Chapter 2 - Microbial Growth and Nutrition

Transcription

1 MICRO 4354 Chap. 2 p 1 Chapter 2 - Microbial Growth and Nutrition I. Microbial nutrition A. Nutritional classification is based on sources of energy, electrons (hydrogen), and carbon 1. Energy a. chemotrophs use chemical energy b. phototrophs use light energy 2. Electrons a. organotrophs use organic molecules b. lithotrophs use inorganic molecules 3. Carbon a. autotrophs use CO 2 b. heterotrophs use organic molecules 4. There is no such thing as a universal medium B. Macronutrients 1. Nutrients needed in high concentrations for four major cellular polymers a. Proteins b. Lipids c. Nucleic acids d. Polysaccharides (carbohydrates) 2. Nutrients needed are carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur (CHONPS) 3. Heterotrophs are more common than autotrophs in industrial microbiology a. [substrate] g/l b. Sugars are very common substrates 4. Hydrogen and oxygen usually obtained from water a. O 2 gas needed for aerobic respiration b. O 2 gas needed for sterol synthesis 5. Nitrogen can make up 15% or more of cell dry weight a. inorganic - ammonium or N 2 b. organic - urea, amino acids 6. Phosphorus and sulfur from inorganic salts a. Phosphorus usually 100 mg/l b. Sulfur mg/l C. Main elements 1. Required at levels of a few milligrams per liter (10-20 mg/l) 2. Calcium iron, potassium, magnesium, sodium D. Trace elements 1. Cobalt, copper, manganese, molybdenum, nickel, selenium, zinc 2. Some organisms may have additional requirments (e.g., Wo) 3. Required at very low levels ( g/l) E. Nutrient uptake 1. membrane-bound transport mechanisms required for uptake of nutrients by microbial cell a. mechanism must be specific (no use to transport unusable compound) b. membrane is selectively permeable c. transport of dilute solutes against a gradient

2 MICRO 4354 Chap. 2 p 2 2. passive diffusion = molecules move from a region of higher concentration to one of lower concentration as a result of random thermal agitation a. rate is dependent on size of concentration gradient b. requires relatively high external concentration c. inefficient, not extensively employed d. gases and small, uncharged molecules 3. facilitated diffusion = diffusion process aided by a carrier a. permeases embedded in membrane function as carrier proteins b. more efficient than passive diffusion (1) more rapid at lower concentrations (2) saturatable (levels off, compared to linear increase with passive diffusion) (3) no energy requirement, still gradient driven (still diffusion) c. permeases may function by changing conformation, allowing buildup of internal concentration (1) allows lipid-insoluble molecules to enter cell (2) can work in reverse if internal concentration is high enough d. does not appear to be very important in procaryotes (used with glycerol); larger role in eucaryotes (for sugar and amino acids) 4. active transport = energy-linked transport of solute molecules a. works against a concentration gradient (1) works well even with low nutrient concentrations in the environment (2) can accumulate nutrients X greater concentration than external environment b. employs carrier proteins (1) high specificity (2) saturatable c. can use ATP, other high energy phosphate compounds, or proton motive force d. many compounds have multiple transport systems (may have selective advantage in different environments) e. typically used with sugars and amino acids f. ATP-binding cassette transporters (ABC transporters) = protein carriers that span the membrane and require energy (usually ATP) for activity (1) specific for molecule to be transported (2) located in the periplasm of gram negative cells or on the outside of the cell membrane of gram positive cells (3) probably involved in chemotaxis g. active transport can also be powered by a proton or sodium gradient + (1) symport = linked transport of two substances in the same direction (H - linked uptake of amino and organic acids) + + (2) antiport = linked transport of two substances in opposite directions (Na /H ) (3) occurs in G- and G+ 5. group translocation = molecule is transported and chemically altered at the same time a. phophenolpyruvate:sugar phosphotransferase system (PTS) best studied (1) transports sugars into procaryotes (2) phosphorylates sugar using phosphoenol pyruvate (PEP) as phosphate donor b. systems are fairly complex, with cytoplasmic and membrane enzymes involved c. present in facultative anaerobes and anaerobes (rare in aerobes, except for some Bacillus species)

