell will receive a copy. 2.) Protein Synthesis (Gene Expression); 2 steps: transcription & translation

III. DNA REPLICATION IN PROKARYOTES


[Remember bacteria have a circular chromosome.]

Replication begins by an enzyme breaking the hydrogen bonds between the nitrogenous bases in the DNA molecule; the double stranded DNA molecule "unzips" down the middle, with the paired bases separating. As the 2 strands separate, they act as templates, each one directing the synthesis of a new complementary strand along its length.

If a nucleotide with thymine is present on the old strand, only a nucleotide with adenine can fit into place in the new strand; if a nucleotide with guanine is present on the old

strand, only a nucleotide with cytosine can fit into place in the new strand, & so on. This is called complementary base pairing. DNA replication is called semiconservative replication since half of the original DNA molecule is conserved in each new DNA molecule. Like other biochemical reactions, DNA replication requires a number of different enzymes, each catalyzing a particular step in the process.

IV. GENE EXPRESSION - PROTEIN SYNTHESIS


A. FROM DNA TO PROTEIN: THE ROLE OF RNA By the 1940's biologists realized that all biochemical activities of the cell depend on specific enzymes; even the synthesis of enzymes depends on enzymes! Remember that the DNA molecule is a code that contains instructions for biological function & structure. Proteins (enzymes) carry out these instructions. The linear sequence of amino acids in a protein determines its 3-D structure & it is this 3-D structure that determines the protein's function. The big question was: How does the sequence of bases in DNA specify the sequence of amino acids in proteins? The search for the answer to this question led to the discovery of RNA (ribonucleic acid), which is similar in structure to DNA (deoxyribonucleic acid).

Three types of RNA: 1. messenger RNA (mRNA) - single stranded; contains codons (3 base codes); mRNA is constructed to copy or transcribe DNA sequences. 2. ribosomal RNA (ribosomes!) (rRNA) - ribosomes "read" the code on the mRNA molecule & send for the tRNA molecule carrying the appropriate amino acid. 3. transfer RNA (tRNA) - clover leaf shaped; at least one kind for each of the 20 a. a. found in proteins; each tRNA molecule has 2 binding sites - one end, the anticodon (also a 3 base code), binds to the codon on the mRNA molecule; the other end of the tRNA molecule binds to a specific amino acid; each tRNA & its anticodon are specific for an a. a.!! Differences between RNA & DNA:

1.

RNA nucleotides contain a different sugar than DNA nucleotides. (ribose vs. deoxyribose).

2. 3.

RNA is single stranded - DNA is double stranded. In RNA, uracil replaces thymine. There is no thyamine in RNA!!! But, there is adenine.

B. TWO MAJOR EVENTS IN PROTEIN SYNTHESIS: 1. Transcription [mRNA copies or transcribes DNA sequences] This process is similar to what occurs in DNA replication. A segment of DNA uncoils unzips. Free RNA nucleotides, are then added one at a time to one end of the growing RNA chain. Cytosine in DNA dictates guanine in mRNA, guanine in DNA dictates cytosine in mRNA, adenine in DNA dictates uracil in mRNA, thymine in DNA dictates adenine in RNA. This complementary base pairing is just like what occurs in DNA replication. An enzyme catalyzes this process. After transcription the mRNA goes out in search of a ribosome. This mRNA molecule will now dictate the sequence of a. a. in a protein in the next step called translation.

2. Translation - actual synthesis of polypeptides or proteins; translate information from one language (nucleic acid base code) into another language (amino acids); remember, the sequence of amino acids (the protein's primary structure) determines what the protein's 3-D globular structure is going to be & structure determines function. a. Initiation - Begins when the ribosome attaches to the mRNA molecule, reading its first or START codon. The first tRNA comes into place to pair with the initiator codon of mRNA (it occupies the peptide site in the ribosome). The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine.

b. Elongation - The second codon of the mRNA molecule is then read and a tRNA with an anticodon complementary to the second mRNA codon attaches to the mRNA molecule; with its a. a. this second tRNA molecule occupies the aminoacyl site of the ribosome. When both the P & A sites are occupied, an enzyme forges a peptide bond between the 2 a. a. & the first tRNA is released. The first tRNA cannot be released until this peptide bond is formed, as it will take its a. a. with it!! The second tRNA is then transferred from the A site to the P site & a third tRNA is brought into the A site. The ribosome continues to move down the mRNA molecule in this fashion, "reading" the codons on the mRNA molecule & adding amino acids to the growing polypeptide chain. c. Termination - Toward the end of the coding sequence on the mRNA molecule is a codon that serves as a termination signal. There are no tRNA anticodons to complementary base pair with this codon. Translation stops and the polypeptide chain is freed from the ribosome. Enzymes in the cell then degrade the mRNA strand.

[In eukaryotic cells, the polypeptide is taken up by the rough e.r. & is modified into a 3-D protein; the proteins are then packaged into transport vesicles (a piece of the e. r. pinches off around the protein); these vesicles transport the proteins to the golgi complex for further modification; the finished protein is pinched off in a piece of golgi membrane (another vesicle) and is transported to the part of the cell where it is needed. In the prokaryotic cell, none of these organelles exist, modification/processing of the polypeptide into a protein occurs in the cytoplasm.]

The genetic code. The mRNA codons for the 20 universal amino acids.
See the table in your text of mRNA codons for the 20 amino acids. The 3-base codons are written to the left and the abbreviations of the amino acids they correspond to are written to the right. The amino acid abbreviations in the table are: Ala - alanine; Arg - arginine;

Asn - apararagine; Asp - aspartamine; Cys - cysteine; Glu - glutamic acid; Gln - glutamine; Gly - glycine; His - histidine; Ile - isoleucine; Leu - leucine; Lys - lysine; Met - methionine; Phe - phenylalanine; Pro - proline; Ser - serine; Thr - threonine; Trp - tryptophan; Tyr - tyrosine; Val - valine.

The code has been proven to be the same for all organisms from humans to bacteria - it's known as the universal genetic code.

Notice that most of the amino acids have more than one code (ex. Arg has 6 codes!). However, each code is specific for an amino acid (ex. UUU only codes for the amino acid Phe).

Three of the 64 codons do not specify amino acids. Instead they indicate STOP or termination of the translation process (they say "This is the end of the polypeptide.")

The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine. Since AUG is the only codon for methionine, when it occurs in the middle of a message, it is ignored as a START codon and is simply read as a methionine-specifying codon. V. MUTATIONS

A. A mutation is any chemical change in a cell's genotype (genes) that may or may not lead to changes in a cell's phenotype (specific characteristics displayed by the organism). Many different kinds of changes can occur (a single base pair can be changed, a segment of DNA can be removed, a segment can be moved to a different position, the order of a segment can be reversed, etc.). Mutations account for evolutionary changes in microorganisms and for alterations that produce different strains within species. Mutations often make an organism unable to synthesize one or more proteins. The absence of a protein often leads

to changes in the organism' structure or in its ability to metabolize a particular substance.

B. Spontaneous mutations occur by chance, usually during DNA replication. Only about one cell in a hundred million (108) has a mutation in any particular gene. Since full-grown cultures contain about 109 cells per milliliter, each milliliter contains about 10 cells with mutations in any particular gene. Because the bacterial chromosome contains about 3,500 genes, each ml of culture contains about 35,000 mutations that weren't present when the culture started growing. Wow, when you think about it thats a lot of mutations in just one ml!

C. Induced mutations are caused by chemical, physical, or biological agents called mutagens. 1. Chemical Mutagens ex. Nitrates and nitrites are added to foods such as hot dogs, sausage, and lunch meats for antibacterial action. Unfortunately these same compounds have been proved to cause similar mutations and cancer in lab animals 2. Physical Mutagens - Include UV light, X-rays, gamma radiation, & decay of radioactive elements; heat is slightly mutagenic.

D. Consequences of Mutations - Most mutations do not change the cell's phenotype. If the mutation changes the codon to another that encodes the same amino acid, the protein remains the same. For example if the DNA code is changed from AGA to AGG, the mRNA codon would change from UCU to UCC. Check your table! The amino acid would not change. The amino acid would stay serine. In this case the genotype is altered, but the phenotype stays the same. Having more than one codon for each amino acid allows for some mutations to occur, without affecting an organisms phenotype. A mutation that changes a codon to one that encodes a different a. a. may alter the protein only slightly if the new a. a. is similar to the original one. However, if a mutation changes an a. a. to a very different one, there may be a drastic change in the structure of the protein, causing major complications for the cell. For example,

if the structure of an enzyme called DNA polymerase was greatly altered, the cell would not be able to replicate its DNA and thus would not be able to multiply.

E. Repair of DNA Damage Bacteria & other organisms have enzymes that repair some mutations.

VI.

GENETIC TRANSFER

Gene transfer refers to the movement of genetic information between organisms. In most eukaryotes, it is an essential part of the organisms life cycle and usually occurs by sexual reproduction. Male and female parents produce sperm and egg which fuse to form a zygote, the first cell of a new individual. Of course, sexual reproduction does not occur in bacteria, but even they have mechanisms of genetic transfer. Gene transfer is significant because it greatly increases the genetic diversity of organisms. Weve already discussed how mutation account for some genetic diversity, but gene transfer between organisms accounts for even more. In recombinant DNA technology, genes from one species of organism are introduced into the genetic material of another

species of organism. For example, human genes can be inserted into the bacterial chromosome.
A. BACTERIAL PLASMIDS & CONJUGATION Most bacteria carry additional DNA molecules known as plasmids: 1. Plasmids are circular DNA molecules, much smaller than the bacterial chromosome. 2. Plasmids can move in and out of the bacterial chromosome. 3. Two important plasmids are fertility (F) plasmids and drug resistant (R) plasmids.

1. The F Plasmid - This plasmids contains about 25 genes, many of which control the production of F pili. F pili are long, rod-shaped protein structures that extend from the surface of cells containing the F plasmid. Cells that lack the F plasmid are known as female (recipient) or F(-) cells. Cells that possess the F plasmid are known as male (donor) or F(+) cells. F(+) cells attach themselves to F(-) cells by their pili and transfer a copy of an F plasmid to the F(-) cells through a pilus. The once F(-) cells are now F(+) and will now produce pili, because they now have the F plasmid that contains the plasmid genes that code for these pili. This transfer of DNA from one cell to another by cell-to-cell contact is known asconjugation and is a form of sexual recombination because new genetic material is introduced into the cell. This is as close to sex as bacteria get! 2. The R Plasmid - In 1959 a group of Japanese scientists discovered that resistance to certain antibiotics and other antibacterial drugs can be transferred from one bacterial cell to another. It was subsequently found that genes conveying drug resistance are often carried on plasmids. Over the last few decades, R factors have proliferated to the point that some infections are difficult to cure with antibiotics.

Note: Plasmids are very important to scientists involved in recombinant DNA research. Genes of interest can be inserted into plasmids. The plasmids are introduced to bacteria and the bacteria take them up by endocytosis. As the bacteria reproduce themselves by mitosis, they replicate the plasmid during interphase and pass it to their daughter cells. The plasmids can then be isolated from all of these bacterial cells and the gene of interest can be excised. In this way a large quantity of a gene of interest can be produced. We'll talk about this more later.

B. TRANSFORMATION - A genetic change in which DNA leaves one cell, exists for a time in the aqueous extracellular environment, & then is taken into another cell where it may become incorporated into the genome. Ex. Extracts from killed, encapsulated, virulent (disease causing) bacteria, when added to living, harmless, unencapsulated bacteria, can convert the latter to the virulent type. By endocytosis, the living, nonvirulent bacteria pick up the DNA from the dead, virulent bacteria and incorporate the DNA into their own DNA. The nonvirulent bacteria now have the genes that code for proteins that transform them into virulent bacteria.

A LITTLE ABOUT VIRUSES Viruses consist essentially of a molecule of nucleic acid (RNA or DNA) enclosed in a protein coat called a capsid. No cytoplasm, ribosomes, or other organelles are present. Viruses move from cell to cell, utilizing the host cell's chromosomes, enzyme systems, and organelles to replicate the viral nucleic acid and synthesize new capsid proteins. They are obligate parasites in that they can't multiply outside the host cell. Viruses that infect bacteria are called bacteriophages.

A virus will attach to a host cell and inject its nucleic acid into the host cell. The viral nucleic acid takes over the host cell's genetic material and causes it to help the virus replicate the viral nucleic acid and to produce its capsids. The new viruses are assembled in the host cell (nucleic acids are inserted into the capsids) and the host cell is lysed to release the new viruses to go and attack other cells. This cycle is known as the lytic cycle. Viruses that infect bacterial cells are called bacteriophages.

C. Temperance or Lysogeny This mechanism gives viruses the capacity to set up long-term relationships with their host cell. Instead of going through the lytic cycle, the virus nucleic acid remains integrated in the host cells chromosome. The virus may remain in this latent stage for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the integrated viral nucleic acid gets replicated along with the host cell's chromosome during cell division & is passed to daughter cells (& then they pass it to their daughter cells, & so on). Something (ex. temperature change, stress) may later trigger these latent viruses to go into the lytic cycle all at once, destroying all of the infected host cells.

D. Transduction - Viruses can serve as vectors or carriers of genetic information from one bacterium to another. During the reproduction of the virus in the bacterium, fragments ofbacterial DNA instead of viral nucleic acid may become accidentally incorporated into a viral capsid. Such "viruses" may be able to infect a new host cell, but they are not able to complete a lytic cycle. The genes they carry from a previous bacterial host may become incorporated into the chromosome of the new bacterial host, possibly giving the bacterium new characteristics (ex. drug resistance).

VII. GENETIC ENGINEERING


Genetic engineering refers to the purposeful manipulation of genetic material to alter the characteristics of an organism in a desired way. One of the most useful of all techniques of genetic engineering is the production of recombinant DNA DNA that contains information from two different species of organisms.

A. PROCESS: For example a particular human gene can be removed from a human chromosome. Recombinant DNA is then constructed by inserting that gene into a bacterial plasmid, which serves as a carrier. The recombinant DNA is then introduced

host bacterium, which takes up the plasmid. The host bacterial cell then divides and its daughter cells divide, producing millions of cells that all contain a copy of the human gene of interest. This process serves at least 2 purposes: 1.) Large quantities of the human gene of interest are produced. 2.) The bacteria can read the human gene of interest, producing the protein coded for on the gene by protein synthesis. The genetic code is universal! We can obtain large quantities of a particular protein using bacteria.

B. MEDICAL APPLICATIONS - (See Table 8.3 p. 211 for a more extensive list) Products such as insulin to treat diabetes, human growth hormone to treat dwarfism, blood-clotting proteins to treat hemophilia, antibiotics, and vaccines have all been produced by bacteria using recombinant DNA technology.

a. Vaccines - Immunization means deliberately introducing an antigen into the body that can provoke an immune response & the production of memory cells. The first injection elicits a primary immune response, which provokes the production of antibodies & memory cells to provide long-lasting protection against disease. Many vaccines are made from killed pathogens (called inactivated vaccines); pathogens can be killed/inactivated by heat or chemicals (such as formaldehyde). Too much heat denatures proteins (changes their shape). When the body comes into contact with the real thing it won't have the right antibodies and memory cells & the person may get the disease. If the pathogens arent heated enough, some live pathogens may be injected in the vaccine! Attenuated (weakened) pathogens are also used in vaccines; these have been cultured in abnormal conditions so that they are no longer pathogenic. Sometimes these organisms cause adverse side effects & can sometimes revert to their pathogenic forms, causing the disease. Using "genetically engineered viruses:" Recently, genetic engineering & recombinant DNA technology has allowed us to use bacteria to produce the protein antigens found in the protein capsids of certain viruses (remember, viruses don't have phospholipid cell membranes - they have proteins coats or capsids). Scientists determine the genetic code for these proteins & insert the gene into the chromosome of bacterial cells. The bacteria produce the proteins

coded for on the inserted genes when they go through their regular process of protein synthesis. These proteins can then be injected as a vaccine (your body doesn't care if the proteins are in the real viral capsid or if they were made by a bacterium; they are the same proteins & your body's immune system will respond to these antigens in the same way). "Genetically engineered viruses" (ex. hepatitis B, influenza, rabies) do not pose the same risks as inactivated and attenuated viruses!

This set of notes goes with the Bacterial unit. Intro. To Taxonomy & The Classification of the Bacteria

I.

Principles of Biological Classification

Organisms are classified into any particular group because they have certain common characteristics. Classification of organisms 1.) establishes the criteria for identifying organisms; 2.) it arranges related organisms into groups based on shared characteristics; and 3.) it provides important information on how organisms evolved.

A classification system based on collecting individuals into groups and groups into progressively more inclusive and broader groups is called a hierarchical scheme of classification. A basic principle of taxonomy is that members of higher-level groups share fewer characteristics than those in lower-level groups. For example, humans have backbones like all other vertebrates, but they share fewer characteristics with fish and birds than with other mammals. Likewise, nearly all bacteria have a cell wall, but in some the wall is G(+) and in others it is G (-).

Taxonomy is the science of classifying organisms.

A Swedish biologist names Carolus Linnaeus devised a taxonomic scheme that was both practical and adaptable to expanding information. The Linnaean scheme remains the basis for biological classification today in 2 regards: 1.) we continue to group organisms hierarchically, & 2.) we use his nomenclature (see below).

Species defined: groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups. Bacteria, with their variety of forms of genetic exchange, do not fit this definition neatly. Microbiologists use the term species, more as a category of convenience (bacteria are grouped based on similarities), existing more in the human mind than in the natural world.

The Linnaean scheme uses the following hierarchical classification scheme: Kingdom Phylum (division) Class Order Family Genus Specific epithet

Example: Humans syphilis) Kingdom Animalia Phylum Chordata [Subphylum - Vertebrata] Class Mammalia

Example: Treponema pallidum (causes

Kingdom Monera (Prokaryotae) Phylum Gracilicutes

Class Scotobacteria

Order Primates Family Hominidae Genus Homo Species - Homo sapiens

Order Spirochaetales Family Spriochaetaceae Genus Treponema Species Treponema pallidum

II. Binomial Nomenclature


Linnaeus introduced a binomial nomenclature (each organism is designated by 2 names). The first name is the organisms genus designation & the second is its specific epithet. Together, the 2 constitute the species name. The species name is always latinized and underlined or italicized. The genus designation is capitalized, but the specific epithet is not. Thus, the proper designation for humans is Homo sapiens (or Homo sapiens ). By convention, the genus designation can be replaced with an initial if the complete genus name has been used recently enough to avoid possible confusion. For example, the bacterium Staphylococcus aureus becomes S.

aureus . All eukaryotes & prokaryotes are named this way. Viruses are not!

III. Artificial & Natural Systems of Classification


1. Artificial - the Linnaean scheme; he grouped organisms on the basis of visible similarities, but does not indicate how closely organisms are related. 2. Natural - based on evolutionary (phylogenetic) relatedness.

IV.

Using a Taxonomic Key Biologists often use a taxonomic key to id. organisms according to their characteristics. The most common kind of key is a dichotomous key, which has paired statements describing characteristics or organisms. Paired statements present an either or choice, such that only one statement is true. Each

statement is followed by directions to go to another pair of statements until the name of the organism finally appears. See example given in class.

V.

The Five Kingdom Classification System

No single classification system is completely accepted by all biologists, but one of the most widely accepted is the five-kingdom system.

Kingdom Monera prokaryotic; unicellular; most have a cell wall; reproduction usually by binary fission; photosynthetic, some chemosynthetic; acquire nutrients from environment by absorption.
Kingdom Protista eukaryotes; most are unicellular (some are organized into colonies); cell wall present in some, absent in others; reproduction mostly asexual, sometimes sexual; some are photosynthetic; acquire nutrients from environment by absorption and ingestion; this group includes the algae (resemble plants), the protozoa (resemble animals), and the euglenoids (resemble both plants and animals). Kingdom Fungi eukaryotic; unicellular or multicellular; cell wall present; sexual and asexual reproduction; acquire nutrients by absorption of organic matter from dead organisms. Kingdom Plantae eukaryotic; multicellular; cell wall present; sexual and asexual reproduction; photosynthetic; acquire nutrients from environment by absorption. Kingdom Animalia - eukaryotic; multicellular; no cell wall; primarily sexual reproduction; acquire nutrients from environment by ingestion (some parasites by absorption).

VI.

The Three-Domain Classification System After the discovery of the archaeobacteria in the 1970s, scientists suggested that these organisms represented a third cell type and they proposed another scheme for the evolution of living things from a universal common ancestor. This common ancestor gave rise to the archaeobacteria, the urkaryotes, and the eubacteria (true bacteria). They hypothesized a group of urkaryotes that gave rise to the eukaryotes directly rather than by way of the prokaryotes. See figure 9.11 on p. 232. In 1990 Woese suggested a new taxonomic category, thedomain, to be erected above the level of kingdom. The three domains Woese proposes are shown in Figure 9.13 on p. 234. The domain Eukarya contains all those kingdoms of eukaryotic organism (animals, plants, fungi, and protists). The traditional kingdom Monera has been divided into 2 domains: the domain Bacteria (true bacteria ) and the domain Arachaea. The Archaea exhibit many differences from the Bacteria: Different cell membrane structure Cell wall present, but not composed of peptidoglycan First amino acid in proteins not methionine like in other bacteria and eukaryotes DNA Contains histone-like proteins similar to eukaryotes (true bacteria have no histone proteins) Live in only extreme environments (groups include extreme halophiles, extreme thermoacidophiles, and methanogens)

VII.

