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STRUCTURE OF BACTERIA
1. Describe the major structural components of bacterial cells. Which are analogous to
those of eukaryotic cells and how do they differ?
Nuclear Structure and Prokaryotes Eukaryotes
Function
3. Describe the Gram stain procedure and its clinical significance; list the Gram-positive
genera.
A. Gram Stain Procedure
i. Crystal Violet--primary stain; positive stain
ii. Iodine--mordant/fixative; forms a complex w/ crystal violet in both +
and - bacteria
iii. Alcohol--decolorizer/destain; differentiates b/w + and -
i. Insufficient application--everything is purple
ii.Excess application--everything is pink
iv. Safranin--counter stain; positive stain
B. Differential, positive staining procedure
i. Gram positive cell wall’s extensive peptidoglycan layer RETAINS crystal
violet-iodine complex
ii. Gram negative’s thin peptidoglycan layer does NOT RETAIN crystal
violet-iodine post alcohol exposure
i. Destained cells can be stained w/ any stain but Safranin is
normally used
iii. Excluding acid-fast and Gram-positive bacteria, ALL OTHER
medically significant prokaryotic genera stain Gram-negative!!
C. Clinical significance--bacteria cannot be identified based on size, shape, cell
arrangement, or Gram Stain reaction alone. However, the results of a gram-stained
clinical specimen:
I. Often determines/influences initial antimicrobial therapy
II. Aids in identification of the etiologic agent
D. Gram-Positive Genera
*Actinomyces
*Bacillus
*Bifidobacterium
*Clostridium
*Corynebecaterium, Propionibacterium, and other diphtheria's
*Enterococcus
*Gardnerella
*Lactobacillus
*Listeria
*Mobiluncus
*Peptostreptococcus
*Staphylococcus
*Streptococcus
Types/Structure *Plain (extend out *Normal pili extend Mucoid like coat
from cell surface out from cell around the cell
into environment) surface into which are: cell
*Endoflagella of environment--types surface associated;
spirochetes I-IV slime layer; both-
(internal structure) *Sex pilus extends cell surface
out from cell associated and a
surface into slime layer
environment
Flagella Pili (Fimbriae) Capsules
1. List and describe those factors which may influence bacterial growth and their clinical
impact. Explain oxygen tolerance and intolerance in some bacteria and its clinical
significance.
a. Organic requirements
i. Order of organic molecules utilized
1. Carbs
2. Protein
3. Lipid
ii. Fastidious vs. Non-fastidious microbes
1. Fastidious--will NOT grow on blood agar--complex growth requirements
2. NON-fastidious--will grow on blood agar
b. Optimum growth temp/temp requirements
i. Optimal growth occurs closer to the MAXIMUM than the minimum temp
range
ii. Temp minimum is determined by REDUCED enzyme activity and
REDUCED membrane fluidity
iii. Temp maximum determed by protein DENATURATION
iv. Categories of bacteria in relation to temp:
1. Mesophiles--20-55* C
a. MOST PATHOGENS--35-36* BEST
2. Thermophiles-->55*
3. Psycrophiles or cryophiles--<20*
a. A few human pathogens are facultative psychrophiles
4. Refrigeration is NOT an acceptable method of maintaining/obtaining sterile
conditions
c. Optimum Oxygen/Oxygen requirements--broad range
i. Obligate aerobic organisms
1. ONLY grow in the presence of O2
ii. Microaerophilic organisms
1. Grow in the presence of REDUCED conc of O2
iii. Facultative anaerobic organisms
1. MANY PATHOGENS
2. Use aerobic respiration for growth when O2 is present and
fermentation when O2 is absent
a. Respiration is MUCH MORE EFFICIENT
b. Early in infection when number of bacteria are low, O2 is plentiful--
