Microbiology Final Exam
CH. 6 MICROBIAL GROWTH
Temperature Requirements & Bacteria Growth
1. Minimum growth temperature: lowest temp. at which sps. will grow; slow metabolism
a. If below this temp. the transport processes become too slow to support cell’s metabolic activity for
sustaining growth  cell dies
2. Optimum growth temperature: temperature at which sps. grow best/fastest
a. All functions/processes in the cell are synchronized
3. Maximum growth temperature: highest temp. at which sps. will grow; slow growth
a. If above this temp. processes fall apart; enzymes denatured, metabolic activity stops  cell dies
Temperature affects lipids and proteins
 Lipids
o High: membrane lipids become too fluid and fail to hold in cellular contents
o Low: membrane lipids become rigid and fragile
 Proteins
o High: H2 bonds break and protein is denatured losing its function; often irreversible
o Low: new H2 bonds are formed and protein stability and structure functions are altered
Mesophiles
 Majority of human pathogens belong to this group
  moderate temperature; optimum temp. is variable, between 20-40°C
 Optimum temp. for growth of most pathogens is close to host, 37°C (98.6°F)
o Clinical incubators for human pathogens are set at 37°C to mimic internal body temp.
 Temperature & Disease
o Mycobacterium leprae: Leprosy (Hanson’s Disease); colonize and cause lesions in cooler areas of
the body  extremities, facial area
 M. leprae is cultured in armadillos foot pads which have a lower body temp than humans
o Treponema sps: Syphilis; colonize and cause lesions in cooler areas of the body  genital area, lips,
throat
 Treatment: deliberate infection of malaria via mosquito  to increase body temperature
 Treponema sps is cultured in rabbit testicles
 Thermoduric Mesophiles (subcategory): can survive very brief periods of high temperatures during
inadequate pasteurization or canning
pH & BACTERIAL GROWTH  cells maintain a constant internal pH (close neutral); 3 groups based on pH factor
1. Neutrophiles: external pH is 6.5-7.5; the majority of pathogenic bacteria
2. Acidophiles: external pH is <5; preferred by many fungi + high salts + sugar
a. Thiobacillus ferroxidans: obligate acidophile; grow between 0-2 pH; oxidize sulfur compounds for
energy
i. Produce energy byproduct + sulfuric acid end product which disassociates into SO4- and H+
 H+ is pumped across the plasma membrane and the pH drops
 Creates its own acidic environment
3. Alkalinophiles: optimum external pH is >8.5
a. i.e. Vibrio cholerae  found in alkaline soils and lakes
Acidic Areas in the Human Body
 Stomach: colonized by acidic tolerant microbes
o Helicobacter pylori: adapted to survive acidic pH by secreting urease and bicarbonate ion; urease
breaks down urea into carbon dioxide and ammonia which neutralizes the immediate surrounding
environment
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Microbiology Final Exam
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Urogenital area of adult female: colonized by fermenting microbes
o Normal microbiota
o Abnormal microbiota
 Dominated by Gardnerella vaginalis and other anaerobic sps: cause bacterial vaginosis
 Bacterial Vaginosis  clinically associate with…
 Poor pregnancy outcome
 Pelvic inflammatory disease
 Post-operative wound infection
 Endomitosis following elective abortion
 Vaginal discharge and increased risk of HIV and STDs
NOTE: in a healthy urogenital system, Lactobacillus acidophilus metabolizes glycogen and breaks
it down to lactic acid which lowers pH
 Low pH of vaginal epithelium prevents bacteria and yeast from establishing
 This is an example of protection of our normal microflora
 Candida sps causes vaginal yeast infection and is an opportunistic fungi present in
the urogenital area
 Normal microflora suppresses its overgrowth; however, when a broadspectrum antibiotic is introduced it eliminates the normal microflora which
leads to Candida sps outgrowth  vaginal yeast infection
OSMOTIC PRESSURE & BACTERIAL GROWTH
 Osmosis: net movement of solvent (water) molecules across a selectively permeable membrane
o Movement if from area of high concentration to an area of low concentration
 To the “dryer” side of the membrane
o Difference in water concentration is required on each side of the membrane
 Osmotic Pressure: pressure exerted on membrane by solutes (chemicals) in solution
o Isotonic Solution: concentration of solute (and H2O) outside and within the cell is the same
o Hypertonic Solution: concentration of solute outside the cell is higher (so there is less water
outside); water inside the cell will move out to the area of lower water concentration
 Plasmolysis: when the plasma membrane pulls away from the cell wall
o Hypotonic Solution: concentration of solute outside the cell is lower (so there is more water
outside); water moves into the cell to the area of lower water concentration
 Osmotic lysis: when the cell bursts
Staphylococcus aureus: opportunist pathogen with high salt tolerance; can tolerate up to 20% salt
 Causes pimples, sties, boils, would infection
 Secretes superantigen TSST  life threatening
CHEMICAL FACTORS
Water
 Reactant in several chemical reactions
 Enzymes and nutrients are dissolved in water
 Dehydrated structures (spores and cysts) can e inhaled and freely dispersed
 Mycobacterium sps cell wall (waxy, lipid, mycolic acid) retains water
o Enables cells to survive dry conditions for an extended period
Trace Elements (micronutrients: Cu, Zn, Mb, Se)
 Required in small amount; acts as cofactor for enzymes
o Enhances chemical reaction
 Sterile tap water is preferred over distilled
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o Contains sufficient amount of ions and satisfies nutrient requirement
Glass is preferred container
Nitrogen
 Required for amino acid synthesis
o NH2 group of amino acids, nitrogenous bases
Phosphorous
 Crucial for needed proteins, nucleic acids, phospholipids, ATP synthesis
Sulfur
 For synthesis of sulfur-containing amino acids (binds to S-S bonds)
 Sulfur-containing vitamins: thiamine, biotin
Other Elements (Ca, Mg, Mn, Fe)
 Iron: sought after element because associated with energy harvesting process  critical role in ATP
synthesis
o In humans, iron is always bound to a protein (in blood, bodily fluids, liver) which limits its
availability to bacteria
 No free iron in the body  limits our susceptibility to iron scavenging bacteria
o Transferrin binds to iron during transport from liver (storage organ) to bone marrow (where RBC
formation occurs)
o Ferritin: excess iron stored in the liver is complexed as Fe-Ferritin complex
o Lactoferritin: has an extremely high affinity for iron and binds to it at very low concentrations
 Present in saliva, mucous, milk
o Siderophore: high affinity iron-binding protein secreted by some bacteria (Enterobactin and N.