Classification of Bacteria

The artificial scheme of classification in Bergey's Manual of Systematic

Bacteriology is widely used. Bergeys Manual disregards evolutionary relationships


because they often group bacteria into assemblages that cannot be easily identified by standard laboratory procedures. Instead, the manual takes a strictly practical approach so that it can be used as a comprehensive & quick reference when accuracy & speed are important, as is often the case in diagnostic labs. Bergeys

Manual divides bacteria into 4 divisions on the basis of their cell wall [G(+) or G(-)],
their lack of a cell wall (mycoplasmas), & walls lacking peptidoglycan (archaeobacteria). Bacteria species in each division are assigned to one or two

sections; sections have no taxonomic standing; they are simply groups of bacteria, which share certain easily identifiable properties.

How do we identify bacteria? 1.) We begin with morphological characteristics (shape, arrangement, etc.), 2.) Rely primarily on physiological characteristics (ability to grow on a selective medium, metabolic end products, etc.). 3.) Knowing the source of the bacterium is also important. 4.) Can also use DNA probes.

THE FOLLOWING IS A LIST OF THE MEDICALLY IMPORTANT MEMBERS OF SELECTED SECTIONS DEFINED IN BERGEYS MANUAL OF SYSTEMATIC

BACTERIOLOGY.

I.

GRAM-NEGATIVE BACTERIA (eubacteria) - have an outer membrane, a periplasmic space, & a thin peptidoglycan cell wall.

A. Section 1 - Spirochetes - Distinguished by their corkscrew shape; possess axial filaments (bundled flagella contained within the periplasm) that enable them to move through viscous environments (mud, mucous). Some live harmlessly in our mouths. Ex. of pathogenic species:

Treponema pallidum syphilis, Borrelia burgdorferi - lyme disease (carried


by ticks)

Leptospira - leptospirosis

B. Section 2 - Aerobic/Microaerophilic, Motile, Helical/Vibrioid Bacteria Helical members are corkscrew shaped, but flagella are ordinary; vibrioid members are comma-shaped. Ex. of species:

Campylobacter jejuni - major cause of diarrhea


of the small intestine]

[hint: the jejunum is part

Helicobacter pylori - cause gastric ulcers in humans

C. Section 4 - Aerobic Rods & Cocci - large & diverse group. Ex. of species:

Bordetella pertussis - pertussis (whooping cough) Neisseria meningitidis - meningococcal meningitis (infection of meninges or
coverings of the brain/spinal cord)

Neisseria gonorrhoeae - gonorrhea Pseudomonas aeruginosa - important opportunistic pathogen; common cause
of infection in weakened hosts, such as burn victims; another species has been used to clean up oil spills.

Brucella brucellosis Legionella - pneumonia and other respiratory infections. Francisella - tularemia

D. Section 5 - Facultatively Anaerobic Rods - Grouped into 3 Families; many can be distinguished by their characteristic fermentation reactions; includes the enterics; Examples:

Salmonella typhi - typhoid fever; other species cause food poisoning Shigella spp. - shigellosis, a form of dysentery Yersenia pestis - bubonic plague

Vibrio cholerae - cholera Escherichia coli - some species cause diarrhea & dysentery; utis Enterobacter cloacae opportunistic infections Proteus vulgaris utis Vibrio cholerae - cholera Haemophilus influenzae - upper respiratory infections (epiglottitis, sinusitis,
ear infections), pneumonia, & meningitis.

Zymomonas - alcoholic fermentation; used to make tequilla Klebsiella pneumoniae - pneumonia; utis

E. Section 6 - Anaerobic Straight, Curved, & Helical Rods - most abundant microbes in mouth & intestinal tract; Example: Bacteroides gingivalis - causes gingvivitis & peridontal disease. Other species cause digestive & respiratory infections, utis, infections of wounds.

F. Section 9 - The Rickettsias & Chlamydias - Once thought to be viruses because of small size. Most species are obligate intracellular parasites & can't be cultivated outside a living host cell. In general, rickettsial pathogens are transmitted by arthropods (ticks, lice, mites, fleas); chlamydiae are spread directly from one infected human to another. Chlamydiae alternate between 2 cell types, elementary bodies and vegetative cells. Elementary bodies are tiny, round structures released when an infected host cell lyses. When phagocytized, they differentiate into rod-shaped vegetative cells that multiply within the host cell [This is different from other bacteria which do not invade the host cell!]. They then differentiate into elementary bodies again before the host cell lyses. Examples:

Rickettsia spp. - typhus (transmitted by body lice & rat fleas), Rocky Mt.
Spotted Fever (transmitted by ticks)

Coxiella Q fever Chlamydia trachomatis - trachoma, sexually transmissible nongonococcal


urethritis or NGU.

Chlamydia psittaci - ornithosis (parrot fever) (a respiratory disease)

II.

MYCOPLASMAS (eubacteria) - Section 10 - All lack a rigid cell wall. To maintain turgor pressure: 1.) their cell membrane contain sterols to add strength (sterols are also found in eukaryotic cell membranes), and 2.) they maintain their cytoplasm at the same pressure as their external environment by actively pumping sodium ion out of the cell. All are parasites of humans, animals, or plants. Almost all are obligate fermenters (they ferment even in the presence of oxygen). Their colonies have a distinctive fried egg appearance. They have various shapes, but when growth conditions are suboptimal, they become distorted, forming long strands that resemble fungi (thus accounting for the name myco , which means "fungus"). Their wall-less structure allows them to squeeze through even the tiny pores in filters used to sterilize liquids. Mycoplasma pneumoniae - common cold & primary atypical pneumonia (walking pneumonia)

III.

GRAM-POSITIVE BACTERIA (eubacteria) - lack an outer membrane & a periplasmic space; have a thick peptidoglycan cell wall.

A.

Section 12 - Cocci - large group. Some examples:

Micrococcus spp. - normal inhabitant of human skin; often contaminants on


agar plates.

Staphylococcus spp. - normal inhabitant of human skin; many species


produce carotenoid pigments, giving colonies characteristic yellow & orange colors

Staphylococcus aureus - major human pathogen; can infect almost any tissue
in the body; causes impetigo, pneumonia, food poisoning; causes many nosocomial (hospital-acquired) infections.

Streptococcus - S. pyogenes causes strep throat, scarlet fever, rheumatic


fever, endocarditis; S. pneumoniae causes life-threatening pneumococcal pneumonia & meningitis; S. mutans causes dental plaque.

B. Section 13 - Endospore-Forming Rods & Cocci - These bacteria are the most heat-resistant living things; they are used as an index of sterilization; location of endospore can be used to distinguish species. Some examples:

Clostridium spp. - all strict anaerobes, inhabiting soil & mud; C. tetani causes
tetanus (fatal rigid paralysis); C. perfringens causes gas gangrene & food poisoning, C. difficile causes iatrogenic (medically induced) diarrhea when antibiotics upset the normal balance of intestinal microbes; C. botulinum causes botulism (food poisoning); some species are harmless.

Bacillus spp. - aerobes, some facultative anaerobes; B. anthracis - causes


anthrax; B. cereus causes food poisoning.

C. Section 14 - Nonsporing Rods - Listeria monocytogenes food poisoning (listeriosis); in young, old, & immunocompromised patients it can cause a form of meningitis.

D. Section 15 - Irregular Nonsporing Rods - Members have irregular shapes (branched, club-shaped, etc.); shapes can change with growth phase of culture.

Propionibacterium acnes - causes acne

Corynebacterium diptheriae - causes diphtheria.

IV.

MYCOBACTERIUM (Section 16) - have a waxy outer layer composed of polysaccharides & mycolic acids; protects against hostile environments & affects staining; identified by the acid-fast stain procedure; Examples: Mycobacterium

tuberculosis - causes tuberculosis; Mycobacterium leprae - causes leprosy

V.

OTHER SECTIONS A. Section 27 - Actinomycetes with Multiocular Sporangia - Bacteria in this group grow as mycelia, masses of branching filamentous cells that resemble a mycelial fungus. They form spores within a multiocular sporangium, a manychambered swelling at the end of a filament. Dermatophilus spp. infects animals & sometimes human skin.

B. Section 29 - Streptomyces & Related Genera - These bacteria are also actinomycetes. Abundant in most soils (important in breakdown of organic matter). Odor of freshly turned soil comes from volatile compounds produced by these bacteria. Colonies have pastel colors, soil-like odor, & are hard & stick into agar. Members of this genus produce most of antibiotics in current use.

Chap. 6 - The Viruses

General Characteristics: virus means poison; someone once called them "a piece of bad news wrapped in a protein;" obligate intracellular parasites (can reproduce/replicate only inside a host cell) not cells; debate over whether or not they are considered alive (see below)

consist of nucleic acids (DNA or RNA) in a protein coat, called a capsid (no cell membrane) they insert themselves into a host cell & direct the host cell's metabolic machinery to make more virus; the virus supplies information (the plan) in the form of its nucleic acid - raw materials and driving force (ATP & reducing power) are supplied by the host cell. all cellular organisms can be attacked by viruses; however, viruses are very specific for the organisms & cells they infect.

Are Viruses Alive? Characteristics of living things: reproduction, metabolism, organized as cells, contain all organic molecules (lipids, enzymes, nucleic acids, carbs), evolution & adaptation to changing environments. Viruses have some of these char's: they can evolve, they contain some macromolecules, they direct their own reproduction; However, they are not cells - they do not have cytoplasm, a cell membrane, organelles, ribosomes, or a nucleus. They have DNA or RNA, unlike prokaryotic and eukaryotic cells, which have both. In addition, they lack a metabolism of their own (they cannot produce ATP, etc.) - raw materials and driving force (ATP & reducing power) are supplied by the host cell.

I. HOW ARE THEY CLASSIFIED? (4 ways: size, structure, host range, life
cycles)

A. Size - range from about 1/10th to 1/3rd the size of a small bacterial cell.

B. Structure - basic structure of a virus is a nucleic acid surrounded by a protein capsid; a membrane envelope may also be present outside of the capsid, but this is acquired from host cell. A complete viral particle (= capsid + nucleic acid + envelope if it is present) is called a virion.

1. Nucleic Acid - Viruses can store their genetic info. in different types of nucleic acid (each virus has only type). Viruses can have DNA or RNA. Their nucleic acid can be double stranded (ds) or single stranded (ss); they can even have double stranded RNA! RNA viruses can have a () sense strand or a (+) sense strand of RNA.

(+) sense RNA acts like mRNA and can be translated into proteins by the host cells ribosomes. (-) sense RNA does not make sense to the host cells ribosomes. After the virus containing this type of RNA enters the host cell, a complementary (+) sense strand is made from its (-) sense strand. Only (+) sense strand RNA can be read by the host cells ribosomes!

2. Capsids - protein coat that surrounds the nucleic acid; the constituent protein molecules making up the capsids are called capsomeres; there are 3 basic shapes based on how the capsomeres are arranged. See diagrams of these shapes!! a. helical - proteins fit together as a spiral to form a rod-shaped structure. b. polyhedral - proteins are arranged in equilateral triangles that fit together to form a geodesic dome-shaped structure; some appear almost spherical; you may have seen architectural structures that have this shape. c. complex - combination viruses with a helical portion (tail) attached to a polyhedral portion (head); ex. many bacteriophages; may also have a tail sheath (participates in injecting the viral nucleic acid into the host cell), plate, pins, & tail fibers (help virus attach to host cell).

3. Viral Envelopes - pieces of the host cell's cell membrane that the virus acquires as it emerges from its host cell; the virus pushes out of the cell

membrane, forming a bud that encloses the virus - then the bud pinches off behind, resealing the cell - as a result the host cell is not lysed. Glycoprotein spikes from the host cells glycocalyx may stick out of the envelope. Viruses that lack envelopes are called naked viruses. Because envelopes are acquired from hosts cell membranes, viruses may be hidden from attach by the hosts immune system. Envelopes also help viruses infect new cells by fusion of the envelope with the hosts cell membrane. On the other hand, enveloped viruses are damaged easily by physical and chemical antimicrobial agents.

C. Host Range - defined as the spectrum of organisms a virus attacks; viruses exhibit considerable specificity for hosts and even cells within that host; viral specificity is determined by whether or not a virus can attach to a cell. Attachment depends on the presence specific receptor sites on the surface of host cell and on specific attachment structures on the viral capsid or envelope. Examples of receptor sites are proteins, LPSs, glycolipids, or glycoproteins.

D. Life Cycles of Bacteriophages (viruses that infect bacteria means

bacteria eating)
1. Replication [= Lytic Cycle] See

diagram

Events:

a.

Adsorption - the virion attaches itself to a specific receptor site on the surface of the host cell.

b. c. happen:

Penetration - the viral nucleic acid penetrates the host cell Uncoating - removing the capsid & envelope; basically 2 ways it can

1.)

during penetration, the virion disassembles so that only the viral nucleic acid enters host cell

2.) later d. components

the entire virion enters the host cell & uncoating occurs

Viral Synthesis (Latent Period) (also called biosynthesis) - more viral

(nucleic acids & proteins for capsids) are synthesized by the host cell. d. Maturation (Assembly) - components are assembled into new viruses f. ways: 1.) If the virus is of the naked type, an encoded protein, lysozyme, dissolves the cell membrane &/or cell wall of the host cell, causing the cell to lyse & releasing the hundreds of viruses inside it. 2.) If the virus is to be an enveloped virus, it pushes out the cell membrane, forming a bud that encloses the virus - then the bud pinches off behind, resealing the host cell; as a result the host cell is not lysed. Release (Burst Period) - hundreds of intact virions exit host cell; 2

2. Lysogenic Cycle (Lysogeny or Temperance) - Temperence involves the capacity of certain viruses to set up long-term relationships with their host cells - the virus remains latent for many cellular generations by becoming integrated into a host cell's chromosome (the integrated viral DNA is called a prophage). In this case no new viral components are synthesized & the host cell is not harmed. The virus may remain latent for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the viral nucleic acid that becomes integrated into the host cell's chromosome gets replicated along with the host cell's chromosome and is passed to daughter cells during cell division. In the prophage state, some viral genes are expressed, which may slightly change the host cell's phenotype (ex. only lysogenic strains of Corynebacterium diphtheriae cause the disease diphtheria because the disease-causing toxin is encoded in the prophage of the infecting virus). Something (ex. temperature change) may trigger prophages to go into the lytic cycle. Released virions cannot infect

cells that are carrying the same prophage - it makes the cell immune to attack by a virion of the same phage.

II.

TAXONOMY
(ex. Retroviridae are called retroviruses). Genus names end in virus - species names are English words. Ex. Retroviridae, Lentivirus, Human Immunodeficiency Virus (HIV)

Family names all end in viridae ; family names are often converted into English

Groupings reflect only common characteristics and are not intended to represent evolutionary relationships.

III.

ANIMAL VIRUSES
A. Cultivating Animal Viruses & Diagnosing Viral Illnesses At one time animal viruses had to be cultivated & counted by infecting animals. In the 1930's it was discovered that embryonated chicken eggs could be used to culture animal viruses; embryonated eggs are inoculated with dilutions of a virus sample to determine the highest dilution that kills the embryo; this procedure was more economical & efficient than using adult animals. In the 1950's cell culture & tissue culture methods were developed. This solved the problem of viral specificity. Ex. Before cell cultures it was impossible to culture viruses in mice or chicken eggs that only infected humans (ex. HIV); continuous cell lines are usually derived from cancerous tissue & grow indefinitely in culture; regular cell lines grow increasingly slowly after 20-30 subcultures & eventually lose their ability to support viral replication; the most famous c.c.l. is the HeLa cell line (named after Helen Lack, the donor - from cervical cancer).

Important Note: Physicians rely on symptoms to diagnose most viral illnesses. Culturing viruses takes too long & antibodies in the blood can usually be detected only after patient has recovered. Viral infections sometimes affect human cells in ways that can be seen under the microscope. For ex. the measles virus causes the membranes of neighboring cells to fuse, creating giant, multinucleated cells. Some virus-infected cells can be id. because they contain inclusion bodies, collections of viral components such as capsids and nucleic acid, waiting to be assembled into new viral particles. For example, the rabies virus produces inclusion bodies called negribodies in infected nerve cells (this is what we look for in suspected cases of rabid animals - have to look for negribodies in brain - animals have to be euthanized).

B.

Replication of Animal Viruses (Lytic cycle) - proceeds through similar stages as bacteriophage replication. 1. Adsorption - Proteins in cell membrane act as receptor sites for a virus; remember, no cell walls in animal cells; adsorption is largely responsible for tissue specificity of animal viruses - only cells with a complementary receptor are attacked by a particular virus. 2. Penetration can occur in 3 ways: a. viral envelope fuses with cell membrane, emptying the rest of the virion inside the cell. b. other enveloped viruses enter by being phagocytized by a host cell c. most naked animal viruses enter as most bacteriophages do - the capsid adsorbs to cell surface & only the viral nucleic acid enters cell. 3. Uncoating - Envelopes/capsids are often removed in the penetration process; viruses that enter the cell partially or completely intact are uncoated inside the cell by the host cell's own hydrolytic enzymes, sometimes those in its lysosomes.

4. Viral Synthesis - The specifics of this process depend on which of the 5 types of nucleic acids is present in the virus. 5. Maturation - Assembly not really understood 6. Release - Enzymes cause lysis of the host cell or viruses "bud." Viruses that kill the host cell by causing lysis are called cytocidal. Viruses that damage the host cell but do not kill it are called cytopathic. Persistent viral infections can last for years, producing new virus particles by budding without killing the infected cell.

C. Latency (similar to temperance or lysogeny) - Sometimes the viral nucleic acid is integrated in the host cell's DNA (called a provirus), allowing the infected animal cells to function normally for years (just as a lysogenic bacteriophage or prophage does).

Ex. Typical of DNA viruses belonging to Herpesvirus family - herpes simplex 1 (causes fever blisters) causes a symptomless latent infection of nerve cells of mouth & lips - infection can be reactivated by a fever, a cold, too much sun, or stress.
Ex. Varicella Zoster (another Herpsevirus) causes chickenpox as the primary infection & shingles as the reactivation. Ex. HIV (Human Immunodefiency Virus) belongs to the Retrovirus family; causes AIDS (Acquired Immune Deficiency Syndrome).

D. Some Animal RNA Viruses

Retroviruses (Retroviridae) large group of RNA viruses; includes HIV (Human Immunodeficiency Virus) which causes AIDS (acquired immune deficiency syndrome); infects T cells (type of white blood cell). capsid contains 2 copies of the same (+) sense RNA molecule (called a diploid virus); capsid also contains the enzyme reverse transcriptase. Retro means "backward." This virus uses the enzyme reverse transcriptase to make DNA from its RNA. This DNA can be integrated into the host cell's chromosome. The proviral DNA can now be transcribed into mRNA and translated into viral proteins to assemble new viruses for release; As with prophages, the provirus can stay in a latent stage in which it is replicated along with host cell DNA, causing the host cell no damage. AZT (azidothymidine), which is used against HIV, helps stop reverse transcription by targeting the enzyme reverse transcriptase.

Flaviviridae enveloped; polyhedral capsid; (+) sense RNA includes Yellow Fever (hemorrhagic fever)

Togaviridae
enveloped; polyhedral capsid; (+) sense RNA includes Rubella virus (Rubella or German measles)

Picornaviridae naked; polyhedral capsid includes Enterovirus (causes polio); Rhinovirus (common cold); Hepatovirus (Hepatitis A)

Orthomyxoviridae - Influenza Viruses Flu viruses; 3 types (A, B, C); A is the most common, infecting many species of animals, including humans; A is responsible for many pandemics (worldwide epidemics); B & C only infect humans & do not cause pandemics; Outbreaks of B occur every 2-3 years; C causes mild cold-like illnesses. enveloped RNA viruses; protein spikes in envelope; its (-) sense RNA is divided into 8 separate pieces, each one packaged in a helical capsid This virus exhibits antigenic shift- sudden changes in properties that id. the virus as a foreign invader to the defenses of the human immune system; occurs from genetic changes that can occur when 2 different flu viruses infect the same cell; when this happens it is likely that the RNA molecules of the 2 infecting virions recombine in various ways among the new virions, producing a virus that is significantly different from either of the original infecting strains. This is why you can get the flu over and over again!

Rhabdoviridae enveloped; helical capsid; (-) sense RNA includes Rabies virus

Paramyxoviridae enveloped; helical capsid; (-) sense RNA includes viruses that cause Mumps, Measles, Viral pneumonia, Bronchitis

Bunyaviridae enveloped; segmented RNA; (-) sense RNA includes Hantavirus (4 corners disease)

Filoviridae
enveloped; filamentous capsid; (-) sense RNA includes Ebola virus

Reoviridae naked; polyhedral capsid; ds RNA includes Rotavirus (most common cause of diarrhea in infants and young children under the age of 2)

E. Some Animal DNA Viruses

Adenoviridae naked; polyhedral capsid; ds DNA mainly responsible for human respiratory diseases; also causes diarrhea in babies and young children

Herpesviridae - enveloped; polyhedral capsid; dsDNA Simplex virus Herpes simplex 1 (oral) and 2 (genital & neonatal) Varicellovirus Varicella zoster chicken pox and shingles Roseolovirus Roseola infantum roseola in infants (rash and fever) Lymphocryptovirus Epstein Barr virus causes infectious mononucleosis and Burkitts lymphoma; also linked to Hodgkins disease.

Poxviridae

enveloped; brick shaped capsid; ds DNA; largest of all viruses includes Orthopoxvirus small pox & cow pox

Papovaviridae naked; polyhedral capsid; ds DNA; replicate in nuclei of hosts cells. Includes Papillomavirus warts (some associated with cervical cancer)

Hepadnaviridae enveloped; mostly ds DNA; hepa = liver Hepatitis B virus

Parvoviridae naked; ssDNA; uses a helper virus to supply necessary component to produce more viruses. Includes Canine parvovirus causes severe and sometimes fatal gastroenteritis in dogs. Also includes Erythrovirus (B19) causes 5th disease (erythema infectiosum) deep red rash on childrens cheeks and ears and both a rash and arthritis in adults; can cross placenta and damage fetus.