respiration/replication
i. As the infection progresses and NUMBERS OF BACTERIA
INCREASE, O2 levels drop --> use of fermentation for continued
growth
iv.Aerotolerant anaerobic organisms
1. Can grow in the presence of O2 BUT GROW BEST in the absence of O2
2. Utilizes ONLY fermentation
v. Obligate anaerobic organisms
1. Can ONLY grow in the absence of O2
2. ONLY utilizes fermentation
vi. O2’s effect on ANAEROBES:
1. All bacteria generate toxic agents in the presence of O2--O2- and H202
a. Can inhibit growth or cause death
2. Oxygen tolerant bacteria (aerobes) produce enzymes that detoxify these
toxic agents
a. If a bacteria is oxygen INTOLERANT (anaerobic) they lack the enzymes
to get rid of the toxic agents --> death of the cell
vii. Special requirements are needed to collect and culture anaerobes
from specimens b/c of death
1. Certain antibiotics are used for anaerobes
d. Inorganic requirements
i. Special ionic requiremens
1. NON-halophiles vs halophiles
ii. HIGH or LOW levels of an ion that is required for a bacteria can TRIGGER
changes in the bacterial phenotypes
1. Low iron --> diphtheria to produce diphtheria toxin
2. Low calcium --> plague --> endotoxins
3. Low magnesium --> s. Aureus --> TSST-1
e. Ph Optimum
i. Phyisiological
2. Define and explain heterotrophic and autotrophic metabolism, aerobic and anaerobic
respiration, and fermentation w/ regard to bacterial metabolism and growth.
a. Heterotrophic metabolism--catabolic paths that utilize organic carbon as sources
of E and carbon
i. All bacteria which cause disease in humans are heterotrophs
ii. Catabolism--enzyme mediated-oxidation of reduced carbon molecules
(glucose) to simpler oxidized carbon compounds and which MAY result in E
production
1. Glycolysis
2. May result in additional E if their end-products become substrates for:
a. TCA cycle--generates reducing power-->ATP and recycling NADH2
i. Completes oxidation of organic carbon into CO2
b. Fermentation path--may generate ATP via substrate level
phosphorylation
i. Sole E source for fermentation
iii. Forms:
1. Respiration
2. Fermentation
b. Autotrophic metabolism--fix CO2; source of building blocks used in synthesis of
cell constituents is CO2
i. Cellular E is obtained from EITHER:
1. Oxidation/reduction of inorganic ions (chemoautotroph)
2. Harvesting light energy (photoautotroph)
c. Respiration--UNIQUE enzymatic process b/c most of the process MUST
occur in a membrane vesicle or sack (enzyme-membrane complex_
i. Function: GENERATE E = ION CURRENT = PMF FOR ATP SYNTHESIS
1. Occurs during the conversion (recycling) of NADH2 to NAD+ (reduced to
oxidized forms)
ii. 2 parts:
1. ETC transfers e- and H+ from NADH2 to the terminal electron acceptor
(TEA) generating BOTH PMF AND REDUCED TEA
a. Oxidase test--determines the presence of an ETC component
(cytochrome C) in SOME bacteria
i. Enterobacteria are oxidase negative
ii. Other gram negative rods are oxidase POSITIVE
2. ATP syntheses used PMF to synthesize ATP from ADP and Pi
iii. Two forms of bacterial respiration; differ in compounds that serve as
TEA for the ETC
1. Aerobic respiration--oxidative phosphorylation
a. O2 serves as TEA which is REDUCED TO H2O
2. Anaerobic respiration
a. Inorganic compounds (nitrate, sulfate, etc) serve as TEA
i. MEDICAL SIGNIFICANCE:
1. MetHB occurs when elevated levels of NO3 occur in drinking
water
a. GI tract normal flora convert NO3 (TEA) to NO2 to MetHb
b. Especially a risk for unborn children
d. Fermentation--catabolic paths in which organic compounds serve as the
electron donors and electron acceptors.