gonorrheae which can steal iron from the body)
 Two fates…
 Iron is made free first and subsequently enters bacterial cell
 Can enter as such, binds to receptor on cell surface and brought inside bacterial cell
Growth Factors (aka organic growth factor)
 Low molecular weight organic compounds that must be supplemented in a medium since bacteria can’t
make it; these are in addition to carbon and energy sources
 Includes: some amino acids, essential vitamins (thiamine, riboflavin), purines, pyrimidines, cholesterol (in
Mycoplasma sps only), heme (which has iron
component required for synthesis of
cytochromes), PABA (synthesis of folic acid)
Carbon Source
 Inorganic carbon (CO2) Autotroph
 Organic compounds  Heterotroph
Energy Source - used for building blocks, motility,
active transport against concentration gradient
 Sunlight  Phototroph
 Metabolism of chemical compounds (lipids,
fatty acids, amino acids, sugar molecules)  Chemotroph
Majority of pathogens are Chemoheterotrophs
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Microbiology Final Exam
Toxic Forms of Oxygen
 Singlet O2
 Superoxide radicals
o Highly reactive, toxic, metabolic product found in cytoplasm
o Neutralized by superoxide dismutase (SOD)
 Hydrogen peroxide and peroxide anion
o Active components of many antimicrobial agents
o Toxic peroxide (generated during normal metabolism); neutralized by…
 Catalase: breaks down hydrogen peroxide to water and oxygen
 Peroxidase: breaks down hydrogen peroxide to water
 Vitamin C and E – antioxidants that provide electrons that reduce toxic forms of oxygen
Oxygen Requirement and Level of Enzymes
 Aerobic: more efficient and more energy  respiration generates maximal amount of ATP; often results in
better, more rigorous growth of bacteria
o Obligate aerobes: SOD and catalase: abundant
The level of enzymes that
 Pseudomonas aeruginosa
each bacterium possesses
o Microaerophiles: SOD and catalase: limited  so limited ability
determines how they will fare
to detoxify
in the presence of oxygen
 Helicobacter pylori (tolerant to 2-10% O2)
o Facultative anaerobes: SOD and catalase: moderate
 Flexible in O2 requirement
 E.coli  more growth in the presence of O2 ; less growth in absence
 Anaerobic/Fermentation: less efficient and less energy  generates less ATP; less growth in bacteria;
metabolic efficiency in compromised in the absence of O2
o Obligate anaerobes: SOD and catalase: absent; bacteria intolerant to O2 (it’s lethal)
 Clostridium and Bacteroides sps
o Aerotolerant (aka obligate fermenters): SOD and catalase: limited
 Don’t use respiratory pathway
 Don’t use oxygen for energy processing and presence of oxygen doesn’t bother them
 S. pyogenes and Lactobacillus sps
Shake Tube Method
 Used to determine oxygen requirement for new
or unknown bacteria
Clostridium perfringens and Gas gangrene
 Anaerobic bacteria that can grow just below
dead tissue because an anaerobic environment
has developed; bacterial fermentation
generates gas which causes swelling
Bacteroides sps
 Present in intestines; slime layer
 B. fragilis and B. theyaiotamicon
o Fermentation of plant sugar derivatives = main energy source
 Significance – 2 imprtant roles; like a function organ
o Break down potential toxins in plant food
 Their fermentation products yield ~15% of caloric value of food
o Remove side chains of bile acids sand return bile to hepatic circulation
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Microbiology Final Exam
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Can cause infection when they escape from the colon (from intestinal perforation or surgery) and invade
surrounding tissue where they form an abscess
o Abscess can also be caused by S. aureus
Media Classification
 Physical State
o Liquid (broth)
o Semisolid
o Solid (agar plate, slant)
 Slants are important because they provide greater surface area and the tube can be stored in
small (limited space area) unlike a petri plate
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Chemical Composition
Both media can be in
o Defined/simple medium: exact composition is known
liquid (nutrient broth)
 Has all required nutrients for target microbe(s)
or solidified form
 May contain vitamins or amino acids in a known quantity
(nutrient agar)
 Buffer – critical component – maintains pH
 ex. Simmons Citrate Medium
o Complex medium: exact composition is unknown; variation from batch to batch
 Nutrients are derived from soy, yeast or beef extract/partial digests
 Extract: provides vitamins and organic growth factors
 Peptones: amino acids provide buffering action
 Supports growth of diverse microbes who nutritional needs are challenging
 Ex. EMB and nutrient agar plates
o Agar: complex polysaccharide derived from marine algae
 No nutritive value; few microbes can degrade it
 Used at a 1.5-1.55% final concentration
 High concentration becomes toxic to cells
 Difficult for bacterial to move around agar
 Dissolves at 100°C and solidifies at 45°C (heat labile nutrients added later)
 Translucent making it easier to visualize colonies
Fastidious organism: N. gonorrhea  picky bacteria; requires 40
additional components, 7 vitamins, 20 amino acids to grow
Versatile organism: E.coli  if vitamin E is not added to the
medium it will still grow; it can produce its own vitamin E
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Functional/Special Types of Media
o General purpose media: used routinely in microbiology (ex. nutrient agar plate)
o Selective media: includes components that inhibit growth of unwanted microbes and encourages
growth of desired microbes
 Thayer Martin Media: used for N. gonorrhea isolation from a clinical samples; cocktail of 3
antimicrobial drugs that inhibit growth of all other microbes except N. gonorrhea
 Sabouraud’s Media: used for fungi isolation; low pH, high salt and sugar inhibit bacteria
o Differential Media: for easier detection and isolation of microbes of interest from others growing
on the same plate
 Often a particular trait of the microbe is exploited
 Ex. Blood agar medium and hemolysin
 RBC sources: sheep (most common), dog, mouse, etc.