3 MICRO 4354 Chap. 2 p 3 6. Polymers (high molecular weight compounds) are hydrolyzed by extracellular enzymes a. Extracelluar enzymes include proteases, amylases, cellulases, lipases, peroxidases, etc. b. Many industrial applications II. Microbial growth kinetics A. General growth 1. growth = increase in cell number or cell mass (usually refers to number in case of bacteria) 2. most bacteria reproduce by binary fission a. cell numbers increase by powers of 2 b. generation time = amount of time required for population to double (reproduce) c. doubling time = generation time 3. growth of filamentous bacteria or those that grow in aggregates are difficult to model B. Batch growth 1. closed system (finite amount of nutrients and accumulation of wastes) a. simple, familiar growth format b. provides data for growth curves c. growth is limited by physiology and medium 2. bacterial growth curve = plot of cell growth over time, usually in a batch culture or closed system a. lag phase = period of little or no cell division (1) "gearing up" phase (2) intense metabolic activity, particularly DNA and enzyme synthesis b. log or exponential phase = period of most rapid growth, where cells are in continual state of cell division (1) generation time reaches a constant minimum based on genetic potential and medium limitations (2) cells most active metabolically (3) microorganisms particularly sensitive to adverse conditions c. stationary phase = period of equilibrium between growth and death of cells (1) depletion of nutrients and accumulation of waste products (2) changes in ph d. death phase = period when more cells are dying than dividing (1) logarithmic decline phase (constant proportion dies with time) (2) population can diminish or become extinct 3. Growth is usually (virtually always) modeled during the exponential phase a. by biomass (X) b. by number (N) c. Growth rate ( ) is dependent on biomass (cell) concentration, generation time is not 4. cells are self-duplicating units, so growth is a first-order reaction a. rate of increase is proportional to number of cells (1) 1st order = reaction dependent on the concentration of one reactant (substrate) (2) zero order = reaction independent of reactant concentration (dependent on catalyst concentration) (3) mixed order = reaction occurs during transition from one phase to another b. dx/dt = X (1) X = cell concentration (2) t = time -1 (3) = specific growth rate (a constant of proportionality), units = reciprocal time (hr )

4 MICRO 4354 Chap. 2 p 4 5. Integration and log conversion linearize the growth equation (y = ax + b)(fig. 2.2 b and c) a. ln(x 2/X 1) = (t 2 - t 1) or X 2 = X. 1 e t b. log 10 X 2 - log 10 X 1 = (t 2 - t 1)/2.303 c. = slope = 2.303(log 10 X 2 - log 10 X 1)/(t 2 - t 1) 6. growth can also be considered in terms of multiplication n a. N 2/N 1 = 2 (1) or ln N 2/N 1 = n ln2 (2) n = number of generations to produce N 2 - N1 b. n ln2 = (t 2 - t 1) so = n ln2/(t 2 - t 1) (1) when n = 1, t 2 - t 1 is the generation time, g or td (2) = ln2/g = 0.301/g 7. Growth rate is related to nutrient concentration in hyperbolic manner analogous to Michaelis- Menton kinetics (developed by Monod). a. = ( max S)/(K s + S) (1) = specific growth rate at limiting nutrient concentration (S) (2) max = growth rate at saturating nutrient concentrations (3) K s = concentration of nutrient at a growth rate of ½ max b. when [S] >> K s, = max c. can be applied to batch or continuous culture 8. diauxy can occur if two subtrates are present and one is used preferentially C. Application and optimization of batch fermentations 1. nutrient concentrations decrease while products increase, so balanced growth never occurs 2. batch systems are easier to run than continuous systems and are much more common 3. down-time is a significant factor in economics with batch processes a. harvesting biomass and spent broth b. cleaning the vessel c. introducing fresh medium d. sterilization e. inoculation f. lag before product accumulation 4. the yield coefficent, Y, is a measurement of biomass or product that results per unit of substrate consumed (g or mole) a. X = Y X/S(S-Sr) where: X = biomass (usually g/l) Y X/S = yield coefficient (g biomass/g substrate consumed) S = initial substrate concentration S r = residual substrate remaining in the fermentation vessel b. Y p/s = yield coefficient for products (g or mole product/g or mole substrate consumed) c. yield coefficients are important because they indicate the efficiency of substrate conversion 5. determination of the maximum specific growth rate, max, is important because primary products are correlated strongly with exponential phase a. the Monod equation (above or 2.15) can be linearized b. double-reciprocal plots (think Lineweaver-Burk) can be used to determine the kinetic constants (1) 1/ = [(K s/ max)(1/s)] + 1/ max (2) other linearizations (e.g., Eadie-Hofstee) work as well D. Continuous growth kinetics