F. Viruses and Cancer 1. Tumors - uncontrolled growth of tissue (cells are dividing out of control); most are benign (non-life threatening); some are malignant (they spread or metastasize to surrounding tissues).

2. Cancer malignant tumors that metastasize to surrounding tissues.

3. Cause Most human cancers arise form genetic mutations or cellular damage caused by environmental factors (chemicals - nicotine, pesticides; radiation - UV, X-rays, etc.; diet). About 15% are attributed to viral infections.

4. Examples: a. Human T-cell leukemia (blood cancer),

b. Epstein-Barr virus causes Burkitt's lymphoma c. Hepatitis B virus causes hepatocellular carcinoma (liver cancer) d. human papillomavirus causes skin & cervical cancers. e. Kaposis sarcoma thought to be associated with Herpesvirus 5. How? Some tumor viruses are retroviruses; they convert a normal cell

to a tumor cell by introducing an oncogene into it (oncogenic provirus). Some researchers believe even normal retroviruses might cause tumors - merely inserting a normal provirus into the host chromosome near a normal gene might alter its expression and convert it to an oncogene.

G.

Viruses and Teratogenesis Teratogenesis the induction of defects during embryonic development. A teratogen is a drug or other agent that induces such defects. Viruses are teratogens that can be transmitted across the placenta and infect the fetus. Cytomegalovirus (CMV), Herpes Simplex virus (HSV), and Rubella account for a large number of teratogenic effects. TORCH series is a series of blood tests used to detect antibodies to these viruses.

IV. VIROIDS

Defined - a circular molecule of ssRNA without a capsid; dont produce proteins

Cause several economically important plant diseases; none known to infect animals

1/10 the size of the smallest plant virus

How it causes disease is a mystery; one theory is that it interacts in some way with the host genome, changing the expression of the host genes to cause disease.

V. PRIONS

Defined: infectious agent composed only of protein

Affect the central nervous system.

Ex. scrapie of sheep, Creutzfeldt-Jakob disease (CJD) of humans, mad cow disease.

Not known exactly how it causes disease.

Chapter 11 - Controlling Microbes (Sterilization & Disinfection)

Some Important Terms Defined: sterilization - treatment to destroy all microbial life (even destroys bacterial endospores and fungal spores); there are no degrees of sterility! disinfection (sanitation) - treatment to reduce the number of pathogens to a level at which they pose no danger of disease; disinfectants are used to kill microbes on inanimate objects (most disinfectants are too harsh for use on delicate tissue); most disinfectants do not kill spores.

antisepsis - kill microbes or inhibit their growth on skin or other living tissue; antiseptics are applied to living tissue. sanitizer typically used on food-handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards (may mean just washing with soap in some cases). -static - treatments that inhibit rather than kill; ex. refrigeration. (bacteriostatic, fungistatic, etc.) -cidal - treatments that kill. (bactericidal, fungicidal, viricidal, etc.) general) (germicidal is a more

chemotherapeutic agents - chemicals, incl. antibiotics, used to treat disease (discussed in Chap. 12)

I. Physical Controls
A. Heat 1. Advantages - simple, inexpensive, effective penetrates to kill microbes throughout the object; best method if material being treated is not damaged by heat. 2. Mode of Action - denatures proteins. 3. Treatments a. Dry Heat Sterilization - ex. flaming loops, tubes in lab & hot air ovens (171oC, 1hr., 160oC for 2 hr., 121oC for 16 hrs.); used to sterilize materials that can withstand high temps. & any materials damaged by moisture. b. Moist Heat Sterilization - ex. boiling or in autoclaves; effective at a lower temperature than dry heat & it penetrates more quickly; disadvantages of boiling - does not kill thermophiles, endospores; autoclave is more effective than boiling- it uses pressure to raise the temperature above that of boiling (121oC, 15psi, for 20 min.); used to sterilize liquids and material easily charred; used in food canning & the lab to sterilize glassware & media. c. Pasteurization - limits growth, but does not sterilize; used to slow spoilage of milk & dairy products, wine, beer; advantage: causes minimal damage to the product; developed by Louis Pasteur; standard treatment: heat to 63oC for 30 min. or 72oC for 15 sec. B. Cold 1. Effect - microbiostatic; does not sterilize; slows down enzymes.

a.

Refrigeration - preserves food because it stops the growth of most species of microbes (slows chemical reactions); most disease-causing microbes are mesophiles, not psychrophiles; an exception is Listeria spp., which causes listeriosis (food poisoning). b. Freezing - kills most bacteria, but survivors can remain alive for long periods in the frozen state; bacteria cultures can be preserved by rapid freezing, sometimes with the addition of a compound called DMSO, milk, or glycerol to protect proteins. C. Radiation 1. Electromagnetic Spectrum - Radiation is classified by wavelength with ionizing and UV light radiation at the short-wavelength end, visible light in the middle, & radio waves at the long-wavelength end. The shorter the wavelength, the greater its energy, & the more lethal it is. Mode of Action: denatures DNA. 2. Two types of radiation that kill bacteria directly are UV (ultraviolet) Light & Ionizing Radiation. The effect of both is sterilization. a. UV Light - bacteria actually have special enzymes that can correct some damage done by UV light!; in the lab mercury vapor lamps (germicidal lamps) are used; disadvantages: kills only on surfaces & these wavelengths can also be harmful to humans.

b. Ionizing Radiation - 2 forms; both cause a chain of ionizations by stripping electrons from atoms, resulting in cell death; disadvantages: technically complex; is being used to sterilize some produce, much to the public's dismay. 1.) X rays 2.) Gamma rays D. Membrane Filtration 1. Effect - physically removes cellular organisms (not viruses - they are too small). 2. Uses - in lab, used with media, antibiotics, & other heat sensitive materials; filtration is replacing pasteurization in some causes, because filtration causes even less damage; you may have heard of the new "cold filtered" beers. E. Drying 1. Defined - the removal of water. 2. Two processes: a. evaporation involving heat - effect - kills many microbes; rarely used in lab because the high heat causes chemical changes (denaturation); is used in food industry.

b. lyophilization [freeze drying] - removes water directly by converting water from a solid state (ice) to a gaseous state; materials are frozen & placed in a chamber to which a partial vacuum is applied; avoids the chemical changes caused by heat drying; effect - stops microbial growth by stopping most chemical reactions (just like regular freezing) frequently used in the microbiology lab to preserve perishable materials such as proteins, blood products, & reference cultures of microbes; used in food industry to make instant coffee, etc.; disadvantage - expensive. F. Osmotic Strength 1. Method - high concentrations of salt or sugar. 2. Mode of action - microbes cannot grow if they are deprived of water; also, crenation or shrinkage can occur (you're placing the microbes in a hypertonic environment). 3. Disadvantage - once added, solutes (such as salt or sugar) cannot be easily removed; not used in lab.

II.

Chemical Control

The effectiveness of a chemical anitmicrobial

agent is affected by time, temperature, pH, and concentration.

A. Testing Germicides 3 ways: 1. Phenol coefficients: Germicides can be tested by comparing their effectiveness to phenol, a traditional germicide. It was phenol that Lister first used - he called it carbolic acid. The procedure involves preparing several dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen) or Staphylococcus aureus (a wound pathogen), incubating the tubes, and then checking for cloudiness in the tubes, indicating growth. The ratio of the effective dilution of the chemical agent to the dilution of phenol that has the same effect is the phenol coefficient. A disinfectant with a phenol coefficient of 1.0 has the same effectiveness as phenol. Less than 1.0 means its less effective. Greater than 1.0 means its more effective. 2. Paper disc method paper discs are saturated with the chemical agent and placed on the surface of an agar plate inoculated with a test organism. Clear zones of inhibition appear around the discs if the chemical agent is effective.

3. Use-dilution test The test microbe is added to dilutions of the chemical agent. The highest dilution that remains clear after incubation indicates a germicides effectiveness. B. Mechanisms of Action

1. Affect Proteins The alteration of protein structure is called denaturation. Denaturation can be permanent (bacteriocidal) or temporary (normal structure can be restored bacteriostatic). Mechanisms of denaturation include: a. Hydrolysis breakdown of a molecule by addition of water b. Oxidation addition of oxygen or removal of hydrogen c. Attachment of atoms or chemical groups ex. heavy metals (mercury), alkylating agents (ex. CH ) 2. Affect Membranes a. membrane proteins denaturation (see above) b. membrane lipids can be dissolved. 3. Affect Cell Wall Formation 4. Affect Nucleic Acid Structure 5. Affect Metabolism C. Types of Germicides 1. Surfactants a. Structure - compounds with hydrophilic & hydrophobic parts. b. Mode of action - Penetrate oily substances in water & break them apart into small droplets that become coated with surfactant molecules. The hydrophobic end of the surfactant stick into the droplets & the hydrophilic end is attracted to the water. The result is an emulsion, a fine suspension of oily droplets in water, which can now be rinsed away. c. Effect of soaps & detergents - wash away microbes, but do not kill them. d. Wetting agents are surfactants that are often used with other chemical agents to help the agent penetrate fatty substances. Surfactants are not germicidal by themselves! e. Quaternary ammonium salts four organic groups attached to a nitrogen atom. Effect: kill all classes of cellular microbes & enveloped viruses by disrupting membranes. Uses: nontoxic & widely used in the home,

industry, labs, & hospitals. Their effectiveness is decreased in the presence of soap. Actually support Pseudomonas growth! Now being mixed with other agents to overcome some of these problems. 2. Phenol & Phenolics a. Structure - compounds with hydroxyl groups (-0H) attached to a benzene ring. Mode of Action - denature cell proteins, disrupt cell membranes. b. Effect - kill most organisms; action is not impaired by organic materials (remain active even in the presence of blood, feces, etc.) c. Examples: 1.) Lysol 2.) Cresol found in creosote; plant derivative used to prevent the rotting of wooden posts, fences, railroad ties. 3.) Hexachlorophene - chlorinated phenolic; effective as an antiseptic; once widely used as an ingredient in soaps & lotions; in 1970's was found to increase risk of brain damage in babies; has now been replaced with chlorhexidine in hospitals good agent for surgical scrubs. Alcohols a. Structure - compounds with a hydroxyl group (-OH). b. Mode of Action when mixed with water disrupt lipids in cell membranes & denature proteins. c. Ethanol & Isopropanol - widely used as skin antiseptics; a 50 to 70% solution in water is the most effective concentration (one of the few exceptions to the rule: increase effectiveness by increasing concentration); does not sterilize skin because it evaporates quickly and does not penetrate deeply enough into skin pores. d. Main disadvantage - do not kill endospores. Halogens a. Mode of Action - inactivates enzymes by oxidation. b. Examples 1.) Iodine antiseptic a.) Tincture iodine in a dilute alcohol solution; one of first skin antiseptics. b.) Iodophor mixture of iodine and surfactants; ex. Betadine and Isodine (used for surgical scrubs and to prepare skin for surgery)

3.

4.

2.) Chlorine disinfectant; ingredient in household bleach; added to drinking water and swimming pools; inactivated by the presence of organic materials. 5. Hydrogen peroxide a. Mode of Action oxidizing agent (denatures proteins) b. Uses of H2O2: antiseptic for cleaning wounds, disinfect medical instruments & soft contact lenses. When H2O2 comes into contact with tissue, it bubbles producing oxygen gas. This is because all aerobes (incl. eukaryotes) produce the enzymes catalase & peroxidase which decompose H2O2 into oxygen & H2O. H2O2 generally kills microbes before it is destroyed by catalase or peroxidase. (You can differentiate between Staphylococcus & Streptococcus using H2O2; Staph is relatively resistant to H2O2 because of the large amounts of catalase & peroxidase it produces.) May be used to clean deep puncture wounds, because the oxygen produced kills obligate anaerobes present in the wound (ex. Clostridium). Heavy Metals a. Mode of Action - heavy metals (mercury, copper, silver) react with the sulfhydryl groups of proteins (denaturation) b. Effect - kills many microbes. c. Examples: 1.) Mercuric chloride - once widely used as an antiseptic; highly toxic; now merthiolate & mercurochrome are used (less toxic); merthiolate is prepared as a tincture; use - basic first aid kit supplies for disinfecting skin & mucous membranes. 2.) Silver Nitrate - once applied to eyes of newborns to prevent gonorrhea; the trend for a while was toward using antibiotics instead, but the development of antibiotic-resistant strains has necessitated the use of silver nitrate again. 3.) Selenium sulfide kills fungi, including spores; commonly used to treat fungal skin infections; included in dandruff shampoos (dandruff is often caused by a fungus). Alkylating Agents 1. Mode of action - they alkylate (attach short chains of carbon atoms) to proteins and nucleic acids. Must not be used where they may effect human cells (these agents are carcinogenic). 2. Formalin - 37% solution. of formaldehyde used to preserve tissues & to embalm; kills all microbes, including spores; lower concentrations are used to inactivate microbes for killed vaccines.

6.

7.

3. Glutaraldehyde - used to sterilize surgical instruments if equipment for heat sterilization is not readily available. 4. Ethylene oxide - gas; advantages: disappears from the object after treatment; disadvantage: extremely toxic to humans so must be used in a sealed chamber; kills all bacteria, including endospores; used to sterilize materials destroyed by heat (plastic, rubber gloves, animal feed, mattresses, telephones). 8. Dyes Ex. Crystal violet blocks cell wall synthesis. It effectively inhibits growth of G(+) bacteria in cultures and in skin infections. It can be used to treat yeast infections.

III. Food Preservation


A. Temperature - Environmental factor most often used to preserve food. Canning is the oldest method. Two factors, time & temp., determine safe heat treatments for canning. Refrigeration is low enough to stop the growth of most microbes. Psychrophilic (ex. Listeria) microbes are the exception. See pg. 1 of this handout for more info. on temp. B. pH - Acidity (low pH) prevents the growth of most microbes, especially in an anaerobic environment. Ex. adding vinegar (acetic acid) to foods. Low pH also increases the effectiveness of heat treatments (ex. acidic foods like tomatoes can be canned merely by boiling). C. Drying - drying & salting do not sterilize but preserve food by making it unable to support microbial growth for lack of water, an essential nutrient. See pg. 1 & 2 of this handout for more info. on drying & freeze drying. D. Chemicals - Various chemical preservatives are added to commercially prepared foods. Ex.: 1. calcium propionate - antifungal agent added to bread. 2. sorbic acid - antifungal agent added to soft drinks, salad dressings, cheeses. 4. sodium benzoate - antifungal agent added to soft drinks, salad dressings, cheeses. 5. sodium nitrate (nitrite) - antibacterial agent that prevents germination of Clostridium botulinum spores when added to bacon, ham, hot dogs.

The History and Scope of Microbiology

Study Outline
I. Microbiology - An Introduction A. Microbiology is the study of organisms too small to be clearly seen by the unaided eye (i.e., microorganisms); these include viruses, bacteria, archaea, protozoa, algae, and fungi B. Some microbes (e.g., algae and fungi) are large enough to be visible, but are still included in the field of microbiology; it has been suggested that microbiology be defined not only by the size of the organisms studied but by techniques employed to study them (isolation, sterilization, culture in artificial media) The Discovery of Microorganisms A. Invisible living creatures were thought to exist and were thought to be responsible for disease long before they were observed B. Antony van Leeuwenhoek (1632-1723) constructed microscopes and was the first person to observe and describe microorganisms accurately The Conflict over Spontaneous Generation A. The proponents of the concept of spontaneous generation claimed that living organisms could develop from nonliving or decomposing matter B. Francesco Redi (1626-1697) challenged this concept by showing that maggots on decaying meat came from fly eggs deposited on the meat, and not from the meat itself C. John Needham (1713-1781) showed that mutton broth boiled in flasks and then sealed could still develop microorganisms, which supported the theory of spontaneous generation D. Lazzaro Spallanzani (1729-1799) showed that flasks sealed and then boiled had no growth of microorganisms, and he proposed that air carried germs to the culture medium; he also commented that external air might be needed to support the growth of animals already in the medium; the latter concept was appealing to supporters of spontaneous generation E. Louis Pasteur (1822-1895) trapped airborne organisms in cotton; he also heated the necks of flasks, drawing them out into long curves, sterilized the media, and left the flasks open to the air; no growth was observed because dust particles carrying organisms did not reach the medium, instead they were trapped in the neck of the flask; if the necks were broken, dust would settle and the organisms would grow; in this way Pasteur disproved the theory of spontaneous generation F. John Tyndall (1820-1893) demonstrated that dust did carry microbes and that if dust was absent, the broth remained sterile-even if it was directly exposed to air; Tyndall also provided evidence for the existence of heat-resistant forms of bacteria The Role of Microorganisms in Disease A. Recognition of the relationship between microorganisms and disease 1. Agostino Bassi (1773-1856) showed that a silkworm disease was caused by a fungus 2. M. J. Berkeley (ca. 1845) demonstrated that the Great Potato Blight of Ireland was caused by a fungus 3. Louis Pasteur showed that the pine disease of silkworms was caused by a protozoan parasite 4. Joseph Lister (1872-1912) developed a system of surgery designed to prevent microorganisms from entering wounds; his patients had fewer postoperative infections, thereby providing indirect evidence that microorganisms were the causal agents of human disease; his published findings (1867) transformed the practice of surgery 5. Robert Koch (1843-1910), using criteria developed by his teacher, Jacob Henle (1809-1895), established the relationship between Bacillus anthracis and anthrax; his criteria became known as Koch's Postulates and are still used to establish the link between a particular microorganism

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and a particular disease: a. The microorganisms must be present in every case of the disease but absent from healthy individuals b. The suspected microorganisms must be isolated and grown in pure culture c. The same disease must result when the isolated microorganism is inoculated into a healthy host d. The same microorganism must be isolated again from the diseased host 6. Koch's work was independently confirmed by Pasteur B. The development of techniques for studying microbial pathogens 1. Koch and his associates developed techniques, reagents, and other materials for culturing bacterial pathogens on solid growth media; these enable microbiologists to isolate microbes in pure culture 2. Charles Chamberland (1851-1908) constructed a bacterial filter that removed bacteria and larger microbes from specimens; this led to the discovery of viruses as disease-causing agents C. Immunological studies 1. Edward Jenner (ca. 1798) used a vaccination procedure to protect individuals from smallpox 2. Louis Pasteur developed other vaccines including those for chicken cholera, anthrax, and rabies 3. Emil von Behring (1854-1917) and Shibasaburo Kitasato (1852-1931) induced the formation of diphtheria toxin antitoxins in rabbits; the antitoxins were effectively used to treat humans and provided evidence for humoral immunity 4. Elie Metchnikoff (1845-1916) demonstrated the existence of phagocytic cells in the blood, thus demonstrating cell-mediated immunity Industrial Microbiology and Microbial Ecology A. Louis Pasteur demonstrated that alcoholic fermentations were the result of microbial activity, that some organisms could decrease alcohol yield and sour the product, and that some fermentations were aerobic and some anaerobic; he also developed the process of pasteurization to preserve wine during storage B. Sergei Winogradsky (1856-1953) worked with soil bacteria and discovered that they could oxidize iron, sulfur, and ammonia to obtain energy; he also studied anaerobic nitrogen fixation and cellulose decomposition C. Martinus Beijerinck (1851-1931) isolated aerobic nitrogen-fixing bacteria, a rootnodule bacterium capable of fixing nitrogen, and sulfate reducing bacteria D. Beijerinck and Winogradsky pioneered the use of enrichment cultures and selective media The Members of the Microbial World A. Procaryotes have a relatively simple morphology and lack a true membranedelimited nucleus B. Eucaryotes are morphologically complex and have a true, membrane-enclosed nucleus C. In a commonly used classification scheme, organisms are divided into five kingdoms: the Monera or Procaryotae, Protista, Fungi, Animalia, and Plantae; microbiologists are concerned primarily with members of the first three kingdoms and also with viruses, which are not classified with living organisms D. Recently a classification scheme consisting of three domains (Bacteria, Archaea, and Eucarya) has become widely accepted; this scheme is followed in this textbook The Scope and Relevance of Microbiology A. Microorganisms were the first living organisms on the planet, live everywhere life is possible, are more numerous than any other kind of organism, and probably constitute the largest component of the earth's biomass B. The entire ecosystem depends on the activities of microorganisms, and microorganisms influence human society in countless ways C. Microbiology has an impact on many fields including medicine, agriculture, food

VIII.

science, ecology, genetics, biochemistry, and molecular biology Microbiologists may be interested in specific types of organisms: 1. Virologists-viruses 2. Bacteriologists-bacteria 3. Phycologists or Algologists-algae 4. Mycologists-fungi 5. Protozoologists-protozoa E. Microbiologists may be interested in various characteristics or activities of microorganisms: 1. Microbial morphology 2. Microbial cytology 3. Microbial physiology 4. Microbial ecology 5. Microbial genetics and molecular biology 6. Microbial taxonomy F. Microbiologists may have a more applied focus: 1. Medical microbiology, including immunology 2. Food and dairy microbiology 3. Public health microbiology 4. Agricultural microbiology 5. Industrial microbiology The Future of Microbiology A. Microbiology has had and will continue to have a profound influence on society. B. In the future microbiologists will be: 1. Trying to better understand and control existing, emerging, and reemerging infectious diseases 2. Studying the association between infectious agents and chronic diseases 3. Learning more about host defenses and host-pathogen interactions 4. Developing new uses for microbes in industry, agriculture, and environmental control 5. Still discovering the many microbes that have not yet been identified and cultured 6. Trying to better understand how microbes interact and communicate 7. Analyzing and interpreting the ever-increasing amount of data from genome studies 8. Continuing to use microbes as model systems for answering fundamental questions in biology 9. Assessing and communicating the potential impact of new discoveries and technologies on society D.