 No human blood because could contain a possible pathogen which lab
worker would be exposed to
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Microbiology Final Exam
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Hemolysin: toxin secreted by some bacteria
Alpha hemolysis: incomplete breakdown of RBCs (partial oxidation of hemoglobin)
 S. pneumoniae (green colonies)
 Beta hemolysis: complete breakdown of RBCs
 S. pyogenes (clear halo around colonies)
 Gamma hemolysis: no effect on RBCs
 Enterococcus faecalis
Anaerobic Media aka reducing media
 Sodium thioglycolate: chemical added to medium which combines with dissolved oxygen
making it unavailable
 Gas Pak chamber: a packet of chemicals (sodium borohydride, sodium bicarbonate ad
pallidum catalyst) placed in a chamber
 The sodium’s combine releasing CO2 and H2; catalyst acts on H2 and O2 which
combine creating H2O, removing all O2 and creating an anaerobic environment
Transport Media: used in clinical settings to transport microbe from a patient to a lab
 Prevents individuals who transport it from getting infected
 Ensures specimen is not contaminated and cells kept alive
 Contains buffer: critical to keep cells healthy and maintain ratios of different microbes in
the specimen
 Contains absorbents to “soak up” metabolic waste generated by cells, so it doesn’t become
toxic
Enrichment Media: includes components that favor the isolation and growth of specific microbes
from low or undetectable level to detectable level
 Ex. V. cholerae can be isolated from a sample in a cold enrichment media because tolerant
to cooler temperatures
Generation Time (aka population doubling time)
 The time is takes for a bacterial culture to double in number
 E. coli has short (fast) generation time – 20 minutes
o In a culture, after every 20 minutes the population doubles (if there are no limiting factors)
 40 cells  after 20 minutes  80 cells  after 20 minutes  160 cells
o Rapid turnover rate of protein
 Mycobacterium sps (i.e. M. tuberculosis) has an extended (slow) generation time – from 24 to 48 hours
o Synthesis of mycolic acid takes a long time because it’s a multistep process
 so division of the cell is also slow
o Results in prolonged tx
 May need to take anti-TB drugs for 6 months to over 1 year
o Rifamycin – anti-TB cocktail/antibiotic
 Targets and binds to bacterial RNA polymerase and stops activity
 Bacteria multiplies at such a slow pace the protein turnover rate is slow
 Drug is very effective in slowing down bacterial growth
 Drug would not be effective in E. coli because it has a rapid turnover rate of protein
Special Culture Techniques
 Animal cell cultures
o M. leprae – cultivated in armadillo foot pad
o Treponema sps – cultivated in rabbit testicles
o Chlamydia and Rickettsias – cultivated in bird eggs and cell cultures; must multiple inside host;
cannot be grown on agar
 Obligate intracellular parasites
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Microbiology Final Exam
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Chlamydia causes sexual and non-sexual diseases; non-traumatic blindness in children
 Shed in swimming pool and gets in child’s eyes
 Rickettsias causes Rocky Mountain Spotted Fever
Low oxygen culture
o Use of CO2 incubators and candle jars
o N. gonorrhoeae – capnophile = CO2 
 Preferred growth conditions are CO2 O2
 Cornstarch is added to the medium to promote growth benefits because it binds to free
fatty acids which would otherwise inhibit gonorrhea growth
Relationships Amongst Organisms
 Antagonistic: harmful; virus “killing” host following replication
 Synergistic: beneficial; symbiotic relationship in which organisms live in close contact (physically or
nutritionally); individually interdependent
o ex. E. coli inhabitation of the intestines – receives food material and provides vitamin K
o Complex relationship (aka biofilm): cooperative; amplified benefits
 Biofilms are responsible for 65% of infections in humans
 Microbes of different species are attached to tooth surface, mucus membrane of
the intestine and other body parts
Biofilms
 Observed in…
o Households – under sink, toilet bowl
o Industry – accumulate in pipes, drains, cooling towers
o Nature – on solid surfaces like rocks (slipperiness of rocks)
o Health – prostatitis, kidney infection, otitis media, cystic fibrosis; infections associated with medical
device implantation (catheters, heart valves, contact lenses, IUD, prosthetic joints)
 Reservoir of pathogens for recurring infections
 Form on surfaces that are in contact with fluids
 Medical implantation devices and bodily fluids
 Develop slimy matrix allowing cells to attach solid surfaces
o Matrix: DNA, proteins, fibers of glycolyces
 Biofilm (with associated matrix) protects members from environmental stress (UV
radiation), pH alterations, temperature/humidity changes, antimicrobial drugs including
antibiotics (challenge for medical treatment)
 Often protection from immune attack
 Cooperative interactions because the different microbes provide a variety of beneficial features
o Allow species to grow in an area they wouldn’t be able to if alone
 The mouth has a heavy concentration of oxygen but anaerobic bacteria can grow there
because the aerobic bacteria use of the oxygen
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Microbiology Final Exam
Quorum Sensing
 Communication between members; cells respond to neighbor cells and secrete hormone-like molecules
o Acyl homoserine lactone diffuses across neighboring cell’s PM and activates transcription factors
 Microorganisms will develop new characteristics
 Plankton Showers
o When a large number of cells leave the biofilm and disseminate throughout body
