Lecture 31 PMB

Frage	Antworten
Physical methods for microbial growth control	--> temperature --> temperature and pressure --> UV/etc --> filters
Chemical methods for microbial growth control	--> disinfectants --> antiseptics --> antibiotics
sterilization	REMOVAL of ALL living cells, spores, and viruses on an object the only method effective against bacterial endospores!
disinfection	REMOVAL of PATHOGENS from INANIMATE surfaces chemical agents: 70% EtOH, bleach, strong ionic detergents do not put any of these in our body!!
antiseptis	REMOVAL of PATHOGENS from surface of LIVING TISSUE (ie. skin) chemical agents: 70% EtOH wipe, hydrogen peroxide, Neosporin, iodine containing compounds
sanitation	REDUCING microbial population to safe levels
autoclave	used for heat/pressure sterilization --> steam achieves high T, rapidly kills everything --> takes about 20-40 minutes for required T to be reached by required time
Pasteurization	precisely controlled heat REDUCES microbial load, pathogen spread prevented --> reduced spoilage, longer shelf life --> does NOT kill all bacteria, just pathogens
Flash Pasteurization	liquids flowing through heat exchanger raised to 71ºC for 15 sec --> rapid cooling --> milk has refrigerated shelf life of ~2 weeks after treatment
Ultra-high temperature Pasteurization (UHT)	liquid is heated to 135ºC for 1-2 sec (using continuous flow heat exchangers) --> also kills endospores --> products with an unrefrigerated shelf life of 6-9 months, if unopened
What reduces the effectiveness of disinfectants/antiseptics?	Organic material (dirt, biofilm, etc) can bind to/react with these to reduce their effectiveness
Antimicrobial drugs/Antibiotics	chemicals used in humans that kill or inhibit the growth of microorganisms --> can be natural or synthetic --> must have SELECTIVE TOXICITY so the host is not harmed
bacteriostatic growth	growth is inhibited, but bacteria are NOT killed OD/direct visual count and viable cell count stay constant inhibitory molecules are at reversible binding equilibrium with a cellular protein, must keep agent at right concentration to constantly prevent growth!!
bactericidal growth	cells are killed, but not lysed OD/direct visual count constant, viable cell count decrease some irreversible damage to a protein or other component --> if agent is washed out/decreased, bacteria will still be dead
bacteriolytic growth	cells are killed and lysed OD/direct visual count decrease, viable cell count decrease usually do damage to the cell wall (PG) or cytoplasmic membrane --> if agent is washed out/decreased, bacteria will still be dead
Minimal Inhibitory Concentration (MIC)	lowest concentration of an agent that completely inhibits growth measured by a series of dilutions within culture medium, turbidity occurs in tubes with concentrations below the MIC compares the effects of different agents (as long as everything is standardized)
Streptomyces antibiotic production	--> production regulated by quorum sensing (G+ peptide system) --> occurs around time of sporulation, triggered by nutrient deprivation --> gives a competitive advantage against soil microbes --> resistant to own antibiotics
spectrum of action	tells us which bacteria are affected narrow spectrum: very specific to harming only a certain species (ie. Isoniazid) broad spectrum : affects many types of microbes (Tetracycline, Sulfonamides, Cephalosporins, Quinolones)
Mechanism of Action (MOA)	tells us the specific molecular target that is bound/the process that is inhibited by the drug typical targets: cell wall, cytoplasmic membrane, DNA synthesis, RNA synthesis (tx), protein synthesis (tl), metabolism
Examples of drugs that target well studied areas	Cell wall synthesis: penicillins Folic acid metabolism: sulfonamides Protein synthesis (30S): Tetracycline DNA gyrase: Cycloflaxin
Target: cell wall biosynthesis	MOA: B-lactam ring of Penicillin is suicide inhibitor of PBPs (perform transpeptidation in PG synthesis) --> BACTERIOLYTIC Spectrum: most effective agains G+ bacteria since they don't always cross OM -can have altered R groups: molecules more acid-stable, or β-lactamase resistant, or better at crossing OM of G- cells
