8 and 9 Control of Microorganisms in the Environment and Antimicrobial Chemotherapy Copyright McGraw-Hill Global Education Holdings, LLC. Permission required for reproduction or display. 1
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Definition of Frequently Used Sterilization Terms destruction or removal of all viable organisms Disinfection killing, inhibition, or removal of disease causing (pathogenic) organisms disinfectants agents, usually chemical, used for disinfection usually used on inanimate objects 3
Sanitization More Definitions reduction of microbial population to levels deemed safe (based on public health standards) Antisepsis prevention of infection of living tissue by microorganisms antiseptics chemical agents that kill or inhibit growth of microorganisms when applied to tissue 4
Antimicrobial Agents Chemotherapy use of chemicals to kill or inhibit growth of microorganisms within host tissue Agents that kill microorganisms or inhibit their growth cidal agents kill static agents inhibit growth 5
-cide -cidal vs. static Agents suffix indicating that agent kills germicide kills pathogens and many nonpathogens but not necessarily endospores include bactericides, fungicides, algicides, and viricides -static suffix indicating that agent inhibits growth include bacteriostatic and fungistatic 6
The Pattern of Microbial Death Microorganisms are not killed instantly Population death usually occurs exponentially Measure of agent s killing efficiency decimal reduction time (D-value) time to kill 90% must be sure persister cells (viable but nonculturable (VBNC) condition) are dead once they recover they may regain the ability to reproduce and cause infection 7
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Conditions Influencing the Effectiveness of Antimicrobial Agent Population size Activity larger populations take longer to kill than smaller populations Population composition microorganisms differ markedly in their sensitivity to antimicrobial agents 10
More Conditions Concentration or intensity of an antimicrobial agent usually higher concentrations kill more rapidly relationship is not linear Duration of exposure longer exposure more organisms killed Temperature higher temperatures usually increase killing Local environment ph, viscosity, concentration of organic matter, etc. can profoundly impact effectiveness organisms in biofilms are less susceptible to many antimicrobial agents 11
Filtration Reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms Also used to reduce microbial populations in air 12
Membrane filters Filtering Liquids porous membranes with defined pore sizes that remove microorganisms primarily by physical screening 13
Filtering Air Surgical masks Cotton plugs on culture vessels High-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets 14
Physical Control Methods Heat Moist heat Steam: Autoclave Pasteurization Dry heat Incineration Radiation Ultraviolet (UV) Ionizing (gamma) 15
Moist Heat Destroys viruses, fungi, and bacteria Boiling will not destroy spores and does not sterilize Degrades nucleic acids, denatures proteins, and disrupts membranes 16
Steam Sterilization Carried out above 100 o C which requires saturated steam under pressure Uses an autoclave Effective against all types of microorganisms (including spores!) Quality control - includes strips with Geobacillus stearothermophilus 17
Pasteurization Controlled heating at temperatures well below boiling Used for milk, beer, and other beverages Process does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present 18
Dry Heat Sterilization Less effective than moist heat sterilization, requiring higher temperatures and longer exposure times items subjected to 160 170 o C for 2 to 3 hours Oxidizes cell constituents and denatures proteins 19
Dry Heat Incineration Bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories 20
Ultraviolet (UV) Radiation Wavelength of 260 is most bactericidal (DNA absorbs) Causes thymine dimers preventing replication and transcription UV limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances Has been used for water treatment 21
Ionizing Radiation Gamma radiation penetrates deep into objects and forms reactive high energy radicals which are destructive to the DNA and proteins. Destroys bacterial endospores; not always effective against viruses Used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food 22
Chemical Control Agents Disinfection Antisepsis Sterilization 23
Chemical Agents should be - Effective against wide variety of infectious agents Selectively toxic Effective at low concentrations Effective in the presence of organic matter stable in storage homogeneous Overuse of antiseptics such as triclosan has selected for triclosan resistant bacteria and possibly antibiotic resistant 24
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Phenolics Commonly used as laboratory and hospital disinfectants Act by denaturing proteins and disrupting cell membranes Tuberculocidal, effective in presence of organic material, and long lasting Disagreeable odor and can cause skin irritation 27
Alcohols Among the most widely used disinfectants and antiseptics Two most common are ethanol and isopropanol Bactericidal, fungicidal, but not sporicidal Inactivate some viruses Denature proteins and possibly dissolve membrane lipids 28
Halogens Any of five elements: fluorine, chlorine, bromine, iodine, and astatine Important antimicrobial agents 29
Skin antiseptic Halogens - Iodine Oxidizes cell constituents and iodinates proteins At high concentrations may kill spores Skin damage, staining, and allergies can be a problem Iodophore iodine complexed with organic carrier released slowly to minimize skin burns 30
