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Sterilization› Destruction or removal of all viable organisms

(including more resilient forms – bacterial spores, mycobacteria, naked viruses, fungi)

› Only on inanimate objects Disinfection

› Killing, inhibition, or removal of pathogenic organisms

› Disinfectants = Agents, usually chemical, used for disinfection (usually used on inanimate objects)

Sanitization› Reduction of microbial population to levels

deemed safe (based on public health standards)

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Antisepsis› Prevention of infection of living tissue by

microorganisms› Antiseptics

Chemical agents that kill or inhibit growth of microorganisms when applied to living tissue

-cidal suffix indicates agents that kill› Germicide

Kills pathogens and many nonpathogens but not necessarily endospores

› Include bactericides, fungicides, algicides, viricides

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Heat (dry heat; moist heat) Filtration Radiation (ultraviolet radiation;

ionizing radiation)

Generally used to sterilize objects and control microbial growth

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Process of heating food or other substance under controlled conditions of time and temperature to kill pathogens and reduce total number of microbes without damaging the substance (i.e. altering taste)

Controlled heating using high temperature short time = temperatures well below boiling (72°C for not less than 16 seconds) + rapid cooling

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 (→ 5 log reduction in viable microbes)

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Most act by causing chemical damage to proteins, nucleic acids or cell membrane lipids

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Cidal vs. Static effect› Bactericidal → kills

bacteria› Bacteriostatic →

only inhibits growth (growth resumes if antibiotic removed)

Spectrum of Activity› Narrow spectrum

→ active against few species only

› Broad spectrum → active against many different species

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Four Main Target Sites for Antibacterial Action› Cell wall synthesis› Protein synthesis› Nucleic acid

synthesis› Cell membrane

function

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Peptidoglycan is:› Vital component of bacterial cell wall› Unique to bacteria (good for selective toxicity)

Beta-lactams:› Penicillin derivatives, cephalosporins › Inhibit cell wall synthesis by binding to penicillin-binding

proteins (PBPs) PBPs are membrane proteins capable of binding to penicillin

that are responsible for final stages of cross-linking of bacterial cell wall structure → Osmotic lysis of bacterial due to incomplete peptidoglycan layer

Non-beta-lactams:› Vancomycin› act at various steps during peptidoglycan synthesis› NOTE: Only cells which are metabolically active and

dividing are affected

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› Aminoglycosides› Tetracyclines› Macrolides › Chloramphenicol

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Quinolones › Inactivate DNA gyrase → no chromosome

supercoiling → no DNA replication› Eukaryotic gyrase is ≈ 1000 X less sensitive to

quinolones Rifampin

› Binds to RNA polymerase → prevents transcription of DNA into RNA, so no proteins are made.

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Toxicity to host› After high-dose, long-term use (ex. tetracycline →

discolored teeth) Allergic reactions

› Penicillin: 1 - 3% of population is allergic Disruption of “normal flora”

› Broad spectrum antibiotics may alter “balance” of bacteria in gut →“Superinfection” Organisms not killed by antibiotic will grow &

predominate → increase in #’s of one species due to absence of competition

Ex. “Antibiotic-associated colitis” due to overgrowth of Clostridium difficile

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Methicillin-resistant Staphylococcus aureus (MRSA)

SA → pneumonia, skin & soft tissue infections, bloodstream

Treatment of choice is methicillin or derivatives (eg. Cloxacillin) -- cost ≈ $30.00 for standard 10-day course

MRSA → resistant to methicillin (+ related antibiotics)

1st seen in Canada in 1981 1995: 0.9 MRSA per 100 SA isolates 2001: 8.2 MRSA per 100 SA isolates Treatment options are limited

› Vancomycin - $200.00 for 10-day intravenous course› Total cost to treat 1 hospitalized isolated MRSA patient

≈ $14,000 Total cost associated with MRSA in Canada: $42-59

million/yr

Continue screening of new bacteria & fungi from environment› Do undiscovered

antibiotics still exist?? Rejuvenate existing

antibiotics› Chemical modifications

to resist bacterial inactivation

Novel non-microbial sources of antibiotics› Plants → anti-bacterial,

anti-tumor drugs› Vertebrates,

invertebrates, insects → antimicrobial peptides

Rational drug design› pick specific bacterial

target & deduce structure / function

› synthesize a chemical which acts against target

Bacteria produce enzymes which break-down antibioticeg. β - lactamase (penicillinase) enzymes → hydrolyze β - lactam ring of penicillins

Direct inactivation of antibiotic

Mutated target no longer recognized by antibiotic

Ex. Aminoglycoside resistance → aminoglycoside-modifying enzymes inactivate the antibiotics

Alteration of antibiotic target

Active efflux through efflux pumps: ABC transportersEx. Tetracycline resistance -- rapid excretion via outer membrane “efflux” proteins → no accumulation of drug in cytoplasm

Reduced uptake across the cytoplasmic membraneEx. Cephalosporin resistance -- altered protein in membrane prevents entry

Prevent uptake or promote excretion of antibiotic

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Spontaneous mutation in DNA (eg. to give altered drug target)› Low frequency events, but presence of drug in

environment exerts selective pressure so that mutant cells persist

Horizontal Gene Transfer: Obtain new resistance genes (eg. gene for β - lactamase)› Often plasmid-mediated› Genetic exchange from donor bacteria with

resistance plasmid› Multiple resistance may be obtained in a single

genetic event (eg. one plasmid carrying several resistance genes)

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A chromosomal mutation (a) can produce a drug resistant target, which confers resistance on the bacterial cell and allows it to multiply in the presence of antibiotic.

Resistance genes carried on plasmids (b) can spread from one cell to another more rapidly than cells themselves divide and spread.

Resistance genes on transposable elements (c) move between plasmids and the chromosome and from one plasmid to another, thereby allowing greater stability or greater dissemination of the resistance gene.