 Have the potential to reform a biofilm community
 ex. cystic fibrosis patients get a plankton showers of Pseudomonas sps
CH 7. MICROBIAL GENETICS
DNA Features
 Genome: cell’s genetic information; mainly composed of DNA on all biological systems
o Exception to DNA: some viruses have RNA genome; called RNA viruses
 HIV, influenza, measles
 Chromosomes: thread-like structures
o Contain DNA and associated proteins  histones or non-histones
 Bacteria only have non-histones
 Gene: unit of genome; a segment of DNA that codes for a specific functional product  a protein with a
defined role
o mRNA is made from the gene and is used for protein synthesis
 Cell Division: 2 processes
o DNA replication: DNA is duplicated and distributed to daughter cells
o Gene expression: occurs following cell division; includes two interdependent processes
 Transcription: DNA s copied to RNA
 Translation: RNA is decoded by ribosomes into proteins
 Central dogma: linear transfer of genetic information from DNARNAProtein
o Universal in all biological systems
 Exception: retroviruses coded with enzyme reverse transcriptase which transcribes
RNADNARNAProtein
 Enzyme is a target for many antimicrobial drugs
 Retroviruses:
 Hepatitis B (DNA virus)
 HIV (RNA virus)
Form helix
backbone
Deoxynucleotides: linear polymer of DNA inside cells
 Three components
o Phosphate group
o Pentose sugar: deoxyribose (5 carbon sugar)
o Nitrogenous bases
 Purines: double ringed organic compound
 Adenine (A) and Guanine (G)
 Pyrimidines: single ringed organic compounds
 Thymine (T) and Cytosine (C)
 Uracil in RNA
 Bases pairing occurs in 2 complementary strands; purines paired with pyrimidines
 A-T, G-C (and A-U in RNA)
 The 2 helix strands are anti-parallel (opposite orientation)
o Held together by hydrogen bonds between bases
 2 bonds between A-T and 3 bonds between G-C
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It is not the number, but sequence of nucleotides t is important
When a cell divides, DNA is distributed equally between daughter cells
DNA is susceptible to denaturation (melting)
o Separation of the 2 strands due to heat (physical factor) or urea (chemical factor)
Triphosphate deoxyribonucleotides
o Building blocks and energy source in DNA synthesis
o dGTP, dCTP, dTTP, dATP
Bacterial Genetics
 Vertical (longitudinal) gene transfer: genes passed from 1 generation to the next
 Horizontal (lateral) gene transfer: genes transferred between members of the same generation
o A donor cell integrates its DNA into a recipient cell’s genome
o Only single strand region replaces homologous genes in recipient cell
o Uncommon but very powerful when it occurs
o Occurs in bacteria in several ways: transformation, conjugation, transduction
 No sexual reproduction in bacteria but they evolve via horizontal gene transfer
 Fred Griffith’s Experiment
o Studied 2 variants of Streptococcus pneumoniae
 Capsulated, pathogen strain “S” (smooth colonies on agar)
 Non-capsulated, nonpathogenic strain “R” (rough colonies on agar)
o He injected a mouse with heat-treated dead cells of strain S and live strain R (both harmless) and
the mouse died; he cultured Streptococcus from the dead mouse and found living capsulated
bacteria
 The nonpathogenic strain R had transformed into deadly strain S
 Strain R acquired the capability to produce capsules by assimilating the capsulecoding gene of strain S cells
 DNA is the transforming agent and carries genetic material
 1st experimental evidence proving DNA is the genetic material
Transformation  direct result of Griffith’s experiment
 DNA transfer is mediated by a competence factor which is synthesized making alterations to the cell wall
and PM so that DNA can be picked up from the environment
 Only possible when a recipient cell is competent  physiological state in cell cycle when it makes
competent factor
 Efficient when donor and recipient cell’s DNA is closely related (Gr+ to G+)
o No physical contact
 Artificial Transformation
o Electroporation: brief high voltage electric current momentarily opens pores in PM and DNA is
taken in
o Chemical treatment: cells exposed to cold CaCl2 followed by brief heat shock which opens pores in
PM d DNA is taken in from environment (test tube)
Transduction
 Transfer of DNA from one cell to another via replicating virus
 Bacteriophage attaches to bacterial host and injects its genome into the cell
 Phage enzymes degrade host DNA; phage control’s host functions directing synthesis of new phage DNA
 Sometimes as cell synthesizes new phages it mistakenly incorporates remaining fragments host DNA
forming transducing phages
 Generalized transduction: random DNA segment transferred
 Specialized transduction: specific sequences and DNA transferred
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Microbiology Final Exam
Conjugation
 Polar/unidirectional transfer`
 Donor cell (F+) attaches to a recipient cell (F-) with its sex pilus which may draw them together
 A single strand of the F plasmid DNA transfers to the recipient beginning with a section called the origin of
transfer
 The F- recipient then synthesizes a complementary strand of F plasmid DNA, becoming a F+ cell
High frequency of recombination generation
 F plasmid integrates into a chromosome by recombination
 The Hfr and F- cells attach via pilus
 DNA transfer begin at the origin of transfer of the F plasmid carrying a copy of the donor’s chromosome
 F- receives a portion of the F plasmid and F+ DNA and remains an FANTIBIOTICS
Antibiotics: antimicrobial agent produced naturally (in minute quantity) by some microbes which inhibit growth or
kill other microbes.
