Control of Microbial Populations: Chapter Bacteria are ubiquitous, we can’t beat them (and in many ways we would not want to even if we could), but we can learn to control where and at what rates they do grow if we keep in mind that bacteria are constantly evolving. Methods of Sterilization: Dry Heat Boiling Moist Heat (Autoclave) Radiation Chemical Disruption Filtration --- Keep in mind that in sterilization you must kill all bacteria, so the technique must be harsh enough to kill even the toughest bacteria that could possibly be present, generally that means the endospore forming bacteria (Bacillus cereus, et. al.)

Dry Heat Basically baking bacteria to death, Not very efficient Works for glass or metal surfaces No good for media or most chemicals Boiling As the name implies, treatment with boiling water Will kill most pathogenic bacteria, viruses, and fungi Won’t kill many of the soil endospore formers Moist Heat: An autoclave is basically a big pressure cooker (at 15 psi water boils at 121 C) Very effective for most liquid and dry materials Keep in mind that heat transfer limits how fast materials reach 121 C autoclave rule of thumb: 20 min/ liter

Radiation Any type of radiation that causes molecular damage (particularly to DNA) can be used to sterilize material, the important things to keep in mind are exposure time and depth of penetration Ultraviolet (UV) good for sterilizing surfaces, will kill most bacteria very effective at damaging DNA (Thymine dimers) Ionizing Radiation much more penetrating power may cause chemical changes in over-exposed material

Filtration Physically remove bacteria and or viruses can separate viruses from bacteria good for heat/ radiation sensitive materials (drugs, antibiotics, etc.) may leave some soluble materials behind (LPS  endotoxins)

Not So Sterile, Sterilization Pasteurization Brief heating kills many, but not all bacteria Most pathogens are less hardy and/or do not grow well outside of the host and if enough are killed they will be too few to initiate a new infection has been very effective for wine, beer, and milk Oddball aside (when aesthetics trump health): CO treatment of meat

Chemical Warfare Disinfectant: kills cells on contact, generally through chemical reaction non-specific will kill any cell Antiseptic: Kills bacteria but does less damage to eukaryotic cells ( example H 2 O 2 ), still relies mostly on basic chemistry generally not used internally Antibiotic: A chemical compound that kills bacteria specifically, usually without reactive chemistry, but by blocking some essential cellular function, can be used internally

Antibiotics Sulfa drugs: A class of molecules that inhibit biosynthetic reactions developed during the 1920 &30’s can be taken internally, but many were not well tolerated some still used today Modern Antibiotics Penicillin was the first discovered in 1929 but was not brought to production until the early 1940’s Many come from the conflict between different bacteria and between bacteria and fungi Over 100 are known, not all are completely selective against bacteria

The Penicillin Story First discovered by Alexander Fleming (a British doctor and researcher) in Fleming had an interest in natural products that could inhibit bacterial infection, he is also known for the discovery of human lysozyme. Fleming was not a chemist and was not successful in producing a molecule that would be useful in medicine. It took Fleming a decade to interest a biochemist in his penicillin project. Howard Florey and his group started on the project in 1938, and by 1940 had a therapeutically useful molecule. It is estimated that penicillin has saved over 200 million lives Collaboration in Science “Chance favors the prepared mind” --- Louis Pastuer

Aminoglycosides: (Streptomycin, Gentamycin) Inhibit protein synthesis by binding to a portion of the bacterial ribosome. Most of them are bacteriocidal (i.e., cause bacterial cell death). Bacitracin: Inhibits cell wall production by blocking the step in the process (recycling of the membrane lipid carrier) which is needed to add on new cell wall subunits. Beta-lactam antibiotics: A name for the group of antibiotics which contain a specific chemical structure (i.e., a beta-lactam ring). This includes penicillins, cephalosporins, carbapenems and monobactams. Cephalosporins: Similar to penicillins in their mode of action but they treat a broader range of bacterial infections. They have structural similarities to penicillins and many people with allergies to penicillins also have allergic reactions to cephalosporins. Chloramphenicol: Inhibits protein synthesis by binding to a subunit of bacterial ribosomes (50S). Glycopeptides (e.g., vancomycin): Interferes with cell wall development by blocking the attachment of new cell wall subunits (muramyl pentapeptides).

Macrolides (e.g., erythromycin) and Lincosamides (e.g., clindamycin): Inhibit protein synthesis by binding to a subunit of the bacterial ribosome (50S). Quinolones: (Novobiocin) Blocks DNA synthesis by inhibiting one of the enzymes (DNA gyrase) needed in this process. Rifampin: Inhibits RNA synthesis by inhibiting one of the enzymes (DNA- dependent RNA polymerase) needed in this process. RNA is needed to make proteins. Tetracyclines: Inhibit protein synthesis by binding to the subunit of the bacterial ribosome (30S subunit). Trimethoprim and Sulfonamides: Blocks cell metabolism by inhibiting enzymes which are needed in the biosynthesis of folic acid which is a necessary cell compound.

Using antibiotics  -lactams are often not prescribed for Gram (-) bacteria, Why not? In general it is best to have at least a good idea of what group of bacteria is involved in the disease. Also, these days, one must consider patterns of resistance that may be common in your geographic area.

Antibiotic Resistance Bacteria evolve, adapting to rapidly changing conditions is the broadest description of their niche. So it should not surprise us that within ten years of its introduction, resistance to penicillin was well documented and widespread. Overuse and misuse of antibiotics plays to the strengths of bacterial adaptation. What does not completely kill them only makes them stronger. The Problem Antibiotics are seen as a “magic bullet” that cures all bacterial problems. We need to focus more on other ways to control bacteria and save antibiotics as a treatment of last resort for acute infections.

Annul Antibiotic use in the United States (2002) 35,000,000 pounds of antibiotics used annually 13% in human medicine 6% therapeutics use in agriculture 78% non-therapeutic use in agriculture 6% use in pets from: Shea., M., K: Pediatrics, vol.112, No.1, July 2003.

Mechanisms of Antibiotic Resistance 1.) Cleavage (penicillinase) 2.) Chemical modification (kanamycin methyl transferase) 3.) Efflux pumps (pump it back out of the cell) 4.) Mutation in the affected protein (mutations in ribosomal proteins can lead to resistance to erythromycin) Potentially any of these mechanism are transferable by HGT

Solutions to Antibiotic Resistance: 1.) Work on overuse (in medicine and agriculture) --- reduce selective pressure 2.) Prevent spread of resistance mechanisms 3.) Massive overkill strategies in medicine (no survivors= no adaptation) 4.) Work with bacterial ecology (competition with innocuous strains, Probiotics, or natural bacterial predators) 5.) In the short term, develop more truly new antibiotics