Microbial Growth Control

1. Microbial Growth Control Chapter 27

2. Terminology “Decrease the microbial load by limiting growth” (inhibition) or reducing the load (killing) Decontamination Inanimate objects; inhibit growth, but not really reduce the initial load; not sterile, but safe to handle Wiping a table after a meal Disinfection Inanimate object; use of a physical agent to inhibit growth and reduce the initial load Using bleach to clean a table Antisepsis Same as disinfection, but on external living tissue Iodine on a wound Antibiosis In vivo killing or inhibition; internal tissues Antibiotic given for an infection

3. More Terminology Sterilization A completely different process than the rest Removing all viable organisms and spores by killing or removing Surgical instruments Bacteriocidal versus Bacteriostatic versus bacteriolytic Viri- Fungi-

4. What to Attack (in general)? Plasma membrane Consequences: PMF, permeability barrier Proteins (enzymes) Metabolism and transport disrupted Nucleic acids Cannot produce enzymes or make new membrane Cell Wall Lysis

5. Physical Methods: Heat Moist heat versus dry heat Denaturation versus oxidation effects Factors that effect sterilization by heat: Time Organism properties Temperature Concentration of microbe Presence of organic material Environmental pH Osmotic pressure Type of heat

6. Heat is bacteriocidal 2 ways of describing Decimal reduction time: time required for 10-fold reduction in population at a given temperature Canning industry looks at 90% reduction It increases for a population as temp. decreases Thermal death time: time at which all cells are killed at a given temperature Important for sterilization procedures Physical Methods: Heat

7. Moist Heat Method Autoclave Bactericidal Heat above boiling (high pressure) Vegetative cells and spores: 121º C (15 lbs/in2) for 10-15 min.

8. A Modern Research Autoclave

9. Moist Heat Method Pasteurization For food Bacteriocidal Kill pathogens and increase shelf life Flash: 71 º C for 15 sec. and rapid cooling Bulk: 63-66 º C for 10-30 min. and slow cooling Both effective, but flash is better for flavor

10. Physical Methods: Radiation Ionizing Ex) gamma rays, X rays, high energy electrons Short wavelength (<1nm) ?high energy Produce ion (hydroxide, hydride, or electrons) Damage to DNA or enzymes (bacteriocidal) Food preservation, medical supply and some pharmaceutical sterilization Some bacteria more resistant (Deinococcus radiodurans)

11. Nonionizing radiation Longer wavelength (>100 nm)?less energy Ex) UV light (best at 260nm) UVC Thymine dimers inhibit replication (bacteriostatic) Initiate the SOS response!!! Lower energy?Low penetration Germicidal lamps and vaccine disinfection Physical Methods: Radiation

13. Physical Methods: Filtration Heat sensitive liquid materials (media, some antibiotics, etc.) Separate microbes from fluid Passage of liquid through a filter pore size of 0.2mm or less and collection in a sterile container Viruses and spores can slip by so sterilization often difficult

14. Filtration

15. Other Physical Methods Low temperatures Water in the cells crystallize below 0?C Refrigeration slows the growth of most cells (not Listeria) Osmotic pressure Old method of preservation Slows the growth of many non-halophilic cells Salt or sugar Molds better suited to these conditions

16. Chemical Methods Antiseptic versus disinfectant Factors affecting efficiency of chemicals Concentration Initial load of microbes Environmental: Temperature, pH, salt Presence of organic matter Time or exposure Organism Spores versus vegetative cells Mycobacterium and lipid rich cell wall Gram negative organisms and outer membrane

17. Chemical Methods Minimum inhibitory concentration (MIC) Not constant (dependent on many factors) Must standardize for comparison Does not distinguish bacteriocidal versus bacteriostatic Tube dilution technique Smallest amount of an agent needed to inhibit the growth of a microbe Agar diffusion method Zone of inhibition Diameter proportional to amount of agent, solubility, diffusion coefficient, and effectiveness Cannot distinguish ability to diffuse from actual effectiveness Does not distinguish bacteriocidal versus bacteriostatic

20. Chemotherapeutic Agents Used to decrease load in tissues Antibiosis Selective toxicity: injure bacteria and no harm to host Synthetic versus antibiotic Synthetic: growth factor analogs Antibiotic: produced by microorganisms

21. Growth Factor Analog: Sulfa Drugs Microbes normally produce folic acid from a para-aminobenzoic acid (PABA) substrate and an enzyme When sulfanilamide is present the enzyme binds to it instead Why is it selectively toxic?

