SECHENOV MOSCOW MEDICAL ACADEMY _______ MEDICAL FACULTY Division for Foreign Students with Instruction Conducted in English

_______ Department of Microbiology, Virology and Immunology

GENERAL MICROBIOLOGY
THEORETICAL & LABORATORY MANUAL

PART II. PHYSIOLOGY & GENETICS OF MICROORGANISMS

N.V. Khoroshko and Ye. V. Budanova

Edited by: Academician V.V. Zverev Special English edited by: Associate Professor I. Yu. Markovina. Computer Illustrations by: A.V. Budanov

M O S C O W - 2008

SECTION 2: PHYSIOLOGY and GENETICS of the MICROORGANISMS The objectives: To study the bacterial nutritional requirements, energy metabolism, stages of bacterial growth, enzymatic activity. To know the methods for isolation of pure cultures of bacteria and their identification. To be able to interpret the results of an antibiogram. To study the microflora of water and air. To know the microflora of human organism and its role in health and disease. To study the environmental factors affecting the microbes. To know the principles of classification of antibiotics, the mechanisms of natural and acquired bacterial resistance to antibiotics. To be familiar with the basic principles of microbial genetics.

PHYSIOLOGY of MICROORGANISMS. PRINCIPLES of CULTIVATION of BACTERIA. ISOLATION of PURE CULTURES of BACTERIA The morphologic simplicity of most microbes makes it difficult to identify them by microscopic examination alone, since many of them look identical. Isolating, culturing, and identifying suspected pathogens from the different specimens may confirm diagnosis of infectious diseases. Quick and accurate diagnosis of infectious diseases depends on our ability to grow pathogenic bacteria in the laboratory. To grow, all microbes must have suitable physical and chemical conditions, nutrients, and freedom from competitors. To survive, every cell on earth needs water, a source of energy to fuel the demands of the cell, and chemical compounds to supply the building blocks from which the cell is composed. One of the very important characteristics of the bacterial growth is the generation time (or doubling time), the amount of time it takes for the population to double. The generation time is usually constant for each organism as long as physical and chemical conditions do not change. (For example, E. coli has generation time of 20-30 min, but M. tuberculosis of 12 hours). The short generation times of most of the microbes facilitate the rapid production of colonies composed of billions of cells in less than a day after inoculation on a solid growth medium. Despite their capacity to reproduce, there are some natural limitations on the microbial growth. These limitations are illustrated by the kinetics of bacterial growth in a batch culture (see Figure 1). The dynamics of bacterial growth can be observed by inoculating bacteria into a liquid culture medium and measuring population sizes at regular time intervals. When these measurements are plotted on a graph, the resulting growth curve shows four distinct phases of growth: (1) lag phase, (2) logarithmic phase, (3) stationary phase, and (4) death phase. A typical growth curve is shown in Figure 1.

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The sum of all the cell-directed chemical reactions is called metabolism. All organisms can be divided into one of two categories according to their energy source. Phototrophs derive energy from sunlight through photosynthesis. Chemotrophs depend on chemical energy harvested by breaking of chemical bonds. Most bacteria are organotrophs, organisms that use organic compounds, such as sugars and aminoacids, as chemical energy sources. Some bacteria are lithotrophs, organisms that obtain chemical energy from inorganic compounds. Bacteria also need construction materials for building more cell material, for cell growth and reproduction, and for repairing damage. The main element for constructing cell material is carbon. Autotrophs use inorganic carbon in the form of carbon dioxide (CO2), as their sole source of carbon. Heterotrophs require a supply of carbon in the form of organic molecules. The majority of bacteria, including all pathogens, are chemoorganoheterotrophs, obtaining both their energy and carbon from organic compounds. In addition to carbon, all cells need hydrogen, oxygen, nitrogen, phosphorus, and sulfur. All cells also require aminoacids for manufacturing proteins, purines and pyrimidines for making nucleic acids, and vitamins, which assist in many enzyme-mediated reactions. Organisms that cannot synthesize these organic compounds from the raw materials in their environment must be supplied with them. (These bacteria are auxotrophs). Some bacteria synthesize all these components within the cell and do not need an external source. (Such organisms are prototrophs). Movement of Materials Across Membranes When the concentration of a substance is stronger on one side of a membrane than on another, a concentration gradient (difference) exists. Materials move across plasma membranes of prokaryotic cells by two kinds of processes, active and passive. In passive processes, substances cross the membrane with the

concentration gradient without any expenditure of energy (ATP) by the cell. In active processes, the cell must use energy (ATP) to move substances against the concentration gradient.
NOTE: for detailes see Topical Lecture "PHYSIOLOGY of BACTERIA".

In addition to nutrients, microorganisms need environmental conditions that are within a certain range to proliferate. Temperature, pH of nutrient medium, oxygen in atmosphere, and osmotic pressure influence survival and growth.
NOTE: Cultures grow best when incubated in environments similar to the physical conditions of the microbes natural habitat.

All organisms can be also characterized by the range of temperatures within which they grow. Bacteria fall into one of three categories according to the temperature at which they grow best (optimum temperature). Human pathogens are mesophiles, adapted to our 37C body temperature. This temperature can be obtained by cultivating microbes in incubator. Some mesophiles can even grow at very low temperatures, although somewhat slowly. Some of these organisms are responsible for food spoilage within the refrigerator (they are called psychrophiles).
NOTE: for detailes see Topical Lecture "PHYSIOLOGY of BACTERIA".

Microbes differ in their response to molecular oxygen. The basic energy-gathering strategy of most chemoorganoheterotrophs is to convert food molecules to glucose or a by-product of glucose metabolism. The chemical energy in glucose is then released while the sugar is disassembled. If an organic compound is the donor but an inorganic molecule, such as molecular oxygen, is the final electron acceptor, the process is termed respiration. If an organic compound is both the electron donor and the final electron acceptor, the metabolic process is called fermentation. Some organisms, called aerobes, require the presence of molecular oxygen for their energy metabolism (respiration). Other organisms can proliferate in either the presence or absence of molecular oxygen. These facultative anaerobes grow best in the presence of oxygen but can use a less efficient alternative mechanism for energy production in anaerobic (oxygen-free) environments. Facultative anaerobes are able to grow in all oxygen concentrations. Microbes of another group, the microaerophiles, appear to require some oxygen, but too much or too little is detrimental to them. Many microaerophiles are also capnophiles, as they require an elevated concentration of CO2. (In the laboratory they can be easily cultivated in a candle jar, a container into which a lit candle is introduced before sealing the airtight lid. The flame burns until extinguished by oxygen deprivation, creating a CO2-rich, oxygen-poor atmosphere).

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There are a lot of bacteria using no molecular oxygen and some may even be killed by its presence. These anaerobes reproduce in oxygen-free environments such is the dead tissue in deep wounds. Although some anaerobes are aerotolerant (they can survive in the presence of oxygen but cannot use it for metabolism). Many other bacteria are the obligate, or strict, anaerobes. They are killed by even the briefest exposure to oxygen. In all such cells, the presence of molecular oxygen results in the production of the lethal by-products hydrogen peroxide and superoxide (an unstable charged atom of oxygen), which quickly oxidize and destroy cytoplasm. Aerobes, facultative anaerobes, and aerotolerant anaerobes have enzymes that immediately destroy these lethal compounds before they damage cytoplasm. One of these enzymes, superoxide dismutase, converts superoxide to hydrogen peroxide, which is then converted by catalases and peroxidases to harmless water and molecular oxygen. Without these enzymes, strict anaerobes are unprotected against oxygens toxic by-products. Many microaerophiles contain limited enzyme defenses, explaining their failure to survive when oxygen approaches normal

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concentrations. Therefore, the gas composition of atmosphere is very important for bacterial cultivation. (It should be always oxygen-free for strict anaerobes). Deep tube method shows the bacterial growth according to their oxygen requirements. Oxygen diffuses into the upper portions of a tube growth medium, but not all the way to the bottom. When inoculated, the bacteria grow in the part of the tube in which oxygen concentrations suit its requirements. This location provides an indicator of the category into which the bacteria falls. (See Fig.2). If an organism is to be successfully cultivated in the laboratory these needs must be satisfied, in most cases by the culture medium. Microbiological Culture Media Microbial cells require a complex mixture of nutrients for replication. These include nitrogenous and carbonaceous substances, vitamins, minerals, and many diverse elements. The majority of microorganisms encountered in the clinical laboratory are heterotrophic in nature since they require complex compounds as carbon and nitrogen sources. Most microbes have simple nutritional needs. Fastidious microbes, on the other hand, have unusual nutritional requirements, making it more difficult and sometimes impossible to grow them in the laboratory. Factors of paramount importance in the formulation of an effective medium for the cultivation of microorganisms include the following characteristics: It should contain essential food elements It should have satisfactory moisture content It should have correct pH (most bacteria are neutrophiles. They grow best at a pH between 6 and 8) The consistency of the medium should be favorable for cultivation It should be sterile It should be isotonic It should be oxygen-free if the cultivation of strict anaerobes is desired. It should be transparent The scheme of classification of culture media is listed in Table 1. Nutrient broth (MPB) is a liquid general-purpose medium. This medium is used for the cultivation of many species of nonfastidious microorganisms. It can be useful in the routine cultivation of microorganisms, in different methods for the examination of water, etc. Nutrient broth consists of meat extract, peptone and purified water. Peptones are the most common sources of nutrients for culture media. These are water-soluble materials derived from proteins by means of hydrolyzing or digesting the source material (meat, milk, etc) by acid, alkali, or specific enzymes. Peptones are the principle sources of organic nitrogen, particularly aminoacids and large-chained peptides. The meat extract contains

water-soluble substances including carbohydrates, vitamins, organic nitrogen compounds, and salts. Table 1 Commonly Employed Microbiological Culture Media
TYPE General purpose media (1) Transport media (2) Enriched media (for fastidious bacteria) (3) Selective media (4) Enrichment media (ONLY LIQUID!) (5) Differential media Defined media LIQUID SOLID and SEMISOLID Nutrient broth (meat-peptone Nutrient agar (meat-peptone agar, broth, or MPB) or MPA) Special purpose media(specialized media) Isotonic solution, Stuarts transport medium, etc. Sugar broth, Serum broth, Ascitic broth (contains native protein), etc. 1% peptone water (for V.cholerae), 10 % bile broth (for Salmonellae), etc. Selenite broth (for Salmonellae and Shigellae), etc. Hiss liquid media --Stuarts transport medium, Transgrow medium, etc Sugar agar, Serum agar, Blood agar, etc. Salt agar, Egg yolk-salt agar (for Staphylococci), etc. --Hiss semisolid media, Endo agar, MacConkeys agar, etc. Synthetic medium; Minimal agar

Nutrient agar (MPA) is used for the cultivation of bacteria and for the enumeration of microorganisms in water, sewage, feces, and other materials. This is a general-purpose medium that supports the growth of a wide variety of microorganisms. It consists of peptone, purified water, meat extract, and agar. Adding the solidifying agent agar to a nutrient solution easily produces a solid or a semi-solid medium. Agar, an extract of seaweed, melts at about 100C and then solidifies the liquid in which it is dissolved when the temperature drops below 45C. The melted medium is usually poured into Petri dishes, specially designed shallow containers, where it is allowed to solidify. If necessary, the MPA can be solidified in a tilted test tube to produce a slanted surface for microbial inoculation. This is called a slant. Both general-purpose media have final pH=6,8-7,2. After preparation, these media should be sterilized by autoclaving at 121C for 15 min. The general-purpose media can be enriched by addition of blood, ascitic fluid, serum, or other special supplements, which greatly enhance the grownpromoting capacity of the media. Such media are termed as complex specialized media. Media can be described according to their function. Transport media are used for the temporary storage of specimens being transported to the laboratory for cultivation. Transport media typically contain only buffers and salts. The lack of organic growth factors prevents microbial

multiplication. Transport media used in the isolation of anaerobes must be free of molecular oxygen. Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. Blood, serum, yeast extract, vitamins supplement the basic nutrients. Selective media support the growth of some organisms while specifically inhibiting the growth of others. When a particular pathogen is suspected, the specimen can be inoculated onto a selective medium that prevents the normal microbial flora present in the specimen from overgrowing the pathogen. Some media are selective by virtue of limited nutrients; that is, all nutrients have been omitted except those that can be used by a small number of microbes. Most selective media contain inhibitors, for example antibiotics that suppress the growth of contaminants. Enrichment broths encourage the growth of a particular organism that would likely be overgrown by competitors in a sample with a mixture of microbes. The broth is designed to give the competitive edge to the desired microbe, which consequently becomes the dominant species. These media hold the normal flora in the lag phase of growth while promoting logarithmic growth of the desired pathogen.
NOTE: The organisms must then be isolated by streaking on a solid medium to obtain colonies.

Differential media contain indicators that distinguish between organisms on the basis of their appearance on the medium. These media allow certain organisms to produce macroscopically distinct colonies, or characteristic zones around colonies, that are helpful in distinguishing these organisms from others in the sample. Differential media can be used for determination of bacterial enzymatic activity. Endo agar, for example, besides basic nutrients contains one sugar (lactose) and fuchsin. Fuchsin turns metallic red when the pH drops below 6.8. Organisms that ferment lactose to an acid end product lower the pH, which causes the colony to become red. Organisms that fail to use lactose produce colorless colonies.
NOTE: There are some enriched complex media that can be used as differential ones. For example, hemolytic activity of bacteria can be detected on the blood agar. (If certain bacteria have the ability to lyse red blood cells, a clear colorless area surrounds the colonies). The lecithinase activity of staphylococci can be observed on the yolksalt agar. (If the microbes produce this enzyme, the turbulent zone with the opalescent halo occurs around the colonies).

Usually pathogenic bacteria must be isolated and grown in a pure culture (one that contains a single species), since specimens often contain a mixture of different bacterial species. Obtaining isolated colonies of the pure culture on plates permits a study of morphological, colonial (cultural) properties, hemolytic

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reactions, and pigmentation of the colony or in the medium surrounding the colony, biochemical, antigenic and other characteristics that are of great importance for bacterial identification.
NOTE: Once a culture is contaminated, it may be impossible to determine which microbes are contaminants and which are from specimen.

Isolation of microbes in pure cultures requires aseptic technique and special methods of inoculation. Aseptic technique allows handling the materials without the introduction of microbial contaminants from air, water, hands, or other nonsterile sources. To prevent microbial contamination, specimens must be collected only with sterile instruments and placed into the sterile containers or transport media. Exposure of the sample to nonsterile environments such as room air must be minimized and contact between the sample and nonsterile objects and surfaces must be avoided. For example, tubes and plates should be kept closed; they should be opened only when necessary. In addition, equipment for transfer, such as wire loops, should be sterilized by flaming. Inoculation Techniques Microbes can usually be isolated in pure cultures by inoculating solid media in a manner that results in the development of single, or isolated colonies (visible aggregates on the solid medium). Since all the cells in an isolated colony are descendants of a single organism, they are identical. Obtaining isolated colonies requires the use of a solid medium, since cells in a liquid medium cannot be separated. Pure cultures can be obtained also by cultivation on selective media and by inoculation into laboratory animals. There are so many microbes in most specimens that direct inoculation of a solid medium would result in overgrowth of the entire surface of the medium. To get well-isolated colonies, the sample must therefore be processed in such a way that cells are deposited on the medium far enough from each other. This can be accomplished by preparing streak plates, pour plates or spread plates. All streaking techniques depend on spreading the inoculum over a large surface area so that the organisms fall off the loop a single cell at a time. Specimens often contain mixed cultures of bacteria and so it is often difficult to isolate an organism in a single attempt, especially if there are many microbes in the specimen. Sometimes, it is necessary to measure microbial concentration in the specimen; plate counts and other methods can then be used. (Quantative cultures help to determine the causative role of the isolated species). Streak plates The streak plate is used primarily for isolating microorganisms in pure culture from specimens containing a mixed flora. Prior to inoculation, it is

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important that the agar surface be smooth and moist, but without excessive moisture which would cause the resultant colonies to merge (confluent growth). Refrigerated plates should be allowed to reach room temperature before use. Organisms can be inoculated onto the surface of a 2 4 - h o u r i n c u b a t io n plate with a wire loop or swab. A series of streaks spreads the inoculum over a large surface area until very few cells are left i on othe a loop. d F i g u r e 3 . S t r e a k i n g t o o b t a i n s l t e The most commonly employed technique is the quadrant streak method. Streak plates have four distinct areas of inoculation. The first quadrant can be inoculated with a loop or a swab. Each of the remaining three quadrants must be streaked with a sterile instrument such as a flamed loop or a fresh swab. (See Fig.3). Loops for inoculation are constructed from a variety of wire materials, such as platinum, aluminum or nichrome. Important characteristics of these metals are durability when bent into the proper configurations, nontoxicity and an ability to cool quickly following the heating (sterilization) process.

