Killing Bacteria Ain’t Easy

Definitions Sterilization Disinfection Sanitization 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)

-cidal suffix indicates agents that kill 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

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

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) Kills microbes responsible for Salmonella food poisoning, tuberculosis, brucellosis, typhoid fever

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

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

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

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

Inhibitors of Protein Synthesis Aminoglycosides Tetracyclines Macrolides Chloramphenicol

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.

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

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

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 ≈ 100 antibiotics known (many cannot be used due to resistance) Only 3 new antibiotics introduced in last 40 years

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

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

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

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)

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. → previously useful antibiotics become obsolete (ex. tetracycline and penicillin for gonorrhea)