1. Killing Bacteria Ain’t Easy

2. 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)

3. -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

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

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

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