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Pour plates and spread plates Another way to obtain isolated colonies is to inoculate an agar medium with a sample that has been diluted in sterile isotonic liquid. In the pour plate technique, a sample of the diluted culture (usually 1ml) is added to 15 ml of melted agar at 45C. Then this mixture is poured into a Petri dish. This results in the growth of many subsurface colonies. Spread plates are prepared by spreading a small volume (0.2 ml) of the diluted sample across the surface of a solid medium by using a sterile bent glass rod. In this case all the colonies grow on the agar surface. Pour plates and spread plates are generally employed for counting microbes. Measuring microbial concentrations Sometimes it is very important to determine the population density (concentrations of microbes) in a sample. Knowing the degree of contamination in a clinical sample often helps physicians to determine whether a particular organism is causing disease or is merely a contaminant from the normal flora. Microbial concentrations can be measured in the laboratory by several direct and indirect methods.

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Direct counts can be performed by examining the microbe-containing liquid 0 , 1 m l deposited on a slide with the 0 , 1 m l brightfield microscope in bacterial counting chamber or with an electronic particle counter. Direct - 3 - 5 - 7 1 0 1 0 1 0 counts do not permit differentiation between living and dead cells and S p e c im e n t o b e c o u n t e d can failed if the microbial ( 1 : 1 0 ) population in suspension is too diluted. Plate count is the most 9 , 9 m l S e r i a l common indirect method. In 0 , 9 % N a d C i l lu t i o n 0 , 2 m l 0 , 2 m reality, each colony represents a l colony-forming unit (CFU). Prior to inoculation, the clinical sample has to be diluted. A series of - 5 - 7 1 0 1 0 dilutions are usually plated to ensure that at least one plate has a countable number of distinct colonies (See Fig.4). Another method for indirect counts is turbidimetric measurements. Turbidity, or optical density, is the cloudiness of a suspension that occurs when bacterial cultures grow in broth. In the laboratory, turbidity is quantified with a spectrophotometer, an instrument that measures the amount of light transmitted through a sample. The turbidity increases as the number of bacterial cells increases in the liquid medium.
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All the inoculated plates should be then incubated under the proper conditions (optimal temperature and gas composition of atmosphere).

Identification Technique (BACTERIOLOGICAL EXAMINATION) To identify a bacterium means to determine its taxonomy (division, family, genus and species). Prior to identification, the microbes are to be isolated in pure cultures. Identification is based on the complex examination of the following bacterial properties:

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1. Morphological and tinctorial (staining) properties Morphological and staining properties are to be examined not only for identification but also for verification of the cultural purity. Except for infrequent mutations, pure cultures show uniform morphologic and tinctorial properties. 2. Cultural characteristics. The appearance of the isolated colonies provides additional clues to the identity of the microorganisms. Cultural characteristics that contribute to identification are size, shape, pigmentation, texture, consistency, and other colonys features. Pigment production by bacterial species depends on the cultural conditions. Pigments may be classified into two groups, extracellular and intracellular, according to their occurrence outside or inside the cell. Some pigments are excreted into the surrounding medium. The main chemical groups to which the pigments belong are the carotenoids, quinones, phenazines, and melanines. 3. Biochemical tests(fermentation reactions, or enzymatic activity) These characteristics can be used to distinguish between microbes that appear morphologically and culturally identical. Microbes produce biochemicals such as acids, alcohols, gases, or specific enzymes, the detection of which may aid in identifying the organism. The identification of bacteria is accomplished by determining the presence of various enzymes, because they are known to be characteristic for individual bacterial species. A series of biochemical tests can provide a microbial fingerprint. An enzyme is a biological catalyst, a substance that accelerates the rate of a specific biochemical reaction without being consumed in the reaction. (Some enzymes, notably those of pathogens, contribute to the ability of the microbes to cause diseases. The damage is directly due to the production of enzymes that attack human tissues, or by interruption of essential metabolic processes in the human organism). The ability to ferment a particular sugar or other substrate depends on the genetic capacity of the organism to produce the necessary enzymes. Like other genetically determined characteristics (such as appearance, staining properties, motility, antigens, etc), fermentation reactions provide valuable clues for identifying microorganisms in the laboratory. This is routinely performed by inoculation of selected or differential culture media containing substrates for the enzymes of taxonomic interest. Most of these biochemical tests result in a decrease in pH due to metabolism of substrate with release of acid, but some tests detect an increase in pH (e.g., urease activity). Standard acid-base dye indicators are incorporated into these media to facilitate detection of acid or alkaline end points. Differential media are applied to demonstrate carbohydratesplitting enzyme activity (sugarlytic properties). When bacteria ferment carbohydrates with acid formation, the color of the medium changes due to the indicator present in it. (To investigate the bacterial sugarlytic properties are commonly used: Hiss media, Endo medium, Levine EMB medium, etc).

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Proteolytic activity of bacterial enzymes can be observed following their inoculating into special culture media, which results in liquefaction of coagulated serum or gelatin column, or milk peptonization. More profound splitting of protein is evidenced by the formation of indol, ammonia, hydrogen sulfide, and other compounds. Several companies now market disposable identification systems for more rapid procedures (API strips, Enterotest, etc). Despite the variety of systems available, they are all based upon the basic biochemical media mentioned. 4. Immunologic (serological) tests. Immunologic reactions can be used to identify microorganisms by determining whether the microbe can combine with specific antibodies. These antibodies usually react only with the same type of organism or molecule that was used to stimulate their production. Thus, reaction with a specific antibody is evidence of a microbial identity. This method is based on determining the antigen structure of bacteria. 5. Genetic tests. Every type of organism contains DNA that is unique to it alone. DNA can be used for identification much like a fingerprint. Such genetic tests use labeled segments of the unique DNA from the known microbes to probe the chromosome of the unknown microbes DNA only if its chromosome contains an identical segment. The label can then be detected. If the DNA probe is unique to another microbe, however, it will not react and no label will be detected. DNA probes are specific and positive reaction is proof of the microbes identity.
NOTE: Sufficient DNA samples for identification of virtually any microbe can be obtained from a specimen even without culturing the organism. When the microbe is difficult to culture, we can use Polymerase Chain Reaction (PCR) for its identification. (For PCR see lesson 9)

6. Epidemiological typing To verify the existence of an epidemic it is necessary to establish that the same etiologic agent causes all cases. This generally requires isolation of the pathogen from the majority of infected people, animals, and even from the inanimate environment. Identification of the pathogens genus and species may not be sufficient to distinguish between independent cases caused by different strains of the same organism (strain is subgroup of microbes within a single species). Marking the strains of the microbes from various sources will help to track down the source of infection. Techniques for epidemiological typing are the following: Biotyping is based on differences in biochemical properties and is used for determination of biological variants (biovars) among the bacteria of the same species

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Serotyping identifies differences in microbial surface antigens, such as the O (cell wall) antigens, H (flagella) antigens, and K (capsule) antigens. Serological variants are termed serovars Bacteriophage typing distinguishes between strains by revealing differences in susceptibility to infection by a number of phages. Variants revealed are termed as phagovars. Toxigenicity. The production of bacterial exotoxins can be determined by serologic reactions with specific antisera (in vitro) or with the help of biological method (in vivo). Bacteriocin typing. Bacteriocins are substances produced by bacteria that inhibit or kill closely related bacteria. Strains of bacteria may be differentiated either by the specificity of the bacteriocins they produce or by the spectrum of bacteriocins to which they are sensitive. Nucleic acid restriction analysis and other genetic tests. Plasmid or chromosomal DNA is isolated from the pure culture and exposed to various restriction endonucleases. Variations in nucleic acid sequences are revealed by differences in the products of enzymatic digestion. This technique is rarely used for routine typing of pathogens. Antibiograms distinguish between similar organisms by revealing different patterns of susceptibility to several antibiotics.

7. Antibiotic susceptibility tests (antibiogram). Once isolated, the microbes drug sensitivities are determined by observing its ability to grow in the presence of the drug. The following methods are used to do that: 1. The Disk Diffusion Method 2. Minimum Inhibitory Concentration (MIC) technique. The resultant antibiograms are used as guidelines to the selection of effective antibiotic therapy.
NOTE: for methodology and procedure of Antibiotic Susceptibility Tests see LESSON 8

Isolation and Identification of Anaerobic Bacteria Obligate anaerobes can exist in the environment; they also make up part of the normal human flora, inhabiting the vagina, intestinal tract, mouth, and other organs. Some of them are serious pathogens that are found in blood, abscesses, abdominal infections, wounds; they are the causative agents of tetanus, gas gangrene, and other severe infections. When an anaerobic infection is suspected, specimens should be obtained by aspirating with a syringe and needle. Then the collected specimen is injected into an oxygen-free anaerobic transport system. If a swab must be used for collecting the sample, it should be placed immediately into a tube of anaerobic

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transport medium to prevent drying. The specimens in anaerobic systems must be processed within 2 or 3 hours after collection (if delayed longer than 3 hours, the specimen should be refrigerated until processed). The use of a battery of nonselective, selective and enrichment media is recommended for the isolation of obligate anaerobic bacteria from clinical specimens. Reducing agents, chemicals that react with and inactivate molecular oxygen, are usually included in the medium. A wide variety of media have been developed for this purpose (for example, thioglycollate medium). Such media contain ingredients such as yeast extract, hemin, vitamin K, L-cystine, 5% sheep blood, etc, to satisfy the nutritional requirements of a wide variety of anaerobes. Prior to inoculation, plated media should be conditioned in an anaerobic system (under anaerobic conditions for 6 to 24 hours). The enriched nutrient broth may be reduced by heating the tubes with plugs loosened in a boiling-water bath to drive off oxygen and cooling to room temperature with tightened plugs or by placing tubes in anaerobic jar. As soon as the plates and tubes are inoculated, they should be incubated under anaerobic conditions. Special equipment helps ensure that oxygen-sensitive microorganisms are protected from even brief exposures to oxygen during laboratory incubation and manipulation. The most reliable (and most expensive) systems are the anaerobic glove boxes, completely enclosed units in which oxygen is replaced by nitrogen or another relatively inert gas. The anaerobes can be also incubated in anaerobic jars, airtight containers that has been flushed with oxygenfree gases after the oxygen has been removed from it by pumping out the air. The oxygen can be also removed from the jar by chemical reaction. (An indicator should be included into the anaerobic jar to demonstrate that anaerobic conditions have been achieved. For example, resazurin in the medium, which is colorless when reduced and pink to lavender when oxidized, provides a visual means to ensure the atmosphere in the jar is oxygen-free). Incubation should ordinarily be at 37C for at least 48 hours before the jar is opened. Sometimes incubation may be continued for 5 to 7 days. Broth cultures should be held for up to 2 weeks. MICROBIAL ECOLOGY. MICROFLORA of AIR and WATER (Microbial examination of materials of sanitary importance) Microorganisms grow abundantly in most soils, oceans, and bodies of fresh water. They also populate the atmosphere close to the earths surface. When they share their environment with other microbes or multicellular organisms, crucial interactions inevitably influence the lives of all. These living individuals make up a community all the organisms that reside in a particular area. These organisms not only interact with one another but are also inseparably dependent on water, air, sunlight, warmth, and other nonliving aspects of the environment. Together, the living community plus the nonliving physical and chemical properties of a particular

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environment form the ecosystem. Understanding ecosystems requires knowledge not only of how the organisms in a community interact with each other, but also of how they affect and are affected by their nonliving surroundings. This knowledge comes from the ecology, the study of ecosystems. The environment for any individual microbe is very small. The conditions in such a small area, or microenvironment, may differ dramatically from those of adjacent environments. Microbial Interactions Microbes usually live in close association with other microbes, plants or animals. This relationship is termed symbiosis (sym = together, bios = life). The relationship between different kinds of organisms that inhabit the same environment may be beneficial, harmful, or neutral for the species involved. To become a permanent member of any community, a microbial species inevitably interacts with and influences other organisms. To survive, it must compete successfully for nutrients and avoid being destroyed by its neighbors. The major symbiotic relationships in which microbes participate are commensalism, mutualism and antagonism (which includes predation and parasitism). Commensalism and mutualism are positive interactions that benefit at least one participant and harm none. Antagonism, on the other hand, benefits one member at the expense of the other. (for example, microbes can produce antibiotics, bacteriocins, and other metabolic products that hinder the growth of competitors. Parasitic microbes can attack other microbes, plants, or animals. They are capable of establishing infection and avoiding host defenses in humans). Different microbes take part in various processes of nitrification, ammonification, sulfur, and phosphorus cycles in soil. The aquatic microbes are the phytoplankton, free-floating cyanobacteria and algae. Not only harmless bacteria exist in the environment, but many pathogens can also exist or can be transmitted by air, water, soil, spoiled food, etc. Evaluating of Water Quality Water that has been contaminated by feces may contain pathogens and transmit serious diseases. Fecal contamination is most commonly detected by assaying water for the presence of fecal coliforms, predominantly Escherichia coli. The only natural source of these bacteria is the intestines of humans and other mammals. Although several strains of E.coli are pathogenic, coliforms are usually not pathogenic when ingested by healthy people. Their presence in water, however, indicates fecal contamination and thus the possible presence of waterborne pathogens. (1) Heterotrophic Plate Count. The most common technique is to measure the total number of microbes per 1ml of tested water. The pour plate method has been the traditional method for counting the number of aerobic and facultative anaerobic heterotrophic bacteria in water. Plate count method consists

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of counting the numbers of colonies formed in pourplated cultures of the water samples in solid nutrient agar. It is measured in CFU/ml. (2) Multiple - Tube Fermentation Technique for Members of the Coliform Group. The coliform group of bacteria is the principal indicator of the presence and degree of water pollution. This technique yelds the Most-ProbableNumber (MPN) Index (Coliform-Index), and Coliform-Titer. The procedure includes a 24-h. Multiple-Tube Fermentation Test and the following direct plating for detection and estimation of coliforms densities. For the 24-h. Multiple-Tube Fermentation Test the row of four tubes with glucose-containing liquid medium and indicator and a special glass float for gas detection is inoculated. Prior to add the test portion of drinking water one should prepare serial dilution of tested sample. After 24 h. incubation at 37C direct plating of Endo agar is performed for detection of lactose-positive bacteria (red colonies with green metallic sheen). The plate with Endo agar is divided onto four sectors and each part is inoculated by the material from the corresponding tube. The final step of Multiple-Tube Fermentation Technique for Members of the Coliform Group is a Catalase Test.
NOTE: Gram-negative rods, which ferment glucose to an acid and gas end products, lactose-positive and catalase-negative are considered to be Coloforms Group members. Coliform-Index (Coli-Index) is the total number of Coliforms in one litre of tested water. It is measured in CFU/l Coliform-Titer (Coli-Titer) is the minimal volume of tested water sample which contains at least one CFU of E.coli or bacteria of Coliform Group. Coliform-Titer (or Coli-Titer) is measured in ml.

(3) Membrane Filter Technique for Members of the Coliform Group. The Membrane Filter Technique is recommended for testing relatively large volumes of water. Bacteria collected on the filter membrane grow when the membrane is placed on an appropriate culture medium (e.g., Endo agar). This test has been supplemented by a Multiple-Tube Fermentation Technique and provides comparable results. The Coliform-Index and the Coliform-Titer can be determined by this test. Examining of Air Contamination It is very important to know how many bacteria exist in the air of operating rooms, wards, etc, because there are a great number of air-borne infections. The most commonly used methods are the following. (1)Sedimentation method (settling plates) Open plates that contain solid culture media are exposed to the air for some periods of time (10 min 30 min 1 hour, etc). Then the plates are incubated at 37C for 24 hours and the number of colonies is then counted. It is known, that microbes from 10 liters of air settle on the surface of the plate (100 cm) within

19

each 5 min. Then it is necessary to count the number of microbes per 1 m (1000 liters) of air, knowing the time of exposure to the air. Quality of the air is characterized by the amount of microbes contaminated 1 m of air. (2)Aspiration method (slit sampler) Since the plate method has many limitations, a more elaborate method, the slit sampler or aspirator, has been introduced. To sample air within open environments special equipment has been developed (for example, Krotovs apparatus). In this, a known volume of air is directed onto a plate through a slit 0.25mm wide, the plate being mechanically rotated so that the microbes are evenly distributed over a nutrient medium. When pathogenic staphylococci and streptococci are looked for, the blood agar plates are used. The inoculated plates are then incubated at 37C for 24 hours. The results of testing air in such a way provide more precise quantification of air-borne contaminants than does the use of settling plates. Table 2 Satisfactory Air Quality (for the air within wards, classrooms, etc): Total number of microbes less than 1500 CFU/m Staphylococcus aureus less than 100 CFU/m Molds and yeasts (totally) less than 20 CFU/m
NOTE: The air in the operating rooms should be less contaminated

HUMAN NORMAL FLORA Microorganisms inhabit the surfaces of living of human and animal bodies. There are ten times as many bacterial cells residing in human colon as there are human cells in a body. In fact, one-third of the dry weight of human feces is bacteria. Such harmless microorganisms comprise the normal flora, those microorganisms that normally live on the human body in a harmonious relationship with their host (Table 3). The term normal microbial flora denotes the population of microorganisms that inhabit the skin and mucous membranes of healthy normal persons. There are no microbes in the cerebrospinal fluid. The blood and tissue fluids of a healthy person are also microbe-free. Urine in the bladder is also sterile, although it becomes contaminated with normal flora organisms during voiding. All the internal organs of a healthy person are free of microbial growth. The microbes of normal flora can be arranged into two groups: 1. The resident flora: relatively fixed types of microbes regularly found in a given area at a given age. If disturbed, it promptly reestablishes itself. 2. The transient flora: consists of nonpathogenic microorganisms that inhabit the skin or mucous membranes for hours, days or weeks. It is derived from the environment and does not establish itself permanently on the surface.