5 MICRO 4354 Chap. 2 p 5 1. open systems, more like natural systems than batch systems are a. nutrients are added, spent media (including cells) are removed b. initial stage is similar to batch culture as cells grow to maximum density c. eventually steady-state conditions are reached where the cell density and growth rate are constant d. bacteria maintained in exponential phase e. growth rate dependent on dilution rate (rate of introduction of fresh medium) f. cell density dependent on concentration of limiting nutrient g. if dilution rate is greater than the growth rate, cells are washed out 2. during steady-state growth, growth rate is equivalent to wash out a. production of cell mass: dn/dt = N b. rate of cell loss through overflow: dn/dt = f/v = DN where: f = flow rate V = culture volume f/v = dilution rate = D c. N = DN or = D when the culture is in steady state (growth = washout) d. the critical dilution rate is when D is greater than max 3. the Monod equation can be applied to continuous culture. a. D = ( max S r)/(k s + S r) (eqn. 2.24) where: K s = Monod constant (substrate concentration at ½ max S r = residual substrate concentration in bioreactor at steady state. b. so S r = (K s D)/( max - D) (eqn. 2.25) (1) states the fundamental relationship between substrate concentration (S r) and dilution rate (D) (2) since S r can be controlled by dilution rate, growth is controlled by a rate-limiting nutrient (3) chemostat = continuous cultivation system where growth is controlled by the rate of a limited nutrient entering the system so cell density is maintained by the concentration of the limiting substrate being consumed (4) turbidostat = continuous cultivation system where growth is controlled by monitoring the turbidity of the culture and adjusting dilution rate to keep cell density constant c. as D increases, increases and S r increases (Fig. 2.5) 4. the concentration of biomass or product during continuous cultivation can be related to the yield coefficient a. = Y x/s(s R - S r) = Y x/s(s R - [(DK s)/( max -D)] where: = biomass or product concentration S R = initial substrate concentration (concentration in input) b. again, during steady state growth, biomass is controlled by substrate feed concentration and dilution rate (1) in the absence of inhibition, cell density is controlled by substrate concentration in the feed (a) D constant (b) S r constant (2) as D increase, S r increases and biomass decreases III. Measuring growth A. Measurements can be direct or indirect, viable or total 1. direct counts cells

6 MICRO 4354 Chap. 2 p 6 2. indirect measures a property or characteristic of cells B. direct, total counts 1. direct microscopic counts with a counting chamber 2. Coulter counter a. automatic counter that counts particles within a size range b. uses changes in resistance as particles in an electrolyte pass between electrodes c. problems with cell clumping and debris C. direct, viable counts 1. plate counting techniques a. pour plates and spread plates (1) assumes that inoculum is homogeneous and cells are not clumped (2) culture is usually serially diluted to provide inoculum with cells b. can take some time for results 2. modifications of standard methods D. indirect, viable counts 1. Most Probable Number (MPN) a. statistical estimation based on number of cultures showing growth in serially diluted series b. useful with cells that won't grow on solid media c. general form of equation: where: x = MPN i = volume tested i = number of total tests = number showing growth i d. tables are readily available for 3-, 5-, and 10-tube MPN setup 2. ATP bioluminometry a. ATP is extracted from cells and reacted with the enzyme luciferase to produce light b. the light output is measured with a bioluminometer c. the method is rapid and sensitive (can detect as little as 1 yeast cell or 10 bacterial cells) E. indirect, total count methods 1. turbidity (spectrophotometry) a. according to the Beer-Lambert Law, -log %T = A b. absorbance is proportional to cell density (doubling density decreases transmittance 10- fold, but absorbance is linear) 6 c. requires cell densities above 10 cells/ml d. can be standardized with viable counts or total counts 2. metabolic activity a. accumulation of product b. depletion of substrate c. useful for ecological studies 3. dry weight a. can be nearly direct, in the case of unicellular organisms (standardized with direct method) b. especially useful for filamentous fungi