i. Simpler than respiration
ii. Results in INCOMPLETE OXIDATION of carbon substrate
1. End products:
a. 1-, 2-, 3-, or 4-C compounds and some CO2
b. Excreted/released from the cell
iii. Uses substrates LESS efficiently but is efficient enough for fermentative
bacteria to GROW AND THRIVE
iv.Paths occur in the cytosol NOT a membrane vesicle
v. DOES NOT DIRECTLY PRODUCE a PMF/ion gradient
vi. MEDICAL SIGNIFICANCE OF MICROBIAL END PRODUCTS
1. Dental caries--lactic acid
2. Acidic pH of vagina/skin
a. Lactobacillus--lactic acid
b. P. Acnes--propionic acid, acetic acid, CO2
3. Abscesses; acidic and anaerobic
a. Many antibiotics are NOT EFFECTIVE AT LOW PH
1. Low pH kills surrounding viable human cells --> release of
compounds that bacteria REQUIRE FOR GROWTH
a. Sulfa drugs are not effective
4. ID bacteria based on the fermentative acidic end products they produce
5. Certain bacteria can ONLY grow fermentatively
a. Lack cytochrome/ETC systems OR cannot use it for E production
b. Do NOT use NAD+ or NADP+; FERREDOXIN IS USED
i. Must still be recycled from reduced to oxidized form
c. Infections by organisms, such as Clostridium perfringens (obligate
anaerobe), which can cause GAS GANGRENE; H2 is generated in
HUGE AMOUNTS
i. H2 gas is INSOLUBLE in tissue
ii. H2 gas tracks along fascial planes
1. Separates mm tissue
2. Collapses b.v --> impede perfusion --> creating anaerobic
conditions
e. Describe enzymes/products of bacterial fermentation/growth and their clinical
significance.
i. Aerotolerant anaerobes streptococcus and lactobacillus:
1. Produce lactic acid and H202
a. H202 from recycling of FERREDOXIN which is used as NAD+/NADP+
are
b. H202 is detoxified by human host produced peroxidase
i. W/out it they CANNOT grow in the presence of air
f. List the bacterial process associated w/ alkaline end-products, describe the
enzymes involved, the products produced and their clinical significance.
i. Proteus spa (bacterium) are responsible for some UTIs which promote the
formation of kidney stones
1. Release the extracellular enzyme urease
a. Hydrolyzes urea in urine --> NH4+ (ammonia) and CO2
b. Ammonia RAISES PH OF URINE which allows proteus to grow
2. Calcium and Ammonia ions form salts which may precipitate at ALKALINE
PH forming renal calcium composed of triphosphate
ii. H. Pylori--causes type b and duodenal ulcers
1. Produces urease-->increased microenvironment pH of stomach
mucous lining so H. Pylori can grow
3. List the steps in DNA replication, transcription, and translation noting the similarities
and differences b/w these processes in prokaryotes and eukaryotes.
a. DNA synthesis/Replication
i. Synthesis
1. REQUIRES DNA dependent DNA polymerase
2. DNA is the template
3. Base pairing b/w dATP/GTP/CTP/TTP is crucial
4. REQUIRES DNA helices--separated d.s. DNA into single strands
a. Generates TREMENDOUS TORSIONAL STRESS
5. REQUIRES PRIMASE--synthesizes shorts ssRNA primers
6. REQUIRES DNA GYRASE (topoisomerase II)--negatively supercoils -->
DECREASED torsional stress
7. REQUIRES TOPOISOMERASE IV--required for decaffeination
ii. Replication
1. Semi-conservative
2. Requires an ORI
3. Must be primed by DNA or RNA for both EUK/PRO
4. 5’-->3’ for both strands and EUK/PRO
5. Semi-discontinuous
a. Lagging strand only
b. Okazaki fragments
6. Bi-directional for both EUK/PRO
7. Timing
a. S-phase in eukaryotes; lag and exponential phase in prokaryotes
iii. RNA SYNTHESIS (TRANSCRIPTION)
1. REQUIRES DNA DEPENDENT RNA POLYMERASE
2. REQUIRES DNA HELICES--separates d.s. DNA into single strands
3. DNA is the template
4. Products:
a. mRNA
i. VERY SHORT HALF LIFE vs eukaryotes (sec/min vs h/days)
ii. Antimicrobial protein synthesis inhibitors are EFFECTIVE
b. tRNA
c. rRNA
5. Sometimes requires DNA gyrase
iv.PROTEIN SYNTHESIS (TRANSLATION)
