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8 and 9 Control of Microorganisms in the Environment and

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1 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.

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3 Definition of Frequently Used Terms
Sterilization 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

4 More Definitions… Sanitization Antisepsis
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

5 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

6 -cidal vs. –static Agents
-cide 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

7 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

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10 Conditions Influencing the Effectiveness of Antimicrobial Agent Activity
Population size larger populations take longer to kill than smaller populations Population composition microorganisms differ markedly in their sensitivity to antimicrobial agents

11 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

12 Filtration Reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms Also used to reduce microbial populations in air

13 Filtering Liquids Membrane filters
porous membranes with defined pore sizes that remove microorganisms primarily by physical screening

14 Filtering Air Surgical masks Cotton plugs on culture vessels
High-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets laminar flow biological safety cabinet

15 Physical Control Methods
Heat Moist heat Steam: Autoclave Pasteurization Dry heat Incineration Radiation Ultraviolet (UV) Ionizing (gamma)

16 Moist Heat Destroys viruses, fungi, and bacteria
Boiling will not destroy spores and does not sterilize Degrades nucleic acids, denatures proteins, and disrupts membranes

17 Steam Sterilization Carried out above 100oC which requires saturated steam under pressure Uses an autoclave Effective against all types of microorganisms (including spores!) Quality control - includes strips with Geobacillus stearothermophilus

18 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

19 Dry Heat Sterilization
Less effective than moist heat sterilization, requiring higher temperatures and longer exposure times items subjected to 160–170oC for 2 to 3 hours Oxidizes cell constituents and denatures proteins

20 Dry Heat Incineration Bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories

21 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

22 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

23 Chemical Control Agents
Disinfection Antisepsis Sterilization

24 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

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27 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

28 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

29 Halogens Any of five elements: fluorine, chlorine, bromine, iodine, and astatine Important antimicrobial agents

30 Halogens - Iodine Skin antiseptic
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

31 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)

32 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

33 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

34 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

35 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

36 Evaluation of Antimicrobial Agent Effectiveness
Complex process regulated by U.S. federal agencies Environmental Protection Agency Food and Drug Administration

37 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

38 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

39 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

40 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

41 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

42 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

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44 Later Discoveries Streptomycin, an antibiotic active against tuberculosis, was discovered by Selman Waksman (1944) Nobel Prize was awarded to Waksman in for this discovery By 1953 chloramphenicol, terramycin, neomycin, and tetracycline isolated

45 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

46 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

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49 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

50 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

51 The E Test 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

52 Antimicrobial Drugs Classified By Their Mode of Action:
Inhibitors of cell wall synthesis Protein synthesis inhibitors Metabolic antagonists Nucleic acid synthesis inhibition

53 1. Inhibitors of Cell Wall Synthesis
Penicillins most are 6-aminopenicillanic acid derivatives and differ in side chain attached to amino group most crucial feature of molecule is the b- lactam ring essential for bioactivity many penicillin resistant organisms produce b- lactamase (penicillinase) which hydrolyzes a bond in this ring

54 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

55 Other Actions of Penicillins
Binds to periplasmic proteins (penicillin- binding proteins, PBPs) May activate bacterial autolysins and murein hydrolases Stimulate bacterial holins to form holes or lesions in the plasma membrane

56 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

57 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

58 Vancomycin and Teicoplanin
Glycopeptide antibiotics Inhibit cell wall synthesis Vancomycin - important for treatment of antibiotic- resistant staphylococcal and enterococcal infections previously considered “drug of last resort” so rise in resistance to vancomycin is of great concern

59 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

60 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

61 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

62 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

63 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

64 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

65 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

66 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

67 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

68 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 alidixic

69 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

70 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

71 Factors Influencing Ability of Drug to Reach Concentrations Exceeding MIC
Amount administered Route of administration Speed of uptake Rate of clearance (elimination) from body

72 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

73 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


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