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Control of Microorganisms in the Environment and

Antimicrobial Chemotherapy

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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

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More Definitions…• Sanitization

– 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

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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

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-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

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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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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

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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

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Filtration• Reduces microbial population or sterilizes

solutions of heat-sensitive materials by removing microorganisms

• Also used to reduce microbial populations in air

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Filtering Liquids• Membrane filters

– porous membranes with defined pore sizes that remove microorganisms primarily by physical screening

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Filtering Air

• Surgical masks

• Cotton plugs on culture vessels

• High-efficiency particulate air (HEPA) filters– used in laminar

flow biological safety cabinets

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Physical Control Methods

• Heat– Moist heat– Steam: Autoclave– Pasteurization– Dry heat– Incineration

• Radiation– Ultraviolet (UV)– Ionizing (gamma)

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Moist Heat• Destroys viruses, fungi, and bacteria

• Boiling will not destroy spores and does not sterilize

• Degrades nucleic acids, denatures proteins, and disrupts membranes

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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

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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

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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

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Dry Heat Incineration• Bench top incinerators are used to sterilize

inoculating loops used in microbiology laboratories

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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

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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

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Chemical Control Agents

• Disinfection

• Antisepsis

• Sterilization

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Chemical Agentsshould 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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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

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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

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Halogens• Any of five elements: fluorine, chlorine,

bromine, iodine, and astatine

• Important antimicrobial agents

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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

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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)

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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

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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

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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

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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

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Evaluation of Antimicrobial Agent Effectiveness

• Complex process regulated by U.S. federal agencies– Environmental Protection Agency

– Food and Drug Administration

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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

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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

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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

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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

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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

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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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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

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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

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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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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

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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

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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

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Antimicrobial DrugsClassified By Their

Mode of Action:

1. Inhibitors of cell wall synthesis

2. Protein synthesis inhibitors

3. Metabolic antagonists

4. Nucleic acid synthesis inhibition

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Factors Influencing Ability of Drug to Reach Concentrations

Exceeding MIC• Amount administered

• Route of administration

• Speed of uptake

• Rate of clearance (elimination) from body

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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

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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