Chap. 13 - Epidemiology

I. SOME IMPORTANT DEFINITIONS:


Epidemiology - study of when & where diseases occur & how they are transmitted in human populations (focuses on groups of people rather than individuals); the modern definition does

not limit this study to that of epidemic diseases; knowing the source of the disease can help prevent transmission even while the causative microorganism (etiologic agent) is still unknown.

Epidemics - a pattern of disease transmission that affects many members of a population within a short time (ex. cholera in South America, flu, etc.).

Pandemic - an epidemic that spreads world wide (ex. AIDS, flu).

Endemic - numbers stay too low to constitute a public health concern (ex. chicken pox).

Sporadic - diseases occurring only occasionally in a population (ex. tetanus, trichinosis).

II. METHODS:

A. Sources of Information 1. vital statistics - birth, death, marriage, & divorce records 2. census data - number of people living in an area & their distribution by age, race, sex, marital status. 3. disease reports - doctors are required to report certain diseases to public health dept.; local public health stats are forwarded to state agencies (ex. Texas Dept. of Health) and theCenters for Disease Control & Prevention (CDC); the CDC prepares the Morbidity & Mortality Weekly Report

(MMWR) for the U.S.; stats included in this report:


a. morbidity rate the number of individuals affected by a disease during a set period in relation to the total number in the population (expressed as number of cases per 100,000 people per year). b. mortality rate the number of deaths due to a disease in a

population during a specific period in relation to the total population (expressed as number of deaths per 100,000 people per year). 4. other sources - surveys, questionnaires, interviews, hospital records.

B. Use of Stats 1. incidence rate number of new cases within a set population during a specified period of time divided by the total number of people in the population; incidence rates measure the growth or spread of a disease; ex. this stat tells us how many people develop AIDS in the U.S. per year. 2. prevalence rate - number of people who have a certain disease at any particular time (old and new cases) divided by the total number of people in the population; ex. this stat tells us how many people currently have AIDS in the U.S.

III. HOSPITAL EPIDEMIOLOGY NOSOCOMIAL INFECTIONS


Among patients admitted to hospitals each year about 10% (2 million) acquire a nosocomial infection; about 20,000 of those infected die from their infection.

A. Organisms Causing Nosocomial Infections

Escherichia. coli, Enterococcus, Staphylococcus aureus, and Pseudomonas are


responsible for one half of all nosocomial infections.

B. Factors Fostering Nosocomial Infections 1. immunocompromised patients people with AIDS, organ transplant recipients (they take immunosuppressants so that the organ will not be rejected by their body), the elderly, cancer patients, patients taking

steroids (ex. those with asthma).

2. invasive medical procedures - ex. blood drawing, i.v.'s, urinary catheters, endoscopes, implants, coronary bypass surgery, hemodialysis, gynecological equipment, tooth extractions, injections

3. antibiotic resistance - many bacteria found in hospitals have developed antibiotic resistance.

C. Types of Nosocomial Infection: (From most common to least common) 1. UTI's (urinary tract infections) - usually E. coli, Proteus, Klebsiella,

Enterobacter; can be from catheterization; more commonly results from


improper hygiene (wiping the wrong way). 2. surgical wound infections - most commonly Staphylococcus aureus & enterics; at least 10% of surgery patients develop an infection despite scrubbing, etc.! 3. respiratory tract (ex. pneumonia) - include Streptococcus,

Staphylococcus, Pseudomonas aeruginosa, enterics.


4. skin infections - particularly in newborns (usually Staphylococcus aureus ) & burn victims (usually

Pseudomonas aeruginosa ).

D. Nosocomial Infection Control 1. hospitals hire hospital epidemiologists. 2. once an epidemic is recognized, take cultures from hospital workers. 4. patient isolation; reverse isolation separates infection-prone patients from sources of infection (ex. the boy in the plastic bubble).

4. 5.

enforce CDC program. treat every patient as if they are infected with AIDS.

IV. PUBLIC HEALTH: PREVENTING DISEASE PROPHYLAXIS


Public health deals with disease prophylaxis (prevention); 2 methods of prophylaxis: 1.) decrease or eliminate the reservoir or interrupt disease transmission. 2.) immunization - artificially augments the body's natural immune defenses.

A. Decrease or Eliminate the Reservoir or Interrupt Disease Transmission 1. Clean Water - diseases such as cholera, typhoid fever, & diarrhea can be spread when human sewage contaminates the water supply. 2. Clean Food - pasteurization, boiling, adequate cooking, refrigeration prevent food poisoning, trichinosis (roundworm), salmonellosis, tapeworm infection, etc. 3. Personal Cleanliness - hand washing of #1 importance. 4. Insect Control - to decrease mosquito populations early programs drained swamps, screened living areas, used mosquito netting, used insecticides such as DDT (until it was found to be carcinogenic to humans!); now efforts concentrate on educating the public to remove stagnant water; biological control is also used - ex. Gambusia, the mosquito fish, was introduced to the U.S. this fish feeds on mosquito larvae 5. Prevention of STD's - public education, limit sexual exposure, use of condoms. 6. Prevention of Respiratory Diseases - isolate infected individuals, wear face masks; most effective way is immunization.

B. Immunization

1. Active Immunization (= Immunization or Vaccination) a. Active Immunization Defined - a person's own immune system is stimulated, memory cells are produced to protect against future natural infection.

b. Vaccine Defined - an agent containing antigen capable of inducing active immunity without causing disease; vaccines must be safe & immunogenic (stimulate an immune response strong enough to confer protection against natural infection); vaccines can be given orally, subcutaneously (below skin), or intramuscularly; some stimulate both Ab & cell mediated immune responses, other stimulate primarily Ab mediated immunity.

c. Types of Active Vaccines 1.) attenuated Live, weakened viruses or bacteria; virus is cultivated in the lab until it loses its virulence; the organism is then injected into a human and allowed to multiply; may cause a limited infection, usually without serious illness; provides strong & long-lasting immunity. Ex. tuberculosis (b), oral Sabin polio (v), mumps (v), measles (v), rubella (German measles) (v). The latter 3 are referred to as MMR. The fairly new chicken pox vaccine is also attenuated. This type of vaccine is not recommended for those who are immunocompromised.

2.) inactivated (killed) - By heat or chemical agents such as formalin, phenol, or acetone; process can destroy the Ag's

that stimulate immunity (ex. heat denaturation of protein Ag's); inactivated microorganisms can't multiply in host so vaccine dose must contain enough Ag to produce a protective immunologic reaction; usually requires a booster; Ex. pertussis (b), typhoid fever (b), rabies (v), Haemophilus

influenzae type B (b) (causes meningitis), injectable Salk


polio (v) (sometime referred to as IPV inactivated polio vaccine), cholera (b), viral influenza

Haemophilus influenzae type B - combined


polysaccharide Ag with a protein to make it more powerful (polysaccharides are weak stimulants of Ab production); called a protein conjugate vaccine.

3.) genetically engineered Genetic engineering & recombinant DNA technology have allowed us to use bacteria to produce the protein antigens found in the capsids of certain viruses and the cell envelopes of bacteria. Scientists determine the genetic code for these antigens & insert the gene into the chromosome of bacterial cells. The bacteria produce the antigens coded for on the inserted genes when they go through their regular process of protein synthesis. These antigens can then be injected as a vaccine (your body doesn't care if the protein antigens are in the real viral capsid or if they were made by a bacterium; they are the same proteins & your body's immune system will respond to these antigens in the same way). These vaccines do not pose the same risks as inactivated and attenuated viruses! Examples:

Pertussis (b) the inactivated vaccine contains many Ag's that contribute to the frequent undesirable side effects of this vaccine; an acellular, genetically engineered vaccine has recently been licensed; it has

fewer side effects, but may not stimulate vigorous immunity.

Hepatitis B (v) - originally produced from recovering viruses from the serum of infected patients, which could have other diseases like AIDS (made people nervous); now a genetically engineered vaccine.

4.) toxoids - for diseases caused by exotoxins rather than the microorganisms themselves, vaccines are made of toxoids (toxins that have been modified by heat or chemical agents to render them harmless); toxoids stimulate the production of Ab's called antitoxins; ex. tetanus (b), diphtheria (b). 2. Passive Immunization

a. Defined - Ab's from an immune person or animal are transferred to a patient; like an Ab transfusion!

b. Preparation 1. gamma globulin, a collection of Ab's from the pooled serum of many different donors; 2. special preparations contain high titers of specific Ab's; ex. varicella zoster (v), (chickenpox & shingles), tetanus (b), mumps (v), measles (v), hepatitis A & B (v), rabies (v), pertussis (b)

c. Advantages - even severely immunosuppressed patients can be

protected & protection is immediate.

d. Disadvantages - protection lasts only as long as the Ab molecules survive in the recipient months if from a human, only weeks if from an animal; also a risk of serum sickness.

Serum sickness - Occurs when proteins from animal serum are used in medical therapy; ex. horse antiserum is used in the treatment of venomous snake bites. Small concentrations of venom are injected into the horse to get it to produce antibody against the toxin. Patients then receive an infusion of these horse antibodies to bind to the snake venom antigen in their blood. The patients may produce antibodies against the horse antibodies, forming large complexes that are deposited in the tissues.

Cha p. 13 Host Micr obe Rela tion ship s and Dise ase Proc esse s

3. Boosters Immunity is not always life-long. Booster shots boost immunity by greatly increasing the numbers of antibody.

V.

NOTIFIABLE DISEASES

These are infectious diseases that are potentially harmful to the publics health and must be reported by physicians to the CDC. Make sure you can list some of these!

I. HOST-MICROBE RELATIONSHIPS

Pathogen parasite capable of causing disease Host an organism that harbors another organism

A.

Symbiosis - two different organisms living

together. 1. commensalism - one organism benefits, the other is neither harmed or benefited. Many bacterial species fall into this category. Many individual bacterial species by themselves don't provide direct benefit to the host. 2. mutualism - both partners benefit. Ex. ruminants (cud-chewing animals) and termites have microbial species that break down cellulose from plant cell walls so that it can be used for energy by the animal; this relationship is essential for the ruminant. Ex. Large numbers of E. coli live in the large intestine of humans. These bacteria release vitamin K, which we use to make certain bloodclotting factors. Ex. Collectively an organisms natural flora protects the host by competing with and edging out many pathogens. This phenomenon is called microbial antagonism.

3. parasitism - host is harmed, the parasite benefits; microbial parasites = pathogens. A narrow definition of parasites would include only eukaryotic pathogens such as protozoa, helminths, and arthropods (ticks, lice, fleas). A broader definition of this term includes viruses, bacteria, and fungi.

B. Contamination , Infection, and Disease A Sequence of Events 1. contamination the microbes are present.

2. infection multiplication of any parasitic organism within or upon the hosts body; growth of normal flora is usually not considered an infection; infection does not always cause disease 3. disease disturbance in the state of health (state of relative equilibrium in which the body's organ systems are functioning adequately); disease is characterized by changes in the host that interfere with normal function.

C.

Types of Flora: (define and give specific examples for each) 1. Resident flora (= normal) - microbial species present in/on human body throughout life; permanent species; coexist with humans in a stable relationship. a.

What does washing do to these guys? Reduces, but does not eliminate.

b. What parts of the body inhabited by normal flora? (external vs. internal surfaces) External - skin, conjunctiva Internal - nose, mouth, intestinal tract, vagina, urethra, ear

2. Transient flora - microbial species that can be cultured from body surfaces under certain circumstances, but are not permanent residents. a. What does washing do to these guys? Usually eliminates. b. Why aren't they part of the body's normal flora? Not well enough adapted to life on human body. c. Noscomial infections (hospital-acquired infections) - Hospital workers have a large transient flora population because of large number of pathogens they are exposed to every day (ex. pathogenic Staphylococcus aureus ); therefore, hospital workers must be extremely careful about hand washing.

3. Opportunists - microbial species that cause disease when the proper opportunity arises, but are usually harmless; infections usually occur when

bacteria get into a place where they dont belong (ex. nonpathogenic bacteria that are part of the normal flora of the colon go crazy when they get into the urinary tract.) a. Name three opportunities for infection - breakdown in immune system, antibiotic treatment, bioimplantation of artificial devices (catheters, pacemakers, artificial joints); b. What's one reason for a vaginal yeast superinfection? Antibiotic treatments reduce numbers of normal vaginal bacterial species; these bacteria usually keep the yeast Candida albicans in check.

D. Changing Flora - Examples: What is one good reason why mothers should breastfeed? Its not important just for the nutrients and the antibodies the baby receives in breast milk. While on breast milk, a baby's intestinal flora is composed mostly of Bifidobacterium, which metabolizes milk sugars into acetic and lactic acid. These acids reduce the pH of the intestine, making it inhospitable to many disease-causing microbes, most importantly those causing diarrhea. The intestinal flora changes when the baby is on formula and the same protection is not provided.

What is the effect of estrogen on the vaginal pH? What effect does this have on the normal flora? Estrogen increases the growth of lactobacilli which produce an acidic vaginal environment, making it inhospitable to disease-causing microbes (ex. E.

coli from feces). Newborns have high estrogen levels from estrogen that crosses
the placenta. In a few weeks, this estrogen level falls off. It doesn't increase again until puberty. This is important for when sexual activity could begin.

II.

ESTABLISHING DISEASE

Pathogenicity is the capacity to produce disease. An organisms pathogenicity depends on its ability to invade a host, multiply in the host, and avoid being damaged by the hosts defenses. Virulence refers to the intensity of the disease produced by pathogens, and it

varies among different microbial species. A pathogen must overcome the following seven challenges if it is to survive on or in a human host & cause disease. Pathogenesis, a microbe's ability to cause disease, depends upon its meeting all of these challenges. The seven challenges are:

A. Maintain a reservoir (a place in which a pathogenic microorganism is maintained between infections). 1. Human reservoirs - ex. pertussis, measles, gonorrhea, common cold. A person who is ill from an infection is a reservoir. Healthy people can also be reservoirs - called carriers. incubatory carrier - in early symptomless stages of illness (most diseases have specific incubation periods associated with them; you may not realize you have an infection, but you can still be contagious). chronic carrier - person who harbors a pathogen for an extended period of time without becoming ill (ex. people who are HIV+ but have not developed AIDS); this group also includes people who recover from an illness, but harbor the pathogen (ex. Hepatitis B); people with herpes are also chronic carriers. 2. Animal reservoirs - ex. for rabies the animal reservoirs are skunks, possums, bats, raccoons, etc.; in this case, the pathogen is spread through the bite of the rabid animal reservoir; insect vectors can also be involved in spreading pathogens from animal reservoirs to humans - ex. lyme disease (Borrelia uses deer and mice as a reservoir; a tick is the vector). Zoonosis - a human disease caused by a pathogen that maintains an animal reservoir. Mutations & genetic variation can occur in the reservoir, causing new strains to emerge. 3. Environmental reservoirs - ex. soil, water, house dust; Clostridium tetani uses soil it's able to survive in this environment because of its ability to produce endospores; Vibrio choleraeuses water.

B. Leave its reservoir & enter the body of a human host. Disease transmission takes place when a pathogen leaves a reservoir and enters the body of a host. Most have a preferred portal of entry. The number of pathogens that reach the portal of entry influences the likelihood of successful disease transmission. The number of microbes that must enter the body to establish infection in 50% of test animals is expressed as theID50 (infection dose). The LD50 (lethal dose) measures fatal infections - the number of microbes that must enter the body to cause death in 50% of test animals. The most common portals of entry for diseasecausing microbes are external & internal body surfaces: skin, conjunctivae (around eyes), nasal cavity & nasopharynx, mouth, intestinal tract, vagina, urinary tract, etc. Others include tissues below the skin in the case of an open wound or the placenta.

Transmission can occur in several ways: 1. Human-to-human (communicable diseases - transmitted from one person to another) a. Respiratory droplets expelled by coughing, sneezing, talking; ex. Bordetella pertussis (whooping cough); more human diseases are transmitted by respiratory transmission than by any other method. b. Direct body contact or person-to-person or horizontal transmission transmission by touching, kissing, sexual intercourse; includes STD's (sexually transmitted diseases); ex. gonorrhea; herpes is most commonly spread by kissing or exchange of saliva (common in young children). c. Vertical transmission - transmission from mother to infant; prenatal transmission - occurs across the placenta; perinatal transmission - occurs during passage through the birth canal; ex. STD's such as syphilis, gonorrhea, HIV, Herpes. d. Fecal-oral route can involve direct contact (ex. a person does not wash his hands after defecating and then shakes hands with someone); this transmission can also be by vehicles such as water, food, fomites, or

vectors (flies, etc.); (crops and water supplies may be contaminated with fecal matter).

2. Airborne Transmission - these pathogens are hardy enough to withstand prolonged drying; can be transmitted across great distances (greater than a meter); can remain viable in dust & reenter the air; ex. Mycobacterium tuberculosis.

3. Vehicle Transmission (objects such as food, water, fomites) Fomites inanimate objects such as cups, towels, bedding, eating utensils, bedding, & handkerchiefs; ex. common cold viruses.

4. Parenteral Transmission - occurs when a biological arthropod vector introduces pathogens during a skin-penetrating bite or when breaks in the skin or mucous membranes provide microbes with access to deeper tissues; can also occur from penetration with a hypodermic needle; ex. HIV, hepatitis B virus, Plasmodium (a protozoan that causes malaria; mosquito vector), Clostridium tetani (causes tetanus when anaerobic conditions are created in deep wounds)

5. Vectors - usually insects or other arthropods (mosquito, tsetse fly, tick, flea, lice, etc.) a. mechanical vector - ex. a fly lays its eggs in dog feces and then lands on your sandwich. b. biological vector ex. the protozoan parasite that causes malaria goes through a stage in its life cycle in the mosquito. Could bacteria use biological vectors?

C. Adhere firmly to the surface of the host's body and thereby colonize it.

Pathogens, like normal flora, attach to specific types of target cells by means of adhesins (protein molecules that are very specific for the receptors that they bind to on target cell surfaces); many adhesins are molecular components of capsules or pili, thus these structures are are responsible for the virulence of many strain of bacteria; some bacteria have a repertoire of adhesins - this versatility makes them extremely virulent.

D. Invade the body in order to enter cells or deeper tissues. Only a few pathogens cause disease by colonizing surfaces; most have additional virulence factor that enable the pathogen to invade tissues (in other words, most pathogens are invasive - they penetrate the body's surface to enter cells or deeper tissues). This ability allows them to escape certain host defenses and to gain access to a nutrient-rich environment that is free of competing microbes. Streptococcus produces the enzyme hyaluronidase that digests hyaluronic acid, a glue like substance that helps hold the cells of certain tissues together. Some pathogens actually enter cells to live and multiply inside them; they are called intracellular pathogens. Ex. of intracellular pathogenic bacteria - Rickettsias (ex. Rocky Mt. Spotted Fever) & Chlamydias. Some bacteria gain entry into cells by adhering to surface receptors that fold into the cell during endocytosis.

Most eukaryotic pathogens do not invade cells. An example of one that does is Plasmodium (a sproozoan protozoan that causes malaria); this parasite enter red blood cells.

E. Evade the body's elaborate defenses against microbial invaders. Here are just a few of the ways:

1. Protection Against Phagocytosis by White Blood Cells.

a. capsules - make bacteria slippery and hard for wbcs to phagocytize; some bacteria are virulent only if they produce a capsule; ex. Streptococcus

pneumoniae, Haemophilus influenzae.


b. surface proteins - interference with phagocytosis; ex. Streptococcus

pyogenes - produces M proteins - hairlike projections on the surface of its cell


wall (kind of makes them prickly); because of these projections, these guys can be phagocytized only if antibodies bind to the bacteria, masking the M proteins. c. living inside the phagocyte (white blood cell) - only pathogens that possess special adaptations can survive the enzymes produced by the phagocyte; ex. Mycobacterium tuberculosis- survives because the phagocyte's enzymes cannot break through its waxy outer layer.

2. Antigenic Variation - some microbes mutate & change their surface antigens; a person may be immune to one strain, but not to another. Ex. Neisseria gonorrhoeae, HIV, cold virus, influenza virus 3. Production of Exoenzymes (enzymes produced and then released by bacteria)
Ex. Coagulase triggers blood clotting mechanism, allowing bacteria protection from immune defenses. Ex. Staphylococcus IgA Proteases - enzymes produced by bacteria that destroy the IgA class of antibody. Ex. Neisseria. Streptokinase dissolves blood clots so bacteria can spread to other tissues.

F. Multiply within the body, perhaps producing toxic products or stimulating host reactions that cause disease.

The 2 most common forms of bacterial pathogenesis are


1. the production of toxins (poisonous products that harm human cells and tissues ) & exoenzymes. 2. stimulation of the body's defenses.

1. Exotoxins Produced by G(+) or G(-) bacteria. Bind to receptors on the surfaces of different types of cells. Specific for the cells they infect (ex. neurotoxins, such as tetanus & botulinum toxins effect only nervous tissue. Enterotoxins, such as those produced by Vibrio cholerae & Shigellaeffect only epithelial cells lining the intestinal tract). After binding, the enzymes enter the cell & disrupt cellular function, usually by inhibiting one specific metabolic reaction. Some exotoxins are unbelievably potent; ex. tetanus toxin - an amount about equal to the size of the period at the end of this sentence can kill an adult! Some exotoxins enter the bloodstream, causing systemic disease or toxemia (tissues throughout the body are affected). Diseases that result from the ingestion of a toxin are termed intoxications rather than infections. Ex. botulism food poisoning is the result of injesting toxins made by pathogens. Exotoxins can be neutralized with special antibodies called antitoxins. Certain exotoxins can be modified in the lab by treatment with heat or chemicals such as formaldehyde to produce toxoids; these molecules that have lost their disease-causing properties, but still stimulate the immune system to produce antitoxins (antibodies against toxin); vaccines can be produced from toxoids (ex. tetanus). In most exotoxin-caused diseases, such as cholera, tetanus, E. coli diarrhea, shigellosis, & pertussis, occur only if the bacteria multiply in the body. In other

diseases, such as botulism (food poisoning caused by Clostridium botulinum ), the toxin is produced outside the body & is ingested with contaminated food called a food intoxication.