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If the resident flora is disturbed, transient microorganisms may colonize, proliferate, and even produce disease. Table 3 Normal Bacterial Flora of Human Organism
Anatomic site SKIN Most common organisms Staphylococci Streptococci Propionobacteria Corynebacteria, etc Streptococci Diphteroids Neisseria Haemophilus sp. Bacteroides Leptotrichiae Spirochaetes, etc Predominantly anaerobes: Lactobacilli Bifidobacteria Bacteroides Clostridia Enterococci Enterobacteria (E.coli), etc Streptococci Lactobacilli Bacteroides, etc Microscopic characteristics Gram Stain Morphology + Cocci + Cocci Rods + Rods + + + + + + + + Cocci Rods Cocci Rods Rods Spiral Spiral Rods Filamentous Rods Rods Cocci Rods Cocci Rods Rods

OROPHARYNX

LARGE INTESTINE

VAGINA

The Role of the Resident Flora The microorganisms of resident flora are commensals. Their flourishing in a given area depends upon physiologic factors of temperature, moisture and the presence of certain nutrients and inhibitory substances. The resident flora of certain areas plays a definite role in maintaining health and normal function. Members of the resident flora in the intestinal tract synthesize sufficient vitamin K and aid in the absorption of nutrients. The microbes of normal flora also take part in cellulose digestion, in transformation of mutagens and carcinogens to harmless substances. The normal gut flora protects the host from most pathogens by stimulating local immune defenses and preventing their establishment on intestinal surfaces. On mucous membranes and skin the normal flora may prevent colonization by pathogens and possible disease through bacterial interference or colonization resistance. Microbes of normal flora form dense layers on the mucous surfaces that act as remarkably effective barriers to the establishment of most pathogens as

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it leave few vacant attachment sites for pathogens. The mechanism of this phenomenon involves competition for receptors or binding sites on the host cells, competition for nutrients, mutual inhibition by metabolic or toxic products, mutual inhibition by antibiotic materials or bacteriocins or by other mechanisms. Sometimes, by-products of fermentation in normal bacterial flora may provide us some protection against infectious disease. For example, the lactic acid produced by the lactobacilli in the adult vagina opposes infection by the lowering the pH below that can be tolerated by many vaginal pathogens. The main role in human colonization resistance play anaerobes and microaerophiles, because these microbes make up the major part of the normal flora, inhabiting the vagina, urethra, mouth, intestinal tract, and even the conjunctiva of the eye. In the colon they may outnumber the aerobes by 1000 to 1. The mouth and the colon are the most heavily colonized regions of the digestive tract. Some of bacteria are able to stick to teeth or mucosal surfaces. Attachment to teeth is not direct, but rather to a coating of sticky macromolecules, mainly proteins dental pellicle. The bacteria themselves produce polysaccharides that help in adherence. They are layered on the pellicle to form a matrix that allows further adherence of other microbes. The result is dental plaque, one of the densest collections of bacteria in the body. (Sometimes the further colonization may result in dental caries or inflammation of gingival tissues). The normal balance of quality and quantity of the microbial species in human microbial flora and positive relationships between the normal flora and the host are called eubiosis. Suppression of the normal flora (for example, as a result of the broadspectrum antibiotic misuse) clearly creates a partial local void that tends to be filled by organisms from the environment or from the other parts of the body. Such organisms behave as opportunists and may become pathogens. The absence of an adequate balance of the microbial species among the resident flora is called dysbiosis. (If the disturbance occurs among the bacterial species of the resident flora it is called dysbacteriosis). Alteration in the normal flora changes in the density or composition of the flora - may permit pathogens to become established. When dysbiosis occurs, the members of the normal flora may themselves produce severe diseases under certain circumstances (usually when microbes penetrate into deeper tissues in immunocompromised host). Special drugs for restoration of the normal flora are called Probiotics. Probiotics Bifidumbacterin, Lactobacterin, Bificol, etc. - contain lyophilized living microbes (for example, E.coli, Bifidobacterium sp., Lactobacterium sp., etc) or growth factors and metabolites that promote the multiplication of normal flora bacteria. These drugs are used to make up for disturbed normal flora (a kind of replacement therapy). Selective decontamination process of selective removing or destroying some species of normal flora microbes in the given anatomic site. (For example, in

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case of abdominal surgery, trauma burns to prevent probable infectious disease, caused by the members of the normal flora). CONTROL OF MICROORGANISMS Physical or chemical control procedures can significantly reduce, and in some cases eliminate the microorganisms in a designated environment or in an infected person. These protective measures are particularly important in hospitals, where there are many sources of pathogenic organisms. Microbial controls are also essential to the safe preparation of food, water, pharmaceutical agents and other products for use in or on the human body. The most appropriate antimicrobial strategy is one that limits the microbial population without damaging the person, animal, plant, or object being treated. When possible, the best way to do this is to avoid microbial contamination. Aseptic techniques are precautions that help prevent contamination of culture materials, equipment, personnel, or the environment. Although the hands cannot be sterilized, most transient organisms can be removed by 30 seconds of proper scrubbing with soap and water. Microbes that reside in sweat ducts and hair follicles of the skin, however, cannot be readily dislodged. These microbes are a threat to patients with reduced defenses, so scrubbing prior to contact with these patients is usually done with antiseptic soap. Hand washing reduces the number of transient organisms on the skin surface. In addition, it is often recommended that washing be supplemented by wearing sterile gloves.
NOTE: Hands should always be washed before and after contact with each patient and after exposure to secretions and excretions, that may be sources of infectious agents.

Sanitization supplements all decontamination procedures with cleaning. This ensures the absence of dirt or organic debris as well as infectious microbes. Sanitization is typically employed for equipment used in food preparation and for reusable instruments in hospitals.

Antimicrobial Effects The control of microorganisms often depends on establishing conditions that cannot be tolerated by microbes. Antimicrobial conditions are created by microbicidal or microbistatic agents. Microbicidal (cide = kill) agents kill microorganisms and therefore have an irreversible and permanent effect. Microbistatic (static = standstill) agents inhibit microbial growth and multiplication, thereby preventing an increase in the number of microorganisms. Microbistatic agents do not kill or eliminate microorganisms. The microbe persists and can resume growth once the agent is removed. Therefore, microbicidal agents are generally preferred over microbistatic ones. Antimicrobial agents perform one or more of the following processes:

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1. Sterilization eliminates all forms of life, including vegetative cells, spores, and viruses. Sterilization treatments also destroy potentially infectious nucleic acids such as viroids. During sterilization, either microbes are killed or they are physically removed from the objects or substances being treated. Sterile is an absolute term there is no such thing as almost sterile. An object or environment is not sterile as long as it contains even a single viable microbe. 2. Disinfection eliminates the vegetative forms of most potentially hazardous and pathogenic organisms but does not ensure the elimination of all microbes. Bacterial spores, tubercle bacilli, and many viruses are particularly resistant to common disinfectants (disinfecting agents). Like sterilizing agents, disinfectants are used only on inanimate objects and never on body surfaces. Disinfection is generally employed if sterilization is either impossible or unnecessary. The purpose of disinfection is to minimize the risk of infection or product spoilage by reducing the number of microbes, especially pathogens, in the inanimate environment. 3. Preservation prevents the deterioration of products from microbial activities either by the addition of a chemical preservative or by establishing a physical environment inhospitable for microbial growth. These techniques retard spoilage and the growth of pathogens in foods, pharmaceutical preparations, and biological products for use in or on the human body. 4. Antisepsis is inhibition or destruction of microorganisms on the surface of living tissue. For these purposes special chemicals antiseptics are used. 5. Chemotherapy is treatment of disease by introducing chemicals into the human (or animal) body. Chemotherapeutic chemicals for treating infectious disease inhibit or kill microorganisms. Most of these agents are antibiotics, chemicals synthesized by microorganisms, usually a bacterium or fungus, which at very low concentrations (micrograms per milliliter) inhibit or kill other microbes. Many drugs similar to antibiotics are chemically synthesized in the laboratory. Since chemotherapeutic agents are used inside the body, they must exhibit selective toxicity for the target microorganisms, with little or no toxicity for human tissues. Factors Affecting Antimicrobial Activity Some antimicrobial agents are microbicidal under one set of conditions and microbistatic under others. They may lose effectiveness as concentrations decrease or as conditions for use become suboptimal. There are several factors that influence the activity of antimicrobial agents. The number of microorganisms: All organisms present in an object do not die simultaneously when a critical exposure is achieved. Death occurs logarithmically a fixed percentage of the population will die during each minute of exposure to the agent. Antimicrobial effectiveness therefore depends on the initial concentration of the microbial population. Removing microbes by washing objects in a detergent and rinsing with water dramatically reduces

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microbial contamination and increases the likelihood that subsequent antimicrobial treatment will be adequate. The concentration or dose of the agent. Diluting microbicidal chemicals usually weakens their antimicrobial activity completely. The antimicrobial effects of temperature or radiation also depend on the intensity of exposure. Low doses may inhibit growth, whereas high doses may sterilize. Length of exposure. Microbial death is a function of time. The longer microbes are exposed to potentially lethal conditions, the greater the number that will be killed. For sterilizing agents, the exposure time must be long enough to ensure that the probability of even a single cell surviving is less than 1 in a million. Environmental conditions. Temperature, pH, and moisture affect the efficiency of most antimicrobial agents. In addition, some chemical agents are absorbed by blood, mucus, feces, tissue, and other organic materials that sharply reduce antimicrobial activity and therefore eliminate them as effective antiseptics. Objects can be rinsed prior to disinfection to prevent interference by organic debris. This is extremely important when treating medical instruments. Physical Agents for Controlling Microbes The most common physical methods of control use moist heat, dry heat, radiation, or filtration. Moist Heat Although any organism can be killed by excessive heat, the lethal temperature depends on the heat resistance of the organism and the amount of water in the environment. Moist heat, especially steam, effectively kills cells by coagulating their proteins (critical enzymes, for example). In an absence of water, heat does not coagulate protein. Dry heat kills cells by oxidizing essential constituents, a process that requires much higher temperatures than can be achieved with moist heat. Three of our most common antimicrobial processes rely on moist heat: pasteurization, boiling, and autoclaving (saturated steam under pressure). Of these, only autoclaving ensures sterilization. 1. Pasteurization. The selected combination of temperature and duration of heating is one of that kills the most heat-resistant pathogens commonly transmitted by that medium without damaging product quality. Pasteurization is used to prevent spoilage of different foodstuffs (for example, milk) and to prevent the transmission of milk-borne diseases. There are different techniques of pasteurization (see Table 4). Table 4
Pasteurization Technique Low Temperature Holding (LTH) High Temperature Short Time (HTST) Ultra High Temperature (UHT) Exposure 30 min 15 sec 3 sec Operating Temperature (in C) 62.8 71.6 this method is followed by rapid cooling 141

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2. Boiling. Heating increases the temperature of water until it reaches 100C, the

temperature at which water boils at normal atmospheric pressure. Most vegetative cells are eliminated by 10 minutes of boiling, but some endospores can survive. This process is considered a procedure for disinfection. Boiling is used when small instruments are to be disinfected or when there is no access to equipment needed for sterilization. Surgical instruments, however, should always be sterilized and not merely disinfected. Although the higher temperature of boiling water kills more effectively than pasteurization, it is not routinely used in food industries because such high temperatures often damage the product. 3. Tyndalization. In some cases, intermittent exposure to steam can be used in fractional sterilization (also called tyndalization, after the person who developed it). This process requires 3 days and is useful only for materials that can support microbial growth. On the first day, the material is steam-heated for 30 minutes, cooled, and incubated at 37C. The high temperature and moist heat kill all vegetative cells and stimulate the germination of heat-resistant endospores (germination = formation of vegetative cell from a spore). A similar treatment on the second day destroys the germinated cells and triggers the outgrowth of any remaining endospores. These bacteria die when the material is once again heated for 30 minutes on the third day. The most common uses of fractional sterilization are for media that cannot withstand the higher temperatures of the autoclave and that cannot easily be sterilized by other methods. 4. Autoclaving (use of steam under pressure). Autoclave is an instrument sterilizes with saturated steam under pressure. The pressure increases the boiling point of water, thereby increasing the temperature to which water can be heated. It is these higher temperatures that destroy cells; the effects of pressure are not lethal. Standard autoclaves are usually operated at 15 lb/in above atmospheric pressure, allowing the temperature to reach 121 C. Spores are killed after 15 minutes at these high temperatures. Other autoclaves use even higher temperatures but for shorter periods of time. Saturated steam heats an object about 2500 times more efficiently than hot air at the same temperature. The only infectious agents known to survive after 4 hours of autoclaving are prions.
NOTE: All items should be packaged to prevent their recontamination when they are removed from the autoclave.

Dry Heat Dry heat is generally used in three ways: flaming, incineration, and baking. 1. Flaming is commonly used to sterilize loops and needles so that microorganisms can be transferred without contamination. The mouths of test tubes and containers are also routinely flamed, although the heat is not sufficient to sterilize these surfaces.

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2. Incineration is useful for destroying heavily contaminated materials or tissues

in the hospital and cultures of pathogenic microbes discarded in the laboratory. 3. Baking in hot-air ovens requires prolonged exposure to temperatures between 150 and 180C. The length of exposure depends on how readily heat can penetrate the material, since all parts to be sterilized must reach critical temperatures. Hot-air ovens are used for sterilizing materials such as glassware or metal instruments that can tolerate prolonged heat exposure. Radiation Cosmic rays, gamma rays, X-rays, ultraviolet light, and visible light are all forms of radiation. When these rays strike an organism, energy may be absorbed by cellular constituents, causing cell damage, or death. Radiation with the shortest wavelengths (wavelengths at or bellow those of ultraviolet light) has the greatest energy and is therefore the most lethal. Ionizing radiation and ultraviolet radiation (UV light) are two types used in microbial control. Ionizing radiation is commonly employed for sterilizing heat-sensitive materials such as disposable plastic products and materials that cannot withstand moisture. UV light is a disinfecting agent for air and surfaces in surgical rooms, wards, and laboratory safety cabinets. Filtration Liquids and gases can be sterilized by passing them through filters (a process called filtration). Membrane filters retain microbes that are too large to fit through the pores of the filter membrane. The filter acts as a strainer, a microbial sieve. Standard bacteriological membrane filters are composed of nitrocellulose, cellulose acetate, or polycarbonate and have pore diameters of 0.2 m, small enough to prevent the passage of most bacteria. Other types of filters (depth filters) are made of porcelain, glass, or fibrous materials that do not contain pores of uniform size but trap cells that are unable to follow the tortuous paths through the filter. Table 5 The Main Physical Methods of Microbial Control
Physical antimicrobial agent Moist Heat Boiling Autoclaving Baking Ionizing Ultraviolet Membrane filters Depth filters Procedure Pasteurization Usual conditions 62.8C for 30 min 71.6C for 15 sec 100C more than 10 min 121C for 15-30 min 170-180C for 50 min 2.5 Mrad 260 nm Pore size as appropriate Effectiveness Disinfection Disinfection Sterilization Sterilization Sterilization Disinfection Disinfection Sterilization

Dry Heat Radiation Filtration

Chemical Agents for Controlling Microbes

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Chemicals used for antimicrobial purposes range from the familiar detergents, antiseptics, and disinfectants people use in their homes to highly toxic agents used to sterilize inanimate objects in hospitals and some industries. Susceptibility of microorganisms varies in their response to different antimicrobial agents (see Table 6). Table 6 Descending order of resistance to antimicrobial chemicals
MOST RESISTANT BACTERIAL ENDOSPORES MYCOBACTERIA NAKED (NONLIPID) or SMALL VIRUSES FUNGI VEGETATIVE BACTERIA ENVELOPED or MEDIUM SIZED VIRUSES

LEAST RESISTANT

Chemicals that Sterilize Only a few chemicals are recommended for use as sterilizing agents: ethylene oxide, hydrogen peroxide, formaldehyde, and glutaraldehyde. When used properly, these agents dependably kill all microbes, including bacterial endospores. All four chemicals kill organisms by damaging their proteins and nucleic acids. Safe chemical sterilization requires special apparatus. Air in its chamber can be replaced by microbicidal gas. Because the gas readily penetrates many materials, packaged items can be sterilized in their containers. Chemicals: Disinfectants and Antiseptics There is a large amount of different chemicals, which provide an antimicrobial activity. These antimicrobial agents have different chemical structure and mechanisms of microbicidal activity: phenolics, alcohols, quaternary ammonium compounds, chlorine, iodine, chlorhexidine, heavy metals, and ozone. Only some of them may be applied both as disinfectants and antiseptics, but in different concentrations. 1. Phenolics, phenol derivatives, are most often used for disinfecting floors, walls, and furniture surfaces. Phenolics kill cells by inactivating cell enzymes. They are effective against most vegetative cells and enveloped viruses. Some of these chemicals are currently incorporated into commonly used antibacterial hand soaps. 2. Quaternary ammonium compounds are bactericidal disinfectants, commonly called quats. They are cationic surfactants (surface-active agents). Surfactants interfere with normal interaction between a cells surface and its environment. They absorb to the negatively charged surface of bacteria, altering plasma membrane permeability and killing the cell. Quats are used as low-level disinfectants and antiseptics (e.g., trichlozan, trichloguard)

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3. Alcohols. Ethanol and isopropanol are used to reduce the number of microbes

4.

