Control of Microbial Growth 2

1. N.B. Due to a seminar, the Monday morning help session on December 5th will be changed to the following: Help Sessions in room M245: Monday, December 5th, 10-11 am & 2-3 pm. Tuesday, December 6th, 11 am-noon & 2-3 pm.

2. 30 November 2005 M.S. Peppler Dept of MMI mark.peppler@ualberta.ca 1-69 Medical Sciences Building Control of Microbial Growth 2

3. Control of Microbial Growth 2 Objectives. After today’s session you will understand: 1. The grouping of antimicrobial chemicals based on application. 2. The main characteristics of antibiotics. 3. Certain fundamental mechanisms of bacterial resistance to anti-bacterial compounds.

4. Objective 1a. Chemical Inhibitors of Microbes. 4. Chemical agents: (no longer talking about food preparation) DEFINITIONS: antimicrobial agents: general term for chemical which kills or inhibits growth of microbes.N.B. These can be selectively toxic (directed against certain organisms) or non-selective (harms host) Recall Lab. bacteriostatic agent: inhibits growth; may allow recovery and re-growth after removed !!! (e.g., inhibits protein synthesis) e.g., many antibiotics. bacteriocidal agent: kills bacteria (at correct concentration) (e.g., binds to macromolecules to stop growth) e.g. heavy metals. bacteriolytic agent: causes death by cell lysis. (e.g., penicillin vs peptidoglycan; e.g., detergents & surfactants).

5. Objective 1b. Chemical Inhibitors of Microbes. General types of antimicrobials: Disinfectants: chemical agents used on inanimate objects, surfaces (e.g., chlorine bleach). Antiseptics: chemicals safe enough for applying to living tissue surfaces (e.g., alcohol wipes, H2O2). Antibiotics: chemicals produced by microbes which act against other microbes; usually for ingestion; USUALLY specific for the pathogen.

6. Objective 1c. Chemical Inhibitors of Microbes. EXAMPLES of disinfectants & antiseptics: Heavy metals: usually alter proteins irreversibly. They are non-selective, therefore are limited to topical use or industrial applications only. Organic or inorganic mercurials (rarely used now). Silver nitrate vs. Neisseria gonorrhoeae (historical treatment) but now featured in antibacterial bandages. Surfactants/detergents: disrupt membranes. Quaternary ammonium compounds: e.g., Roccal used in Lab; foamy mouthwash.

7. Objective 1c. Chemical Inhibitors of Microbes. EXAMPLES of disinfectants & antiseptics: Oxidizing agents: liquids, gases. • phenols (e.g. Lysol; active ingredient o-phenylphenol (recall Lab Exercise; phenol reference) • peroxide (recall toxic effects of O2 from lecture) • ozone (GAS: using a toxic form of O2 to advantage) • chlorine compounds (hypochlorite [Cl– bleach], Cl2 gas) • iodine solutions (Red Cross blood donor clinic). Alcohols: e.g., 70% ethanol. Rubbing alcohol for piercings; dehydrate bacteria, damage membranes, (therefore are more effective against Gram negative organisms.

8. Objective 1d. Chemical Inhibitors of Microbes. EXAMPLES of disinfectants & antiseptics: TV ads for household cleansers: “kills 99% of household bacteria” i.e., 2 log reduction. Is this significant???? if N0 = 106/cm2 What about contact time?? (≥5 minutes?)

9. Objective 2a. Characteristics of Antibiotics. • We now take antibiotics for granted: • how many have taken an antibiotic? • how many might have died without one? • How might history have changed with antibiotics? • plagues and European history; cholera; TB • How is it changing NOW because of antibiotics? • Relatively recent discovery (grandparents) • Until 1940's, only a few antimicrobial agents (i.e., produced chemically, not biologically) were available. • Antibiotic "era" began with penicillin (discovered in 1928 by Fleming), upon large-scale production from P. chrysogenum (mouldy canteloupe) during WWII by Florey and colleagues.

10. Objective 2b. Characteristics of Antibiotics. Penicillin was needed for treating infections of war casualties. • started with cultures producing 60 milligrams/L • isolated over-producing mutants • now 20 grams/L (300-fold improvement) • 24,000 tonnes produced annually; considered a "bulk chemical" • < US$5/billion of units (bou) in Jan 2005 for penG (used for semi-synthetics). But there are more antibiotics than penicillin! Antibiotics work on the principle of selective toxicity, i.e., Kill the bug, not the person with the bug!

11. Objective 2c. Characteristics of Antibiotics.

12. Objective 2d. Characteristics of Antibiotics. POINTS TO NOTE: Antibiotics differ from antiseptics, etc. because they are specific with respect to: • the producing organism • the site of action in the target organism • target organism types (selective toxicity) i.e., certain organisms are sensitive to certain antibiotics. Therefore, there is no universal antibiotic that will work on all types of pathogens. Some antibiotics are broad-spectrum i.e. work vs. Gram +ve and Gram -ve others are narrow-spectrum. Recall PROBLEMS natural flora with using broad-spectrum antibiotics: e.g., yeast infections.

