2. 933 MCB 3020, Spring 2005 Microbial Growth Control and Antibiotic Resistance

3. 934 Chapter 18 Microbial Growth Control: I. Microbial growth control II. Measuring antimicrobial activity III. Food preservation IV. Antimicrobial drugs V. Antibiotic Resistance

4. 935 I. Microbial Growth Control A. uses B. autoclave C. radiation D. filters E. chemical agents TB

5. 936 I. Microbial Growth Control Sterilization Killing or removal of all living organisms and their viruses Inhibition limiting microbial growth TB

6. 937 A. Uses Food preservation Laboratory work Disease prevention Disease treatment TB

7. 938 B. Autoclave Machine that uses steam under pressure for sterilization. Items are heated to 121°C for 10-15 minutes. kills endospores Autoclave TB

8. 939 C. Radiation 1. Ultraviolet (220 to 300 nm) poor penetrating power used to disinfect surfaces and air TB 2. Gamma and X-rays good penetration used for food preservation and sterilization of surgical supplies ionizing radiation

9. 940 D. Filters Used to sterilize heat-sensitive solutions and gasses A pore size of 0.22 micron will remove most bacteria. TB Will it remove most viruses?

10. 941 Antimicrobial agents Chemicals that kill or inhibit the growth of microorganisms Cidal agents Chemicals that kill (bacteriocidal, fungicidal, viricidal) E. Chemical Agents

11. 942 Bacteriostatic agents chemicals that inhibit growth, but do not kill frequently are inhibitors of protein synthesis Bacteriolytic agents kill cells by lysis eg. penicillin

12. 943 1. Disinfectants Chemicals used to kill microbes on inanimate objects. Chlorine Phenolic compounds TB

13. 944 2. Antiseptics Chemicals used to kill microbes on living tissue. Alcohol (70% on skin) Hydrogen peroxide TB

14. 945 II. Measuring antimicrobial activity A.Tube dilution assay B. Agar diffusion assay TB

15. 946 A. Tube dilution assay 1. inoculate tubes containing several concentrations of test compound with test organism and incubate. TB

16. 947 1.1.01.00110 MIC bacterial growth MIC (minimum inhibitory concentration) The lowest concentration of a substance that inhibits growth of a test organism TB

17. 948 B. Agar diffusion assay agar plate lawn of test bacteria filter paper soaked with test compound zones of inhibition (no growth) TB

18. 949 III. Food preservation A. Common spoilage organisms B. Preservation methods TB

19. 950 A. Common food spoilage organisms Meat enteric bacteria Escherichia coli Salmonella Milk products lactic acid bacteria Fruits and vegetables Erwinia Pseudomonas TB

20. 951 1. Pasteurization Heat treatment to reduce the number of viable organisms. 71°C, 15 sec., or 63-66°C, 30 min milkjuicewineetc. Used on foods the would be ruined by higher temperatures. B. Preservation methods TB

21. 952 2. Temperature control refrigeration and freezing 3. Sterilization canning 4. Controlling water availability adding salt (eg. ham) adding sugar (eg. jelly) TB

22. 953 5. pH control pickling fermented foods 6. Chemical preservatives Na propionate Na benzoate TB

23. 954 IV. Antimicrobial drugs B. Growth factor analogs C. Antibiotics D. Antivirals and antifungals A. Selective toxicity

24. 955 Toxicity for the pathogen, but not for the host. Something to think about: What is the basis for selective toxicity? TB

25. 956 B. Growth factor analogs A substance structurally related to a growth factor that blocks its use. TB

26. 957 1. Sulfanilamide Growth factor analog structurally related to p -aminobenzoic acid (PABA) Inhibits microbial growth by inhibiting folate synthesis TB

27. 958 H2NH2NSO 2 NH 2 sulfanilamide H2NH2N COOH PABA N HN H2NH2N N NCH 2 N H C O R folate TB

28. 959 Sulfanilamide is nontoxic to humans because we take up folate from our diet. TB

29. 960 C. Antibiotics Substances produced by microbes that kill or inhibit the growth of microbes bacteriostatic agents inhibit growth bacteriocidal agents kill TB

30. 961 1. inhibitors of cell wall synthesis penicillin vancomycin 2. inhibitors protein synthesis erythromycin (50S ribosomal subunit) tetracycline (30S ribosomal subunit) streptomycin (30S ribosomal subunit) TB