5.

6.

7.

on skin and for disinfecting small medical instruments. Alcohol diluted with water to concentrations of 50 to 90 percent kills cells by coagulating essential proteins. Alcohol is not used at full strength because water is necessary to prevent dehydration of the cell (dehydration actually protects cells by retarding protein coagulation). Because they evaporate quickly from surfaces, alcohols are useful only for short-term disinfection or antisepsis. Chlorine and iodine are some of the oldest and still most useful microbicidal agents. Chlorine oxidizes and thus inactivates critical enzymes of bacterial cells. Iodine is used in dilute solutions of water or alcohols, which rapidly inactivates microbes by irreversibly combining with bacterial proteins. Iodine and iodofors (complex of iodine and surfactants) are commonly used in disinfecting small medical implements, to prepare skin sites for surgery, as a presurgical hand wash, for treating cuts and wounds, etc). Chlorhexidine is a popular antiseptic, active against both gram-positive and gram-negative bacteria and some fungi but is useless against mycobacteria, bacterial spores, and viruses. It kills cells by interfering with plasma membrane permeability and essential membrane-associated enzymes. Chlorhexidine is used as a component of antiseptic lotions for surgical scrubs, in cleaning the skin and mucous membranes and in decontaminating wounds. Heavy metals. Ions of heavy metals readily bind with inactive proteins. Their effect is not selective for microbes, so, as antiseptics, they must be used in dilute concentrations and only topically. The most common of these antiseptics are the mercurials (compounds containing the heavy metal mercury) mercurochrome and merthiolate; and the silver-containing agents: silver nitrate and silver chloride. Silver nitrate drops are added to the eyes of all newborn children to prevent blinding Neisseria gonorrhoeae and Chlamydia infections acquired during the birth process. Ozone (O3), a form of oxygen, is a gas used in water and wastewater treatment. It effectively kills bacteria, viruses, fungi, and protozoa.

ANTIBIOTICS Antibiotics are chemicals usually produced by microorganisms that in very low concentrations selectively kill or inhibit the growth of microbes and tumor cells. Antibiotics that are nontoxic to human cells can usually be safely introduced into an infected person to combat pathogens. There are natural antibiotics, semisynthetic derivatives, and synthetic compounds with antimicrobial activities. Natural antibiotics are metabolic products of fungi, bacteria, plants, or eukariotic cells. The most effective medically important natural antibiotics were isolated from the molds Penicillium and Cephalosporium and from members of bacterial genera Streptomyces and Bacillus.

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Semisynthetic derivatives are agents with additional useful properties. One part of their structure is the result of fungal or bacterial metabolism and the other part is the result of chemical synthesis in the absence of the organism. These agents are more resistant to bacterial enzymes, inactivating antibiotics and usually have broad-spectrum activity. Synthetic compounds are synthesized chemicals with antimicrobial activity. They do not exist in nature.

Antibiotics may be classified by their chemical structure as follows: Natural antibiotics and their semisynthetic derivatives: 1. Beta-lactams: Penicillins: Natural (e.g., Penicillin, etc.) Semisynthetic (e.g., Ampicillin, Oxacillin, etc.) Cephalosporins (e.g., Cefazolin, Cefaclor, etc.) Newer Beta-lactams (e.g., Amoxicillin/Clavulanic acid, Imipenem, Ticarcillin, Ampicillin/Sulbactam, etc.) 2. Aminoglycosides (e.g., Gentamicin, Streptomycin, etc.) 3. Tetracyclines (e.g., Tetracycline, Doxycycline, etc.) 4. Macrolides (e.g., Erythromycin, etc.) 5. Polyenes (e.g., Nystatin, Amphotericin B, etc.) 6. Polypeptides (e.g., Polymyxin, etc.) 7. Rifamicins (e.g., Rifampin, etc.) 8. Glycopeptides (e.g., Vancomycin, etc.) 9. Lincozamides (e.g., Lincomycin, Clindamycin, etc.) 10. Group of nonclassified antibiotics (e.g., Chloramphenicol) Synthetic compounds: Agents against cellular microorganisms: 1. Sulfa drugs (e.g., Trimethoprim Trimethoprim/Sulfamethoxazole, Sulfisoxazole, etc.) 2. Azoles: Imidazoles (e.g., Clotrimazole, etc.) Nitroimidazoles (e.g., Metronidazole, etc.) 3. Nitrofurans(e.g., Nitrofurantoin, etc.) 4. Quinolones: 8-oxy-quinolines (e.g., Nitroxoline, etc.) quinolines (e.g., Nalidixic acid, etc.) Floxquinolones(e.g., Norfloxacin, Ciprofloxacin, etc.)
Agents against noncellular microbes: Antiviral agents

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Today, scientists seek new sources of natural antibiotics and use modern tools of genetic engineering and molecular biology to achieve effective antimicrobial agents. Antimicrobial drugs are used prophylactically or therapeutically. The administration of drugs to prevent infectious disease is called chemoprophylaxis (for example, before and during surgery on heavy contaminated body sites to reduce the likelihood of post surgical infection; in the individuals with depressed immunologic responses, etc). An antimicrobial treatment of an existing infectious disease is called chemotherapy. Different antibiotics are also used for treating several oncologic processes. Antimicrobial Mechanisms Microbistatic chemotherapeutic agents impede microbial growth until the host defense mechanisms can eventually destroy the pathogens. B A C T E R I A L C E L L Most microbicidal agents can kill N u c l e i c A c i d P S o y t ne t i hn e S s yi s n t h e s i s r N a l id ix ic a c i dA m i n o g l y c o s pathogens with no assistance from i d e s ( i n h i b i t s D N A C r he lp o l ri ca am t i po hn e) the c host defenses. These agents n i o l R if a m p in T e t r a c y c l in e s ( i n h i b i t s t r a n Es cr yr i t p h t r i o m ) y c i n function by selectively disrupting n M e t r o n i d a z o Cl e l i n d a m y c i n (1)cell wall synthesis; (2)cell membrane function; (3)protein C e l l M e m b r a n e P o ly m y x in s synthesis; (4)nucleic acid synthesis, or (5)other specific metabolic reactions. (The microbial targets of the major drugs are identified in Fig.5). C e l l W a l l M e t a b o l i c r e a c t i o n s
P e C e V a C y

M T

B
F i g o t h ( B )

Target: Bacterial Cell Wall Agents that prevent the A synthesis of peptidoglycan produce E U K A R Y O T I C C E osmotically fragile bacterial cells L L that lyse unless kept in a medium e t a b o l i c R e a Nc u i c ln e s i c A c i d t o that prevents the influx of water. r im e t h o p r in - s u l f a G r i s e o f u lv in C h l o r o q u i n Bacteria with defective cell walls e F l u o r o c y t o (for eexample, L-forms) survive s in M e t r o n id a z o l e in the human body. Q u i n a c r i n e poorly Because these agents cannot C e l l M e m b r a n e destroy existing peptidoglycan, P o ly e n e s I m i d a z o l e s they are effective only against actively growing bacteria. Gramu r e 5 . M i c r o b i a l t a r g e t s o f c h e m positive bacteria a are tsensitive l tod r u g e r a p e u t ic a g e n t s . ( A ) A n t i b c e r i a D r u g s u s e d a g a i n s moste uof k thesey oagents; p gram- o g e t a r t i c a t h

n p n c

ic i llin ( c o m p e h a l o s p o i rn i nh si b i t c o m y c in u lf a S d l o s e r i n e T rB i m a ce i t t hr

t i t i o n r u g ao c p

i v e ) s i rn i m

e n

z y m

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negative bacteria usually are not. The outer membrane of most gram-negative cell walls prevents the antibiotic from penetrating to its site of action. Eukaryotic cells lack peptidoglycan and are completely resistant to penicillins and cephalosporins, which are among the least toxic of all antibiotics. -lactam antibiotics penicillins and cephalosporins Penicillins and cephalosporins are the most common of the -lactam antibiotics, which are similar in structure and activity (Fig.3). Each of these antibiotics contains a ring structure called -lactam ring. Many bacteria produce enzymes called -lactamases that can break the -lactam ring of these antibiotics. Some of the newer -lactam antibiotics, such as carbapenems and monobactams, are unaffected by most of these enzymes. Carbapenems are effective against a broad range of gram-positive and gram-negative bacteria, whereas monobactams show activity only against gramnegative bacteria. The -lactam drugs have several antibacterial mechanisms. Their effect on the cell is determined by binding proteins, termed penicillin-binding proteins, in the plasma membrane or periplasmatic space. Depending on the type of binding protein with which it couples, the antibiotic can cause inhibition of peptidoglycan synthesis, activation of autolytic enzymes in the cell, cellular filamentation, or other abnormal configurations that interfere with growth. In some species, the drugs tend to cause bacteristatic changes rather than cell lysis. Although penicillins are among the least toxic antibiotics, approximately 1 in 20 patients who receive the drug experiences some allergic reaction to it. Cephalosporins are less likely than penicillins to evoke an allergic reaction. However, because of the chemical similarities between the antibiotics, a person who is allergic to penicillins is usually not treated with cephalosporins. Usually the symptoms are mild rashes, but the occasional severe reaction can kill a person due to anaphylaxis. Bacitracin, vancomycin, cycloserine Bacitracin kills gram-positive bacteria, but because of its systemic toxicity, it is used only for topical applications. Vancomycin inhibits linear strands of peptidoglycan from forming, as bacitracin, but this drug is not so toxic. It is used to treat gram-positive bacterial infections as an alternative to penicillin in allergic individuals or in cases where the bacteria are penicillin-resistant. (It is especially effective drug in treating Clostridium difficile the etiologic agent of intestinal disorder associated with continued use of antibiotics) Cycloserine is also a peptidoglycan inhibitor but is rarely used in chemotherapy, due to its severe toxicity to the host. Target: Cell Membranes Some antibiotics kill cells by interfering with the normal permeability of the plasma membrane. The most important inhibitors of bacterial membrane

32

function are polymyxins. Polyene antibiotics and azoles react with sterols of eukaryotic membranes (antifungal drugs). Polymyxins The polymyxins bind to the phospholipids in bacterial membranes and alter their permeability. This cases leakage of small molecules and cell death. Polymyxins are especially valuable as topical agents for controlling gram-negative bacterial infections. Their toxic side effects, however, prevent their widespread systemic use. Polyenes The most therapeutically valuable of the polyenes are those that bind with ergosterol, the sterol in fungal membranes, creating membrane pores and causing leakage of cell metabolites. Human cells contain cholesterol instead of ergosterol and are therefore not as susceptible as fungi to these agents. The sterols in human cells, however, are somewhat affected by the polyenes. Toxicity for the patient, therefore, should be considered when using these antibiotics. Amphotericin B and nystatin are most commonly used polyenes. Azoles The azoles are another group of synthetic compounds that interfere with the cell membrane of fungi. Unlike the polyenes, these compounds inhibit the synthesis of ergosterol. One group of azoles, the imidazoles, are effective broadspectrum antifungal drugs with few side effects. Target: Protein Synthesis Some antibacterial chemicals can selectively inhibit bacterial protein synthesis by disabling prokaryotic ribosomes while causing little deleterious effect on eukaryotic ribosomes. The selective toxicity of these agents is often enhanced by their ability to pass through bacterial membranes more readily than through eukaryotic membranes. Aminoglycosides The aminoglycosides are a group of structurally similar antibiotics that attach to the bacterial ribosome and interfere with accurate translation of the genetic code. Unfortunately, they also affect the small ribosomes in the mitochondria of eukaryotic cells, which perhaps accounts for their severe side effects (renal damage, deafness, etc). These antibiotics are used against serious gram-negative infections. Tetracyclines The tetracyclines display the broadest spectrum of all antibiotics. They are active against both gram-negative and gram-positive bacteria. Because tetracyclines readily penetrate cell membranes, they are the drugs of choice for treating intracellular bacterial infections caused by chlamidias, rickettsias, and Brucella. Tetracyclines prevent the binding of transfer RNA to 70 S ribosomes. Since 80 S ribosomes are not affected, the inhibition is specific for prokaryotes.

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Tetracyclines are broad-spectrum antibiotics and their extended use disrupts the normal flora and encourages secondary infection by resistant microbes. In addition, these antibiotics discolor developing teeth and may retard normal growth of bones. Tetracyclines should therefore not be prescribed for pregnant women (they cross the placenta) or for young children. Erythromycin, Clindamycin, and Chloramphenicol Several ribosome inhibitors prevent peptide bond formation during bacterial protein synthesis. They do no structural damage to ribosomes, so they are all bacteristatic agents. Erythromycin is active against gram-positive bacteria, Clindamycin is used for treating infections, caused by gram-negative anaerobic bacteria, and Chloramphenicol is a broad-spectrum antibiotic. Target: Nucleic Acids Most chemotherapeutic agents that interfere with nucleic acid synthesis are synthetic compounds and not natural antibiotics. Only a few agents in this category are selective enough for safety treating infectious diseases. Rifampin is a semisynthetic derivative of a natural product of Streptomyces. This drug inhibits transcription of mRNA from DNA by binding to and activating bacterial mRNA polymerase. Rifampin is especially effective against Mycobacterium species that cause tuberculosis and leprosy. Nalidixic acid belongs to a family of compounds called quinolones that inhibit DNA gyrase, an enzyme needed for replication of bacterial chromosomes. High concentrations of nalidixic acid are excreted in the urine, making it useful for treating urinary tract infections. Newer quinolone derivatives (floxquinolones) are broad-spectrum antibiotics. Some antibiotics of this group interfere with nucleic acid replication in yeasts by incorporating into fungal DNA. It does not have the similar effect on humans because only fungi contain the enzyme that activates these antibiotics. They are often used in conjunction with amphothericin B for treating systemic Candida and Cryptococcus infections. Gryseofulvin also interferes with DNA replication in some fungi. It is used for treating dermatophyte infections of skin, nails, and scalp. Metronidazole is commonly imployed to treat vaginal infections caused by the protozoan Trichomonas vaginalis but is also effective against Giardia and parasitic amoebas. This synthetic drug is also one of the most active agents against anaerobic bacteria. Almost no anaerobic bacteria are affected. Sensitive organisms metabolize metronidazole to a compound that binds to DNA and rapidly kills the cell. Target: Bacterial Metabolism (inhibition of intermediary metabolic pathways) Several important chemotherapeutic agents called antimetabolites competitively inhibit bacterial metabolic reactions. The antimetabolites are usually substrate analogues, compounds with structures that closely resemble the substrate

34

of an enzyme and therefore compete for the enzymes active site. If the concentration of the inhibitor is high enough, it successfully competes with the substrate and prevents its conversion to products. Inhibitors that interrupt reactions essential to microbial growth but not need for human metabolism may be used for chemotherapy. Sulfa drugs, for example, are analogues of para-aminobenzoic acid (PABA). Normally PABA is enzymatically converted to folic acid, a coenzyme essential for growth. Sulfa drugs react with the enzyme but are not converted to products. They tie up the active site and prevent the production of folic acid. Bacteria that must synthesize their own folic acid (they cannot absorb folic acid from the medium) are inhibited by the sulfa drugs. Human cells obtain all their folic acid from external sources and are therefore not affected by inhibitors of folic acid synthesis. Consequently, the sulfa drugs are selectively toxic for bacteria. Because inhibitor binding is reversible, sulfa drugs are bacteriostatic agents. Trimethoprim blocks a later step in the folic acid pathway. Trimethoprim and sulfa drugs interact synergistically and are often prescribed together. Isoniazid (INH) is another antimetabolite that inhibits mycolic acid synthesis in mycobacteria. It is the most widely used drug for treating tuberculosis. Unlike the sulfa drugs, INH is bactericidal. Since INH resistance is common among mycobacteria, this antimetabolite is usually used in combination with rifampin and other antibiotics. Chloroquine has been used for over 50 years in treating malaria. This drug interferes with the enzymatic digestion of hemoglobin during the erythrocytic phase, when Plasmodium parasites have invaded red blood cells. Chloroquine prevents the detoxification of the hem breakdown products, which then kill the protozoa inside the erythrocytes. Antiviral Agents Viruses theoretically present a variety of targets for chemotherapy during their replication cycle. For example, drugs may interfere with attachment, virusspecific transcription and translation, replication, or assembly mechanisms. Currently, however, only a few chemotherapeutic chemicals are licensed for the treatment of a limited number of viral diseases, and not all of these are both safe and effective in treating existing viral disease. The drug amantadine blocks the uncoating of type A influenza virus and the release of viral RNA into cells but has little or no effect on the virus once it is replicating. Amantadine may prevent influenza infection and can shorten the duration of symptoms by 24 hours if administered within 2 days of disease onset. Acyclovir inhibits DNA synthesis in certain herpesviruses. The drug, an analogue of guanine, must be converted to a triphosphate form to be active. When the activated drug becomes incorporated into viral DNA, it causes the termination of DNA synthesis.