7 MICRO 4354 Chap. 2 p 7 c. can be extended to cellular components (1) protein (2) DNA (3) membrane lipids d. cell yields can be determined for a limiting nutrient Y = (mass of cells)/(mass of substrate consumed) (1) basis of microbial assays for vitamins and other growth factors (2) can be extended to Y ATP if pathways known IV. Effects of environmental conditions on microbial growth A. temperature 1. every organism has a minimum temperature, a maximum temperature, and an optimum temperature 2. psychrophile opt. 15 o C o a. oceans (avg temp 5 C) b. organisms growing under ice in polar regions (snow alga) c. membranes rich in unsaturated fatty acids 3. mesophile opt about 35 o C a. best studied group b. corresponds to pathogens 4. thermophile opt about 60 o C a. hot springs b. water heaters c. compost heaps and digestors d. membranes rich in saturated fatty acids e. changes in enzyme structures 5. extreme thermophile opt 85 o C a. archeans b. thermal vents c. steam vents d. increased %G+C e. high intracellular potassium concentration with the counterion 2,3-diphosphoglycerate 6. eukaryotes rarely grow above 55 o C B. ph 1. each organism has a ph range where growth is possible, along with an optimum ph 2. most life occurs around neutrality, but ranges from acidophiles live at low ph 4. alkalinophiles live at high ph 5. neutraphiles live around ph 7 6. internal ph tends to be around neutrality C. water activity 1. describes the availability of water to the organism 2. a w = ratio of vapor pressure of air over a substance or solution divided by vapor pressure of water at the same temperature 3. high water activity = dilute environment 4. low water activity inhibits bacterial growth a. high solute concentration b. method of preservation

8 MICRO 4354 Chap. 2 p 8 5. halotolerant = able to survive with salt, but less than optimal growth 6. halophiles = require salt for growth a. often archeans b. modified cell walls and lipids 7. osmophiles = live in environments with low water activity (could be due to non-salt solutes) 8. xerophiles = live in dry environments 9. osmohiles, halophiles, and xerophiles usually contain a compensating solute to balance the osmotic strength of the external solute a. polyols (1) arabitol in fungi (2) glycerol in alga b. potassium c. amino acids (1) glutamic acid (2) proline d. other organisms may use different internal solutes D. oxygen concentration 1. bacteria vary in their need for and tolerance 2. obligate anaerobes are killed by oxygen 3. aerotolerant anaerobes can grow in the presence of oxygen but cannot use it as an electron acceptor 4. facultative anaerobes can use oxygen, but can also grow without it 5. microaerophiles require oxygen, but at lower levels than in air (2-10%) 6. aerobes require oxygen for growth 7. toxic forms of oxygen a. superoxide anion, O - 2 (1) occurs during reduction of O 2 to H2O (2) highly reactive (3) can cause oxidative destruction of lipids and other biochemical components b. peroxide, O 2-2 (1) formed during respiratory processes (2) often used as a disinfectant c. hydroxyl free radical, OH. (1) formed by ionizing radiation (2) formed by reaction of superoxide anion with peroxide 8. several enzymes to protect against oxygen toxicity a. catalase (1)HO HO 2 2 2H2O + O2 (2) present in aerobes and facultative anaerobes b. peroxidase (1) H O + NADH + H 2H O + NAD (2) no oxygen produced c. superoxide dismutase (1) O 2 + O 2 HO O2 (2) present in aerobes, facultative anaerobes, and aerotolerant anaerobes