Halogens - Chlorine Oxidizes cell constituents Important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant Destroys vegetative bacteria and fungi, Chlorine gas is sporicidal Can react with organic matter to form carcinogenic compounds THMs (trihalomethanes) 31
Heavy Metals e.g., ions of mercury, silver, arsenic, zinc, and copper Effective but usually toxic Combine with and inactivate proteins; may also precipitate proteins 32
Quaternary Ammonium Compounds detergents that have antimicrobial activity and are effective disinfectants amphipathic organic cleansing agents most likely disrupts cell membrane cationic detergents are effective disinfectants kill most bacteria, but not M. tuberculosis or endospores safe and easy to use, inactivated by hard water and soap 33
Aldehydes Commonly used agents are formaldehyde and glutaraldehyde Highly reactive molecules Sporicidal and can be used as chemical sterilants Combine with and inactivate nucleic acids and proteins 34
Sterilizing Gases Used to sterilize heat-sensitive materials Microbicidal and sporicidal Ethylene oxide sterilization is carried out in equipment resembling an autoclave Betapropiolactone and vaporized hydrogen peroxide Combine with and inactivate DNA and proteins 35
Evaluation of Antimicrobial Agent Effectiveness Complex process regulated by U.S. federal agencies Environmental Protection Agency Food and Drug Administration 36
Phenol Coefficient Test Potency of a disinfectant is compared to that of phenol Useful for screening but may be misleading Phenol has a residual effectiveness Protocol involves adding disinfectant and organism to the same tube which is different than how disinfectants are really used 37
Other Evaluation Methods Use dilution test determines rate at which selected bacteria are destroyed by various chemical agents Protocol involves contaminating a bead and then disinfecting Normal in-use testing testing done using conditions that approximate normal use of disinfectant 38
Biological Control of Microorganisms Emerging field showing great promise Natural control mechanisms predation by Bdellovibrio viral-mediated lysis using pathogen specific bacteriophage, currently limited use in food industry but expanded toxin-mediated killing using bacteriocins 39
Chemotherapeutic Agents Chemical agents used to treat disease Destroy pathogenic microbes or inhibit their growth within host Most are antibiotics microbial products or their derivatives that kill susceptible microbes or inhibit their growth 40
The Development of Chemotherapy Paul Ehrlich (1904) developed concept of selective toxicity identified dyes that effectively treated African sleeping sickness Sahachiro Hato (1910) working with Ehrlich, identified arsenic compounds that effectively treated syphilis Gerhard Domagk, and Jacques and Therese Trefouel (1935) discovered sulfonamides and sulfa drugs 41
Penicillin First discovered by Ernest Duchesne (1896), but discovery lost Accidentally discovered by Alexander Fleming (1928) observed penicillin activity on contaminated plate did not think could be developed further Effectiveness demonstrated by Florey, Chain, and Heatley (1939) Fleming, Florey, and Chain received Nobel Prize in 1945 for discovery and production of penicillin 42
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Later Discoveries Streptomycin, an antibiotic active against tuberculosis, was discovered by Selman Waksman (1944) Nobel Prize was awarded to Waksman in 1952 for this discovery By 1953 chloramphenicol, terramycin, neomycin, and tetracycline isolated 44
General Characteristics of Antimicrobial Drugs Selective toxicity ability of drug to kill or inhibit pathogen while damaging host as little as possible Therapeutic dose drug level required for clinical treatment Toxic dose drug level at which drug becomes too toxic for patient (i.e., produces side effects) Therapeutic index (high TI is best) ratio of toxic dose to therapeutic dose Minimum toxic dose to the host/minimum effective microbial lethal dose 45
General Characteristics of Antimicrobial Drugs Side effects undesirable effects of drugs on host cells Narrow-spectrum drugs attack only a few different pathogens Broad-spectrum drugs attack many different pathogens Cidal agent - kills microbes Static agent - inhibits growth of microbes 46
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General Characteristics of Antimicrobial Drugs Effect of an agent may vary with concentration, microbe, host Effectiveness expressed in two ways minimal inhibitory concentration (MIC) lowest concentration of drug that inhibits growth of pathogen minimal lethal concentration (MLC) lowest concentration of drug that kills pathogen 49
Kirby-Bauer Method Standardized method for disk diffusion test Sensitivity/resistance determined using tables relating zone diameter with microbial resistance Table values plotted, used to determine if effective concentration of drug in body can be reached 50
Convenient for use with anaerobic pathogens Similar to disk diffusion method, but uses strip rather than disk E-test strips contain a gradient of an antibiotic Intersection of elliptical zone of inhibition with strip indicates MIC The E Test 51
Antimicrobial Drugs Classified By Their Mode of Action: 1. Inhibitors of cell wall synthesis 2. Protein synthesis inhibitors 3. Metabolic antagonists 4. Nucleic acid synthesis inhibition 52