35

Gancyclovir is a similar drug that is more readily activated by cellular enzymes, and its spectrum of activity includes cytomegalovirus. Foscarnet is a structurally unrelated drug that is used to treat infections due to acyclovir- resistant herpesviruses or gancyclovir- resistant cytomegaloviruses. Ribavirin is an analogue of guanine that prevents DNA and RNA synthesis and translation of viral messenger RNA. Ribavirin displays antiviral activity against influenza viruses, respiratory syncytial viruses, and a variety of viruses found mainly in Asia and Africa. There are several other antiviral drugs (for example, Azidothymidine) that inhibit viral DNA-replication or inhibit reverse transcriptase activity in human immunodeficiency virus (HIV). Commercially produced interferon, a product of recombinant DNA technology, inhibits viral replication by inducing host cells to produce proteins with antiviral activity. Despite interferons broad antiviral activity, its general use as an antiviral drug is limited because, in most cases, it is more effective in preventing disease than in curing it. Antibiotic Resistance Chemotherapeutic effectiveness depends upon the sensitivity of the pathogen to the agent. Some microorganisms are naturally resistant to certain antibiotics because they lack the target that the antibiotic affects, or because the drug cannot reach the site of action. (Fungi, protozoa, and viruses for example, contain no peptidoglycan and are naturally resistant to penicillin and other inhibitors of bacterial cell wall synthesis). Sensitive microbes may become resistant due to the development of antibiotic resistance. Antimicrobial resistance is acquired either by mutations in the pathogens chromosome or by direct transfer of some chromosome genes or R-factor plasmids from antibiotic-resistant strains to sensitive recipients by conjugation or transformation. R-factors usually carry genes for multiple resistance, fortifying the bacterial recipient with protection from a number of drugs. The overuse of antibiotics favors these plasmid-carrying strains, establishing a reservoir of R-factors in the normal flora. Since plasmids can be transferred among different species, drug-sensitive pathogenic bacteria may acquire R-factors from the normal flora bacteria, and a patients disease suddenly becomes untreatable by antibiotics that would have been effective prior to the plasmid transfer. Such plasmid promiscuity allows for extensive spread of antibiotic resistance throughout a heterogeneous population of bacteria.
NOTE: Antibiotics do not create resistant cells or cause mutations that produce resistant organisms. They do, however, selectively favor the survival and proliferation of drug-resistant strains, which otherwise are only a small subpopulation within the vast majority of sensitive cells. Therefore, prolonged exposure to even a single antibiotic may favor the proliferation of bacteria resistant to several drugs.

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Mechanisms of Acquired Antibiotic Resistance R-factors and several chromosome genes encode the bacterial cell ability to: 1. Inactivate or destroy the antibiotic by producing extracellular enzymes (for example, penicillinases) 2. Alter their own membranes so that they are no longer permeable to the agent and its uptake is decreased 3. Alter the target site so that it is no longer affected by the drug (for example, amonoglycosides cant bind to modified bacterial ribosomes) 4. Develop a mechanism to bypass the target metabolic reaction, that is some bacteria acquire resistance to antimetabolites by bypassing the metabolic step inhibited by the drug. Cross Resistance Sometimes an organism develops cross-resistance, an insensitivity to several related antibiotics. For example, a single cellular modification may provide resistance to all the tetracyclines. Penicillinase-producing bacteria inactivate several types of penicillins. For this reason, when an organism is resistant to an antibiotic, the alternative agent is usually selected from an unrelated group of antimicrobial compounds. Antibiotic Susceptibility Tests Ideally, before any antibiotic is administered, a clinical specimen is collected from the infected patient, the infectious agent is isolated and identified, and the drugs to which the pathogen is susceptible are determined. The pathogens drug sensitivity can be determined by observation the microbes ability to grow in the presence of the drug. This can be done with a help of the following methods: 1. The Diffusion Methods (for example, the disk diffusion method) 2. Minimum Inhibitory Concentration-test (MIC-test) 3. Automated Methods The Disk Diffusion Method All the parameters used in this procedure are carefully standardized. Antibiotic-impregnated disks are placed on the surface of a solid medium that has been seeded with the isolated pathogen. After 24 hours of incubation, each antibiotic has diffused into the agar. Antibiotics that inhibit microbial growth produce a clear zone around the disk in which no organisms grow. The diameter of the zone of inhibition indicates whether the pathogen is resistant or sensitive to the drug in the disk (see Fig.6). The organism is designated sensitive only if its growth is inhibited by a concentration of the drug that can be achieved at the site of infection. This sensitivity pattern is called an antibiogram. (Since pathogens

37

generally react similarly to closely related antibiotics, an antibiogram predicts

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susceptibility to more drugs than are actually used in the test).

Minimum Inhibitory Concentration (MIC) A more accurate method of determining a pathogens drug sensitivity is to measure the MIC, the smallest amount of the drug that inhibits the multiplication of the pathogen. MIC is usually determined by a broth dilution method either in test tubes or in panels of small wells (see Fig.7). A standard inoculum of the pathogen is incubated in a series of tubes or wells containing decreasing concentrations of the antibiotics being tested. If the drug inhibits the microbe at the concentration in the tube, no growth appears; the organism grows only in concentrations below the one required for inhibition. Therefore, the highest dilution (the lowest concentration) showing no visible growth is the MIC. Cells from the tubes showing no growth can be subcultured in media lacking antibiotics to determine if the inhibition is reversible or permanent. In this way, the minimum bactericidal concentration (MBC) is determined. The MBC is nearly always higher than the MIC, since it usually requires more antibiotic to kill an organism than to merely inhibit its growth. Monitoring drug level

F i e f c o a n o f m ( M g r m t h

g u r e 7 . M e a s u r in g a f e c t i v e n e s s . T h e M I C n c e n t r a t i o n i n a s e r i t im ic r o b i a l a g e n t t h a t h e t e s t o r g a n i s m . T in i m u m b a c t e r i c id a l B C ) , m a t e r i a l f r o m t o w t h i n M I C t e s t s a r e d iu m t h a t l a c k s a n t a t h a v e b e e n k il le d f

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Knowing the concentration of antibiotics in body fluids can help in ascertaining whether therapeutic levels have actually been reached at the site of infection. This is particularly important when the chemotherapy fails to promote patient recovery even when the pathogen was shown to be sensitive to the agent used. The drug should have a serum level that is between 2 or 8 times the MIC of the organism to ensure that the tissue concentrations, which are lower than those in the serum, will reach the MIC. Drug concentrations are also monitored to prevent harmful side effects. The diffusion Method is usually used for monitoring antibiotic level in different biological samples. Choosing the Best Chemotherapeutic Agent No single antimicrobial drug is safe in all patients or effective against every infectious disease. Several factors influence the therapeutic value of antimicrobial drugs and must be considered if the patient is to receive the most effective chemotherapy. These factors are the following. The selective toxicity of the drug, that is, if it inhibits or kills the microbe without producing undesirable side effects in the person being treated. Eukaryotic human cells are usually lack of targets for most of antibiotics, which provides the basis for the drugs selective toxicity. The susceptibility of the pathogen to the chemotherapeutic agent The drugs spectrum of activity (There are broad-spectrum drugs, which affect a wide number of microorganisms, and narrow-spectrum drugs, which are more limited in the types of cells they affect. Usually the ideal agent has the narrowest spectrum that is effective against the identified pathogen, since broad-spectrum agents disrupt the normal flora) Possible adverse reactions to the drug (Antibiotics may have mild to fatal side effects. These may include general symptoms: chills, fever, headache, nausea, or rash. More severe toxic reactions may damage the liver, kidney, or nervous system. Some of these drugs can pass through the placenta and cause fetal damage in pregnant women. The broad-spectrum antibiotics usually disrupt the normal flora, which causes the development of secondary infections. Many antibiotics affect the immune systems functions and may cause hypersensitivity reactions (for example anaphylaxis) ,and immunodeficiency The site of infection and the drugs ability to reach those tissues (Antimicrobial agents have no therapeutic effect unless they reach the site of infection in concentrations high enough to incapacitate the pathogen. For example, antibiotics that cannot cross the barrier between the blood and the central nervous system, are useless for treating meningitis unless injected directly into the cerebrospinal fluid) Metabolism of the drug in the body (Many drugs change their antimicrobial effectiveness within the body. Some of them are destroyed by the low pH of the stomach, others are bound and inactivated by serum proteins. Therefore, the

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amount of effective antibiotic in the body may be considerably lower than the amount administered) Duration of treatment (Most drugs are metabolized or excreted before the infectious agents are eliminated. These drugs must be periodically readministered to maintain therapeutic levels) Interaction with other drugs the patient may be taking (Several antimicrobial agents are sometimes administered together to increase the spectrum of activity against mixed infection. These combinations are synergistic. There are some antibiotics which antagonize each other and should not be used in combination). PRINCIPLES of CULTIVATION of the OBLIGATE INTRACELLULAR PARASITES Viruses and some bacteria (e.g., Rickettsia and Chlamydia) cannot be cultured on artificial bacteriological culture media. They are obligate intracellular parasites-they need living host cells for propagation. Human and animal viruses, Rickettsia and Chlamydia can be grown in laboratory animals, cell cultures or in fertile eggs (usually, embryonated chicken eggs) under strict controlled conditions. Bacteriophages, for example, can be propagated on susceptible bacterial cultures. Diagnostic laboratories attempt to recover viruses and bacteria which are obligate parasites from clinical samples to establish disease etiologies. Research laboratories cultivate viruses, as the basis for detailed analyses of viral expression and replication.
NOTE: Precautions must be taken by laboratory personnel to prevent the transmission of the pathogens. Specimens should be transported to the laboratory in special doubling mailing containers with a secure lid to prevent leaking during transport. The procedure of inoculation of animated models and the further isolation and identification of the pathogens must be performed only in Biohazard Cabinets by highly quialified vaccinated personnel.

Cell culture, the growth of animal or human cells on artificial media, is the most common system for detecting and cultivating human viruses and some bacteria. These cells are grown in a monolayer, a uniform layer one cell thick on the inner surface of a bottle or test tube. The availability of cells grown in vitro has facilitated the identification and cultivation of newly isolated viruses and the characterization of previously known ones. There are three basic known types of cell culture. Primary cultures (trypsin-treated primary cell cultures) are made by dispersing cells (usually with trypsin) from freshly removed host tissues. Since cells of most primary cultures remain viable for several generations, they may be repeatedly subcultured (passaged). But, in general, they are unable to grow for more than a few passages in culture, as secondary cultures. Diploid cell lines are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages) but which retain their normal chromosome pattern. Human

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diploid cells are highly sensitive to numerous viruses and are extensively used in virology. Continuous cell lines are cultures capable of more prolonged, perhaps indefinite growth that have been derived from diploid cell lines or from malignant tissues. They invariably have altered and irregular numbers of chromosomes. The type of cell culture used for cultivation depends on the sensitivity of the cells to a particular virus or bacteria. Cell cultures are cultivated in glass or plastic bottles and tubes of various size and shape, preferably disposable, with sterility strictly observed at all stages of cultivation. Sterile nutrient media for cell cultures contain the whole range of amino acids, vitamins, and growth factors. The Eagles medium, medium 199, lactic albumin hydrolysate are most commonly used commercially available artificial culture media for cell cultures. Before the medium is used, antibiotics are added to it in order to prevent bacterial contamination. Using buffer systems the pH of the medium is maintained at 7.2-7.6. An indicator (commonly, phenol red) which becomes orange-yellow in acid medium or crimson in alkaline medium is also added to the culture media. Although cell culture is the preferred method of viral cultivation, some viruses and many bacteria (e.g., Rickettsia, Chlamydia, causative agent of tularemia, etc.) can be grown in living experimental animals. Growth of obligate intracellular parasites in animals is still used for the primary isolation of certain viruses or bacteria, and for studies of the pathogenesis of viral and bacterial diseases. The animals that can be taken for cultivation of obligate intracellular parasites range from mouse to chimpanzee and other mammalians. The choosing of the best animal model depends on the viral or bacterial species type. Viruses, Rickettsia, Brucellae and some other bacteria can be cultivated in 6-15-day-old chicken embryos. The material to be tested is introduced with sterile syringe onto the chorioallantoic membrane (CAM), into the yolk sac, or into the amniotic or allantoic cavity. Before the inoculation of embryo, the eggshell is treated with iodine and alcohol solutions. The procedures of inoculation of chicken embryos are as follows: A. For inoculation into the allantoic cavity the tested material is introduced through the lateral opening of the shell above the air sac 10-15 mm deep. The damaged site of the shell is then coated with heated paraffin. B. When the amniotic cavity is to be inoculated, the virus-containing specimen is injected through the opening at the obtuse end of the egg. The needle should be directed toward the embryonic body so as to ensure penetration of the virus into different organs and tissues of the embryo. One should control the procedure of inoculation of amniotic cavity with the help of ovoscope. Puncture site is sealed with paraffin. C. To infect the CAM, the eggshell is treated with iodine and alcohol solutions as usual, punctured above the air cavity (sac) on the obtuse end of egg and 2x2 mm opening is made laterally at the place of the greatest vascular ramification. Without destroying the shell-underlying membrane, 1-2 drops of virus-

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containing material are placed onto the CAM with a short thin needle. The damaged sites of the shell are coated with sterile paraffin. Then, the embryo is put into an incubator at horizontal position. The infected embryos are stored in an incubator at 35-37oC for 48-72 h, depending on the species of microbe suspected. Then, the eggs are cooled at 4oC for 18 h for maximum construction of the embryonic blood vessels and opened under sterile conditions. The amniotic and allantoic fluids are aspirated with a syringe and the membranes and embryo are transferred into sterile Petri dishes for the further examination. All the tested samples and specimens taken from the previously infected embryo are examined with unaided eyes for the detection of presence some changes in emryo: hemorrhages, pustules, plaques, etc. The scheme of the following examination of tested material with obligate intracellular parasite suspected depends on the type of the microbe.
NOTE: the scheme of the following examination of tested samples to identify the pathogen will be discussed next semester in the SPECIAL MICROBIOLOGY course.

PRINCIPLES of BACTERIAL GENETICS. METHODS of MOLECULAR GENETICS for IDENTIFICATION of BACTERIA The study of bacterial genetics has contributed enormously to our understanding of the genetics of all organisms. Many of the mechanisms discovered in relatively simple bacterial systems are very similar to corresponding mechanisms in humans. Bacteria are useful scientific models for studying the mechanisms of genetics for several reasons. 1. They can be propagated so rapidly that dozens of generations can be studied in a short time. 2. Large populations of essentially identical bacteria can be cultured from a single parental cell (essential for genetic homogeneity). 3. Compared to eukaryotes, bacteria are genetically simple organisms. E.coli, for example, possess a single chromosome that contains almost 5,000 genes. Human cells, with their 46 chromosomes and 100,000 genes are much more complex and difficult to characterize genetically. 4. Genetic material is readily transferred from one bacterial cell to another, that is why we can experimentally investigate the mechanisms of gene function. 5. Bacteria require much less laboratory space than plants and animals. Genes The terms genetics is derived from the word "generation" and means the study of heredity and variation among generations of organisms. The fundamental units of heredity are genes, which are linearly arranged along the chromosomes. Genes direct the synthesis of all the organism's traits, most of which are products essential to the organism's growth and survival.

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The entire genes composition of an organism is called its genotype, the organism's genetic potential. The genetic characteristics expressed at any given time constitute the organism's phenotype. The phenotype can change dramatically. In contrast to alterations in phenotype, changes in genotype, called mutations, are relatively infrequent. The Bacterial Chromosome and Plasmids In prokaryotes, genetic information is contained in DNA arranged as a circular chromosome. Bacteria are haploid organisms; that is their cells contain a single copy of the chromosome (although during rapid growth, when DNA replication is faster than cell division, each cell may contain two identical copies of the chromosome). The bacterial chromosome is approximately 1000 times as long as the cell containing it, but it occupies only about 10 percent of the cell volume. This long molecule is packed into the cell as a highly condensed supercoil. The DNA circle folds upon itself, and its twists and turns give it an extremely compact form. Supercoiling and untwisting of the DNA are controlled by enzymes called DNA gyrases. Most of the information in the bacterial chromosome directs the synthesis of enzymes and structural proteins. Most bacteria also contain smaller pieces of circular molecules of DNA that are not part of the chromosome. These extrachromosomal circles of DNA are called plasmids. Although certain plasmids can integrate into the chromosome, most plasmids remain autonomous in the cytoplasm. The genes carried on plasmids specify different traits from those on the chromosome, traits that normally are not essential to the organism (see Table 7). Plasmids do, however, contribute some advantages. Genes on the plasmid may encode information for such advantageous traits as an increase in metabolic options, resistance to antibiotics, or the capacity to synthesize compounds toxic to competing bacteria. Table 7 Representative Plasmid Genes Functions
Antibiotic resistance; Antibiotic production; Catabolic enzymes Toxin production; Heavy metal resistance; Plant tumor induction; Conjugation (plasmid transfer).