how B-lactam ring binds to PBP	B-lactam ring is cysteine + valine, resembles D-ala-D-ala piece of PG --> PBP transpeptidase domain binds to this COVALENTLY, enzyme is inactivated peptide x-linking during PG synthesis blocked
different steps of cell wall biosynthesis to be blocked	D-Ala ligase - Cycloserine bactoprenol recycling - bacitracin transpeptidation - Penicillins, cephalosporins (D-Ala mimics) and Vancomycin (binds D-Ala-D-Ala)
target: DNA supercoiling/replication/integrity	MOA: Quinolones interact w/ bacterial DNA gyrase --> stabilize covalent intermediate between enzyme and DNA --> gyrase-DNA complex is roadblock for replication + creates broken ends --> irreversible damage to DNA, BACTERICIDAL Spectrum: both G+ and G- Ex: Ciprofloxacin, more soluble in fluorinated form
DNA gyrase	a topoisomerase II, makes ds breaks and introduces negative supercoiling found in ALL bacteria quinolone stabilizes covalent intermediate between DNA and gyrase --> blocks DNA replication
target: Metabolism	MOA: Growth factor analog, resembles PABA and blocks synthesis of folic acid; binds reversibly --> BACTERIOSTATIC Spectrum: G+ and G- bacteria Ex: Sulfanilamide
folic acid and PABA	Cells need folic acid as cofactor for single-carbon reduction rxns (used in nucleotide synthesis), and can't import it through bacterial cell membrane --> synthesize it from PABA, pteridine, and glutamic acid Sulfanilamide binds enzyme and attaches to pteridine, acts as a competitive inhibitor
target: protein synthesis	MOA: binds reversibly to 30S subunit of ribosome at A site --> prevents charged tRNA from entering --> BACTERIOSTATIC Spectrum: G+ and G- Ex: Tetracycline
Napthacene ring system and protein synthesis	Binds reversibly to 30S subunit of ribosome at the A site Tetracycline resembles this with two different R groups --> spectrum of activity (slightly diff drugs bind ribosomes of diff bacteria + diff drugs can taken up into cytoplasm of diffbacteria)
all steps of protein synthesis and corresponding drugs	premature peptide termination: Puromycin (mimics tRNA) peptide bond formation: Chloramphenicol (binds to 50s RNA) movement along mRNA: Erythromycin (binds to 50S rRNA) incoming tRNA: Tetracycline (binds to A site) mRNA reading accurately: Streptomycin (changes shape of 16S RNA)
antibiotic resistance mechanisms	1) Slightly alter the target so that the drug no longer binds (EX: point mutation in rpsL for protein S12) 2) Modify the antibiotic so that it no longer binds the target (EX: nptI phosphorylates kanamycin, cat acetylates chloramphenicol, B-lactamases cleave B-lactam ring) 3) Pump the drug out of the cell (EX: tatA catalyzes exchange of tetracycline out for protein in)
Triclosan	thought to be non-specific, disrupting membrane structure/function used in toothpaste, soaps, scrubs low water solubility but can be used in high concentrations in hydrophobic substances
Triclosan resistance: experimental methods	perform genetic selections to identify mutant genes: make a genomic library from each resistant mutant --> select for plasmids that would confer increased resistance on WT strain
results of Triclosan experiments	resistant mutants had pt. mutations --> a.a. subs in FabI (carrier protein reductase ENR involved in fatty acid (FA) biosynthesis that makes new cell membranes) MOA: Triclosan binds active site of FabI --> blocks FA biosynthesis --> arrests growth at LOW concentrations (0.1-20 μg/ml)
Problems with Triclosan today	Triclosan is used at HIGH 2-20 mg/ml conc. in products and toothpaste + it’s a stable compound --> it’s accumulating in the environment + could disrupt the endocrine systems of fish and mammals + some bacteria could become resistant by upregulating the expression of efflux pumps
Triclosan effect on efflux pumps	efflux pumps that have relaxed specificity could help bacteria pump out OTHER antimicrobials used to treat disease --> really bad

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