 Most are synthesized by soil fungi or soil bacteria – high competition for nutrients in soil
Drug: agent that affects physiology
Semisynthetic antibiotic: chemically altered; synthesized in a lab and a microbe
 Doxycycline, Methcillin, ampicillin, amoxycillin
 Advantages
o Longer lasting (extended half life)
o Easier to administer (often orally)
o More effective (against Gr- bacteria)
Synthetic Drug: antimicrobial agent completely synthesized in lab
 NOT antibiotic
 Isoniazid – used for TB treatment (Mycobacterium)  targets mycolic acid synthesis
 Sulfa drug
Ideal Properties of Antimicrobial Agents
 Selective Toxicity: must be more toxic to pathogen than host
o Exploit key aspects of microbe physiology that’s different from eukaryotes
 Structure (cell wall), metabolism, protein synthesis
o Therapeutic Index (aka chemotherapeutic index): lowest dose toxic to patient divided by
therapeutic dose
 Therapeutic dose: minimum dose (per kg body weight) that stops pathogen growth
 High TI: less toxic to patient; targets unique feature of bacteria
 Sulfa drugs – targets folic acid synthesis
 Penicillin – targets peptidoglycan cell wall
 Low TI: dose must be carefully monitored so as not to reach a toxic level
 Chloramphenicol – targets bacterial 50S larger ribosomal subunit
o Has serious side effects so given as a last choice
 Agent Should Be Bio-available
o Able to penetrate host tissue and reach microbial community
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o Must be effective at a low, nontoxic concentration to host
Antibiotic Action
o Bacteriostatic: slows bacterial growth – then body’s defense cells are able to eliminate
o Bactericidal (aka cidal): kill bacteria
Stability
o Should be stable in bodily fluids and exhibit therapeutic affect
 Needs to be absorbed efficiently
o Should NOT be degraded by stomach acids
 Some antibiotics are inactivated in gastric environment and should be given iv or im
Spectrum of Action – drug action on various pathogens
o Broad Spectrum: targets bacteria from both taxonomic groups – Gr+ and Gr Critical in many life threatening situations
 Disadvantage: disturbs normal microflora which are necessary for preventing pathogen
colonization by outcompeting them for nutrients and attachment sites
 Reduction of normal microflora opens the door for secondary infections or
superinfections (caused by transient pathogens)
o Narrow Spectrum: antimicrobial action limited to only a few pathogens
 Isoniazid affects Mycobacterium sps only
 Penicillin affects Gr+ bacteria only (vs. ampicillin)
Tissue Distribution, Metabolism and Excretion
o Antimicrobial agents differ with regards to these properties
o A bacterial infection of the eyes or CNS is a challenge to treat because the brain blood barrier is
highly selective and semipermeable restricting entry or antimicrobials
o Antibiotics that are quickly removed from circulation are a good choice for treatment of a bladder
or UTI but poor choice for a heart infection
o Some antibiotics are detoxified by the liver and excreted by the kidneys
o Half Life: time it takes to eliminate ½ the original agent dose
 Some antibiotics, like penicillin, have a short half life – easily degraded/rapid removal
 For liver and kidney problems, dose needs to be adjusted because associated with slow
metabolism and excretion of drug
Combinations of Antimicrobials
o Synergism: when 2 drugs are taken simultaneously, one enhances the antimicrobial effect of the
other
 Streptomycin + penicillin – inhibition of cell wall formation by penicillin allows for easier
entry into cell for Streptomycin which interferes with protein synthesis
 Trimethoprim + sulfamethoxazole (Septra/Bactrim)– for UTI and AIDS cocktail
o Antagonism: when 2 drugs given simultaneously, actions interfere with each other
 Tetracycline (bactericidal) + penicillin (bacteriostatic)
Stops bacterial growth/kills cells | Slows growth of living cells
o Additive: drug combination is neither synergetic nor antagonistic
Side Effects Should be Minimal
o Side effect: undesirable/adverse effect on host; can limit clinical usefulness of an agent; 3 main
categories
 Allergies
 Penicillin allergy – 300 deaths/year; associated with rash, fever, anaphylactic shock
 Violet/life threatening
 Toxicity
 Ciprofloxacin: targets gyrase enzyme of bacterial DNA replication complex; can
withstand chemical composition of urine (like sulfa drug)
 Chloramphenicol: inhibits peptidyl transferase of large ribosome subunit
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Polymyxin B: toxic to kidneys; never taken orally
Tetracycline: causes soft, discolored teeth that could fall out and affects strength
and shape of bones due to calcium malabsorption
 Streptomycin: binds to small ribosome subunit and stops protein synthesis
 Metronidazole: causes harmless black hairy tongue resulting from breakdown
products of RBCs deposited on the tongue; targets DNA synthesis
 Disruption of normal microflora:
 Long term use of a broad-spectrum antibiotic may reduce or eliminate normal
microflora leading to an overgrowth of Candida sps
 Yeast infection in the vagina (vaginosis) and mouth (thrush)