13. Objective 2f. Characteristics of Antibiotics. Some problems can be encountered with antifungal and antiparasitic agents because of host toxicity. (e.g., related membranes, ribosomes, etc.) One problem is that bacteria grow (multiply) very quickly, and that resistance can arise within a normally susceptible population so that these "magic bullets" become ineffective. “Antimicrobials” incorporated into soaps, kitchen sprays, toothpaste, mouthwash, laundry soap, kitchen cutting boards, kids’ plastic toys……… Ample opportunity for natural microbiota to be exposed to antimicrobials (including antibiotics) and become resistant……and now antimicrobial residues are being detected in the environment……… Do we really need to be so clean????? Maybe around sick people and newborns, but otherwise NO.

14. Objective 3a. Microbial Resistance. Bacteria can be RESISTANT to different antibiotics for different reasons: Reminder of terms: WE (the host) do NOT become RESISTANT to antibiotics; the antibiotics should not have any effect on US; they are directed against microbes. rather, the pathogens we are infected with become resistant to the antibiotics: i.e, the antibiotics no longer are effective in killing or inhibiting the bacteria. This occurs by one or more different mechanisms:

15. Objective 3b. Microbial Resistance. INTRINSIC RESISTANCE • impermeability (e.g. penicillins vs. Gram -ves) overcome by chemical modification; e.g. ampicillin - a semisynthetic antibiotic • lack of target site e.g. penicillins vs. mycoplasmas • alter/lack biochemical pathway target e.g. sulfa drugs & folic acid pathway PABA analogue

16. Objective 3c. Microbial Resistance. 2) ACQUIRED RESISTANCE • mutation of target site (formerly susceptible) e.g. altered 30S ribosome e.g. altered enzyme in pathway • enzymatic inactivation of antibiotic • efflux pumps to excrete antibiotic quickly How do they "acquire" resistance? they become genetically altered either by a)mutation of existing genes or b)acquisition of new genes.

17. Objective 3d. Microbial Resistance. (a) spontaneous mutation e.g., gentamicin binds to 30S subunit, changes ribosome conformation, mRNA is read incorrectly --> cell dies. by RANDOM/CHANCEmutation of the gene for the ribosomal protein on the chromosome (e.g., point mutation, typical frequency of 1 x 10-6), causes change in shape of ribosome so that it functions correctly in the presence of Gm. This mutant is now resistant to Gm (GmR) (and some even come to require Gm for growth) Another example: rifampicinR DNA gyrase mutates)

18. Objective 3e. Microbial Resistance. NOTE that this GmR mutant has, through chromosomal mutation, acquired resistance to only ONE antibiotic (or to possibly close structural relatives, e.g. other aminoglycosides in this example = “cross resistance”). It can pass this resistance on • ONLY to its daughter cells, (i.e., vertical transmission of resistance). • NOT to its contemporary "neighbors" (i.e., horizontal transmission of resistance). i.e., the cell has to GROW and DIVIDE in order to spread resistance to other cells. Also NOTE that, as long as Gm is present, the mutant has an advantage over sensitive (non-mutated) cells, and will come to predominate in the culture/infection. That is, the continued presence of Gm can ENRICH for the resistant mutant.

19. Objective 3f. Microbial Resistance. N.B. The antibiotic does not CAUSE the mutation, it just provides the SELECTIVE PRESSURE for the mutant to proliferate in the flora.

20. Objective 3g. Microbial Resistance. Another mechanism of acquired resistance is to (b) acquire a gene which encodes an enzyme to modify or destroy the antibiotic. i.e., cannot arise by spontaneous mutation – this is acquisition of a new function, needing a new gene. e.g., penicillinases (ß-lactamases) enzyme breaks the ß-lactam ring of penicillins to inactivate the penicillin. e.g. gentamicin & chloramphenicol Enzymes add chemical groups onto the antibiotic to inactivate it (different enzymes for each). Penicillin G

21. Objective 3h. Microbial Resistance. NOTE: that these acquired genes MAY be encoded on plasmids called R (resistance) PLASMIDS (more on these later). The plasmids will be passed on to daughter cells i.e., vertical transmission of resistance. • Especially in the presence of selective pressure (as with the spontaneous mutations). • May be lost in the absence of selective pressure. but ALSO the plasmids MAY be mobilized, disseminated i.e. horizontal transmission of resistance.

22. Objective 3i. Microbial Resistance. HOW?? • between contemporary cells (same species) • between species by >> conjugation between cells or >>transformation with naked DNA (i.e. lyse resistant cell, releasing plasmid, but nearby cell takes up the plasmid); or by >>transduction through phage transfer of genes i.e., resistance can spread in a mixed population of microbes, even without growth of the resistant organisms.

23. Next time: Efflux pumps, R plasmids and the problem of antibiotic resistance.