9 MICRO 4354 Chap. 2 p 9 9. enzymes, especially those with reduced metal (iron) groups, may be sensitive to oxygen V. Control of growth A. Terminology 1. sterilization = destruction of living cells, viable spores, viruses, viroids 2. disinfection = killing, inhibition, or removal of organisms 3. sanitization = reduction of microorganisms to safe health levels 4. antiseptic = chemical agents applied to tissues to kill or inhibit pathogens 5. -cide = kills organisms 6. -lytic = lyses organisms 7. -static = inhibits growth of organisms B. Effectiveness of antimicrobials 1. population death is generally exponential or logarithmic 2. efficiency of antimicrobials influenced by at least 6 factors a. population size - larger population requires more time to die b. population composition - different degrees of resistance between different organisms or structures (1) spores more resistant than vegetative cells (2) Mycobacterium tuberculosis (acid-fast) more resistant than most bacteria (3) young cells more readily destroyed than older cells c. concentration of antimicrobial (1) usually greater concentration = greater effectiveness (2) 70% ethanol more effective than 95% ethanol d. exposure duration (1) longer exposure = greater death (2) sterilization = reduction of survival probability to 10-6 e. temperature; increase usually increases effectiveness of chemical f. local environment (1) heat kills better at acid ph (2) efficiency higher with lower organic matter C. Heat 1. moist heat a. boiling kills viruses, bacteria, fungi b. 10 minutes boiling kills vegetative cells, not endospores c. thermal death time (TDT) = shortest period of time to kill all organisms at a specific temperature under defined conditions d. decimal reduction time (D) or D value = time to kill 90% (1) important to food industry 12 0 (2) usually assumed population of 10 cells, reduced to 10 o (3) if D for C. botulinum spores is minutes at 121 C, 12D = 2.5 minutes (4) z value = increase in temperature required to reduce D to 1/10 its value (a) for C. botulinum, z = 10 o C o (b) at 111 C, D = 2.04 minutes, 12D = 24.5 minutes e. pressurized steam (autoclave) (1) autoclave = fancy pressure cooker (2) combines wet heat with pressure; allows temperatures above 100 o C o (3) temperatures above 100 C required to destroy endospores o (4) chamber filled with saturated steam for 121 C, 15 psi

10 MICRO 4354 Chap. 2 p 10 (5) materials exposed for 15 minutes f. pasteurization (1) reduces microorganism numbers but retains flavor of foods, especially dairy, beer, and other beverages (2) brief heating, followed by rapid cooling o o (3) C for 30 minutes (batch or older method) o (4) flash pasteurization = 72 C for 15 seconds g. tyndallization = discontinuous boiling or fractional steam sterilization o o o (1) heat material to C for 30 minutes on 3 consecutive days, incubated at 37 C (2) 1st heating destroys cells but leaves endospores (3) 2nd and 3rd heating destroys germinating endospores 2. Dry heat sterilization o o a C for 2-3 hours b. cell constituents oxidize c. less effective than moist heat 3. Filtration a. physical removal of microorganisms (1) excellent for heat sensitive materials (2) can be used with gases b. depth filters = thick layers of fibrous or granular material (1) twisting channels of small diameter (2) microbes removed by physical entrapment and adsorption to filter material c. membrane filters = thin (0.1 mm) membranes (1) made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene chloride, or other synthetic materials (2) vegetative cells removed with 0.2 m pore size 4. Radiation a. alters DNA, causing lethal mutations b. UV= ultraviolet light (260 nm) (1) doesn't penetrate glass, dirt films, water, many plastics (2) often used to sterilize cabinets or entire rooms c. ionizing (e.g., gamma) (1) penetrates objects (2) also called cold sterilization (3) widely used with food D. Chemical methods 1. phenolics a. early use by Lister b. Lysol contains a mixture of phenolics c. denature proteins and disrupt cell membranes d. excellent for surfaces, but can cause skin irritation 2. alcohols a. bactericidal and fungicidal, but not sporicidal b. denature proteins and dissolve membrane lipids 3. halogens a. iodine most common, followed by chlorine b. tincture of iodine = 2% iodine in water/ethanol solution of potassium iodide