DNA stores specific genetic information that ultimately determines all the characteristics of an organism. Biological differences between organisms are due primarily to differences in the information encoded in their chromosomal DNA. The biological tasks of DNA are threefold: 1. Storage of genetic information. DNA is the cell's blueprint and contains all the information needed to produce and maintain a unique organism.

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2. Inheritance. Genetic information is precisely transmitted to all the organism's descendants. 3. Expression of the genetic message. Information stored in DNA is used to direct the protein synthesis by a cell. The manner in which genetic information is stored, inherited, and expressed is basically the same in all organisms. Transposable Genetic Elements Although most bacterial genes are fixed in one spot on the bacterial chromosome, a few can move about the genome. When these mobile transposable elements insert themselves into new regions of the DNA, they often cause mutations. Insertion may occur anywhere on the bacterial chromosome or on plasmids. When a transposable gene is inserted into the middle of another gene, that gene can no longer synthesize a functional gene product. Two types of transposable elements are commonly found in prokaryotes. Insertion sequences are composed of genes whose only activity is to transpose themselves, whereas transposons contain additional "passenger" genetic information, such as genes for 1 antibiotic resistance. Transposons D N A f r a g m e n t can shuttle these genes around the f r o m d o n o r c e ll chromosome and into plasmids. Genetic Transfer in Bacteria Genes are transferred among 2 bacteria in one direction - from le a v a g e b y donor to recipient. In most cases, D N a s e only part of the DNA is transferred. Once this DNA fragment is in the recipient, it may recombine by 3 breakage of the host chromosome E x c i s e d f r a g m e n t o f h o s t D N A and union of the free ends with the newly received DNA fragment (Fig. 4 8). Recombination, therefore, results D i s c a r d e d in the stable incorporation of the new D N A genes into the recipient's chromosome. R e c o m b i n a n t h o The three mechanisms for s t c h r o m o s o m gene transfer between bacteria are e F ig u r e 8 . I n t e g r a t i o n transformation, gtransduction, Aand o f f o r e i n D N . N e w l y r e c e iv e d D N A i s s t a b ly in s e r t e d in t o t h e e x i s t in g c h r o m o s conjugation. o f t h e o m e r e c i p ie n t
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Transformation Destruction of a cell does not necessarily destroy its genetic material. When bacterium is lysed, its DNA is often released into the surrounding medium. This DNA retains its ability to direct the synthesis of specific proteins if introduced into a viable bacterial cell. Recipient cells absorb pieces of the released DNA onto their cell surface. The DNA is then transported across the cell wall and plasma membrane into the cytoplasm and is incorporated by recombination into the cell's chromosome. The recipient cell often acquires new characteristics as a result. This transfer of genetic information by free, extracellular DNA is called transformation. Transformation may be an important natural mechanism of genetic transfer among bacteria. It occurs among streptococci and bacilli and certain gramnegative organisms, notably Neisseria and Haemophilus. These cells, however, are not always capable of being recipients in transformation. They must be competent in order to bind double-stranded DNA to their surface and transport the fragment into the cytoplasm. Competence is a property of the type of cell and is also related to the medium and to growth conditions. Plasmids are rarely transferred in nature by transformation. This genetic exchange is prevented because plasmids are circular, and transforming DNA is taken up as a linear molecule. Transduction In bacteria, virus-mediated gene transfer is called transduction. Bacterial genes can become accidentally enclosed within a bacteriophage (a bacterial virus) during replication of the virus in a host cell (Fig. 9). This happens because, during viral infection, the host chromosome may be degraded into small pieces, and occasionally a fragment similar in size to the replicating viral chromosomes is packaged in the viral protein coat. Transducing particles, viral particles that contain bacterial genes, are liberated with normal progeny viruses during lysis of the host cell. When a transducing particle subsequently infects a susceptible host cell, the genes from the previous bacterial host are introduced into this new cell. Because viral genes have been replaced by bacterial genes, no bacteriophages are synthesized. The newly introduced bacterial genes may replace the homologous segment of the bacterial chromosome. Since any portion of the bacterial chromosome can be transferred, this type of transduction is reffered to as generalized transduction (Fig. 9). Another type of transduction is mediated by bacteriophages that insert their DNA into the host chromosome during their infection cycle. These bacteriophages remove their DNA from the host chromosome prior to replication and the assembly of new viral particles. Sometimes the viral DNA accidentally picks up a few adjacent bacterial genes as it leaves the host chromosome. This phage-host DNA hybrid replicates and becomes packaged, so all the progeny virions in the cell are transducing particles. When they infect a new host cell and integrate into

45

the chromosome, the recipient cell acquires the characteristics specified by the "passenger" genes from the previous host. This type of gene transfer is called specialized transduction because only those genes that lie adjacent to the phage integration site can be transferred.
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Conjugation Some bacteria have the ability to attach to other bacterial cells and transfer genetic material. This process, called conjugation, is mediated by certain plasmids. One type of conjugative plasmid, the R-factor (resistance factor), contains genes that convert drug-sensitive bacteria to antibiotic-resistant bacteria. Conjugation occurs in both gram-positive and gram-negative cells, but the mechanisms are quite different.

46

Conjugation in gram-negative bacteria. Externally, gram-negative donor cells differ from the recipients by the presence of at least one sex pilus extending from the cell surface. The sex pilus connects the two cells during conjugation (Fig.10). A conjugation tube develops through which DNA is transferred.
F 1 . C o n j u - f a c t o r g a t i o 2n . R e p l i c a t i o n a n d t r a n s f e r o f F - f a c t o r 1 . M o b i l i z a t i o n o f c h r o m o s o m e s 2 . T r a n s f e r o f f o r r e p l i c a t i o n b a c t e r i a l c h r o m o s o m F - f a c t o r F + ( d o n F o r ) C h r o m o s o m ( r e c i p i e n t ) e

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Although conjugation superficially resembles some aspects of sexual reproduction in eukaryotes, it has several important differences. It is not a method of reproduction (no fertilization occurs) but rather a means of genetic transfer. Genetically, the donor may differ from the recipient only by the presence of a plasmid called F-plasmid (F = fertility). Genetic information on the F-plasmid provides a bacterial cell with everything needed to be a donor, including the capacity to synthesize the sex pilus. Conjugation may have one of two outcomes depending on whether the F-plasmid is free or integrated. 1. F+ donors. When the F plasmid is not integrated, recipient (F-) cells are readily converted to donor (F+) cells (Fig. 10A). The donor does not sacrifice its F plasmid but replicates it and gives away the copy. In this way, a single F+ cell introduced into a culture of F- recipients can lead to the rapid conversion of all

47

the cells to F+. The F+ donors do not transfer any chromosomal genes, only the F plasmid. 2. Hfr donors. If the F plasmid integrates into the bacterial chromosome, the resulting donor is called an Hfr cell. The term Hfr denotes high frequency of recombination, referring to the fact that, unlike the F+ donors, Hfr cells commonly donate copies of genes on the bacterial chromosome (Fig. 10B). Chromosomal replication begins at the origin (the site adjacent to the integrated F plasmid). The replicated genes pass through the conjugation tube until the cells separate. Due to the fragile nature of the attachment, conjugation rarely lasts the 90 minutes required to transfer an entire copy of the donors chromosome; thus, because the F plasmid is always the last thing to be transferred, recipients are rarely converted to donor cells. In the recipient, the newly acquired bacterial genes become integrated into the chromosome. As a result, the recipient may receive new genetic traits from the donor. Conjugation in gram-positive bacteria. Conjugation among gram-positive bacteria is neither mediated by pili nor driven by an F plasmid. Donor bacteria do, however, contain conjugative plasmids that mediate conjugation between donor and recipient bacteria, during which a copy of plasmid DNA is transferred. Genetic information contained in the donors plasmid directs the synthesis of a surface substance that promotes binding to recipient cells. This binding substance is produced only in the presence of recipient cells. In some cases, the recipient releases a chemical that induces the plasmids binding substance gene. Gene Amplification - Polymerase Chain Reaction A new in vitro method for the synthesis of DNA has recently revolutionized the field. This procedure, called polymerase chain reaction (PCR), is a rapid method that can replicate DNA over a millionfold in less than 8 hours. The PCR reaction replicates DNA in a manner similar to that which occurs in the cell (Fig.11). 1. Double-stranded DNA is separated into single strands. In PCR, heat, not an enzyme, is used to denature the helix, that is, to separate the DNA into singlestranded molecules. 2. Short nucleotide segments serve as starters (called primers) for replication. The PCR primers are synthesized from deoxynucleotides. They are complementary to sequences that border the gene(s) of interest. A primer bonds with the 3 end of each of the complementary DNA strands. 3. DNA polymerase extends the primers. The products of this semiconservative replication are two progeny strands. The advantage of PCR is that after each round of replication the doublestranded progeny are denatured, more primers are added, and the process begins again. (The DNA polymerase is derived from a thermophilic bacterium, such as

48

Thermus aquaticus, and retains its activity during the high-temperature denaturation step). Thus a single piece of DNA can be amplified 1000-fold over the course of only 10 cycles.
O r ig d o u b l D N a t u r a t i n a l e - s t r a n d e A i o n 3 A d d p r i m P d 5 e r s r im 5 F o u r d o u b le 3 ) R s t r a n d s i d e n t ic a l t o o r i g i n a l4 ) H e a t d e n a t u r a t i o n , p r i m e r s a d d e d 3 e r 3 e p l i c a t i o n P r im e r

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e r a s e n c e i s n t h e s i z e r a t u r e t h e o p a s e e x - s t r a n d m o le c is r e p n d o f r r o f t e m e .

After replication, the abundant DNA is sufficient for examination. The DNA is digested with restriction enzymes, and the resulting fragments are separated by size and composition using gel electrophoresis (movement of molecules through a porous gel in response to an electric current - smaller fragments move faster than large fragments). This DNA pattern is then compared to that of the subject in custody. PCR has been used to generate nucleic acid probes for identification of microorganisms. The selected DNA region is one that is specific for the organism.

49

It can detect small numbers of pathogens in clinical samples and thus aid in the diagnosis of diseases caused by agents that are difficult to culture, such as the AIDS virus, the slow-growing Mycobacterium tuberculosis, or the intracellular pathogen Chlamydia. Genetic Engineering (Recombinant DNA Technology). Genetic manipulation can introduce foreign genes into bacterial cells where they exist on plasmids or recombine with the chromosome and become stable incorporated. These foreign genes may come from other species, even from eukaryotic cells (since the genetic code is universal for all cells). For example, insulin, which was formerly available only by extraction from animal cells (often an expensive, low-yield process), is now produced inexpensively by genetically engineered bacteria that have been given the human insulin gene. The transfer of DNA from eukaryotic cells to prokaryotic cells, however, does not occur naturally. Viruses are being genetically engineered to create new piggyback vaccines to protect against infectious diseases. Genes for a pathogens surface antigen are incorporated into the DNA of the vaccine virus, historically used as a vaccine against smallpox. Vaccinia is an ideal vaccine vector because it can accept large pieces of foreign DNA and has been proven safe for use in humans and animals. An engineered vaccinia virus will reproduce with no ill effect on the host and, while growing in an inoculated persons local tissues, will synthesize the surface antigens of the pathogen. Thus, the inoculated individual will build an immunity to the disease with no risk to being infected by the viable pathogen. ***** Recommended reading: 1. Topical lectures 2. Textbook 1: Mechanisms of Microbial Disease/edited by Moselio Schaechter, Gerald Medoff, Barry I. Eisenstein. - 2nd ed. 3. Textbook 2: Medical Microbiology & Immunology. Examination and Board Review./W. Levinson, E. Jawetz.-5th ed. 4. Manual (Part 2)

50

PRACTICAL PART LESSON 6 PRINCIPLES of CULTIVATION of BACTERIA. ISOLATION of PURE CULTURES of BACTERIA. IDENTIFICATION of PURE CULTURES. STERILIZATION. MICROFLORA of AIR and WATER Prelab conference. Topics for discussion: 1. Principles of cultivation of bacteria. 2. Microbiological culture media. 3. Classification of media by composition and consistence. Application of media based on bacterial metabolism. 4. Types of respiration of bacteria (energy metabolism). 5. Pure culture of bacteria. Mechanical dissociation of microbial cells and biological bacterial attributes. 6. Principles of cultivation of anaerobes. 7. Determination of the amount of bacteria in a tested sample. 8. Sterilization (methods and equipment). 9. Microflora of air and water. Methods of analysis. PRACTICAL WORK PART 1. Bacteriological Examination of Tested Sample. (the first day). (a) Preparation of pure bacterial culture to achieve isolation and separation of bacterial colonies from a mixed culture Clinical specimen often contain more than one type of microbes and it is essential to separate these into pure culture before any attempts at identification and further study can be made. Pure cultures (isolated colonies) can be practically obtained by streaking on solid nutrient media. A broth culture of bacteria (or bacterial suspension) will be supplied. Each student should streak one plate for isolation by the following method: METHODOLOGY:
1. Flame the entire length of the wire loop. Allow the wire to cool for a few

seconds prior to its contact with the test material.

NOTE: If the wire is not sufficiently cooled, spattering of infectious material or death of the microbes in the specimen can occur. 2. Remove the plug from the test specimen container. 3. Select a representative part of the sample, choosing purulent-appearing

material. Remove a thin loopful of the specimen while avoiding contact of the wire with potentially contaminated surfaces.
NOTE: The amount of the inoculum removed should not be too much, because great amount of inoculum makes it difficult to obtain pure culture.

51

4. Replace the plug in the container and place it aside. 5. Remove the agar plate cover, put it aside on the work surface of the table and

lift up the bottom of the plate. Hold it in one hand during the inoculation step. Streak approximately one-fourth of the surface with the test material on the loop.
6. Sterilize the loop in the flame of the burner; allow the wire to cool. 7. Rotate the plate a quarter turn and streak again, overlapping the originally

streaked area
8. Sterilize the loop again and allow it to cool. 9. Rotate the plate and streak the remaining area as above. Return the cover on

the plate. Sterilize the loop.

10. Label the inoculated plate with your name and your group number 11. Incubate the plates (agar side up!) under the appropriate conditions until the

next laboratory period.

NOTE: One must be very cautious during the inoculation process to protect himself from aerosols containing infectious organisms produced during the transfer process. The aerosols can result from the spattering of material from a too-hot loop. Another cause may be improper handling of infectious agents during their removal from or introduction into culture media containers. Microorganisms of high virulence should be manipulated only within a biological safety cabinet. Allow the instructor to examine the inoculated plate. He/she will decide

whether the streaking attempt has been successful. If it is not satisfactory, it must be repeated until successful streak-plate isolation has been achieved by the student. (b) Demonstration of the serial dilutions technique. Your instructor will demonstrate the preparation of serial dilutions of the examined specimen. Then the spread plates technique will be demonstrated. PART 2. Examining of Air Contamination Examine the microbial contamination of the air in the classroom by sedimentation and aspiration techniques. Sedimentation method.

52

Open the Petri dish with a sterile MPA and place it on the table. After 10minutes exposure close the plate with a cover; label it for content and place it into incubator or a place designated by your instructor until the next lesson. Aspiration method. Your instructor will demonstrate the application of Krotovs apparatus (air slit sampler). PART 3. Evaluating of Water Quality (Demonstration) The Procedure of Evaluating of Water Quality Prepare the serial dilutions of the water sample in the sterile water: 1:10, 1:100, and 1:1000 Put 1 ml of each dilution of water sample into empty plates and pour them with 15 ml of melted MPA each. Cover the Petri dishes, label them for content and put them into the place designated by your instructor until the next lesson. After 24h incubation at 37oC results are estimated. of the tested water. To do this: 1. Count the number of CFU on the plates with the inoculated dilutions of water tested (1:10, 1:100, 1:1000. 2. Determine the concentration of microbes per 1 ml of water tested by multiplying the number of colonies on the plate by the dilution factor (* dilution factor = 1/ dilution) 3. Take the average concentration by dividing the sum of the concentrations (CFU/ml) in the three inoculated water samples by 3. 4. Fill in Table 6-2 in the protocol notebook. PRACTICAL TASKS Prepare streak plate inoculation of the tested sample. Prepare pour plate inoculation of the water tested and two dilutions of water sample. Write down in your protocol notebooks the scheme of bacteriological examination of tested sample (for aerobic and facultative anaerobic bacteria) (see Table 6-1). Perform sedimentation inoculation of air in the classroom.

A. B. C. D.

Examine the results of demonstration and determine microbial contamination

1. 2.

3.

4.