1. OCCURS IN THE CYTOPLASM
2. Ribosomes are FREE--NOT membrane bound
3. Requires 70s ribosomes + accessory components
4. Uses charged-tRNAS (ammoniacal-tRNA syntheses)
5. mRNA is the template
a. Requires accurate codon-anticodon base pairing
6. rRNA catalyzes peptide bond formation
7. Product--polypeptide
8. Protein turnover (half life) is MUCH SHORTER in prokaryotes vs
eukaryotes so antibiotics that INHIBIT PROTEIN SYNTHEIS are
EFFECTIVE
4. List the steps in peptidoglycan synthesis.
a. Synthesis of amino sugars/peptidoglycan subunits
i. Uracil Diphosphate (UDP)--cytoplasmic/cytosolic tag which allows for
DIRECTED synthesis of these subunits
ii. NAG/NAM synthesis occurs while bound to UDP
b. Assembly and translocation of peptidoglycan subunits through the CM
i. The complete peptideglycan subunit (NAM---NAG) is ASSEMBLED,
BOUND TO BACTOPRENOL, and TRANSLOCATED ACROSS CM
1. Sequential transfer of components from UDP to bactoprenol w/
release of UMPs
c. Addition of subunit to GROWING END of peptidoglycan chain
(TRANSGLYCOSYLATION)
d. Final cross linking (TRANSPEPTIDATION) of peptide stems is done by
enzymes--transpeptidases or penicillin binding proteins (PBP)
i. 2 AA subunits are COVALENTLY BONDED
ii. Completes syntheis of fabril shell around the bacteria --> mechanical strength
and rigidity
5. Define bacterial growth, binary fission, and generation time.
a. Bacterial growth--Dependent on many factors:
i. Nutrient conditions
ii. Cultivation conditions
iii. Genotype that encodes for catabolic and anabolic paths
iv. The growth of a population of a PURE CULTURE of bacteria in a liquid
medium can be GRAPHICALLY ILLUSTRATED by plotting the log of the
number of bacteria produced vs the time required to grow them
1. Four phases:
a. LAG PHASE--cell volume and mass increase, chromosome/DNA
replication/synthesis begins, but NO CELL DIVISION TAKES PLACE
AND THERE IS NO CHANGE IN CELL NUMBERS
b. EXPONENTIAL/LOG PHASE--balanced growth occurs
i. Cell number, mass, volume, and cell component amounts
INCREASE by the same exponential factor
1. Logarithmic incrases in cell number
ii. Expressed as generation time
c. STATIONARY PHASE--NO NET INCREASE in cell numbers
d. DEATH PHASE--log DECREASES in cell number
i. Bacteria autolyse
b. Binary fission--
c. Generation time--time required for one bacterium to divide into 2 cells; time
required for a cell to replicate
d. How may the growth curve of pathogenic bacteria relate to acute and chronic
forms of disease?
i. Rapidly growing bacteria:
1. ACUTE disease
2. RAPID ONSET AND PROGRESSION (FULMINANT) DISEASE
3. HIGH Ag dose in host
4. STRONG IMMUNE RESPONSE
ii. Slow growing bacteria
1. CHRONIC diseases
2. SLOW onset and progression
3. LOW Ag dose
4. WEAK IMMUNE RESPONSE
iii. Bacteria with a SHORT GT often produce:
1. Rapidly progressive (fulminant) disease w/ high Ag dose in host
iv. LONG GT Bacteria often produce:
1. Chronic diseases w/ low Ag disease in the host
v. Selection of antibiotics
1. INHIBITORS of protein/peptidoglycan syntheis are MORE EFFECTIVE
against fast-growing bacteria
2. Organisms in STATIONARY PHASE also less sensitive to
antimicrobials
BASIC VIROLOGY