 Antibiotic associated pseudomembranous colitis caused by Clostridium difficile
(C. diff); severe intestinal disease
 When antibiotic eliminates normal microflora, C. diff colonizes intestines
and secretes toxins killing epithelial cells and causing pseudomembranes
(lesions) which are composed of dead intestinal epithelial cells, clotted
blood, and inflamed defense cells
 Associated with severe abdominal pain and bloody stools
Route of Administration
o External: topical/local application; directly on skin
o Internal
 Orally: simple, self-administered; no needle or provider help
 Disadvantage: patient may not follow prescribed time table
 TB treatment is over an extended period and some people stop too soon;
the WHO created DOT requiring healthcare workers to observe and record
treatment
 Intramuscularly: slow diffusion via blood vessels
 Disadvantage: concentration of drug in blood is not high
 Intravenously: via needle or catheter
 Disadvantage: high concentration is limited; rapidly diminishes as liver and kidneys
remove it from circulation
 Best option to bypass limited time is a continuous diffusion via antibiotic drip
Safety
o Prescribing an antibiotic often involves assessing the risk and benefits (TI)
o 2nd drug use may have toxic effect that was not there when the 1st drug was taken alone
o 1 drug may neutralize the intended effect of the other
 Some antibiotics dampen effectiveness of contraceptive pill
 hypersensitivity towards penicillin
o pregnant women can only receive antibiotic considered safe by the FDA
 no/minimal risk to developing fetus
 some antibiotics should never be given in the 1st and 2nd trimester
Drug Susceptibility vs. Sensitivity
o Microbe may be susceptible to drug action, but humans can develop sensitivity to the drug
 Ex. Penicillin Allery
Penicillin Allergy
 After penicillin is administered it is degraded by the liver into
 In individuals with an allergy, penicilloyl covalently binds to serum proteins leading to formation of
HAPTENS which induce an IgE reaction which facilitates a hypersensitivity
 Mast cells are activated and release a massive amount of histamines which trigger anaphylaxis
Page 12 of 19
Microbiology Final Exam
MECHANISM OF ANTIMICROBIAL DRUGS 7 & ANTIFUNGAL DRUGS– SEE EXCEL SPREADSHEET
PROTOZOA
 Microscopic, unicellular eukaryotes; all functions
integrated into 1 cell
 Majority are free living, found in aquatic
environments
o Require large amounts of moisture; water
constantly seeps inside cell
 Lowest form on food chain – eaten by fish and
plankton
o Exception: parasitic human pathogen
 Cytoplasm divided into
o Ectoplasm
o Endoplasm: confines membrane-bound organelles
 Vacuoles: membranous organelles
o Contractile: regulate fluid within cells; pump out water; avoid cell bursting
o Food: fuses with lysosomes inside cell; contain hydrolytic enzymes, break down food particles
 Lack rigid cell wall which normal prevents water entry
o May have pellicle (flexible outer covering)
 Nuclei
o Micro: required for sexual reproduction; contain normal chromosomes
o Macro: derived from micronuclei; helps in routine cellular functions; asexual reproduction
 Cilia and flagella present for motility
 Complex life cycle – more than 1 habitat/host
o Invertebrate host and non-invertebrate host
 Polymorphic
o Morphologically distinct stages during life cycle
o Polymorphic forms are either…
 Trophozoite: vegetative or feeding stage
 Cyst: resting form with dormant cytoplasm
 Means of survival and dispersal to new host
 Equivalent of endospores but NOT resistant to heat or harsh conditions
 When they encounter favorable conditions, they become trophozoite
 Several trophozoites out of a single cyst
 Cyst formation is triggered by:
 Limited nutrients
 Low temperature
 Low moisture
 Radiation
 Presence of toxic compounds
FUNGI
 Heterotrophs: obtain carbon, energy and nutrients from organic compounds
 Saprophytes: obtain energy/nutrients from dead, organic material
 Some are parasites
 Cell wall: chitin (strong and flexible)
 Most are aerobes; many yeasts are facultative anaerobes (flexible in oxygen requirement)
o Some are obligate anaerobes – in cattle or deer intestines
Page 13 of 19
Microbiology Final Exam
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Prefer to grow in environment that is: acidic, 5pH, high salt and sugar, slightly moist, relative humidity of
70%
Composed of individual filaments called hypha which form the filamentous body called mycelium
Hyphae are well adapted to absorb food because…
o High surface to volume ratio
o Release enzymes to breakdown food and act as a repellent against competition
o Vegetative hyphae: segment which obtains nutrients; remains underground
o Aerial hyphae: projects above ground; bears reproductive spores
o Specialized hyphae
 Haustoria: associated with parasitic fungi
 Grow on skin; penetrates tissue of host to withdraw nutrients
Two Classes of Hyphae (dependent upon presence of septa)
o Septa – cross walls that divide cells within hyphae
 Referred to as woromi bodies
o Septate hyphae: have microscopic openings; mobilize cellular resources right to the point of growth