22. Other Analogs Nucleic Acid analogs Similar to nucleic acids DNA/RNA mutations Ex: 5-bromouracil is a thymine analog Isoniazid is a growth analog effective only against Mycobacteria Interferes with synthesis of mycolic acid

23. Antibiotics Produced by microbes; kill or inhibit other microbes Mostly produced by fungi or streptomycetes (a group of bacteria) Spectrum of microbial activity Broad spectrum: affect broad range of bacteria Ex: Tetracyline Also destroys normal flora (why is this a problem?) Narrow spectrum: effective against single group Superinfection: overgrowth of an organism that is either not sensitive to an antibiotic or is resistant Ex: Candida albicans

24. Antibiotics: Cell Wall Penicillin G Narrow (Gram +) Inhibit cell wall synthesis No transpeptidation Cannot replace peptidoglycan lysed by autolysins during cell wall synthesis

25. Antibiotics: Proteins Inhibition of protein synthesis Selective toxicity?difference in ribosomes Bacteria= 70S ribosome (50S and 30S portions) Ribosomes are key players in protein synthesis Chloramphenicol (bacteriostatic and broad spectrum) 50S portion Inhibits formation of peptide bonds Tetracycline (bacteriostatic and broad spectrum) 30S portion Interference with attachment of tRNA, which carries amino acid Streptomycin (bacteriocidal and broad spectrum) 30S portion Changes shape of 30S portion and genetic code read wrong

26. Antibiotics Injury to plasma membrane Changes cell permeability Loss of metabolites or cell bursting Ex: Polymyxin B attaches to phospholipids (bacteriocidal and Gram neg.) Inhibition of nucleic acid synthesis More difficult to find b/c of lack of selective toxicity Ex: Rifampin inhibits mRNA synthesis (bacteriocidal and Mycobacterium tuberculosis)

28. Antibiotic Resistance Organisms that produce them are resistant to them Pass these resistance genes on to others Resistance has always existed Recent problems due to selection Replacement of sensitive bacteria in a population by resistant bacteria* Resistance is genetically coded Plasmids* Genomic

29. Plasmids: self replicating, extra chromosomal DNA, which can be passed on to others (typically between similar organisms).

30. Mechanisms of Drug Resistance Lack a target structure Cell wall for Mycoplasma–penicillin resistance Destruction or inactivation Some Staphylococcus have enzymes that inactivate Penicillin Impermeable Gram – organisms and penicillin resistance Modify the target site Modify the ribosome to become erythromycin resistant Rapid efflux Able to pump out the antibiotics Genetic change in metabolic pathway Resistance to sulfonamides: take up folic acid rather than make it

31. Reasons for Resistance Problems Lack of prescriptions Improper use (headaches and viruses) CDC estimates percentage of wrong prescriptions 30% ear infections, 50% sore throats, 100% colds Weak forms select for resistant bacteria Not taken long enough

32. Drug Combinations In case the organism is resistant to one drug Synergism: drug assistance Ex: Penicillin and Streptomycin for bacterial endocarditis Penicillin disrupts cell wall and Streptomycin gets in faster Antagonism: One drug makes other ineffective Ex: Penicillin and Tetracycline Tetracycline interferes with Penicillin

33. Viral Control (HIV) Difficult to be selectively toxic Nucleoside analog—AZT Azidothymidine is analog of thymine Inhibits elongation of viral nucleic acid chain Host toxicity Protease inhibitors Viral protease enzyme used to cut up larger polypeptides to assemble new viruses Binds to active site of protease enzyme Inhibits viral maturation and processing of viral polypeptides Interferons Made by uninfected host cells to prevent viral infection Host specific (not viral specific) Clinical aspects: needs to be in high concentration in local area Neuraminidase inhibitors--Tamiflu®

34. Protease Inhibitors and Neuraminidase Inhibitor

35. Chemotherapeutic Future Modify existing drugs Extend spectrum Prevent resistance New drugs New targets of activity Novel organisms