Some are enzymes. Ex. Hemolysins which lyse red blood cells. Alphahemolysin partially breaks down hemoglobin (the oxygen-carrying protein in rbcs), leaving a greenish halo around colonies grown on blood plates. Betahemolysin completely breaks down hemoglobin, leaving a clear ring around colonies. Hemolysins are produced by streptococci and staphylococci.

Leukocidins produced by stretptococci and staphylococci damage or destroy certain kinds of white blood cells. While most diseases are characterized by an elevated white cell count, some may result in a decrease in numbers of wbc's.

May be named after the part of the body they affect. Ex. neurotoxin, enterotoxin

2. Endotoxins

Released only by G(-) bacteria; all G(-) bacteria produce endotoxins. Endotoxins are lipopolysaccharides molecules (LPSs) in the outer membrane. Its effects include: fever, increased or decreased #'s of wbc's, shock, death, diarrhea

Endotoxins are not secreted by bacteria, but are released into the environment when the bacterial cell dies.

Unlike exotoxins, endotoxins are not proteins, so they are relatively heat stable.

Unlike exotoxins, endotoxins don't stimulate the immune system to produce antibody; therefore, toxoid vaccines would be useless.

Unlike exotoxins, endotoxins are not specific for the cells they effect.

Normally clinically significant only when large numbers of dying bacteria are circulating in the bloodstream; paradoxically, agents that kill G(-) bacteria (antibiotics, etc.) may actually increase endotoxin-mediated damage.

3.

Stimulation of the Body's Defenses Ex. Streptococcus pneumoniae - when it multiplies in the lungs, phagocytes (white blood cells) come to combat the infection; however, this bacteria is protected by a capsule so more and more phagocytes arrive to help; dead bacteria & phagocytes accumulate in the lungs, impairing normal gas exchange & making breathing difficult.

G. Leave the body and return to a reservoir &/or enter a new host. The anatomic route through which a pathogen usually leaves the body of its host is called its portal of exit.

For most respiratory pathogens, the portal of exit is the same as the portal of entry. For most gastrointestinal pathogens, the portal of entry is the mouth & the portal of exit is the anus.

STD's exit the same way they enter - across the mucous membrane surfaces of the genital tract.
Parenterally transmitted pathogens exit the same way they enter - in the blood. III. TYPES OF INFECTIOUS DISEASE

Acute disease develops rapidly and runs its course quickly Chronic disease develops more slowly, is usually less severe, and persists for a long

period. Latent disease characterized by period of inactivity (ex. Herpes) Local infection confined to a specific area Systemic infection generalized infection; affects most of the body. Septicemia pathogens are present in and multiply in the blood Primary infection initial infection in a previously healthy person. Secondary infection follows a primary infection (ex. a bacterial infection following a cold). Superinfection secondary infection that results from the destruction of normal microflora and often follows the use of broad-spectrum antibiotics. Mixed infection - caused by several species of organisms present at the same time. IV. STAGES OF INFECTIOUS DISEASE

A. INCUBATION PERIOD Time between infection and the appearance of signs and symptoms. Although the infected person is not aware of the presence of an infectious agent, she can spread the disease to others. Each infectious disease has a typical incubation period.

B. PRODROMAL PHASE (prodromos = forerunner) - Short period during which nonspecific, often mild, symptoms such as malaise and headache sometimes appear. You feel like youre coming down with something.

C. INVASIVE PHASE Period during which the individual experiences the typical signs and symptoms of the disease (fever, nausea, rash, cough, etc.). During the acme part of this phase, signs and symptoms reach their greatest

intensity. In some diseases this phase may be fulminating (sudden and severe), in others it may be persistent or chronic. A period of chills followed by fever marks the acme of many diseases. The battle between pathogens and host defenses is at its height during this stage.

D. DECLINE PHASE Symptoms begin to subside as the host defenses and the effects of treatment if being administered finally overcome the pathogen. Secondary infections may occur during this phase.

E. CONVALESCENCE PERIOD Tissues are repaired, healing takes place, and the body regains strength and recovers. Individuals no longer have disease symptoms, but they may still be able to transmit pathogens to others.

Chapter 14 Nonspecific Defense Responses

NONSPECIFIC DEFENSE RESPONSES - 1st & 2nd LINES OF DEFENSE

[Nonspecific defenses are general attack responses; the response is the same, no matter who the "invader" is.]

I.

The Body's First Line of Defense: Structural, Mechanical, & Chemical Defense Responses on Internal & External Body Surfaces:

A. Skin & mucous membranes (epithelial surface tissues) 1. Cells are tightly joined together, preventing bacteria from invading deeper tissues.

2. Sloughing of dead cells prevents microbial population from continually increasing.

3. The protein, keratin, fills the cells in the outer layers of the epidermis. These cells then contain little water, making the skin dry & inhospitable to many microbes.

4. Ciliated, mucous membranes (ex. in the respiratory tract) trap microbes, dust etc. in mucous & cilia move mucous toward mouth, where it is coughed up and swallowed.

B. Normal flora - Normal bacterial inhabitants of the skin, gut, & vagina - the "natives" outcompete the "foreigners" for resources. Also, some normal bacteria produce acid from sugar fermentation, creating an acidic environment that keeps other populations in check (ex. lactic acid produced by bacteria in the vagina keep the yeast Candida albicans under control).

In the vagina, low estrogen concentrations in prepubertal and postmenopausal women result in a decrease in bacterial numbers in the vagina; this can lead to vaginal yeast infections. Yeast infections can also result from antibiotic treatments (broad spectrum antibiotics kill the pathogen and the normal flora) & douching.

C. Movement of body fluids dislodges microbes. Ex. urine, tears, saliva. Peristalsis in digestive tract causes food & digestive juices to sweep microbes away. (Urine itself is not microbiocidal!)

D. Secretions:

1. Tears, perspiration, & saliva contain lysozyme, an enzyme that destroy the bacterial cell wall. Lysozyme is especially destructive to G(+) bacteria because they lack an outer membrane.

2. Perspiration also contains high concentrations of salt, creating a hypertonic environment.

3. Bile, produced by the liver, also disrupts the bacterial cell wall. Bile is secreted into the small intestine to aid in the digestion of lipids. It passes from the small intestine into the colon in feces; the bacterium E. coli , which is part of the normal flora of the colon, is resistant bile. Remember that bile salts are an important ingredient in some selective media that select for G(-) bacteria and against G(+) bacteria.

4. Hydrochloric acid produced in the stomach (pH of the stomach is 2!).

5. Fatty acids are contained in the oil secreted from oil glands in the skin. It makes the skin slightly acidic.

II.

The Bodys Second Line of Defense What Happens Once the Microbes Get Past the Surface Defenses:

First a little about the types of white blood cells (called leukocytes): a. macrophages - phagocytic b. eosinophils - phagocytic

c. neutrophils - phagocytic d. basophils - release histamine; involved in the inflammatory response. e. lymphocytes - 3 types: B cells, T cells, Natural Killer cells. (Be careful not to get the terms leukocyte and lymphocyte confused!)

A. Natural Killer Cells - Type of lymphocyte (type of wbc); most lymphocytes are involved in specific defense responses (ex. B & T lymphocytes). NK cells are unlike other lymphocytes in that they lack antibodies & antigen receptors (well talk about these under the specific defenses); they are like a specific type of T lymphocyte called a killer T cell in that they releaseperforins [chemicals that cause lysis of the bacterium - they perforate or punch holes in the cell envelope of bacterium].

B.

Phagocytic White Blood Cells (Phagocytes) - Phagocytosis occurs in 3 phases: (remember CAI) 1. Chemotaxis - the chemical attraction of phagocytes to a particular location; chemotactic chemicals that attract phagocytes include bacterial toxins components of damaged tissue cells, complement proteins, & antibodies.

2. Adherence or Attachment - Because of certain microbial defenses, adherence of the phagocyte cell membrane to the surface of the microbe may be difficult (for example some bacteria produce a slimy outer capsule that makes them slippery). Opsonization of microbes by complement proteins and antibody facilitates phagocytosis.

3. Ingestion - The phagocyte engulfs the microbe with its cell membrane. The engulfed microbe moves into the cytoplasm of the phagocyte inside a vesicle (sac); these vesicles fuse with lysosomes containing digestive enzymes; phagocytes include the wbcs such as neutrophils, eosinophils, & macrophages;

phagocytes circulate within blood vessels, & are also located in the lymph nodes, spleen, liver, kidneys, lungs, joints, skin, red bone marrow, & brain.

Fever - When phagocytes ingest certain bacteria, the phagocytes secrete a type of interleukin, which circulates to the hypothalamus & causes it to secrete prostaglandins; these chemicals "reset" the hypothalamic thermostat at a higher temperature; temperature-regulating mechanisms (vasoconstriction, increased metabolism, shivering) act to bring the core body temperature to this new setting. [Aspirin, ibuprofen, & acetaminophen inhibit the synthesis of prostaglandins.] A low grade fever has a beneficial effect on the body: 1.) It inhibits the growth of some microbes. 2.) It increases the heart rate so that white blood cells, etc. are delivered to infection sites more rapidly. 3.) B cell & T cell proliferation (division) increases. 4.) Heat speeds up chemical reaction rates.

High grade fevers are dangerous - they can denature the body's own enzymes & other proteins.

Whats low grade? What is considered low grade in infants is much lower in adults. This is due to a babys higher surface area to volume ratio. Basically, a baby has more surface area compared to her volume than an adult does. So, its easier for heat to reach the skin and dissipate into the air. Heat does not dissipate as easily from an adults body (too much volume for it to move through) and so it does more damage to internal organs.

D. Interferon (IFN) - Interferons are proteins that are produced by certain viralinfected cells (particularly macrophages). Once interferons are released from viral-infected cells, they diffuse to neighboring uninfected cells & bind to their

surface protein receptors. This binding induces the uninfected cells to synthesize antiviral proteins that interfere with or inhibit viral replication. In other words, interferons serve as a red flag to warn uninfected cells that there's a "stranger among us" & the uninfected cells take action to protect themselves.

Certain interferons also enhance the activity of phagocytes & natural killer cells.

Certain interferons also inhibit cell growth & suppress tumor formation. Ex. Alpha-IFN is approved in the U.S. for treating Kaposi's sarcoma, a cancer that often occurs in patients infected with HIV; it is also used for treating genital herpes & hepatitis B & C.

E. Complement System - When certain microorganisms invade the body, about 20 complement proteins in blood plasma & on cell membranes interact as a system. When activated, these proteins "complement" or enhance certain immune, allergic, & inflammatory reactions; therefore, the complement system enhances the effectiveness of both nonspecific & specific defense responses. Complement proteins respond to the binding of antibodies to the cell membrane of the invading microbe; the complement proteins are activated one after another in a "cascade" of reactions [one reaction catalyzes the next.] These reactions have the following results: (use the acronym COLA to remember them!)

1. Chemotaxis - They act as chemotactic chemicals to attract phagocytes to the scene. 2. Opsonization - Complement proteins bind to the surface of the microbe & then interact with receptors on phagocytes to promote phagocytosis. In this way complement proteins give macrophages a "foot hold." 3. Lysis - Other complement proteins kill the microbe by causing lysis.

4. Activation of Inflammatory response (See below).

F. Inflammatory Response - Many cells, the complement system, & other substances take part in this response. This response is a series of events that destroys invaders & restores damaged tissues to normal. The 4 major symptoms of inflammation are redness, heat, swelling, & pain (think about what happens when a bee stings you or a cut gets infected). Inflammation is a nonspecific defense - the response of a tissue to a cut is similar to the response that results from a burn, radiation, or microbial invasion. The inflammatory response involves the following events:

1. Vasodilation & Increased Permeability of Blood Vessels Vasodilation is an increase in diameter of the arterioles. Arteriole dilation enables white blood cells & other substances to more easily penetrate the tissues. Increased permeability means that an increased amount of material is allowed to pass out of the blood vessels. Blood vessels dilate & become more "leaky" due to the release of histamine by basophils, which are activated by the complement system. Prostaglandins, released by damaged cells, intensify the effects of histamine. Within minutes after injury, dilation & increased permeability of blood vessels produces heat, redness, & swelling. Warmth & redness occurs from the large amount of warm blood flowing through the area. Temperatures will continue to rise due to the release of heat energy from chemical reactions (increased metabolic activity). Fluid seeping from "leaky" capillaries causes swelling. Pain can result from injury of nerve fibers, from irritation by toxins produced by microbes, from increased pressure due to swelling, or from prostaglandin release.

2. Phagocytosis Fluid seeping from "leaky" arterioles causes local swelling & delivers more complement proteins to the tissues (remember, proteins are large molecules - they would normally stay in the blood vessels & couldn't get into the interstitial spaces between cells). Phagocytes, following increased concentrations of complement proteins to affected tissues, engulf foreign invaders & damaged cells. Eventually phagocytes die. Within a few days a pocket of dead phagocytes & damaged tissue forms (called pus).

3. Tissue Repair Platelets initiate clotting mechanisms, help wall off the pathogen, & help repair tissues.

Chapter 15 - Specific Defenses

THIRD LINE OF DEFENSE - SPECIFIC DEFENSE RESPONSES: THE IMMUNE SYSTEM


When general attack responses are not enough to stop the spread of an invader & illness follows, three types of white blood cells (macrophages, T cells, & B cells) will counterattack. Their interactions are the basis of the immune system. Two important characteristics of the immune system are its specificity & its memory.

Summary Table: White Blood Cells Involved in Specific Defense Responses: 1. Macrophages - phagocytic; involved in inflammatory, antibody-mediated, & cellmediated responses (nonspecific responses); important not only for phagocytosis, but also for antigen presentation.

2. ones)

Lymphocytes (these are not all of the lymphocytes, but some of the important

a. B cells - produce antibodies (Ab) [Y-shaped protein molecules which bind to specific targets (antigens) & tag them for destruction by phagocytes or the complement system]. b. Cytotoxic T cells - involved in the cell-mediated response; directly destroy body cells already infected by certain viruses or parasitic fungi. c. Helper T cells - involved in the antibody-mediated & cell-mediated responses; they stimulate the rapid division of B cells & cytotoxic T cells by producing compounds called interleukins. d. Memory cells - certain B cell & T cells, which are produced during a first encounter with a specific invader (primary immune response), but are not directly involved in this first attack; they circulate freely & respond rapidly to any subsequent attacks (secondary immune response) by the same type of invader.

Recognition of Self & Nonself Among the surface proteins on your own body cells are MHC markers ("self" markers), which are normally ignored by your own white blood cells; MHC markers are unique to each individual (no one has the same kinds, except in the case of identical twins); microbes, etc. also have markers [called antigens (Ag) because they are foreign to your cells] on their surfaces which are not ignored by white blood cells; antigens are usually surface proteins with distinct configurations that trigger immune responses.

I. PRIMARY IMMUNE RESPONSE:

A first-time encounter with an antigen elicits a primary immune response from the lymphocytes & their products. We will consider an antibody-mediated immune response & a cell-mediated immune response to such an encounter:

A. Antibody-Mediated (Humoral) Immune Response - The main targets of this type of response are extracellular organisms: bacteria, extracellular phases of viruses, some fungal parasites, & some protozoans. Antibodies can't bind to antigen if the invader has already entered the cytoplasm of a host cell!! The following events involve a bacterial infection:

1. Macrophages - When bacteria enter the body, their invasion triggers a general inflammatory response, & macrophages engulf some of the bacterial cells by phagocytosis. The engulfed bacteria move into the cytoplasm of the macrophages inside vesicles. These vesicles fuse with lysosomes (vesicles containing digestive enzymes) & enzymes digest the bacterial cells, but do not destroy their surface antigens. At the same, the cell synthesizes MHC markers & is packaging them into vesicles in its golgi apparatus. The vesicles containing the antigen & the vesicles containing the MHC markers fuse. Inside the vesicle, the antigen binds to the MHC markers (now called antigen-MHC marker complexes). The vesicle containing the complexes undergoes exocytosis & the complexes are inserted in the cell membrane of the macrophage. Macrophages can now present the antigen to other white blood cells (ex. helper T cells).

2. Helper T cells - When the appropriate helper T cells make contact with the macrophages, some of their membrane-bound antigen-receptors bind to the macrophage antigen-MHC complexes (these receptors are specific for these particular complexes - they won't bind to any other type!). This binding causes macrophages to secrete a compound calledinterleukin that stimulates the helper T cells to secrete their own interleukins. The helper T cell interleukins will cause activated B cells to start dividing (see below).

3. B cells

a. B cells "mature" in bone marrow. While each B cell is maturing, it makes many copies of just one kind of antibody (each B cell is unique in that it will only make one kind of antibody that no other B cell makes - each kind of antibody will only react to one antigen). While the B cell is maturing, some of the antibodies it is producing become positioned at the cell's surface, where they will later bind to a specific antigen. The "tail" of each antibody is embedded in the cell membrane, & the "arms" stick out above the cell membrane's surface. From the bone marrow, B cells migrate to the lymph nodes, the spleen, or lymphatic tissue in the g.i. tract.

b. When a B cell is released from the bone marrow into circulation, it is known as a "virgin" B cell because its antibodies have not yet made contact with antigen. c. A virgin B cell with the right antibodies binds to a specific antigen. Some of the antigen is then taken into the B cell, combined with MHC markers, & moved to the B cell surface (see how macrophages do this above). The B cell is now said to be activated; it's no longer virgin - it's come into contact with a specific antigen. d. If an activated B cell interacts with the appropriate interleukinproducing helper T cell (see above under helper T cell), the B cell will start dividing quickly, giving rise to aclonal population of identical B cells. e. Part of the B cell clonal population differentiates into plasma cells, which secrete thousands of copies of the particular antibody that had been produced by the virgin B cell (the antibody actually leaves the plasma cells!). Antibodies have different effects on antigen: 1.) Neutralizing - the binding of Ab with AG blocks or neutralizes the damaging effect of some bacterial toxins and prevents attachment of some viruses to body cells. 2.) Immobilization - If Ab forms against cilia or flagella of motile bacteria, the Ab-Ag complex may cause the bacteria to lose their motility, limiting their spread into nearby tissues.

3.) Agglutination - Because antibodies have tow or more sites for binding to Ag, the Ab-Ag reaction may cross-link pathogens to one another, causing agglutination (clumping together); this enhances phagocytosis. 4.) Activation of complement system- complement proteins cause lysis of microbe; complement also cause opsinization, which enhances phagocytosis. 5.) Opsinization - Antibodies enhance phagocytosis by coating the microbe (remember that complement protein can also be opsonins). f. Some of the B cell clones differentiate into memory B cells, which are involved in a secondary response (will be discussed later).

How do B cells produce the millions of different antibodies required to detect all of the millions of possible antigens? Part of each arm of an antibody is a polypeptide chain made up of amino acids, folded into a groove or cavity, which "fits" with the antigen (there are poor fits & better fits - the better the fit the better the immune response). All B cells have the same genes for coding the amino acids in the chain, but each maturing B cell shuffles the genetic code into one of millions of possible combinations, so that the sequence of amino acids then gets shuffled (this changes the shape of the protein, thus changing the shape of the antibody). So B cells can give rise to virtually unlimited chain configurations. Therefore, when an antibody comes into contact with an antigen for the very first time, the right antibody just happened to be there! Your immune system did not produce the virgin B cell antibodies in response to a particular antigen! It is our genes that determine what specific foreign substances our immune system will be able to recognize & resist! (The same rule applies to T cell Ag receptors.) The 5 Classes of Antibodies (Ab): (Ab's are part of the immunoglobulin (Ig) family of proteins)

1.) IgG - largest class, activates the complement system; effective opsonin (enhances phagocytosis); only Ab that can cross the placenta (protects the newborn for several months after birth); predominates in secondary immune responses; all antitoxins belong to this class (remember antitoxins are antibodies made against exotoxins made by G(+) & G(-) bacteria); found in blood & extracellular fluids. 2.) IgA - second largest class; found in blood and body secretions (saliva, milk, mucus, tears); protects mucosal surfaces, especially preventing attachment of viruses; its presence in colostrum defends the g.i. tract of newborn humans against infection. 3.) IgM - extremely effecting in fixing complement; sometimes called early Ab, because it is the first Ab to form during a primary immune response; its structure allows it to build complex Ag-Ab lattices that clump, forming a visible precipitate; its found in blood & extracellular fluids. 4.) IgD - main type of Ab displayed on the surface of B cells. 5.) IgE - fixed to the surface of basophils; stimulates the microbe to release histamine when the basophil binds to Ag; basophils then releases histamine; this can contribute to allergies

B. Cell-Mediated Immune Response (Cellular Immunity) - This type of response deals with viruses & other pathogens that have already penetrated host cells (they are intracellular!), where they remain hidden from antibodies. In the cell-mediated immune response, the host cells are killed by cytotoxic T cells (killer T cells) before the pathogens can replicate & spread to other cells. The following events involve a viral infection:

1. Cytotoxic T cells or Killer T cells - Cells in the bone marrow give rise to forerunners of killer T cells, which travel to the thymus gland, where they mature into killer T cells. Each T cell produces antigen receptors that become positioned at its surface (these receptors are not antibodies, but are similar!!!). Ag receptors recognize specific Ag-MHC marker complexes. When

an Ag enters the body, only a few T cells have receptors that can recognize & bind to the Ag. 2. Killer T cells are released into circulation by the thymus gland. When a virus infects a cell, viral proteins become associated with MHC markers on the host cell's surface. 3. The antigen receptors of killer T cells, bind to the antigen-MHC complexes of macrophages, infected cells, etc. 4. Cytotoxic T cells secrete perforins (proteins that punch holes in the infected cell's cell membrane). 5. This kills the infected cell, but prevents the virus from replicating & spreading to other cells.