11 MICRO 4354 Chap. 2 p 11 (1) effective antiseptic (2) stains and may damage skin (3) iodophor = complex of iodine + organic carrier (a) water soluble, stable, nonstaining (b) slow release to prevent skin burns and irritation c. chlorine disinfectant of choice for municipal water supplies and swimming pools (1) added in many forms; forms hypochlorous acid (2) oxidizes several materials, destroying cells but not endospores (3) Halzone tablets used for personal drinking water (4) excellent household disinfectant (a) 1:100 dilution of household bleach (1.3 oz/gal) + 0.7% nonionic detergent (1 oz/gal) (b) cleans and kills bacteria 4. heavy metals a. Hg, Ag, As, Zn, Cu used to be common germicides (1) most heavy metals are bacteriostatic, not bactericidal (2) currently using less toxic, more effective germicides (3) 1% silver nitrate added to eyes of infants to prevent ophthalmic gonorrhea; being replaced by erythromycin, which is also effective against Chlamydia and Neisseria (4) silver sulfadiazine used on burns (5) copper sulfate used as algicide in lakes and swimming pools b. combine with proteins (sulfhydryl groups), inactivating them 5. quaternary ammonium compounds a. detergents = organic molecules (non-soaps) that serve as wetting agents and emulsifiers (1) amphipathic molecules (2) effective cleansing agents b. cationic detergents more antimicrobial than anionic detergents (1) quaternary ammonium compounds most popular (2) positively charged quaternary nitrogen with long hydrophobic aliphatic chain c. disrupt membranes and may denature proteins d. kill most cells, but not endospores or M. tuberculosis e. often used as disinfectants for food utensils, small instruments, and skin antiseptics 6. aldehydes a. formaldehyde and gluteraldyde most common b. combine with and deactivate proteins c. 2% glutaraldehyde commonly used to disinfect hospital equipment 7. gases a. ethylene oxide is both microbicidal and sporicidal (1) combines with cell proteins (2) penetrates packing materials, even plastic wraps b. explosive, usually done in special sterilizer E. Evaluation of agents (EPA regulates disinfectants, FDA controls antiseptics) 1. phenol coefficient a. potency compared with phenol (1) dilutions inoculated with Salmonella typhi and Staphylococcus aureus and incubated (2) tubes subcultured at 5 min intervals (3) phenol coefficient obtained from highest dilution that kills bacteria after 10 min, but

12 MICRO 4354 Chap. 2 p 12 not after 5 min (4) reciprocal of concentration divided by that for phenol for phenol coefficient value b. can be misleading because conditions of test are not realistic (1) clean medium (2) pure cultures 2. use dilution test more realistic than phenol coefficient a. stainless steel cylinders are contaminated with bacterial mixtures, dried briefly, immersed in test disinfectants for 10 min, transferred to culture media, and incubated 2 days b. disinfectant concentration that kills all organisms with 95% confidence level is determined 3. disinfectants can also be tested under "in use" conditions VI. Antimicrobial chemotherapy A. General mechanisms of activity 1. pathogen damage can occur through several mechanisms a. most selective antibiotics interfere with cell wall synthesis b. high therapeutic index since cell walls not sound in eucaryotes 2. Cell wall synthesis inhibition a. penicillin, ampicillin, carbenicillin, methicillin, cephalosporins b. inhibit enzymes for peptidoglycan cross-linking; activate cell wall lytic enzymes c. bacitracin inhibits CW synthesis by interfering with lipid carrier that transports precursors across the plasma membrane 3. protein synthesis inhibition a. streptomycin, gentamicin; bind to 30S ribosome subunit and causes misreading of mrna b. chloramphenicol binds to 50S ribosomal subunit, inhibits peptidyl transferase, blocking peptide fond formation c. tetracyclines bind to 30S, interfere with aminoacyl-trna binding d. erythromycin binds to 50S, inhibits peptide chain elongation e. high therapeutic index because drugs differentiate between procaryotic and eucaryotic ribosomes 4. nucleic acid synthesis inhibition a. rifampicin b. inhibits DNA-dependent RNA polymerase, blocking RNA synthesis c. often toxic to eucaryotic systems also 5. Cell membrane disruption a. polymyxin B b. binds to cell membrane, disrupts structure and permeability 6. metabolic antagonism (antimetabolites) a. sulfa drugs compete with PABA, inhibits folic acid synthesis b. trimethoprim inhibits dihydrofolate reductase, blocking tetrahydrofolate synthesis c. dapsone interferes with folic acid synthesis d. isoniazid may disrupt pyridoxal or NAD metabolism and functioning; inhibits synthesis of mycolic acid "cord factor" 7. Several factors determine effectiveness of antimicrobial drugs a. drug must reach site of infection, so delivery system important (1) penicillin G unstable in stomach acid (2) gentamicin (aminoglycosides) not well absorbed through gut and must be injected intramuscularly (3) parenteral routes = non-oral administration