53

Table 6-1 The Scheme of Bacteriological Examination

Day of examination First day Manipulations Isolation of pure culture: Using a loop, streak the material onto the solid medium in a Petri dish. Place the inoculated dishes in a 37C incubator for 18-24 hours or until the next lab period. NOTE: if the tested specimen is contaminated with microbes of normal flora, it is necessary to use selective media for primarily inoculation. If the material to be studied is lack of pathogens or contains them in low concentration it is inoculated into the liquid enrichment medium and incubated for 24 hours. Then it can be transferred to the Petri dishes with a solid nutrient medium to get the isolated colonies. Investigation of the cultural properties Examination of morphological and tinctorial properties, control of the purity of the isolated colony Subculturing of pure culture on slant agar Results and Conclusions This ensures mechanical separation of microorganisms on the surface of the nutrient medium, which allows for their growth in isolated colonies.

We have got ___ types of isolated colonies

Second day

Fill in the Table Cultural properties of isolated colonies(see LESSON 7, Table 7-1)

Third day

Forth day

Fill in the Tables Checking of the isolated culture purity Biochemical properties of Investigation of biochemical properties: (a) the isolated colonies, sugarlytic and (b) proteolytic Determination of antibiotic Determination of antigenic properties susceptibility of the isolated Determination of toxigenic activity culture, and Determination Study of phagosensitivity, phagotyping, of the MIC colicinosensitivity, and other properties (see LESSON 8 and 9) Determination of antibiotic susceptibility by Disk Diffusion method and MIC. CONCLUSION: Isolated microbes are identified as ______________________ and ______________________ . The____________ is mostly sensitive to antibiotics: ______________________. The____________ is mostly sensitive to antibiotics: ______________________.

5.

Familiarize yourself with serial dilutions technique and spread plates method of inoculation. Get acquainted with the application of Krotovs apparatus for slit sampler technique (aspiration method) for determining of air contamination.

6.

54

7.

Enter the results of demonstration of Evaluating of Water Quality and Fill in the Table: Determination of water contamination (Table 6-2). Table 6-2 Determination of Water Contamination
1:10 1 ml 15 Water dilutions 1:100 1 ml 15 1:1000 1 ml 15

Inoculated volume of water MPA (ml) Number of colonies on the plate (CFU) CONCLUSION: The average concentration of microbes in the water sample is ____CFU/ml

8.

Get acquainted with the methods of anaerobic bacteria cultivation (Fortner's method, artificial culture media for anaerobes, anaerobic jar application, etc.)
NOTE: your instructor will demonstrate you special equipment for strict anaerobes culturing and will show you culture media for anaerobes.

LESSON 7 ISOLATION of PURE CULTURES of BACTERIA. IDENTIFICATION of PURE CULTURES BASED on CULTURAL and TINCTORIAL PROPERTIES (continued from lesson 6). MICROFLORA of the HUMAN ORGANISM Prelab conference. Topics for discussion: 1. Cultural properties of bacteria. Bacterial growth on liquid and solid media. 2. Bacterial colonies. Characterization criteria. 3. Pigments of bacteria. 4. Normal microflora of human organism. Intestinal microflora. 5. Eubiosis and dysbiosis. Dysbacteriosis, reasons for its development. 6. Colonization resistance of human organism. Selective decontamination. 7. Eubiotics: drugs for restoration of normal microflora. PRACTICAL WORK PART 1. Bacteriological Examination (the second day) Following a 24-hour incubation, the cultural properties of bacteria (nature of their growth on solid and liquid media) are studied. A. Register all the colony properties in the protocol notebook (Table 7-1). Macroscopic examination of colonies Turn the dish with its bottom to the eyes and examine the isolated colonies in transmitted light, reflected light and under the microscope, using a 10x

55

objective. In the presence of various types of colonies count them and describe each of them. The following properties are paid attention to: Size of colonies (large, 4-5mm in diameter or more; medium, 2-4mm; small, 1-2mm; minute, less than 1mm in diameter) Configuration of colonies (regularly or irregularly rounded, rosetteshaped, rhizoid, etc) Degree of transparency (nontransparent, semitransparent, transparent) Color of the colonies (colorless, or pigmented and the color of the pigment) Nature of the surface (smooth, glassy, moist, wrinkled, dry, lusterless, or lustrous, etc) Position of the colonies on the nutrient medium (protruding above the medium, submerged into the medium; flat, at the level of the medium; flattened, slightly above the medium) Structure of the colonies (homogeneous or amorphous, granular, fibrillar, etc) The nature of the colonies edges (smooth, wavy, jagged, fringy, etc) Table 7-1 Cultural Properties of the Isolated Bacteria
# of colony Size, mm Transparency Surface Configuration Structure Color Position on the medium edgesNature of Morphology and tinctorial properties (Gram stain) Suspected bacterium

B. Use some portion of the isolated colony to prepare the Gram-stained smears

for microscopic examination. Determine the morphological and tinctorial properties of the colony and check of the purity of the isolated culture.
C. In the presence of uniform bacteria, carefully pick up with the loop the

remaining portion of the colony used in the Gram stain and transfer it to an agar slant for obtaining a sufficient amount of pure culture (use the streaking technique). Place the test tubes with the inoculated medium into a 37C incubator for 18-24 hours or until the next lab period. This is called a subculture. PART 1b. Determination of the Concentration of Bacteria in a Sample (total viable count in the serial dilutions method) Count the number of colonies on the plates with the inoculated serial dilutions of the tested sample (10-5, 10-6 , and 10-7 ) see Table 7-2.

1.

56

Determine the concentration of microbes per 1 ml in each dilution by multiplying the number of colonies on the plate by the dilution factor, and by 5. (Or by dividing the number of the colonies by 0.2, because only 0.2 ml of the tested sample has been inoculated) 3. Count the average concentration by dividing the sum of the concentrations in the three inoculated dilutions by 3 4. Draw and fill in Table 7-2 in the notebook. Table 7-2 Total Viable Count (Determination of Microbial Concentration in the Specimen)
2.

Inoculated volume of sample (ml) Number of colonies on the plate (CFU) Number of the colonies per 1 ml (CFU/ml) CONCLUSION: The average concentration of microbes in the sample tested is ____CFU/ml

10-5 0.2

Sample dilutions 10-6 10-7 0.2 0.2

1. 2.

3.

PART 2. Determination of Air Contamination Characterizing of growth following incubation, examine the plates for the types and numbers of colonies present. Interpret the results according to the limitations for satisfactory air contamination (see the Manual). Make a conclusion (if the air contamination is satisfactory or not). Fill in Table 7-3 in the protocol notebook: Table 7-3 Determination of Air Contamination

Time of Volume of the CFU Description of the exposure tested air (m) (totally) colonies Sedimentation method 5min 0.01 Aspiration method 4 min 0.1 CONCLUSION: The average concentration of microbes in the classroom air is ____CFU/m

1. 2. 3.

PART 3. Examination of the Human Normal Flora (a)Sampling personal skin flora With a glass-marking pencil draw a line across the bottom of a agar-containing Petri dish. Label one side before and the other side after. Place the fingers of your right hand gently but firmly on the surface of the agar half labeled before Wash your hands with soap and water, as you would do ordinarily. Then place the fingers of the washed right hand on the agar half labeled after.

57

4.

Incubate the plate in the inverted position at 37C until the next laboratory period.

(b)Examination of the microbes in personal dental plaque. (Bacteria of the indigenous oral flora). 1. With the wooden stick take a portion of your dental plaque (or dental pellicle), and put it on the slide glass. 2. Prepare a smear and stain it with the Gram technique. 3. Examine the stained smear under the microscope. 4. Draw the results of your observations in the notebook. (c)Sampling personal oral flora. (Bacteria of the indigenous throat flora). With the cotton swab touch to the tonsil surface and remove a portion of a mucus. Using streak technique, inoculate a plate with 5% blood agar. Incubate the plate bottom side up at 37C until the next laboratory period. PRACTICAL TASKS Examine and register in the notebook the cultural, morphological, and tinctorial characteristics of the two types of isolated colonies (Table 7-1). Subculture the isolated colony on the slant. Examine your personal skin flora. Examine the microflora of your own dental plaque. Draw in the notebook the results of your observations of the Gram stained dental plaque smear. Examine your personal throat flora. Fill in Tables 7-2 and 7-3 with the results of experiments. Draw in the notebook the demonstration of the bacterial pigments. Draw in the notebook the demonstration of the Deep Tube method in the liquid media.

1. 2. 3.

1.

2. 3. 4.

5. 6. 7. 8.

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LESSON 8 ISOLATION of PURE CULTURES of BACTERIA. IDENTIFICATION of PURE CULTURES BASED on BIOCHEMICAL ACTIVITY (continued from lesson 7). DETERMINATION of BACTERIAL SENSITIVITY to ANTIBIOTICS Prelab conference. Topics for discussion: 1. Enzymes of bacteria. Classification. 2. Differential diagnostic media in identification of bacteria. 3. Antibiotics. Classification according to chemical structure, spectrum and mechanism of action. Sources of antibiotic production and ways of manufacturing. 4. Methods of determination of bacterial sensitivity to antibiotics. PRACTICAL WORK PART 1. Bacteriological Examination (the third day) (A) Checking of the Purity of the Culture. Using the subculture, which has grown on the agar slant, prepare smears, and stain them by the simple method. Such characteristics as homogeneity of the growth, form, size, and staining of microorganisms allow checking of the purity of the culture.
(B) Determination of Enzymatic Activity.

To identify the isolated pure culture, supply the study of morphological, tinctorial, and cultural properties with determination of their enzymatic activity, phagosensitivity and antibiogram. (Determination of the antigenic attributes, toxigenicity, and other properties characterizing specificity of the microbial species will be discussed in the next section of the Course) To demonstrate the carbohydrate-splitting enzymes (sugarlytic activity), Hiss media are utilized. Inoculate the tested pure culture by pricking into the several semisolid Hiss media containing glucose, lactose, and mannitol. Incubate the inoculated tubes until the next lab period. Profound splitting of protein (proteolytic activity) is evidenced by formation of indol, ammonia, hydrogen sulfide, and other compounds. To detect the gaseous substances, inoculate the tested pure culture into a nutrient broth (MPB) and put special indicator papers between the test tube and the plug so that they do not touch the inoculated medium. Incubate the tube until the next lab period. (C). Phagotyping test. Draw three squares on the bottom of the plate with nutrient agar (MPA) and label them with the names of applied bacteriophages. Prepare the suspension of the pure culture with 2 ml of the sterile 0.9% sodium chloride solution. Inoculate the bacterial suspension onto the solid medium with the

59

spread plates method. Transfer the drop of each phage onto the corresponding square. Incubate the inoculum until the next lab period. (D). Determination of bacterial sensitivity to antibiotics. The pathogens drug sensitivity can be determined by observation the microbes ability to grow in the presence of the drug. a. The Disk Diffusion Method 1. Prepare the suspension of the subculture growth on the slant with 2 ml of sterile 0.9% sodium chloride solution. 2. Seed the plate with the suspension by spread plate technique. 3. Place the antibiotic-impregnated disks on the surface of the inoculated solid medium with the pincers. The disks should be placed far enough from each other. 4. Incubate until the next lab period. b. Serial dilutions for measuring Minimum Inhibitory Concentration (MIC) A standard inoculum of the Staphylococcus aureus is incubated in a series of tubes containing decreasing (two-fold) concentrations of penicillin. If the drug inhibits the microbe at the concentration in the tube, no growth appears; the organism grows only in concentrations below the one required for inhibition. Therefore, the highest dilution (the lowest concentration) showing no visible growth is the MIC. No visible growth occurs if antibiotic inhibits the multiplication of the microbe. You have to determine the last tube with transparent broth in the presence of an intensive growth in the control one. METHODOLOGY: Nutrient broth was poured by 1-ml portions into 8 test tubes. 1 ml of prepared antibiotic solution containing 2 g/ml was added into the first tube. Following thorough mixing, 1 ml of the given antibiotic broth solution was transferred into the next tube and so on until the 7th tube was reached. The 8th tube containing no antibiotic serves as a control of culture growth. One loopful of the tested microbe (S. aureus) was inoculated into each tube of the row beginning from the control one. The results of the experiment are read following incubation of the tubes at 37C for 18-20 hours.
NOTE: Cells from the tubes showing no growth can be subcultured in media lacking antibiotics to determine if the inhibition is reversible or permanent. In this way, the minimum bactericidal concentration (MBC) is determined. To determine MBC one may also prepare spread plates, inoculating antibiotic solutions from the tubes tested. The MBC is determined as the lowest concentration that inhibits the growth of S.aureus on the nutrient agar.

Examine the demonstration of Measuring the MIC. Notify the tube which contains the MIC of penicillin solution. Register the results in Table 8-1.

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PART 2a. Examination of Human Skin Flora 1. Examine the skin microflora growth on the plates with your fingerprints. 2. Observe the types and number of the colonies; prepare a simple stained smear from the most interesting colonies. 3. Compare the results of the both parts of the plate. PART 2b. Examination of Human Throat Flora 1. Examine the throat flora growth on the blood agar plate. 2. Observe the types and number of the colonies. Note the possible clear zones around some colonies (hemolysis zones). Prepare a simple stained smear from the most interesting colonies. PRACTICAL TASKS 1. Check the purity of the subculture on the slant (see Part 1, A).
2.

Familiarize yourself with the demonstration of Commercial Identification Systems (your instructor will show you API-20E, Enterotest, Enterotube, etc. that are employed for biochemical identification of bacteria).
NOTE: Commercial Identification Systems.

Routine bacterial identification is usually based on the results of biological tests that reflect the genetic composition of the microbe. Such information is complied in a practical guide called Bergeys Manual. Biochemical identification of bacterial pure culture is made by comparing the reactions of an unknown microbe in a series of tests with the known reactions of all possible microbes. It usually takes 10 to 30 tests to identify a bacterium. (Thus, these tests are actually examining a very tiny portion of the organisms properties). So, isolates with noncharacteristic responses often are identified with limited certainty, and additional tests may have to be performed to confirm their identification). Commercial identification systems (API-20E, Enterotest, Enterotube, etc) are composed of a limited series of biochemical tests. These systems test the substrate-using versatility of bacteria by measuring growth of the suspension of bacterial pure culture in the battery of differential culture media contained in small wells in a single plastic plate or strip. After incubation of the pattern under optimal conditions, the results of the tests are estimated. All the test results are entered in the appropriate spaces on the special Encoding form. Then the numerical value for each positive test result is noted according to that printed above the test results on the Encoding form. These sums are the Code for the microbe to be identified. This code is the key to the information listed in the special Manual (for example, Octal code in Micro-Id System). Automated Identification systems. Several methods relying on computer analysis have been developed to aid in the rapid identification of bacteria. These systems collect data on specific characteristics of the unknown isolate and compare this information to a computerized database of known bacteria.

61

3.

Inoculate the Hiss media and MPB to characterize the enzyme-splitting activity of isolated bacteria (see Part 1, B). Perform the phagotyping (see Part 1, C). Perform the Disk Diffusion method for determination of bacterial susceptibility to antibiotics (see Part 1, D). Register the results of MIC Determination in your notebook (see Table 8-1). Table 8-1 Determination of Minimum Inhibitory Concentration
# of tubes 1 1 2 2 1 1 3 1 0.5 4 5 6 7 1 1 1 1 0.25 0.12 0.06 0.03 One loopful in each tube 8 control 1 ---

4. 5.

6.

MPB (ml) Penicillin (g/ ml) Staphylococcus aureus Results (visible growth): CONCLUSION: MIC of penicillin is _________ g/ ml

7.

Examine the skin microflora growth on the solid medium.

8. Examine the throat flora growth on the blood agar plate.

LESSON 9 ISOLATION of PURE CULTURES of BACTERIA. IDENTIFICATION of PURE CULTURES. DETERMINATION of BACTERIAL SENSITIVITY to ANTIBIOTICS (final lesson on the theme). PRINCIPLES of BACTERIAL GENETICS. METHODS of MOLECULAR GENETICS for IDENTIFICATION of BACTERIA Prelab conference. Topics for discussion: 1. Identification of bacteria by the complex of morphological, tinctorial, biochemical and other properties. 2. Antibiogram. Interpretation. 3. Principles of cultivation of the obligate intracellular parasites. Cultivation of viruses. 4. Recombination of bacteria. 5. Mechanisms of transfer of the genetic information in bacteria. 6. Plasmids. Properties of plasmids. 7. Transposable genetic elements. 8. Polymerase chain reaction (PCR). 9. Plasmid profile of bacteria. Restriction analysis.