Note: As in antibody-mediated immune responses, macrophage-stimulated helper T cells stimulate killer T cells to divide by secreting interleukins. This creates a clonal population of killer T cells, all with the same antigen receptor as the original killer T cell (these cells are not called plasma cells!!!!!). As with B cells, some of the clones become memory T cells & will be involved in a secondary immune response. (When the body rejects a tissue graft or an organ transplant, cytotoxic T cells are one of the reasons why. They recognize MHC markers on the grafted cells as being foreign. Organ recipients take drugs to destroy cytotoxic T cells, but this compromises their ability to mount immune responses against pathogens.)

II. TYPES OF IMMUNITY

1. Active - a product of a person's own immune system. a. naturally acquired - comes from infections encountered in daily life. b. artificially acquired - stimulated by vaccines.

2. Passive - Ab's produced elsewhere are given to a person.

a. naturally acquired - refers to Ab's transferred from mother to fetus across the placenta & to the newborn in colostrum & breast milk. b. artificially acquired - consists of Ab's formed by an animal or a human & administered to an individual to prevent or treat infection; ex. hepatitis A, diphtheria.

III. SECONDARY IMMUNE RESPONSES:


A secondary immune response to a previously encountered antigen can occur in 2 or 3 days. It is greater in magnitude than the primary response & of longer duration. This is because some of the B & T cells of the clonal populations do not get involved in the primary response attack. They circulate for years as memory cells. When a memory cell encounters the same type of antigen that initiated the primary response, it divides at once (no helper T cells are needed to stimulate cell division!). A large clonal population of active B or T cells can then be produced in just a matter of days.

IV.

IMMUNIZATIONS:
Defined: Immunization means deliberately introducing an antigen into the body that can provoke an immune response & the production of memory cells. The first injection elicits a primary immune response. A second injection (the "booster shot") elicits a secondary response, which provokes the production of even more antibodies and memory cells to provide long-lasting protection against the disease. We will discuss the types of vaccines in chapter 15.

Chapter 16 & 17 - Pracatical applications of immunology & Immune Disorders

There are many practical applications that one can use in the diagnostic immunology laboratory with respect to disease detection and disease monitoring. Many of the same processes that occurr in the human body, such as Ag-Ab interactions, can be utilized in a laboratory setting to augment disease conditions. The following 3 laboratory based detection methods are examples of very common diagnostic tools used in clinical settings, public health labs, and in research settings. Be aware that there are MANY more of these types of detection methods. I have chosen to describe the following 3 because of there

"basic" principles behind each technique and due to their popularity with respect to high sensitivity and specificity. 1) Agglutination reactions occurr when an Ab and Ag are specific for each other and a "lock and key" lattice of Ab-Ag forms to allow for an observable agglutination reaction. An example is the Rapid Plasma Reagin (RPR) kit used for the detection/screening of syphillis caused by the bacterium, Treponema pallidum. The IgM antibody is often referred to as early Ab or the 1st Ab to appear in an infection. It is also known as the Ab of agglutination because of it's molecular make-up. IgM is typically arranged as a pentamer (5 Ab's bound together which allows for 10 sites of Ag attachment). An IgM pentamer (see figure in text) allows for many Ag's to bind and form a complex lattice that will become insoluble in solution and be visible to the naked eye. Thus, one can use specific Ab for syphillis to bind to specific Treponema pallidum Ag's and form a complex (this is typically called a "reactive"). The test is easy and low cost....because of these features, it makes an RPR test a useful and cheap screening test for this disease. 2) Fluorescent Antibody (FA) assays are one of the most common kits available to diagnostic labs for the detection of a variety of microbes. A kit will simply have an Ab that has been "labeled" with a fluorochrome such at FITC. The FITC will fluoresce when exposed to a uv light source on a microscope and "light up" as apple green fluorescence. If the reaction on a slide is not specific (ie. the Ag doesn't match the labeled Ab) then there is no fluorescence. See text for illustrations of this test. 3) Enzyme Linked Immunoabsorbent Assays (ELISA) are also a very common kit available for diagnostic labs. The HIV screening test is an ElISA based test. The kit will have a specific Ab for HIV in a plastic coated well. A technician will then add serum from a patient. If the serum has HIV Ag, it will bind to the Ab in the plastic coated well. This step is followed by another Ab that is labeled with an enzyme. Then a substrate is added to the "sandwich" ELISA. If the labeled enzyme has attached in the previous step, the substrate will fit into the enzymes active site and a colorimetric reaction will take place. So, a typical color like yellow will be produced with a positive test. See the text for illustrations of this test. Immunological disorders are caused when the immune system malfunctions, producing either an inappropriate or inadequate immune response. Hypersensitivity - a misdirected response in which either Ab's or T cells cause damage. While there are 4 types of hypersensitivities, I have chosen to discuss only those that are related to responses to microbes. You will probably learn about the others in an Anatomy & Physiology course.

A. Types of Hypersensitivity: 1. Type III (Immune-complex ) hypersensitivity - antibody mediated; IgM & IgG antibodies react with a person's own antigens - bind to antigens that are free in circulation (in Type II reactions, Ab binds to Ag on tissues & cells); AbAg binding creates complexes that remain soluble in body fluids; complexes can lodge in capillaries, activating complement & provoking an inflammatory response & attracting phagocytes; Ex: a. viral hepatitis - chronic infection from long-term exposure to microbial antigens b. farmer's lung - contracted by people who continually inhale certain antigens from molds, plants, or animals c. serum sickness - occurs when proteins from animal serum are used in medical therapy; ex. horse antiserum is used in the treatment of venomous snake bites; patients receive an infusion of horse antibodies to bind to the snake venom antigen; the patients may produce antibodies against the horse antibodies, forming large complexes. 2. Type IV (Cell Mediated or Delayed) hypersensitivity - does not involve Ab's; a T cell encounters an Ag that matches its Ag receptors; this stimulates the T cell to divide; when sensitized or activated T cells encounter the Ag again they release interleukins that stimulate macrophages and initiate inflammation, causing tissue damage. Ex.: organ transplants (tissue grafts); immunosuppressive drugs are used; cyclosporine is an improvement - a product of a fungus, it interferes with T cell function, but not with B cell function. b. tuberculosis, leprosy - damage is caused by a granulomatous reaction (when a macrophage cannot completely destroy microbial Ag, it persists within the cell; these persistent Ag's cause T cells to release interleukins that stimulate the production of a granuloma, a nodule of activated macrophages; the continuing inflammation that goes on within & around the granulomas displaces & destroys normal cells). B. Immunodeficiency Disorders Immunodeficiency - failure to mount an adequate immune response 1. Congenital - inherited or develop before birth & usually appear early in life; Ex.: a. severe combined immunodeficiencies (SCID) - B & T cell immunity are disabled. 2. Acquired - occur later in life; caused by infection, cancer, or the side effects of immunosuppressive medication (ex. steroids); Ex.: a.

a. AIDS - acquired immunodeficiency syndrome C. Cancer & the Immune System Cells become cancerous when they under transformation, a process leading to uncontrolled division that produces large numbers of undifferentiated cells. These primitive, rapidly dividing cells form large growths called tumors that crowd and eventually kill normal neighbor cells. Transformed cells often express new surface antigens that are not found on normal cells & mark the cancer cells for destruction by T cells, NK cells, & macrophages. Cell transformation occurs frequently, but the immune system eliminates most malignant cells before they cause cancer. AIDS patients are at an increase risk for cancer (ex. Karposi's sarcoma) because they lack the white blood cells to kill off the malignant cells. As immune defenses weaken with age, the elderly are also at an increased risk for cancer. Many anticancer drugs are highly toxic because they are nonspecific (nonspecific tumor destruction) - they kill normal rapidly dividing cells as well as cancer cells (ex. hair cells, intestinal cells, bone marrow stem cells). Cancer immunotherapy would be more effective. If we can determine unique antigens on the surface of cancer cells, then we can target only those cells for destruction.

Chapter 18 (skin/eyes) & 23 (Urogenital tract) (See Chp 5 for parasitic/fungal/helminthic infections) Some Bacterial & Viral Diseases of the Skin, Mucosa, Eyes, Wounds, Urogenital System, & STDs

I. Some Diseases of the Skin, Mucosa, Eyes, Wounds

A. Bacterial
1. Scalded Skin Infections (scalded baby syndrome) Staphylococcus aureus; caused by exotoxins called exfoliatins; toxins travel through bloodstream causing upper skin layers to separate and peel; most common in infants; high fever present; septicemia and death can occur.

2. Abscesses, Boils, Sty strains of staphylococci

3. Impetigo caused by S. aureus or S. pyogenes; a highly contagious pyoderma (pus producing skin infection); occurs almost exclusively in children; easily treated with penicillin; usually heals without scarring, but pigment can be permanently lost.

4. Scarlet Fever Streptococcus pyogenes; bacteria harbor a prophage that codes for production of erythrogenic (red producing) toxin that causes the scarlet fever rash; can lead to rheumatic fever and kidney problems; was once a lifethreatening illness; today's cases are mild.

5. Acne caused by bacteria feeding on sebum(oil); Propionibacterium acne

6. Conjunctivitis (pink eye) bacterial conjunctivitis is extremely contagious; can be caused by S. aureus, S. pyogenes, Haemophilus influenzae, Pseudomonas sp.,

Neisseria gonorrhoeae; conjunctivitis is more commonly caused by viruses;


bacterial conjunctivitis is usually more pyogenic than viral.

7. Trachoma Chlamydia trachomatis; trachoma means pebbled or rough; scarring of eyelids causes eyelashes to point inward; leading cause of preventable blindness worldwide; uncommon in US.

8. Hansens Disease (Leprosy) Mycobacterium leprae; 2 forms of disease: 1.) 2.) tuberculoid areas of skin lose pigment and sensation lepromatous nodular form where a granulomatous response causes enlarged, disfiguring skin lesions called lepromas.

This is the only bacterium known to destroy peripheral nerve tissue; it likes cooler parts of body (nose, ears, fingers, etc.); as it progresses, disease erodes bone and it deforms hands and feet, pits develop in skull; until recently, patients were isolated in special hospitals; now, the disease can be arrested and people can live nearly normal lives (must sleep in separate bedrooms and cannot live with children).

9. Plague Yersinia pestis; zoonosis; spread from infected rodents to humans by fleas; incidence of disease is low; bacilli travel in lymphatics to lymph nodes where they cause enlargements caused buboes (especially in armpits and groin); hemorrhages turn skin black (Black Death); deaths can be prevented with antibiotic treatment.

10. Tularemia Francisella tularensis zoonosis; reservoir: usually cottontail rabbits (number of causes rises during rabbit-hunting season); first isolated in Tulare County, CA in 1911; low incidence in US; disease is an occupational hazard for taxidermists; transmission: breaks in skin, bite from arthropod vector (tick, deer fly), inhalation of aerosols during skinning, consumption of contaminated meat; clinical signs: fever, severe headache, and buboes; one form can lead to a septicemia that resembles typhoid fever; vaccine does exist but frequent boosters are required.

11. Lyme Disease - Borrelia burgdorferi (spirochete); transmitted by deer ticks (Ixodes damini); white-tailed deer are a reservoir; develop flu-like symptoms; listed here because of the rash erythema chronicum migrans, which characterizes the disease (bulls-eye rash concentric rings around initial site of tick bite); other symptoms include arthritis, myocarditis, etc.; there is a vaccine for dogs; treat with antibiotics.

12. Burn infections usually Pseudomonas aeroginosa

13. Wound infections a. Gas Gangrene Clostridium perfringens and other species; often a mixed infection; causes a snap, crackle, and pop sound in crepitant tissue (distorted tissue caused by gas bubbles). b. Tetanus (lockjaw) Clostridium tetani; spores from dirt are deposited deep in tissues (ex. puncture wound from stepping on nail); spores germinate in anaerobic environment; spastic paralysis results from exotoxin; paralysis leads to death; make puncture wounds bleed to flush out spores or flush with hydrogen peroxide (oxygen kills bacteria); disease treated with antitoxin.

B. Viral
1. Rubella (German Measles) - rash first appears on trunk

2. Measles (Rubeola) - febrile disease (causes fever); Kopliks spots (white spots with central bluish specks) appear on upper lip and cheek mucosa; sometimes accompanied by conjunctivitis; rash begins on forehead and spreads to upper extremities, then trunk, and then lower extremities.

3. Chicken pox & shingles already discussed

4. Small pox already discussed

5. Warts caused by papillomaviruses; some warts are malignant (cancerous) some are associated with cervical cancer; grow on skin, genital and respiratory tracts, and oral cavity; transmitted by direct contact or my fomites; no cure for infected but warts can be removed - treated with cryotherapy (freezing) or caustic chemical agents.

6. Roseola caused by a herpesvirus; seen in infants; several days of high fever, followed by a rash. (Be careful not to confuse with Rubella and Rubeola)

7. Fifth Disease (Erythema infectiosum) virus destroys cells in bone marrow that give rise to blood cells; infected children have a bright red rash on the cheeks (slapped cheek rash) that may spread to the trunk and extremities, low grade fever; serious danger to those having chronic hemolytic anemia (ex. sickle cell anemia); virus can travel across the placenta; probably spread via respiratory route.

19th century list of 5 childhood diseases: 1.) scarlet fever, 2.) rubeola, 3.) rubella, 4.) epidemic pseudoscarlatina (type of sepsis), and 5.) erythema infectiosum.

II.

Urogenital Tract Infections & STDs (Chapter 23)


Urinary system: kidneys, ureters, bladder, urethra Reproductive system: Female: external genitalia, vagina, uterus, fallopian tubes, ovaries Male: testes, penis, glands (produce seminal fluid), specific ducts (vas deferens, etc.)

Understand anatomy of urogenital system:

A. Urogenital Diseases Usually Not Transmitted Sexually

1. infections

UTIs Cystitis bladder

Urethritis inflammation of urethra

Ureteritis inflammation of ureter infection

Pyelonephritis kidney

Diagnosis: urinalysis; look for presence of nitrates & proteins. Prevention: drink lots of water; urinate frequently; drink cranberry juice; urinate after sexual intercourse; avoid bubble bath, etc. that could irritate urethra; wipe front to back. Treatment: antibiotics

2. TSS (Toxic Shock Syndrome) S. aureus; incidence rose sharply in late 1970s; rise associated with new superabsorbent, but abrasive, tampons, which were left in vagina for long periods of time; tampons caused small tears in vaginal wall and provided appropriate conditions for bacteria to multiply; organism enters blood and produce exotoxins; fever, low blood pressure (shock), and a red rash on the trunk; deaths are due to shock.

3. Vaginitis usually caused by opportunistic organisms that multiply when the normal vaginal microflora are disturbed by antibiotics or other factors (pregnancy, use of contraceptive pills, menopause, douching); most bacterial infections caused by Gardnerella vaginalis.

B. STDs (Sexually Transmitted Diseases)

1. Syphilis Treponema pallidum (spirochete) can be transmitted in saliva; see disease stages on p. 579; has periodic latent stages that make patients sometimes think that they do no have an STD; called the great imitator because its symptoms mimic those of so many other diseases; heart valves, blood vessels, and meninges can be affected; internal gummas destroy neural tissue, external gummas destroy skin tissue; mental illness accompanies neural damage; congenital syphilis occur when bacteria cross the placenta from mother to baby infant may show signs such as notched incisors (Hutchinsons teeth), a perforated palate, a deformed tibia (shin bone), an aged face with a saddle-shaped nose; treated with antibiotics

2. Gonorrhea (flow of seed a Greek physician in 130 AD mistook pus for semen) Neisseria gonorrhoeae; can survive in dried masses of pus for 6-7 weeks; possess pili that allow them to attach to epithelial cells and to sperm; produce an endotoxin that damages the mucosa in reproductive tract and produce proteases that destroy IgA antibody; some people are asymptomatic; can be a carrier for 5-15 years postinfection; contraceptive pill alter vaginal conditions in favor of gonococcal growth; females can develop PID (pelvic inflammatory disease which leads to sterility); can also cause infections of pharynx, rectum, arthritis, blindness); treatable with antibiotics; silver nitrate administered to the eyes of newborns to prevent infections

3. Nongonococcal Urethritis (NGU) most cases caused by Chlamydia

trachomatis; prevalence is greater than any other STD; bacteria has an


intracellular life cycle; symptoms are similar to gonorrhea, but milder; disease my be difficult to detect (80% of women and 10% of are asymptomatic); females can develop PID; infants can become infected when passing through birth canal; silver nitrate does nothing.

4. AIDS (Acquired Immune Deficiency Syndrome) caused by HIV (Human Immunodeficiency Virus); most cases in U.S. caused by type I.

a. About the virus: This virus is called a retro virus (retro means backward). This virus uses the enzyme reverse transcriptase to make DNA from its RNA. This DNA can be integrated into the host cell's chromosome (now called a provirus). The provirus can stay in a latent stage in which it is replicated along with host cell DNA, causing the host cell no damage. AZT (azidothymidine), which is used against HIV, helps stop reverse transcription by targeting the enzyme reverse transcriptase. b. Symptoms: infection typically causes flu-like symptoms, followed by an asymptomatic period of months or years during with the patient can transmit the disease; HIV specifically targets and destroys T helper cells and macrophage, thus affecting both the B cell and cytotoxic T cell responses, so person can die of a secondary infection; see page 520 for symptoms; most patients develop Kaposis sarcoma (tumors of blood vessels seen as purplish spots on skin, but occur throughout the body); AIDS has become the leading cause of death among 25 to 44 yr. olds! c. Transmission: sexual contact (all forms), sharing of needles, blood transfusions, mother to infant; development of a vaccine is difficult due to high mutation rate of virus

5. Hepatitis B Virus (called AIDS Twin and serum hepatitis); transmission is the same; can create a carrier state (cant ever donate blood); symptoms are similar to those of hep A, except that liver cells are frequently destroyed; some patients develop fulminant hepatitis, a condition of total liver failure; about half of infected people are asymptomatic; treatment relieves some symptoms, but does not cure disease; has been linked to liver cancer (increases chances of developing liver cancer by as much as 300 times); now part of routine infant vaccination.

6. Hepatitis C not identified until 1989; mostly transmitted by blood or sexual contact; usually mild or inapparent infection, but can be severe in compromised individuals.

7. Herpes

a. herpes simplex type 1 oral herpes; causes fever blisters or cold sores

b. herpes simplex type 2 genital herpes

Oral can become genital and genital can become oral (usually due to oral sex).

Latency is a hallmark of herpes infections. More than 80% of the adult population worldwide harbors these viruses, but only a small proportion experience recurrent infections. Within 2 weeks of an active infection, viruses travel via sensory neurons to ganglia, where they become latent. They can be activated by fever, UV, stress, hormone imbalance, menstrual bleeding, trauma, etc. When reactivated, virus moves along neuron to the epithelial cells, where it replicates and causes lesion.

Neonatal herpes babies become infected when passing through birth canal; rare, but can become infected in utero; neonates with disseminated infections usually have central nervous system damage and die within 10 days.

Disease can affect mucous membranes of mouth, eyes, lungs (herpes pneumonia); can also affect skin in places other than the mouth and genitals (a herpetic whitlow is a lesion on a finger that can result from exposure to oral, ocular, or genital herpes lesions).

Treatment: cannot eradicate virus, but acyclovir can reduce reoccurrence of lesions.

8. Cytomegalovirus (CMV) - Cytomegalic Inclusion Disease - virus is a member of the herpesvirus group; establishes a latent infection that can be reactivated; cells infected with virus swell and develop inclusion bodies; usually transmitted sexually, but also by the exchange of saliva and infected blood; infection in adults & healthy children is usually asymptomatic, but it may a cause brief, mononucleosis-like illness; can cause stillbirths, spontaneous abortions, birth defects; causes systemic infections in AIDS patients.

Chp 19 Bacterial and Viral Diseases of the Brain and Meninges (See Chp 5 for parasitic/fungal/helminthic infections)

I.

Bacterial Meningitis - Meningitis is an inflammation of the meninges, the membranes that cover the brain and spinal cord

Meninges (spinal & cranial) protective coverings around the brain and spinal cord. a. dura mater - "tough mother;" outermost layer of the meninges; tough, fibrous connective tissue; gives structural support. b. arachnoid mater- "spider layer;" middle layer of the meninges; weblike appearance; arachnoid villi capillaries function in the absorption of

cerebral spinal fluid (CSF) (CSF is the fluid that circulates around the brain and spinal cord to provide nutrients and oxygen and to take waste products away). d. subarachnoid space - between arachnoid & pia mater; where cerebral spinal fluid circulates. e. pia mater - "delicate mother;" innermost layer of the meninges; transparent fibrous membrane that adheres to the surface of the spinal cord & brain; it contains numerous blood vessels.

The Disease: Meningitis causes necrosis, clogging of blood vessels, increased pressure within the skull from edema (swelling), decreased CSF circulation, impaired central nervous system function. Early symptoms are headache, fever, and chills. Death occurs from shock and other serious complications.

