Killing Bacteria Ain’t Easy

1. Killing Bacteria Ain’t Easy

2. Definitions • 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)

3. 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

4. Physical Methods in Control • Heat (dry heat; moist heat) • Filtration • Radiation (ultraviolet radiation; ionizing radiation) • Generally used to sterilize objects and control microbial growth

5. Pasteurization • 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)

6. Chemical Control Agents • Most act by causing chemical damage to proteins, nucleic acids or cell membrane lipids

7. 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

8. Mechanisms of Action of Antibiotics • Four Main Target Sites for Antibacterial Action • Cell wall synthesis • Protein synthesis • Nucleic acid synthesis • Cell membrane function

9. Inhibitors of Cell Wall Synthesis • 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

10. Inhibitors of Protein Synthesis • Aminoglycosides • Tetracyclines • Macrolides • Chloramphenicol

11. Inhibitors of Nucleic Acid Synthesis • 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.

12. Side Effects of Antibiotic Use • 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

14. 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

15. New Sources of Antibiotics • 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

16. Direct inactivation of antibiotic • Bacteria produce enzymes which break-down antibiotic • eg. β - lactamase (penicillinase) enzymes → hydrolyze β - lactam ring of penicillins

17. Alteration of antibiotic target • Mutated target no longer recognized by antibiotic • Ex. Aminoglycoside resistance → aminoglycoside-modifying enzymes inactivate the antibiotics

18. Prevent uptake or promote excretion of antibiotic • Active efflux through efflux pumps: ABC transporters • Ex. Tetracycline resistance -- rapid excretion via outer membrane “efflux” proteins → no accumulation of drug in cytoplasm • Reduced uptake across the cytoplasmic membrane • Ex. Cephalosporin resistance -- altered protein in membrane prevents entry

19. How do bacteria acquire resistance? • 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)

20. 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.