62

PRACTICAL WORK PART 1. Bacteriological Examination (the fourth day). (A). Examination of the results of enzymatic activity of isolated bacteria Characterizing of growth following incubation examine the tubes with the Hiss media and the indicator paper strips in MPB-tube. 1. Watch for the pH indicator in the Hiss media to change color. This occurs due to the acid fermentation products. Some bacteria produce gas, which becomes trapped in the inverted tube in a liquid Hiss media, or looks like bubbles in a semisolid media.
NOTE: In an actual laboratory situation, many more sugars than three should be tested. 2. Examine the indicator paper strips in the MPB-tube. When Indole is released,

3.

the lower part of the paper strip turns pink as a result of its interaction with oxalic acid in the indicator paper. Hydrogen sulfide is detected by means of an indicator paper strip saturated with lead acetate solution. Upon interaction between hydrogen sulfide and lead acetate the paper turns black as a result of lead sulfide formation. Register the pattern of sugar and protein fermentation in the following table in the notebook (see Table 9-1). Table 9-1 The Pattern of Sugar and Protein Fermentation
Sugarlytic activity Glucose Lactose Maltose Proteolytic activity Indole Hydrogen sulfide

Staphylococcus aureus Micrococcus luteus Bacillus cereus Escherichia coli

(B). Examination of the results of Phagotyping. Determination of culture sensitivity is based on bacteriolytic effect of the definite phages. In this case a clear zone appears on the medium, while the rest part of the medium is covered with visible bacterial growth. Notify the name of the bacteriophage, which lyses the given bacteria. (C). Determination of the antibiogram (the results of the Disk Diffusion method). After 24 hours of incubation, each antibiotic has diffused into the agar. Antibiotics that inhibit microbial growth produce a clear zone around the disk in which no organisms grow. The diameter of the zone of inhibition indicates whether the pathogen is resistant or sensitive to the drug in the disk. The organism is

63

designated sensitive only if its growth is inhibited by a concentration of the drug that can be achieved at the site of infection. 1. Register the sensitivity pattern of the tested microbe (fill in the following table in the notebook): Table 9-2 Antibiogram
Antibiotics 1. 2. 3. 4. 5. 6. Diameter of inhibition zone (mm)

1.

(D). Making the conclusion. Draw a conclusion about the genus and species the isolated bacterium belongs to, according to the results of its complex biological examination. Choose the agent, to which the bacterium is mostly sensitive. Write your conclusion in the notebook (see Table 6-1). PART 2. Principles of Cultivation of the Viruses Your instructor will demonstrate you the technique of inoculation of viruscontaining material into the allantoic cavity of embryonated chicken egg.

1.

PART 3. Principles of Bacterial Genetics 1. Study the results of experiments of gene transfer between bacteria by transformation, transduction and conjugation (observe the demonstration plates). PRACTICAL TASKS Perform the final steps of bacteriological examination and register the results.

1. 2.

Fill in your protocols (Tables from lessons 6-9) with the results of bacteriological examination.
3.

Familiarize yourself with the scheme of the chicken embryo structure and different methods of its inoculation listed on the diagram in your classroom.

4. Get acquainted with the procedure of chicken embryo inoculation.

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Draw in your protocol notebook the schemes of experiments on gene transfer between bacteria and the bacterial growth as a result of recombination processes (see Fig. 12).
4.

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Review. Questions for discussion on topic "Principles of bacterial genetics": 1. What do the terms "genotype" and "phenotype" mean? 2. How do bacterial plasmids differ from the chromosome? 3. What functions are performed by different regions of the bacterial chromosome and plasmids? 4. What are the "transposable genetic elements" ("insertion sequences" and "transposons")? 5. What is the significance of genetic transfer in bacteria? 6. How do the properties of the donor cell, the mechanism of transfer and the fate of the transferred DNA within the recipient cell, differ in transformation, transduction and conjugation? 7. What are the components of the Polymerase Chain Reaction (PCR)? 8. What are the advantages PCR compared to the routine bacteriological method? 9. What are the practical tasks of Genetic Engineering (Recombinant DNA Technology)? LESSON 10

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FINAL LESSON ON SECTION 2: PHYSIOLOGY and GENETICS of MICROBES (written TEST on the SECTION and ORAL TEST). Topics for discussion. PHYSIOLOGY of MICROORGANISMS 1. Types of bacterial metabolism. Bacterial nutritional requirements. Mechanisms of transport and movement of materials across bacterial membranes. 2. Types of respiration of bacteria (energy metabolism). 3. Enzymes of bacteria. Classification. Differential diagnostic media in identification of bacteria. 4. Bacterial growth. Generation time. The dynamics of bacterial growth in a liquid culture media. 5. Principles of cultivation of bacteria. Principles and methods of cultivation of anaerobes. 6. Microbiological culture media. Specific requirements. Classification of media by composition and consistence. Application of media based on bacterial metabolism. 7. Pure culture of bacteria. Mechanical dissociation of microbial cells and biological bacterial attributes. Inoculation techniques for bacterial pure culture isolation. 8. Bacteriological examination. Identification of bacteria. GENERAL VIROLOGY 1. Viruses. Distinguishing characteristics. 2. Classification of viruses. Viral structure. 3. Types of viral replication. Stages of viral infection. 4. Principles of cultivation of the viruses. Isolation of the viruses in animals, embryonated eggs and in tissue cultures. 5. Bacteriophages. Structure. Reproduction. 6. Virulent and temporal bacteriophages. Lysogeny. 7. Bacteriophages. Application in microbiology, medicine and biotechnology. BACTERIAL GENETICS 1. Bacterial genome. Genotype and phenotype. 2. Mutations and modifications in genome (genetic alteration). 3. Recombination of bacteria. 4. Mechanisms of transfer of the genetic information in bacteria (transformation, transduction and conjugation). 5. Plasmids. Properties and functions of plasmids. Types of bacterial plasmids. 6. Plasmid profile of bacteria. Restriction analysis. 7. Transposable genetic elements: insertion sequences and transposons. 8. Gene amplification. Polymerase chain reaction (PCR). Using for identification of microbes. 9. Genetic engineering and biotechnology - the aims and application. Progress and current research. MICROBES in the ENVIRONMENT

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Human normal flora. Composition and functions. Normal microflora of human organism. Intestinal microflora. Eubiosis and dysbiosis. Dysbacteriosis, reasons for its development. Eubiotics: drugs for restoration of normal microflora. 4. Microbial ecology. Microflora of air and water. Methods of analysis. 5. Microflora of water. Evaluating water quality: the total number of microbes, coli-titer and coli-index. 6. Microflora of air. Examining of air contamination. Quality characteristics. Methods of examination: sedimentation and aspiration methods. 7. Control of microorganisms. Aseptic technique. Sanitizaion. Disinfection. Sterilization. 8. Sterilization (methods and equipment). ANTIBIOTICS and CHEMOTHERAPY 1. Antibiotics and chemotherapeutic agents. Antimicrobial effects: microbicidal and microbistatic effects. 2. Antibacterial agents. History of discovery. Classification according to chemical structure, spectrum and mechanism of action. Sources of antibiotic production and ways of manufacturing. 3. Antibiotics. Side effects. Choosing the best chemotherapeutic agent. 4. Antibiotic resistance: natural and acquired. Cross resistance. 5. Methods of determination of bacterial sensitivity to antibiotics: the diffusion methods and MIC-test. Antibiogram. Interpretation.
1. 2. 3.

WRITTEN TEST for Review on the SECTION: PHYSIOLOGY of the MICROBES Task . Choose the appropriate answer: A if only 1, 2 and 3 are correct B if only 1 and 3 are correct C if only 2 and 4 are correct D if only 4 is correct E if all are correct 1. Which of the following microorganisms can destroy hydrogen peroxide and superoxide? 1. Facultative anaerobes 2. Capnophiles 3. Aerobes 4.Strict anaerobes 2. The proper conditions for culturing of bacteria in laboratory are which of the following: 1. Nutrient medium 2. Optimum temperature 3. Suitable atmosphere 4. Time of exposure
3. The very important characteristics of

cultural media are which of the following? They should 1. have appropriate pH 2. be isotonic 3. be sterile 4. contain essential nutrients 4. Point out the methods to measure microbial concentrations in tested samples: 1. Spread technique

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2. Pour plates technique 3. Streak plates technique 4. Serial dilution method 5. Cultural properties of bacteria are which of the following: 1.Morphology of the colony 2. Shape and size of bacterial cells 3. Color of the colony 4.Staining characteristics of bacterial cells 6. Bacteriological examination consists of which of the following steps: 1. Inoculation of the specimen 2. Isolation of pure culture 3. Identification of pure culture 4. Antibiotic susceptibility test 7. The main role in human colonization resistance play which of the following bacteria: 1. Staphylococcus sp., 2. Bifidobacterium sp. 3. Candida 4. Lactobacillus sp. 8. Point out differential nutrient media: 1. Hiss media 2. Sugar broth 3. Endo agar 4. MPA 9. All of the following are the passive processes of material movement across membranes, EXCEPT: 1. Simple diffusion 2. Group translocation 3. Facilitated diffusion 4. Transport against concentration gradient 10. Which of the following are the favorable conditions for successful isolation of strict anaerobes? Use of: 1. oxygen-free anaerobic culture media 2. oxygen-free transport system 3. anaerobic jar 4. Endo agar

11. The normal balance of quality and quantity of the microbial species in human microbial flora is reffered to as which of the following? 1. Dysbiosis 2. Colonization resistance 3. Selective decontamination 4. Eubiosis 12. Which of the following microorganisms are common in normal microbial flora of human skin? 1. Propionobacteria 2. Escherichia coli 3. Staphylococci 4. Herpesvirusis 13. Drugs used to restore the normal flora are reffered to as: 1. Antibiotics 2. Probiotics 3. Antiseptics 4. Eubiotics 14. Which of the following drugs can be used to restore the disturbed balance of normal flora? 1. Penicillin 2. Bifidumbacterin 3. Bacitracin 4. Bificol 15. Eubitics can be administered for: 1. selective decontamination 2. chemotherapy 3. identification of eubacteria 4. treatment of dysbacteriosis 16. The members of the human intestinal normal flora are which of the following: 1. Bifidobacterium sp. 2. Escherichia coli 3. Lactobacillus sp. 4. Mycobacterium sp. 17. Physical methods used for sterilization of infectious material are which of the following: 1. Autoclaving

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2. Pasteurization 3. Tyndalization 4. Boiling 18. Antimicrobial agents that prevent the synthesis of peptidoglycan are which of the following: 1. Tetracycline 2. Penicillin 3. Amphotericin B 4. Vancomycin 19. All of the following drugs are antibacterial preparations, EXCEPT: 1. Azoles 2. Polymyxins 3. Tetracyclines 4. Polyenes 20. Which of the following methods can be used to determine the pathogens sensitivity to antibiotics? 1. The disk diffusion method 2. Phagotyping 3. MIC-test 4. Sedimentation method 21. The possible mechanisms of acquired antibiotic resistance of pathogen are all of the following, EXCEPT: 1. Mutations in the chromosome 2. -lactamase production 3. R-factor plasmids 4. Catalase production 22. The broad-spectrum antibiotics are which of the following: 1. Cephalosporins 2. Polyenes 3. Floxquinolones 4.Polymyxins 23. Physical methods used to control microbes are all of the following, EXCEPT: 1. Radiation 2. Filtration 3. Moist heat 4. Chemotherapy

23.

The natural antibiotics are which of the following: 1. Sulfa drugs 2. -lactam antibiotics 3. Quinolones 4. Polymyxins Nystatin belongs to which of the following group of drugs: 1. -lactam antibiotics 2. Polymyxins 3. Aminoglycosides 4. Polyenes

24.

26. Natural antibiotic resistance of a pathogen depends on which of the following? 1. Production of penicillinases 2. Presence of R- plasmids 3. Environmental conditions 4. Lacking the target 27. Antibacterial agents that can inhibit the bacterial protein synthesis are which of the following: 1. Tetracycline 2. Penicillin 3. Erythromycin 4. Trimethoprin 28. Microbicidal agents can: 1. stimulate the endospore production 2. produce osmotically fragile bacterial cells 3. inhibit cell division 4. kill most of pathogens 29. All of the following properties are characteristics of plasmids, EXCEPT: Plasmids are 1. small circular molecules of DNA 2. temperate bacteriophages in bacterial chromosome 3. extrachromosomal circles of DNA 4. fragment of cytoplasmic membrane 30. Transformation is the process of: 1. gene transfer between bacteria

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2. formation of L-forms of bacteria 3. integration of donors DNA into the recipients cell 4 synthesis of pigments. 31. All of the following statements characterize bacterial genome, EXCEPT: 1. It consists of bacterial chromosome and plasmids 2. It has haploid gene composition 3. It is readily transferred from one bacterial cell to another 4. It possesses DNA-associated histones 32. Lysogeny is characterized by which of the following: 1. integration of temperate bacteriophage into bacterial genome 2. lysis of bacterial cell 3. formation of prophage 4 synthesis of new progeny of viruses 33. Point out the properties of plasmids. They:

1. can integrate into the bacterial chromosome 2. carry gene, that are not essential to bacteria 3. can encode the resistance to antibiotics 4. can replicate independently of bacterial chromosome 34. Polymerase chain reaction is usually used to: 1. transfer genes between bacteria 2. generate the microbial nucleic acid samples 3. determine the biochemical properties of bacteria 4. identify the microbes without cultivation 35. The conjugation is characterized by which of the following: 1. virus-mediated gene transfer 2. presence of at least one sex pilus 3. integration of viral DNA into the bacterial chromosome 4. presence of F-plasmid

Match the nutrient medium with its characteristic feature: 36. Egg yolk-salt agar A. General-purpose medium 37. Kligler iron agar B. Enriched medium 38. Endo agar C. Selective medium 39. Sugar broth D. Enrichment medium 40. MPB E. Differential medium 41. 10% bile broth Match the characteristics with the bacteria respiration type: 42. Require the presence of small oxygen concentration in A. Strict anaerobes atmosphere B. Microaerophiles 43. May require the presence of CO2 in atmosphere C. Both A and B 44. Can be reproduced only in oxygen-free atmosphere D. Neither A no B 45. May detoxify superoxide 46. Must be cultivated in the anaerobic jar Match each of the following processes of material movement across membranes with its characteristic: 47. It is a passive process A. Active transport 48. Transported molecule is modified B. Group translocation 49. Permease helps to move the molecule C. Both A and B

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50.

Bacterial cell needs to expend energy

D.

Neither A nor B

Match each of the following characteristics with nutrient medium: 51. It is used only for the storage of specimens A. Thioglycollate medium 52. It is used for determination of sugarlytic Endo agar activity of bacteria B. Sugar broth 53. It is used for the isolation of strict C. 1% peptone water anaerobes E. Isotonic solution (as a transport medium) Match each of the term listed below with its definition: 54. Transformation A. Virus-mediated gene transfer between bacteria 55. Transduction B. Incorporation of donors supercoiled DNA into the recipients 56. Conjugation cell C. Attachment of F+ donor bacterial cell to F- recipient cell to transfer genetic material D. The acquisition of new properties following lysogeny Match the antimicrobial mechanisms listed below with the appropriate antibiotic group: 57. Inhibit the synthesis of peptidoglycan A. Tetracyclines 58. Inhibit the cytoplasmic membrane function B. Quinolones 59. Inhibit the bacterial protein synthesis C. Sulfa drugs 60. Inhibit the DNA gyrase D. Penicillins E. Polymyxins Match the statements listed below with the appropriate antibiotic susceptibility test: 61. The test in which antibiotic-impregnated disks are A. Disk diffusion method used B. MIC-test 62. The test in which antibiotic is diluted in nutrient C. Both A and B broth D. Neither A no B 68. The test in which the lowest inhibitory concentration of antibiotic can be determined REFERENCES 1. Handbook on Microbiology. (Ed. by: Yu.S.Krivoshein). //Mir Publishers.-Moscow.-1986. 2. High-Yield Immunology.(Arthur G. Johnson). //Lippincott Willams & Wilkins.-1999. 3. High-Yield Microbiology and Infectious Diseases.(Louise B. Hawley.) //Lippincott Willams & Wilkins.-2000. 4. Jawetz, Melnick and Adelbergs Medical Microbiology.(G.F.Brooks, J.S.Butel, L.N.Ornston.21th ed.) //Appleton & Lange.-1998. 5. Larry McKane. Microbiology: Essentials and Applications. (L.McKane, J.Kandel. -2nd ed.) //McGraw Hill, Inc.-1996. 6. Manual of BBL Products & Laboratory Procedures. //Becton Dickinson Microbiology System.1988. 7. Mechanisms of Microbial Disease (Ed. by: M.Schaechter, G.Medoff, B.I.Eisenstein. - 2nd ed.) //Wiliams & Wilkins- 1993. 8. Medical Microbiology & Immunology. Examination and Board Review.(W.Levinson, E.Jawetz.- 5th ed.) //Appleton & Lange.-1998. 9. Medical Microbiology. (2nd ed.; Edited by: C.Mims, J.Playfair, I.Roitt, R.Williams, D.Wakelin). //Mosby Int. LTD, 1998.

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Microbiology. (Kingsbury D.T., Wagner G.E. -2nd ed.) //Wiliams & Wilkins.-1990. 11. Microbiology and Infection. A clinically-oriented core text with the self-assessment. (T.J.J.Inglis). //Churchill Livingstone.-1998. 12. Oxford Textbook of Medicine. (3d ed.; Edited by: D.J.Weatherall, J.G.G.Ledingham, & D.A.Warrell). //Oxford University Press.-1996.
10.

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