o Coenocytic hyphae – not divided into cells by cross walls; cytoplasm is continuous; multinucleate
Have enzymatic lysosomes
o Digest damaged cells
o Help parasitic fungi to invade host
 Fungi growing on skin go deeper into body cells
Dimorphism
o Grow as yeast or as mycelia
o Human pathogenic fungi: thermally dimorphic
 Change with temperature difference inside vs. outside
 Inside host – yeast
Or vice versa
 Outside host – mycelia
Fungal Reproduction/Life Cycle
Page 14 of 19
Microbiology Final Exam
BACTERIAL CELL WALL
Peptidoglycan
 Macromolecule providing rigidity and mechanical stability due to its highly cross-linked structure
1. Backbone: linear polymer of repeating disaccharide units
 Units made of alternating NAG and NAM (modified glucose molecules) connected by
O-glycosidic linkage
 NAG: N-acetylglucosamine
 NAM: N-acetylmuramic acid (attachment site of D and L amino acids)
2. Tail: tetrapeptide chains composed of alternating D and L amino acids
 D and L amino acids: stereoisomers of amino acids
 L: common in environment
 D: rare
 Attaches to NAM sugar via a complex linkage
 Provides an extended, rigid peptide chain and minimizes tendency to form helices
3. Bridge (aka connecting chain/peptide interbridge): cross-link between parallel tetrapeptide side
chains that reinforces stability and 2/3-dimensional configuration
 Composed of either:
 Short chain of amino acids (indirect bridging)  Gr(+)
 Directly bonded amino acids (direct bridging)  Gr(-)
 Links 3rd amino acid of one chain (tail) to 4th amino acid of adjacent tetrapeptide chain
Backbone NAG-NAM-NAG-NAM-NAG-NAM
Page 15 of 19
Microbiology Final Exam
Tail

Indirect
Bridge
Direct
Bridge
PBP: Penicillin Binding Protein
o Binds to a specific protein
o Facilitates cross-bridging in both Gr(+) and Gr(-) cell walls
Gr(+) Cell Wall
 Stratified peptidoglycan permitting free passage of substances (sugar, ions amino acids)
 Little protein associated with cell wall
 Possess typical peptidoglycan structure (backbone, tail, bridge) with 4 unique features:
1. 3rd amino acid in the tetrapeptide tail is always L-lysine
2. Wide variation in bridge amino acid sequence
 Typically, about 5 amino acids (ex. Glycine)
3. Cross-bridge is a short chain (indirect)
4. Teichoic acids are found associated with cell wall
 Mycobacterium sps cell wall
o Includes mycolic acid which provides a waxy nature to cell wall
 Cannot stain using conventional gram stain
 Teichoic Acid: polymer of sugar/glycerol phosphate with amino acid  D-alanine
o Covalently bonded to NAM sugar where the tail attaches (anchoring base)
o Two Classes/Types
1. Lipoteichoic Acid: spans thick peptidoglycan layer & attached to plasma membrane lipids
2. Wall Teichoic Acid: confined to peptidoglycan layer and reinforces complete multilayered
peptidoglycan
o Functions of Teichoic Acids
a) Binds and regulates movement of positive charged ions into and out of cell
 TA has a negative charge
b) Furnish attachment site for many bacteriophages: viruses that have evolved to use TA as an
attachment site
 Bacteriophages: viruses that target/infect bacteria
c) Provides antigenic specificity
 Humans do NOT have TA so body’s defense cells recognize bacteria as non-cells
 PAMPs: pathogen associated molecular pattern
 Like a “barcode” for Gr(+) bacterial cells
 Examples: capsule components  poly-D-glutamic acid, D amino acids, TA
 PRR: pathogen recognition receptor  present on host cells
o Acts as a “barcode reader” and checks PAMPs
 Recognizes molecular patterns on cell surface
Gr(-) Cell Wall
 Does NOT contain teichoic acids
 Consists of:
o Periplasmic space containing periplasm
o Few (1-2) layers of peptidoglycan
o Outer membrane unique in Gr(-) cell wall
 Periplasm
o Fluid in periplasmic space between inner (plasma membrane) and outer membrane (which
surrounds the cell’s periplasmic space and contains the peptidoglycan within)
Page 16 of 19
Microbiology Final Exam
o
o
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No presence of phospholipids in periplasm (they are found in the outer membrane)
High concentration of:
a) Degradative proteins/hydrolytic enzymes
 Involved in nutrient acquisition/absorption by breaking down large nutrients into
smaller units
b) Transport/binding protein
 Specific role in transport of substances
c) Oligosaccharides (sugar molecules)
 Play role in osmoregulation
Peptidoglycan
o Thin layered (1-2) suspended in periplasmic space
o Linked to outer membrane via lipoproteins; stably anchored, not free floating
o Possess typical peptidoglycan structure (backbone, tail, bridge) with two changes:
1. The 3rd amino acid in the tetrapeptide tail is always diaminopimelic acid
 a modified L-lysine (NOT a true amino acid)
2. The cross bridge is directly bonded (vs indirect as in Gr(+))
Outer Membrane (aka Lipopolysaccharide layer)
o Surrounds periplasmic space which contains the peptidoglycan which in turn is linked to the OM
o Asymmetric bilayer composed of:
 Inner leaflet phospholipids
 Outer leaflet Liposaccharides
o Functions:
a) Provides Gr(-) bacteria extra protection from antimicrobial agents