A. Meningococcal Meningitis Neisseria meningitidis causes 2000-3000 cases per year in the US. Mortality is 85% if untreated, but only 1% if treated; Organism colonizes nasopharynx, spread to blood, and to meninges; cocci can invade all parts of body and cause endotoxin shock and death within hours (WaterhouseFrederichsen syndrome); vaccine is available, but is not very effective.
B. Haemophilus Meningitis Hameophilus influenzae type B (Hib) causes 2/3 of bacterial meningitis cases during the first year of life; children can be carriers; human are exposed to bacteria early in life and rapidly acquire immunity, so disease is rare in adults; almost always fatal if not treated (about 1/3 die even with treatment); leading cause of mental retardation in US and worldwide; Hib vaccine has drastically reduced incidence. C. Streptococcus Meningitis leading cause among adults; organism spread via the blood from lungs, sinuses, and ear infections.

D. Can also be caused by E. coli and Staphylococcus species.

II.

Viral Diseases of the Brain and Meninges

A. Viral Meningitis unlike bacterial meningitis, which is always fatal if untreated, viral meningitis is usually self-limiting and nonfatal; can be caused by enteroviruses or mumps virus; cannot be treated with antibiotics.
B. Encephalitis

The Disease Causes an inflammation of the brain. 4 types (all caused by a different virus): Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), Venezuelan Equine Encephalitis (VEE), and St. Louis encephalitis (SLE). Named because they infect horses more often than humans.

Transmission usually from a mosquito to a bird, back to a mosquito, and to a horse, human, or other mammal.

Clinical Signs Fever, headache; convulsions sometimes occur. EEE is the most serious (causes sever necrotizing infection of the brain). VEE resembles the flu in humans. SLE occurs in late summer epidemics about every 10 years and is severe in elderly patients.

Treatment only alleviates symptoms. Vaccines are available for horses, but are not used on humans for fear of inducing a virulent form of the disease.
C. Rabies

Reservoirs dogs, bats, foxes, coyotes, skunks, raccoons; bats can be asymptomatic carriers.

Transmission bite (viruses are shed in saliva)

Diagnosis IFAT (immunofluorescent antibody test) developed in 1958; animal is killed and its brain is examined for rabies antigens; prior to 1958, brain was examined for Negri bodies (inclusion bodies-clusters of viruses in neurons).

The Disease virus replicates in injured tissues and then slowly migrates to nerves where it eventually reaches the central nervous system (brain/spinal cord); the length of time required for symptoms to appear is proportional to the distance between the wound and the brain and is affected by the accessibility of nerve fibers (think about a face bite vs. a foot bite).

Clinical Signs headache, fever, nausea, partial paralysis near the bite site (persist for 2-10 days, then worsen); paralysis then becomes more general, throat muscles undergo painful spasms, confusion and hallucinations occur; 1014 days after onset, patient goes into a coma and dies.

Treatment Ordinarily there is sufficient time for the bitten individual to be vaccinated and to respond by making enough protective antibodies to prevent onset of the disease; once symptoms have occurred it is too late to vaccinate and death usually follows quickly. No longer administer 20 abdominal injections/day to treat rabies! The current vaccine is given intramuscularly on days 0, 3, 7, 14, and 28. Hyperimmune globulin (antibody) is also placed deep in the wound and infiltrated around the wound (remember passive immunization).

III. Bacterial Nerve Diseases


A. Hansens Disease already discussed B. Tetanus already discussed

C. Botulism & Infant Botulism already discussed

IV.

Prion Diseases of the Nervous System


Prion Defined: infectious agent composed only of protein (no DNA or RNA!).

Includes Kuru and Creutzfeldt-Jakob Disease (CJD) of humans, mad cow disease, scrapie of sheep, chronic wasting disease of elk and mule deer. These diseases are referred to collectively as transmissible spongiform encephalopathies because they give brain tissue a spongy appearance.

Kuru (Laughing Death) Spread through breaks in the skin; occurred mainly in New Guinean women. Discovered that they prepared the bodies of the dead for cannibalistic consumption and smeared their own bodies with the raw flesh of the corpses. Since cannibalistic rites have been stopped, the disease has disappeared.

CJD in most cases, no source of prions has been identified; there seems to be a genetic predisposition in some families.

Mad Cow Disease reached a peak in Britain in the early 1990s; spread through the practice of boiling down animal remains for livestock feed. The US has banned the import of British beef, cattle, and beef products.

Prions have clearly been transmitted from one species to another in lab trials.

Could play a role in Alzheimers and Parkinsons diseases.

Chp 21 Bacterial & Vial Infections of the Respiratory System See Chp 5 for parasitic/fungal/helminthic infections

I. The Respiratory System


A. Structure & Function - 2 Regions: 1. Upper Respiratory System a. nasal cavity - incoming air is filtered by mucus (which traps dust particles) and cilia of columnar epithelial cells (which move the particles to the throat for elimination) called the mucociliary defense. b. pharynx - extends from the internal nares and extends partway down the neck, where it opens into the esophagus (food tube) and the larynx; 3 regions of the pharynx: nasopharynx (uppermost portion; contains 2 internal nares & 2 openings that lead into the Eustachian tubes to equalize ear, nose, & throat air pressure), c. oropharynx (middle portion; serves as a common passageway for air, food, drink), laryngopharynx (lowest portion; just above the esophagus and larynx). d. associated structures: 1.) sinuses - air-filled cavities; one fxn.: produce mucus. 2.) tonsils - nodules of lymphatic tissue that form a ring at the junction of the oral cavity & oropharynx & at the junction of the nasal cavity and nasopharynx; the single adenoid tonsil is embedded in the posterior wall of the nasopharynx. 3.) epiglottis - large , leaf-shaped piece of cartilage lying on top of larynx; during swallowing the larynx elevates, causing the epiglottis to fall on the glottis (opening into larynx) like a lid, closing it off - this prevents food from entering the windpipe (trachea).

2. Lower Respiratory System a. larynx (voice box) - mucous membranes are composed of ciliated columnar epithelial cells. b. trachea (windpipe) - air passageway extending from the larynx; it divides into right and left primary bronchi; mucous membranes are composed of ciliated columnar epithelial cells; windpipe is supported by hyaline cartilage rings. c. bronchi - the trachea divides in to a right primary bronchus (goes to right lung) and a left primary bronchus (goes to left lung); bronchi are similar in structure to the trachea; upon entering each lung the primary bronchi divide to form smaller bronchi the secondary bronchi; secondary bronchi continue to branch into smaller bronchi, called tertiary bronchi, that then divide into bronchioles, which divide into even smaller terminal bronchioles.

As the tubes get smaller, several structural changes occur: Cartilage rings are replaced by cartilage plates and then the cartilage disappears all together. The mucous membrane changes from ciliated columnar epithelium to cuboidal epithelium (any debris reaching these smaller tubes must now be removed by white blood cells). As the cartilage decreases the amount of smooth muscle increases during asthma attacks the muscles go into spasm and because there is no supporting cartilage the spasms close off the air passageways - asthma medication such as albuterol targets this smooth muscle tissue).

d. lungs - lobes contain lobules (small compartments in the lungs); each lobule contains a lymphatic vessel, an arteriole, a venule, and

a branch from a terminal bronchiole;terminal bronchioles subdivide into microscopic branches called respiratory bronchioles, which subbdivide into several alveolar ducts; around each alveolar duct arealveolar sacs containing alveoli (cup-shaped projections lined with epithelium); the exchange of respiratory gases between the lungs and blood takes place by diffusion across the alveolar and capillary walls; the lungs are surrounded by a membrane called the pleura.

B. Defenses & Normal Flora 1. Defenses a. mucociliary defenses in upper respiratory system, larynx, trachea, & bronchi (cilia on that columnar epithelial cells whips up mucous. b. macrophages in the bronchioles & alveoli. c. IgA (antibody) protects mucous membranes. 2. Normal Flora - includes species of streptococci, lactobacilli, & some Gram negatives in the upper respiratory system; the lower respiratory system (as well as the sinuses & middle ear) is normally sterile.

C. Clinical Syndromes 1. Rhinitis - nasal inflammation; most common of all respiratory syndromes. 2. Adenoiditis - infection of adenoid tonsil 3. Pharyngitis - infection of the throat; called tonsillitis if tonsils are primarily infected; symptoms include sore throat, sometimes fever, throat may be covered by a milky white exudate, ulcers, blisters, or even a grayish membrane. 4. Sinusitis - sinuses fill with fluid & become infected. 5. Otitis media - middle ear infection; middle ear fills with fluid & becomes infected.

6. Epiglottitis - infection of the epiglottis; can cause the epiglottis to swell to many times its normal size; can cut off respiration and cause sudden death; on rare occasions laryngitis (infection of the larynx) & laryngotracheobronchitis (croup - produces a barking cough) may also cause this to occur; a severe airway narrowing near the epiglottis or the larynx causes stridor (whistling sound heard as the person breathes in). 7. Bronchitis - infection of the bronchi; produces a thick, infected mucous; cough brings up infected phlegm; fever is another symptom; complete obstruction does not occur because the bronchi are so numerous. 8. Bronchiolitis - infection of the bronchioles; inflammation narrows these tiny collapsible airways; air can enter, but has difficulty getting out; clinical signs include wheezing(musical noise heard during expiration) & trachyapnea (rapid breathing). 9. Pneumonia - infection of the lungs, with fluid & microbes replacing the air that normally fills the alveoli; normal gas exchange cannot take place; clinical signs include fever, trachyapnea, labored breathing, & a cough that may produce infected secretions; if pneumonia involves the pleura, it causes pleurisy, associated with painful breathing; caused by bacteria, viruses, and fungi.

II. Upper Respiratory Infections


A. Bacterial Causes of URI's (Bacteria are the most virulent of the u.r. pathogens, but the great thing is that they can be treated with antibiotics!)

1. Haemophilus influenzae a. General G(-) rod needs a growth factor present in human red blood cells - hence the name Haemophilus ("blood loving")

once thought to cause influenza - IT DOES NOT CAUSE INFLUENZA (the flu)!! major cause of virulence is the production of a capsule. b. URI's: 1.) epiglottitis - caused by H. influenzae type B (encapsulated) adults produce Ab's, but young children are at risk; ampicillin not effective due to plasmid-borne antibiotic resistance; conjugate vaccine (Hib) now available; babies are routinely immunized against this pathogen. 2.) sinusitis - caused by H. influenzae (nonencapsulated) strains 3.) otitis media (middle ear infection) - caused by H.

influenzae (nonencapsulated) strains.

c. H. influenzae type B (encapsulated) also causes: 1.) meningitis - infection of membranes covering the brain & spinal cord; before the vaccine this bacterium was the leading cause of meningitis and mental retardation in children. 2.) cellulitis - infection of skin & subcutaneous tissues. 3.) conjunctivitis (pink eye)

2. Streptococcus pyogenes a. General: G(+) cocci pyogenes means "pus forming" do not produce the enzyme catalase (distinguishes them from the Staphs)

medically important strains are classified by their hemolytic & serological properties [see lab manual for beta vs. alpha hemolysis; S. pyogenes is usually beta hemolytic - clear zones of hemolysis] b. URI's: 1.) pharyngitis (strep throat) - transmitted by respiratory droplets or contaminated food/drink; clinical signs: severe sore throat, fever, chills, headache, inflamed pharynx, tender lymph nodes in neck; whitish exudate on tonsils; diagnosis: latex agglutination kit that detects Ag in throat swab. c. Complications of strep throat: 1.) scarlet fever - some strains produce an erythrogenic exotoxin that causes scarlet fever; the toxin kills cells and causes intense inflammation; was once a life-threatening illness; today's cases are mild (due to a decrease in virulence). 2.) septicemia - bacteria spread to into the blood stream 3.) rheumatic fever - occurs after the infection is over (postinfection complication); causes inflammation of joints, skin, brain, heart valves (endocarditis); leading cause of heart disease among children in developing countries; bacteria have an Ag similar to that on heart cells wbcs become sensitized to the bacterial Ag, then attack the heart cells; disease can be prevented if strep throat is treated with penicillin within first 10 days; r.f. patients should receive a monthly penicillin injection - they are in danger if they contact another strep infection. d. Other diseases caused by Streptococci: 1.) Impetigo (pyoderma) highly contagious; occurs almost exclusively in children; usually different strain than those that cause strep throat; easily treated with penicillin; usually heals without scarring, but pigment can be permanently lost; also caused by staphylococci.

3. Corynebacterium diphtheriae a. General: G(+) irregular rod produce an exotoxin; gene for exotoxin is carried by a temperate bacteriophage, so only strains infected by this virus can produce toxin & cause diphtheria; toxin interferes with protein synthesis in eukaryotic cells.

b. Diphtheria transmission: respiratory droplets; bacteria is noninvasive, but deeper tissues are affected because they absorb the toxin clinical signs: infected throat swells & becomes covered by a tough, grayish pseudomembrane composed of dead human cells & microbes [membranous pharyngitis]; swollen tissue & pseudomembrane can obstruct airway, leading to death by suffocation; fatal complications can also result if toxin enters the blood stream and damages other organs; treatment: horse antitoxin (serum sickness is a potential risk) prevention: toxoid vaccine - produced by treating toxin with formaldehyde - part of DPT (diphtheria-pertussis-tetanus) series given to infants; immunization does not confer lifelong immunity adults should have a booster every 10 years!

4. Bacterial causes of the common cold: Mycoplasma pneumoniae, Coxiella burnetii. Remember that most colds are viral.

B. Viral Causes of URI's - more common & less serious than bacterial infections; treatment is a challenge (antibiotics are worthless). (See Chapter 10 on these groups of viruses)

1. Rhinoviruses a. General RNA viruses named for portal of entry - rhino means "nose" primary cause of commn cold - causes 1/4 to 1/2 of colds. about 100 different serotypes (have different antigens in capsids), with new types continuing to be identified. b. Common Cold - clinical signs: sneezing, rhinorrhea (excess nasal mucous), nasal congestion, sore throat, fever, headache; malaise (feeling of general discomfort) due to interferons produced to combat the infection; cold typically lasts 1 week; transmission mainly by direct contact (hand to hand) or fomites; also by respiratory droplets; treatment: none - recovery depends on individual's immune system antibiotics are useless; over-the-counter medications only help alleviate symptoms.

2. Coronaviruses - also cause the common cold. a. General named for prominent spikes on their outer surface difficult to isolate in cell culture cause 10-15% of colds in adults (also cause pneumonia & intestinal infections).

3. Other viral causes of the common cold: coxsackieviruses, echoviruses, adenoviruses, myxoviruses.

III. Lower Respiratory Infections


A. Bacterial Causes

1. Streptococcus pneumoniae [pneumococcus] a. General: G(+), lancet-shaped diplococci (paired diplococci with pointed ends) do not produce catalase; optochin sensitive; alpha hemolysis part of normal flora of 10% of population capsule is critical factor in virulence causes 90% of acute bacterial pneumonias

b. Pneumococcal Pneumonia 1.) transmission - respiratory droplets 2.) clinical syndrome: bacteria in the lung trigger an intense inflammatory response; leaky capillaries allow fluid, blood cells, & serum proteins to flow into the alveoli, filling them; the affected region becomes consolidated, giving the impression of a solid organ when tapped, listened to, or penetrated by x-rays; difficult & labored breathing occur; sputum is bright with blood; 30% of untreated patients die - suffer from unrelenting fever & worsening respiratory problems; pneumococcal sepsis (bacteria enter the bloodstream) can occur - particularly in patients with no spleen spleen filters out bacteria for macrophages to engulf and process Ag (Ag presentation in antibody-mediated response).

3.) treatment - penicillin is the standard treatment, although some resistant strains do exist. 4.) vaccine: Pneumovax - polyvalent (multiple-Ag) vaccine. 5.) bacteria that cause the same clinical syndrome: Haemophilus

influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, E. coli .

2. Mycoplasma pneumoniae a. General lack a cell wall adhere to epithelial cells; do not invade deeper tissues grow slowly in lab grows in trachea; transmitted in respiratory droplets

b. Mycoplasma Pneumonia (Primary Atypical Pneumonia or "Walking Pneumonia")

1.) clinical syndromes: occurs most frequently in school-age children


& teens; comes on gradually; mild; causes headache, low-grade fever, persistent dry cough; shows a patchy pattern in chest xrays rather than dense consolidation of an entire lobe; rarely fatal; viruses, chlamydiae, & rickettsiae cause similar atypical pneumonias.

2.) treatment: tetracycline; erythromycin for pregnant women &


young children; not sensitive to penicillins & cephalosporins because they lack a cell wall.

3. Chlamydia psittaci a. General

obligate intracellular parasites; can only be cultured in lab in chick embryos; therefore, infections are usually diagnosed serologically. psittaci means "parrot" b. Ornithosis, Psittacosis, or Parrot Fever 1.) transmission - microbe commonly infects all types of birds; usually does not produce illness in bird; disease spreads when infected bird become stressed and microbe is excreted in bird's droppings; humans inhale microbe from droppings; occupational hazard for bird handlers or those working in poultry industry. 2.) clinical syndrome - fever, headache, chills, cough; can progress to persistent high fever, mental confusion, & marked shortness of breath. 3.) prevention - antibiotic supplements in feed & antibiotic treatment of imported birds have prevented many infections. 4.)

Coxiella burnetii

a. General rickettsia - life cycle requires both insect (tick) & vertebrate hosts; obligate intracellular parasite "Q" stands for query, as etiologic agent was unknown. Another species causes trachoma, a blinding conjunctivitis. b. Q Fever 1.) transmission - humans become infected by inhaling the microbe from infected animal placentas, feces, amniotic fluid, or milk (they can survive the pasteurization process).

2.) clinical syndrome: atypical pneumonia; can't be distinguished


clinically from mycoplasmal pneumonia or parrot fever; rare disease; rarely causes death.

5. Legionella pneumophila a. General In 1976, 183 American Legion conventioneers in Philadelphia became ill with a mysterious form of pneumonia. this flagellated microbe can be seen only by means of special stains, such as silver-impregnation stains or IFA. can live inside macrophages; multiply as an intracellular pathogen.

b. Legionellosis or Legionnaires' disease 1.) transmission - lives in natural & artificial water supplies; survives heat & chlorination; infections occur when waterborne microbes become aerosolizes & are inhaled (water-cooled air conditioning systems). 2.) clinical syndrome - minor symptoms in most; some develop a virulent pneumonia - sudden onset, weakness, headache, high fever, cough, shaking chills; x-rays show consolidation of an entire lobe; smokers & alcoholics are particularly susceptible.

3.) treatment - resistant to penicillin & cephalosporins; early


diagnosis is critical in order to treat with erythromycin.

6. Bordetella pertussis a. General G(-) coccobacillus produces exotoxins pertussis means "intensive cough"

b. Pertussis (Whooping Cough) 1.) transmission - respiratory droplets; highly contagious

2.) clinical syndrome - uncontrollable fits of coughing (paroxysms) 3.) treatment - by the time diagnosis occurs, the toxin has caused considerable damage, so antibiotic treatment does little to shorten the illness; supportive nursing care is the only treatment. 4.) prevention - infants are protected by the DPT immunization series; vaccine has side effects (fever, convulsions in some); an acellular vaccine genetically engineered vaccine is being developed.

7. Mycobacterium tuberculosis a. General rod-shaped obligate aerobe; id. by Robert Koch waxy cell capsule contains mycolic acids; called "acid fast;" require special staining techniques; waxy capsule allows microbe to survive prolonged drying and resists digestion by lysozymes inside a phagocyte; can remain viable for as long as 8 months upon entering the air when a patient coughs. other species also cause tuberculosis.

b. Tuberculosis - leading killer among infectious diseases today; can also affect the lymphatic, the genitourinary, the skeletal, & the nervous systems. 1.) transmission - respiratory droplets 2.) clinical syndrome: a.) primary infection - microbes enter the lungs & are phagocytized by alveolar macrophages, but are not killed; several weeks later, T cells are activated, eliciting a cell mediated immune response & tuberculin hypersensitivity (Type IV, delayed); cellular immunity helps control the infection - hypersensitivity causes most

of the tissue damage associated with severe cases; the bacteria are isolated within nodules called tubercules or granulomas (dense collections of activated macrophages & lymphocytes); if host cells in the center of a tubercle die, the dead tissue looks dry & crumbly, like cheese (called caseation necrosis "cheeselike death"); bacteria can persist in these granulomas for many years; progressive primary infection can occur - cellular immunity fails to control the microbes and they spread to other parts of the body; miliary tuberculosis is a life threatening form of the disease infection sites are so numerous, they look like seeds of millet (grain) scattered throughout the body. b.) secondary (reactivation) infection - walled-off bacilli inside old tubercles may escape; the ensuing hypersensitivity reaction can destroy the lungs; patients suffer a chronic cough (consumption); without 3.) prevention 1.) BCG vaccine - Bacilllus Calmette-Guerin - made from attenuated M. bovis; not entirely reliable; not widely used in U.S., but is used in other parts of the world. 2.) Skin testing 4.) treatment - people who have a positive skin test but do not have active tuberculosis are treated with a single drug (ex. isoniazid) for 1 yr. (prevents reactivation infection); people with active infections receive multiple drug therapy for 2 years. treatment, the disease is fatal.

Important: The number of cases in the U.S. is on the rise. New drug-resistant strains have been identified; have emerged in patients who did not finish their full course of medication and are lost to medical follow-up.