13 MICRO 4354 Chap. 2 p 13 b. concentration must exceed MIC (1) dependent on amount administered, (2) speed of uptake, (3) rate of elimination from body (4) best if drug is absorbed slowly over a long period and excreted slowly c. infecting organism (1) dormant bugs less susceptible (2) pathogen must have proper target site d. many agents less effective due to resistance mechanisms, spread quickly via plasmids B. Classes of antibiotics 1. sulfa drugs a. structural analog (similar to metabolic intermediate) b. similar to PABA, necessary for synthesis of folic acid 2. quinolones a. synthetic drug, broad spectrum, bactericidal b. inhibits DNA replication and repair, transcription c. nalidixic acid, fluoroquinolones (ciprofloxacin, norfoxacin, ofloxacin) 3. penicillins a. -lactam ring is common feature (side chains vary) b. penicillinase destroys ring c. block peptidoglycan cross-linking, leading to lysis d. many people are allergic 4. cephalosporins a. originally isolated from Cephalosporium (fungus) b. -lactam ring, like penicillins c. useful for people allergic to penicillin d. broad spectrum 5. tetracyclines a. naturally produced by Streptomyces or semi-synthetic b. bind to 30S ribosomal subunit, inhibiting protein synthesis c. bacteriostatic d. broad spectrum 6. aminoglycoside antibiotics a. Streptomyces make streptomycin, kanamycin, tobramycin b. Micromonospora purpurea synthesizes gentamicin c. bind to small ribosomal subunit, inhibiting protein synthesis d. bactericidal, most effective against gram negatives e. quite toxic to humans f. widespread resistance 7. erythromycin a. macrolide, synthesized by Streptomyces erythraeus b. broad spectrum, bacteriostatic, most effective against G+ c. bind to 23S rrna of 50S ribosomal subunit, inhibiting protein elongation d. macrolides have 12- to 22-carbon lactone rings 8. chloramphenicol a. synthetic, but originally from Streptomyces venezuelae b. acts like erythromycin

14 MICRO 4354 Chap. 2 p 14 c. broad spectrum, bacteriostatic d. quite toxic to humans C. Mechanisms of drug resistance 1. drug cannot enter cell a. G- unaffected by penicillin G because it can't penetrate the outer membrane b. changes in binding proteins render cells resistant 2. chemical modification a. penicillinase hydrolyzes the -lactam ring b. groups can be added which inactivate drugs 3. modification of target a. changes in 23S rrna protects against chloramphenicol or erythromycin b. change binding site for sulfanilamide 4. genes for drug resistance can be chromosomal or on plasmids a. spontaneous mutations in chromosome are rare b. chromosomal changes usually result in changes in drug receptors, preventing binding c. R plasmids (resistance plasmids) often code for enzymes that destroy or modify drugs (1) implicated in resistance to aminoglycosides, penicillins, cephalosporans, erythromycin, tetracyclines, sulfonamides, chloramphenicol, and others (2) plasmids transferred rapidly through populations (3) single plasmid can carry resistance to many drugs 5. Overuse of antibiotics has led to many resistant strains a. increase drug concentrations to destroy susceptible and spontaneous mutants b. use two drugs together c. limit use, especially broad-spectrum antibiotics D. Antifungal drugs 1. eukaryotic, so drugs often toxic to humans 2. most fungi have efficient detoxification mechanisms 3. often target membrane sterols or cell walls E. Determining activity levels 1. dilution susceptibility tests a. a series of broth tubes containing a range of antibiotic concentrations inoculated with test organism b. minimal inhibitory concentration (MIC) = lowest concentration that prevents growth (no growth after hr) c. minimal lethal concentration (MLC) = lowest concentration that kills the organism (no growth in subculture) d. cidal drugs usually kills at 2-4x MIC, static drugs kill at much higher concentrations (if at all) 2. disk diffusion tests a. antibiotic impregnated disks are placed on agar previously inoculated with the test bacterium (1) antibiotic diffuses, forming a gradient (2) resistant organisms grow up to the disk (3) susceptible organisms grow some distance from the disk, displaying a clear zone around the disk (a) wider the clear zone = more susceptible (b) zone width is a function of initial concentration, solubility, diffusion rate,

15 MICRO 4354 Chap. 2 p 15 susceptibility of organism (c) zone width cannot be used to compare 2 antibiotics b. Kirby-Bauer most used disk diffusion test (1) Mueller-Hinton agar inoculated with lawn of bacteria (2) disks placed on surface o (3) incubation at 35 C for hr (4) diameters of zones measured and compared to tabulated values to determine degree of microbial resistance (a) plot MIC vs zone diameters for different strains (b) determine from plot if treatment dosage would result in MIC