b) Has ability to pump out antimicrobial preparations; Gr(-) bacteria are resistant to and
cannot be treated by antibiotics (which cannot cross the outer membrane)
c) Has porin molecules interspersed throughout
 Allows substances to pass through outer membrane to periplasm
 Cells express different porins
1. Dilute Environment: where nutrient concentrations are diluted
 Upregulates expression for porins of large size to maximize
nutrient uptake
2. Rich Environment: as inside a host
 Downregulates expression for porins of smaller size to select
smaller nutrients and avoid toxin uptake
o Lipopolysaccharide Molecule  outer leaflet
 Serves as molecular fingerprint during identification process
 Identification tool
 LPS  NOT a target for antibiotics, NOT used for attachment of bacterial cells and has NO
role in permeability of outer membrane (no transport/nutrient absorption)
 Made of two components:
1. Polysaccharide portion
 Polymeric sugars with O-glyosidic bonds
 Composition varies amongst Bacterium sps
 E. coli K12 strain: in humans intestines
 E. coli 0157:H7: in cattle intestines; secretes a shiga-like toxin
o H7: Flagella protein
 Play's role in resistance against complement activation and MAC lysis
 MAC: membrane attack complex (outcome of complement
activation)
o Inserted into microbe’s membrane
Page 17 of 19
Microbiology Final Exam


Complement proteins are part of innate immunity
Neisseria gonorrhaeae has serum resistanct strains that resist MAC
mediated lysis due to an extended Liposaccharide
2. Lipid portion
 Lipid A  Endotoxin
 Essential for insertion of polysaccharides into outer leaflet
 Released during bacterial cell lysis due to:
a) Phagocytosis
b) Treatment by antibiotics
c) Activation of complement system and MAC formation
 Plays important role during pathogenesis of many microbes
o Medical Significance of Lipid A: antibiotics may cause the release
of Lipid A in the bloodstream harming the patient by inducing
fever, inflammation, diarrhea, hemorrhaging, blood coagulation
and shock
 Shock: sudden drop in BP due to vasodilation leading to
increased permeability of blood vessels and fluid leakage
o Causes DIC (Disseminated Intravascular Coagulation)
 Due to clotting activation blocking smaller blood vessels
 Consequences of DIC:
a) Tissue necrosis due to lack of O2 and nutrient supply
b) Hemorrhage due to using up clotting factors
c) Vital organs functionality is severely compromised as
they demand a threshold of BP
 Endotoxin produces the same S/S to a different degree (irrespective of the
microbe)
 Same mode of action (irrespective of the microbe)
 Fever Induction & Role of Interluekin-1
1. Macrophage ingests Gr(-) bacterium
2. Bacterium degraded releasing endotoxins that induce macrophage
to produce IL-1
3. IL-1 released into bloodstream and travels to the hypothalamus
4. IL-1 induces hypothalamus to produce prostaglandins which reset
body’s thermostat to a higher temperature, producing fever
 Tylenol acts on COX enzyme to reduce fever and return body temperature
to normal setpoint
 Fever significance: forces individuals to rest/conserve energy + increased
temperature may slow bacterial growth; increased temperature enhances
cellular reactions of host defense cells in eliminating pathogens
 TNA alpha, IL-1, 16, 18 (pro-inflammatory cytokines)
o Promote inflammation and elimination of invading pathogens
 TLRs (toll-like receptors), a type of PRR (pathogen recognition receptor),
are activated by the presence of Gr (-)pathogens in the body
COX enzyme
(cyclooxygenase)
is activated by IL-1
arachidonic acid
derived from phospholipid
membrane of Gr(-) bacterium
prostaglandins
reset body’s thermostat
to a high set point
Page 18 of 19
Microbiology Final Exam
Cell Wall Destabilization & Antimicrobial Compounds
 When peptidoglycan wall structure is compromised it leads to cell burst
1. Penicillin
 Interferes with peptidoglycan synthesis when cells are growing
 Synthesis occurs inside bacterial cell during binary fission
 Interferes with cells ability to create protective, rigid wall
 Binds with PBP
 More effective in Gr(+) cells because Gr(-) have an outer membrane acting as a protective
barrier
2. Vancomycin
 Binds to 4th amino acid of tetrapeptide tail preventing/disabling cross-bridging
3. Lysozyme
 Present in tears, saliva and bodily fluids
 Breaks bond between NAG and NAM backbone units
 Provides natural defense (chemical barrier of innate defense)
NAG-NAM-NAG-NAM-NAG-NAM
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Protoplast: Gr(+) cells stripped of cell wall
Spheroplast: Gr(-) cells partially stripped of cell wall
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Gr(+) Cell Wall
Multilayered peptidoglycan
Indirect cross-bridge
3rd amino acid in tail is L-lysine
Wide variation in bridge amino acids
Little protein associated with wall
Contains teichoic acids
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Gr(-) Cell Wall
Thin (1-2 layers) peptidoglycan
Directly bonded cross-bridge
3rd amino acid in tail is diaminopimelic acid; a
modified L-lysine (not a true amino acid)
Wall linked to outer membrane via lipoproteins
Does NOT contain teichoic acids
Page 19 of 19