Penicillins 1. Inhibitors of Cell Wall Synthesis most are 6-aminopenicillanic acid derivatives and differ in side chain attached to amino group most crucial feature of molecule is the β- lactam ring essential for bioactivity many penicillin resistant organisms produce β- lactamase (penicillinase) which hydrolyzes a bond in this ring 53
Penicillins Mode of action blocks the enzyme that catalyzes transpeptidation (formation of cross-links in peptidoglycan) prevents the synthesis of complete cell walls leading to lysis of cell acts only on growing bacteria that are synthesizing new peptidoglycan 54
Other Actions of Penicillins Binds to periplasmic proteins (penicillinbinding proteins, PBPs) May activate bacterial autolysins and murein hydrolases Stimulate bacterial holins to form holes or lesions in the plasma membrane 55
Penicillins Naturally occurring penicillins penicillin V and G are narrow spectrum Semisynthetic penicillins have a broader spectrum than naturally occurring ones Resistance to penicillins, including the semisynthetic analogs, continues to be a problem ~1 5% of adults in U.S. are allergic to penicillin allergy can lead to a violent allergic response and death 56
Cephalosporins Structurally and functionally similar to penicillins Broad-spectrum antibiotics that can be used by most patients that are allergic to penicillin Four categories based on their spectrum of activity 57
Vancomycin and Teicoplanin Glycopeptide antibiotics Inhibit cell wall synthesis Vancomycin - important for treatment of antibioticresistant staphylococcal and enterococcal infections previously considered drug of last resort so rise in resistance to vancomycin is of great concern 58
2. Protein Synthesis Inhibitors Many antibiotics bind specifically to the bacterial ribosome binding can be to 30S (small) or 50S (large) ribosomal subunit Other antibiotics inhibit a step in protein synthesis aminoacyl-trna binding peptide bond formation mrna reading translocation 59
Aminoglycoside Antibiotics Large group, all with a cyclohexane ring, amino sugars Neomycin, gentamicin, streptomycin, tobramycin Bind to 30S ribosomal subunit, interfere with protein synthesis by directly inhibiting the process and by causing misreading of the messenger RNA Resistance and toxicity 60
Tetracyclines All have a four-ring structure to which a variety of side chains are attached Are broad spectrum, bacteriostatic Combine with 30S ribosomal subunit inhibits bind of aminoacyl-trna molecules to the A site of the ribosome Sometimes used to treat acne 61
Macrolides Contain 12- to 22-carbon lactone rings linked to one or more sugars e.g., erythromycin broad spectrum, usually bacteriostatic binds to 23S rrna of 50S ribosomal subunit inhibits peptide chain elongation Used for patients allergic to penicillin 62
Chloramphenicol Now is chemically synthesized Binds to 23s rrna on 50S ribosomal subunit and inhibits peptidyl transferase reaction Toxic with numerous side effects so only used in life-threatening situations 63
3. Metabolic Antagonists Act as antimetabolites antagonize or block functioning of metabolic pathways by competitively inhibiting the use of metabolites by key enzymes Are structural analogs molecules that are structurally similar to, and compete with, naturally occurring metabolic intermediates block normal cellular metabolism 64
Sulfonamides or Sulfa Drugs Structurally related to sulfanilamide, a paminobenzoic acid (PABA) analog PABA used for the synthesis of folic acid and is made by many pathogens sulfa drugs are selectively toxic due to competitive inhibition of folic acid synthesis enzymes 65
Trimethoprim Synthetic antibiotic that also interferes with folic acid production Broad spectrum Can be combined with sulfa drugs to increase efficacy of treatment: Bactrim or Septra combination blocks two steps in folic acid pathway Has a variety of side effects including abdominal pain and photosensitivity reactions 66
Nucleic Acid Synthesis Inhibition A variety of mechanisms block DNA replication inhibition of DNA polymerase inhibition of DNA helicase block transcription inhibition of RNA polymerase Drugs not as selectively toxic as other antibiotics because bacteria and eukaryotes do not differ greatly in the way they synthesize nucleic acids 67
Quinolones Broad-spectrum, synthetic drugs containing the 4- quinolone ring: Cipro (ciprofloxacin) and Levaquin Nalidixic acid first synthesized quinolone (1962) Act by inhibiting bacterial DNA-gyrase and topoisomerase II Broad spectrum, bactericidal, wide range of infections 68
Factors Influencing Antimicrobial Drugs Ability of drug to reach site of infection Susceptibility of pathogen to drug Ability of drug to reach concentrations in body that exceed MIC of pathogen 69
Ability of Drug to Reach Site of Infection Depends in part on mode of administration oral some drugs destroyed by stomach acid topical parenteral routes nonoral routes of administration Drug can be excluded by blood clots or necrotic tissue 70
Factors Influencing Ability of Drug to Reach Concentrations Exceeding MIC Amount administered Route of administration Speed of uptake Rate of clearance (elimination) from body 71
Drug Resistance An increasing problem once resistance originates in a population it can be transmitted to other bacteria a particular type of resistance mechanism is not confirmed to a single class of drugs Microbes in abscesses or biofilms may be growing slowly and not be susceptible Resistance mutants arise spontaneously and are then selected 72
Overcoming Drug Resistance Give drug in appropriate concentrations to destroy susceptible Give two or more drugs at same time Use drugs only when necessary Possible future solutions continued development of new drugs use of bacteriophages to treat bacterial disease 73