B. Viral Causes

1. Influenza virus a. General orthomyxovirus family, enveloped virus composed of 8 separate pieces of RNA; this and its ability to infect an already infected cell enables the virus to undergo genetic recombination (antigenic shift); this contributes to the virus's genetic variability & potential to cause epidemics. different strains: A (most severe; responsible for pandemics), B (common cause In children), & C b. Influenza (Flu) 1.) transmission - animal reservoirs are critical; Type a is widespread in birds, which transmit it to pigs, who transmit it to humans (you may have heard of the "swine flu"). 2.) clinical syndrome - fever, headache, muscle aches, cough; tracheobronchitis; person becomes prone to secondary bacterial infections because ciliated columnar epithelial cells are killed; can also cause pneumonia. 3.) treatment - antibiotics only prevent secondary bacterial infections; antiviral agent amantadine can speed recovery if it is administered during the first 2 days of illness. 4.) prevention - immunization (only 70% of those vaccinated are protected); changing Ag's required a new vaccine every year. 2. Parainfluenza virus a. General paramyxovirus family; RNA viruses; enveloped some cause common cold

b. Croup 1.) transmission - respiratory droplets 2.) clinical syndrome - laryngotracheobronchitis causes the airway to narrow at and below the vocal cords; extremely severe cases can resemble epiglottitis, but the illness is usually milder and more gradual in onset; common in toddlers; loud, barking cough; symptoms are worse at night when mucous accumulates (take them outside in the cold - cold air constricts blood vessels; cool mist humidifier thins mucous). 3.) treatment/prevention - supportive nursing care; no immunization.

3. Respiratory Syncytial Virus (RSV) a. General paramyxovirus infected respiratory tissues develop syncytia (large, abnormal cells with multiple nuclei) b. Bronchiolitis - most common cause of fatal lower respiratory infection in young children/infants. 1.) transmission - hand to hand; respiratory droplets; nosocomial infections. 2.) clinical syndrome - infants under 6 mo. suffer the most; wheezing; rapid breathing. 3.) treatment/prevention - supportive nursing care; no immunization.

4. Hantavirus

a. General - arboviruses; named for Hantaan River in North Korea b. Hantavirus Pulmonary Syndrome - appeared mysteriously in the early 1990's in the 4 Corners area of the American Southwest. 1.) transmission - virus occurs in the long-tailed deer mouse; the mice shed the virus in their urine, feces, & saliva, and people contract the disease by inhaling aerosolized viral particles. 2.) clinical syndrome - fever, muscle aches, respiratory distress; 70% of cases result in death within 5-6 days; death caused by catastrophic lung failure; capillaries leak profusely & fluid fills the air spaces.

IV. Other Viral Diseases (Portal of Entry is the Respiratory System, but Usually Affect other Systems)

1. Mumps - paramyxovirus; transmitted in saliva or respiratory secretions; enters a new host through the respiratory system; infects salivary glands; swelling of glands results, along with mild pain and sometimes fever; sometimes enters bloodstream and infects other tissues; in adult males may infect the testes (inflammation called orchitis); in rare cases it infects the inner ear, causing deafness; part of MMR (mumps, measles, rubella) vaccine given at 15 mo.

2. Rubeola Measles (= Measles) - paramyxovirus; one of most communicable diseases known; virus is inhaled; virus multiplies in respiratory tract, then spreads throughout the body, multiplying in lymphoid tissue; virus fuses cell to one another - can be seen under the microscope; symptoms: fever, cough , runny nose, conjunctivitis ("pink eye"), Koplik spots appear around mouth; rash appear first on face and gradually spreads downward to cover the entire body; complications such as ear infections, pneumonia, & encephalitis can occur; part of MMR vaccine given at 15 months.

3. Rubella measles (German Measles) paramyxovirus; virus is inhaled; incubation period is 2 weeks; symptoms: mild fever, rash that lasts less than 3 days, swollen lymph nodes; complications are rare; not as communicable as measles or chickenpox; infection during first 3 months of pregnancy can cause a miscarriage or birth defects; part of MMR vaccine given at 15 months.

4. Varicella Zoster (Chicken pox & Shingles) - like herpes, this virus establishes a latent infection in nerve cells that can be reactivated later; people infected for the first time develop a generalized infection called varicella or chickenpox, that produces blisters all over the body; recovery is complete, but the virus remains latent in neurons; adults who come down with chickenpox can contract a life threatening viral pneumonia; chickenpox during pregnancy is dangerous; reactivation of a latent varicellla zoster infection is called shingles; it is usually brief, but can be painful; passive immunization with varicella zoster immune globulin (VZIG) greatly decreases the severity of chickenpox if administered within 3 days of exposure; an active vaccine has been approved in the U.S. & is currently being administered. 5. Smallpox (Variola virus) - virus is inhaled; less communicable than measles, but very hardy; disease was eradicated in 1979; a closely related poxvirus, vaccinia, is used for smallpox immunization; severity of disease depends on strain; produces a high fever and a severe blistering rash, killing about half of its victims.

6. Infectious Mononucleosis ("Kissing Disease") - caused by Epstein-Barr Virus (EBV); associated with lymphatic system; virus establishes a latent infection in B cells symptoms: fever, fatigue, sore throat, swollen lymph nodes, enlarged spleen (spleenomegaly - appears 1-2 mo. after infection); in most patients, the illness lasts 4-6 weeks; EBV is one of the few viruses proved to be oncogenic (Burkitt's lymphoma, nasopharyngeal carcinoma, B cell lymphoma).

Chp 22 Intestinal system - Bacterial & Viral Infections of the Digestive System

see Chp 5 for parasitic/fungal/helminthic infections Parts of the digestive system: oral cavity, esophagus, stomach, small intestine, large intestine (colon), associated structures (salivary glands, liver, pancreas)

Clinical syndromes: Gastro = stomach; entero = small intestine; col = colon or large intestine Gastritis inflammation of the stomach; causes pain and occasional bleeding Gastroenteritis diarrhea, nausea, vomiting, crampy abdominal pain; can be caused by viruses or bacterial enterotoxins; also caused by intoxications (toxins produced outside of the body in food and are then ingested) Colitis involves colon; involves significant cellular damage (unlike gastroenteritis); diarrhea contains blood and mucous; called enterocolitis (dysentery) if it involves both coon and some of lower small intestine. Dental caries cavities or tooth decay Periodontal disease or periodontis destruction of gum and bone tissue Parotitis infected parotid salivary glands (over jaw, below ear) Hepatitis liver damage; symptom is jaundice (the liver filter bilirubin out of the blood and secretes it into bile that is dumped into the small intestine; if the liver is not functioning, then bilirubin remains in the blood stream; bilirubin is a yellow pigment that comes from the breakdown of hemoglobin, the pigment in red blood cells responsible for the transport of oxygen in the blood.) Food-borne intoxication toxin is produced in food and is then ingested; disease is not caused by bacteria multiplying inside the body; antibiotics are useless!

I. BACTERIAL DISEASES ACQUIRED THROUGH THE DIGESTIVE SYSTEM

Streptococcus mutans
Disease dental caries

Mechanism of pathogenesis; What makes this strep cariogenic? Bacteria possess adhesins on its pili that allow it to cling firmly to tooth enamel; it produces a glucan mesh from sucrose (mesh + bacteria + debris = dental plaque); it also produces lactic acid which damages dental enamel.

Susceptibility determined by consumption of sugar, genetic factors, fluoride (makes enamel stronger)

Prevention future vaccine; introduce antibodies (passive immunization); modify mouths normal flora (introduce a species to compete with S. mutans)

Bacteroides gingivalis
Disease gingivitis & periodontal disease; leading cause of tooth loss in adults; dkamages tissue that surround and support teeth; most infections affect the gingiva and then spread to periodontal structures.

Shigellosis (Bacillary Dysentery) Etiologic agent Shigella (G- rod; nonlactose fermenter; does not produce H2S)

Clinical Signs fever; enterocolitis (stools are streaked with blood and contain strings of mucous composed of many neutrophils); toxin may also contribute to watery diarrhea; toxin may affect other organs in body; causes convulsions in children; can be life threatening due to dehydration. Mechanism of pathogenesis/invasiveness adhesin proteins on pili bind to human colon cells; colon cells phagocytize the bacteria; they are taken into the cytoplasm where they multiply and inhibit protein synthesis; bacteria then cause lysis of the

host cells; produce patchy areas of destruction and inflammation called microabscesses; shiga toxin causes damage to blood vessels in intestinal wall and intense inflammation.

Epidemiology Transmission fecal oral route (flies, food, water, fomites are vehicles) Who? Children are more affected than adults; used to be known as asylum dysentery due to massive outbreaks in mental institutions. Treatment fluid therapy; antimicrobial therapy

Typhoid Fever Etiologic agent Salmonella typhi (G- rod; nonlactose fermenter; produce H2S)

Clinical Signs high fever (>104) continues for days or weeks; some develop rose spots (faint rash); majority recover (about 10% die); some develop a chronic gallbladder infection that makes them persistent carriers (may not work around children or food).

Mechanism of pathogenesis/invasiveness invasive (unlike Shigella); produce an endotoxin (accounts for high fevers)

Treatment antibiotics (chloramphenicol is drug of choice, but has toxic side effects) Salmonellosis Important: classified as a food poisoning, but is a true infection caused by bacteria multiplying in the bowel not a foodborne intoxication. Etiologic agent Salmonella enteritidis & Salmonella cholerasuis (G- rod; nonlactose fermenter; produce H2S)

Clinical Signs diarrhea (from enterotoxin), abdominal cramps, fever, nausea, vomiting, can develop into severe dehydration or systemic bloodborne infection; onset of illness is usually 1-2 days.

Epidemiology Transmission contaminated food (poultry, unpasteurized milk, eggs); estimated that about 1 in 4 chickens are contaminated; major health concern.

Prevention using nonporous cutting boards; disinfecting cutting boards and cooking utensils; thoroughly cooking foods; washing hands.

Treatment antibiotic treatment of uncomplicated salmonellosis is medically inadvisable (treatment may cause them to become chronic carriers); growing drug resistance among strains due t widespread use of antibiotics in animal feed.

E. coli G- rod, lactose fermenter; Most abundant facultative anaerobe in large intestine of humans part of normal flora; most strains are harmless; also important pathogen of urinary tract. Diseases - Travelers Diarrhea, Dysentery, Epidemic Diarrhea in Nurseries, Hemolytic-uremic syndrome

Clinical signs may involve nausea, vomiting, diarrhea, bloating, malaise and abdominal pain; a typical case of t.d. causes 4-5 loose stools per day for 3-4 days; the toxin causes excessive water and electrolyte secretion; can invade intestinal epithelium and cause dysentery; deadly outbreaks of strain 157:H7 have been

attributed to undercooked hamburgers several cases involved the kidneys and resulted in the condition known as hemolytic-uremic syndrome.

Mechanism of pathogenesis causes a dysentery syndrome almost identical to shigellosis; produce proteins that allow bacteria to invade human cells; its Shigalike toxins inhibit protein synthesis; not as virulent as Shigella.

Prevention of t.d. some travelers take antibiotics prophylactically not recommended (not effective and contributes to development of mutant strains); a better practice is to keep an antidiarrhea medicine available and us it only after symptoms appear. Cholera Etiologic agent Vibrio cholerae(short, curved G- rod; flagellated)

Pandemics in the 18OOs led to the adoption of modern systems of sewage disposal and public sanitation; The current pandemic is caused by the El Tor strain. It began in Indonesia in 1958 and is still rampant in parts of Africa (remember the thousands of Rwandan refugees that contracted it?), South America, and Asia. There was a small outbreak in Alabama (Dauphin island) in 1991 officials had to close an oyster reef they prevented an epidemic.

Clinical Signs dehydration from diarrhea (lose as much as a liter an hour); characteristic rice water stools (water flecked with small particles of mucous); dehydration is sudden and dramatic; a person can die in a day.

Mechanism of pathogenesis cholera exotoxin causes epithelial cells to secrete large quantities of chloride into intestine, causing water, sodium and other electrolytes to follow and leave body as diarrhea.

Epidemiology Transmission fecal-oral (contaminated water, infected shellfish, fish) Prevention sanitation; current vaccine is not effective.

Treatment replacing lost fluid; administer water & electrolytes intravenously; tetracycline.

Campylobacteriosis Etiologic agent Campylobacter jejuni (slightly curved G- rod); not recognized until the 1970s because they are so difficult to cultivate in the lab.

Clinical Signs frequent episodes of bloody diarrhea, abdominal pain, fever; major cause of diarrheal illness and dysentery; causes over 2 million illnesses in the U.S. each year (more thanSalmonella or Shigella).

Mechanism of pathogenesis/invasiveness destroys epithelial lining; produce a toxin and invade cells.

Epidemiology Transmission grows in intestinal tract of cattle, sheep, poultry, dogs, cats; human infection probably occurs from ingesting contaminated meat or milk; direct person-to-person transmission may occur.

Treatment usually non-life threatening and self-limiting (lasts about a week). Peptic ulcers Etiologic agent Helicobacter pylori(G- rod); has only been associated with ulcers since 1993!

How do the bacteria survive the HCL in stomach? Produces an enzyme that converts urea to ammonia, raising the pH in its vicinity.

Treatment antibiotics!!!!!

Botulism (a food-borne intoxication) Etiologic agent Clostridium botulinum (G+ rod, spore-former, anaerobic); toxin production depends on a prophage; bo-tox is the most poisonous natural substance known (as little as .000005 micrograms can kill a mouse one oz. would kill the entire US. Population!)

Clinical Signs flacid paralysis; muscle paralysis starts with the eye muscles; no fever; usual cause of death is respiratory paralysis

Mechanism of pathogenesis produces a neurotoxin that affects the nervous system.

Epidemiology Transmission usually from improperly home-canned nonacid foods

Treatment - antitoxin

Infant Botulism (not a food-borne intoxication bacteria produce toxin while multiplying inside body) Etiologic agent - Clostridium botulinum (G+ rod, spore-former, anaerobic); disease first recognized in 1976.

Clinical Signs infant becomes lethargic and loses the ability to suck and swallow (disease is sometimes called floppy baby syndrome); may be cause of some infant deaths attributed to SIDS.

Epidemiology Transmission associated with feeding honey to infants (10% of honey contains botulism endospores; endospores germinate and grow in the immature digestive tract of infants)

Prevention do not give honey to a child under the age of 12 months.

Pseudomembranous colitis C. diff Diarrhea Etiologic agent Clostridium difficile produces a type of iatrogenic (antibiotic induced) diarrhea; antibiotics kill off normal flora, but C. difficile is resistant; can be life-threatening; often occurs in hospitals (nosocomial).

Epidemiology Treatment - vancomycin

Clostridium perfringens food poisoning (food-borne intoxication)

Clinical Signs mild gastroenteritis; diarrhea; illness lasts less than a day; seldom reported; noticeable illness occurs only if high numbers of spores are ingested.

Epidemiology Transmission lives in the g.i. tract of animals, humans, & is common in feces-rich soil; spores usually contaminate meat; ; when food is left unrefrigerated after cooking, spores germinate and new cells produce toxin which is then ingested.

Staph. aureus food poisoning (foodborne intoxication)


Grapelike clusters of G+ cocci; can survive in foods with a high sugar or salt content.

Clinical Signs vomiting, diarrhea, crampy abdominal pain; onset of illness is rapid (2-6 hours).

Mechanism of pathogenesis produce a heat stable enterotoxin; toxin cannot be destroyed by refrigeration or cooking.

Epidemiology Transmission most frequently reported food poisoning in the U.S. occurs in large outbreaks at picnics or social gathering; most occur because of lapses in food prep or storage; usually introduced into food from body of person preparing it (remember that path Staph are found in many healthy people).

Treatment illness is usually brief and self-limiting; fluid replacement may be necessary.

Bacillus cereus food poisoning (foodborne intoxication)


G+ anaerobe; produces endospores.

Clinical Signs gastroenteritis is usually mild and brief; there are 2 forms of illness associated with 2 enterotoxins; one form causes vomiting, the other causes diarrhea.

Epidemiology Transmission present in soil, water, g.i. tract of humans and animal, so often found in food; when food is left unrefrigerated after cooking, spores germinate and new cells produce enterotoxins which are then ingested.

Listeriosis Etiologic agent Listeria monocytogenes; G+ rod; psychrophilic

Now a leading cause of infection in kidney transplant patients. Can cross the placenta and cause miscarriage and stillbirth; responsible for many cases of fetal damage.

Epidemiology Transmission by improperly processed milk, cheese, meat (hotdogs, lunch meat), and vegetables.

II. VIRAL DISEASES ACQUIRED THROUGH THE DIGESTIVE SYSTEM

Rotavirus Major cause of viral enteritis (water diarrhea) among infants and young children; number of cases rises during winter months in U.S. (helps to distinguish it from bacterial diarrheas; transmission is fecal-oral; viral capsids resemble little wheels.

Norwalk Agents Virus is named for a 1968 outbreak in Norwalk, Ohio; responsible for nearly half of all U.S. outbreaks of acute infectious nonbacterial enteritis; affects older children and adults more often than infants or preschoolers; outbreaks occur throughout the year; characterized by 1-2 days of diarrhea, vomiting, or both; immunity does not follow an attack. Poliomyelitis Caused by 3 strains of polioviruses that have an affinity for motor neurons of the spinal cord and brain.

Clinical signs High fever, back pain, and muscle spasms can occur. Most infections are inapparent, or mild and nonparylitic. In less than 1% of cases, partial or complete paralysis of muscles can occur; nature and degree of paralysis depends on which neurons in the spinal cord and brain are damaged. Any paralysis remaining after several months is permanent.

Epidemiology Transmission fecal-oral route and from pharyngeal secretions; danger of fecally contaminated swimming pools

Vaccines:

1.) Salk vaccine injectable, inactivated (killed) with formalin; became available in 1955; may not produce immunity in all recipients (may require boosters); has to be refrigerated (unlike oral) 2.) Sabin vaccine oral, attenuated (live); provides longer-lasting immunity, but in a small number of cases (about 6 each year in the US), viruses have mutated into virulent viruses; cannot be administered to immunocompromised patients.

Absence of animal reservoirs plus availability of effective vaccines have made health authorities choose polio as the next disease to eradicate from the planet.

Hepatitis A Virus (HAV) Transmission is fecal-oral; occurs most often in children and young adults; shellfish can be contaminated; outbreaks due to contaminated food in fast-food restaurants have been on the rise; symptoms include malaise, nausea, diarrhea, abdominal pain, and jaundice; jaundice is caused by impaired liver function (the liver normally filters out hemoglobin from worn out red blood cells and breaks it down into the yellow pigment bilirubin; bilirubin is normally deposited in bile and is eliminated from the body in feces; if liver function is impaired, bilirubin builds up in the blood stream); disease is usually self-limiting; chronic infections are rare; no treatment except for alleviating symptoms; vaccine is now available.

Chp 20 Diseases of the blood (Bacterial & Viral Diseases Transmitted by Arthropod Vectors) See Chp 5 for parasitic/fungal/helminthic infections

A. BACTERIAL: 1. Plague Yersinia pestis; zoonosis; spread from infected rodents to humans by fleas; incidence of disease is low; bacilli travel in lymphatics to lymph nodes where

they cause enlargements caused buboes (especially in armpits and groin); hemorrhages turn skin black (Black Death); deaths can be prevented with antibiotic treatment.

2. Tularemia Francisella tularensis zoonosis; reservoir: usually cottontail rabbits (number of causes rises during rabbit-hunting season); first isolated in Tulare County, CA in 1911; low incidence in US; disease is an hazard for taxidermists, hunters, trappers; transmission: breaks in skin, bite from arthropod vector (tick, deer fly), inhalation of aerosols during skinning, consumption of contaminated meat; clinical signs: ulcer formation at the entry site, fever, severe headache, and buboes (enlargement of lymph nodes); one form can lead to a septicemia that resembles typhoid fever; vaccine does exist but frequent boosters are required.

3. Relapsing Fever Borrelia recurrentis (spirochete) and other species of this same genus; transmitted by ticks and human body and head lice; lice are crushed and their body contents scratched into the skin; ticks transmit bacteria in their salivary secretions several days of high fever, respites, and shorter periods of fever due to changes in organisms antigens; can cross placenta; treat with antibiotics; syphilis patients were once purposely infected with relapsing fever to kill syphilis bacteria.

4. Lyme Disease Borrelia burgdorferi (spirochete); transmitted by deer ticks (Ixodes damini); white-tailed deer are a reservoir; develop flu-like symptoms; the rash erythema chronicum migrans characterizes the disease (bulls-eye rash concentric rings around initial site of tick bite); other symptoms include arthritis, myocarditis, etc.; there is a vaccine for dogs; treat with antibiotics. 5. Typhus Fever occurs in a variety of forms:

a. Endemic (murine) typhus murine refers to rats and mice; Rickettsia

typhi (obligate intracellular parasite); does occur in Texas; transmitted by fleas


(fleas defecate while biting, infecting the humans); fever, chills, crushing

headache; disease is self-limiting and lasts about 2 weeks if untreated; mortality is low.

b. Epidemic typhus (classic or louseborne typhus) Rickettsia

prowazekii (obligate intracellular parasite); epidemics were halted with


discovery of pesticide DDT during WWII; transmitted by body lice (when a louse bites, it defecates; as victim scratches bite, they inoculate the microorganisms into the wound); fever and headache; rash starts on trunk and spreads to extremities (rarely affects palms or soles); vaccine is available; treat with antibiotics; mortality can range to 40% if untreated.

c. Brill Zinsser disease (recurrence of epidemic typhus) disease has milder symptoms, in shorter in duration, and often does not cause a skin rash; caused by reactivation of latent microorganisms harbored in lymph nodes.

6. Rocky Mountain Spotted Fever Rickettsia rickettsii (obligate intracellular parasite); transmitted to humans by dog ticks; fever, headache, weakness; rash begins on ankles and wrists and progresses toward trunk (reverse of progression in typhus); rash is prominent on palms and soles; blood vessel damage cause hemorrhages throughout the body; treat with antibiotics.

7. Rickettsial pox Rickettsia akari (obligate intracellular parasite); transmitted by mites fond on house mice; disease is mild and lesions resemble those of chickenpox; often misdiagnosed as chicken pox.

B. VIRAL: 1. Yellow Fever monkeys are reservoirs of the disease; transmitted to humans by mosquitoes; many victims suffer severe liver damage and become jaundiced.

2. Encephalitis discussed under nervous system diseases; transmitted by mosquitoes.