31. 962 H N O N S CH 3 H COO- H beta-lactam ring R natural penicillin R = CH 2 -CO- Prevents transpeptidation in cell wall

32. 963 CH 3 OCH 3 H3CH3C H3CH3C H2CH2C H2CH2C OH O H3CH3C O HO O CH 3 HO O N CH 3 H3CH3C O HO OH O O Erythromycin (macrolide) macrolide ring 50S ribosomal subunit

33. 964 R4R4 R2R2 R3R3 N CH 3 H3CH3C R1R1 H OHO O CO NH 2 Tetracycline: R 1 =H, R 2 =OH, R 3 =CH 3, R 4 =H Broad spectrum Target: 30s ribosomal subunit

34. 965 H 2 C-NH 2 O OH HO O NH 2 O OH O NH 2 CH 2 OH O NH 2 OH HO Kanamycin (aminoglycoside) Target: 30 s ribosomal subunit

35. 966 3. inhibitors of DNA gyrase naladixic acid novobiocin 4. inhibitors of RNA synthesis rifampin TB

36. 967 Antivirals and Antifungals Chemicals rifampin azidothymidine (AZT) Interferon inhibits viral RNA synthesis A. Antivirals TB

37. 968 B. Antifungals Ergosterol inhibitors polyenes azoles Selective toxicity is more difficult to obtain with antivirals and antifungals Important point Why? TB

38. 969 V. Antibiotic Resistance A. the problem of resistance B. resistance mechanisms C. development of resistance D. enzymes that inactivate antibiotics

39. 970 A. The problem of resistance Vancomycin-resistant Staphlyococcus aureus Penicillin-resistant Streptococcus pneumoniae Quinolone-resistant Salmonella enterica Examples of drug-resistant bacteria

40. 971 Why so much resistance? Overuse of antibiotics in inpatient and outpatient settings. Increased use of quinolones, tetracyclines, and glycopeptides in agriculture, the poultry industry, veterinary practice, and marine biology. Newer, implantable cardiovascular and orthopedic devices that necessitate prophylactic antibiotics.

41. 972 B. Resistance mechanisms lack of target site impermeability chemical modification of the antibiotic pump antibiotic out of cell

42. 973 C. Development of resistance 1. Mutation target site modification 2. Gene transfer R-plasmids

43. 974 R-plasmids (resistance plasmids) Plasmids that carry antibiotic resistance genes. Antibiotic resistance genes usually encode enzymes that inactivate antibiotics

44. 975 3. Tetracycline pump pumps tetracycline out of the cell D. Enzymes that inactivate antibiotics 1. Chloramphenicol acetyltransferase acetylates chloramphenicol  beta-lactamase cleaves the beta-lactam ring

45. 976 Interactions of antibiotics with alcohol in humans Antibiotics that are affected by alcohol are chloramphenicol, cephalosporins, metronidazole, and others. These produce "disulfiram-like" reactions.

46. 977 Disulfiram-like reactions Disulfiram is a drug to treat alcoholism. Some antibiotics cause a reaction similar to disulfiram reactions. Inactivates the enzyme aldehyde dehydrogenase. Causing accumulation of acetaldehyde in blood. Symptoms are flushed face, severe headaches, chest pains, shortness of breath, vomiting, and sweating.

47. 978 Ethanol NAD+ NADH NAD+ NADH Acetaldehyde Acetate Acetyl CoA Alcohol dehydrogenase Aldehyde dehydrogenase X Disulfiram

48. 979 Study objectives 1. Know how the following are used to control microbial growth: autoclaves, radiation, filters, disinfectants, antiseptics. Contrast cidal agents, bacteriostatic agents,and lytic agents. Know the examples presented in class. 2. Compare and contrast the tube dilution assay and the agar diffusion assay. Understand how each is used to measure antimicrobial activity of chemicals. 3. What is MIC? How does it correlate with antimicrobial activity of an inhibitor? 4. Memorize the common food spoilage organisms covered in class. 5. Know what pasteurization is and what types of foods are pasteurized. 6. Memorize the food preservation methods and examples presented in class. 7. What is selective toxicity? Understand the basis of selective toxicity of growth factor analogs, antibiotics, azidothymidine, interferon, and antiviral agents. 8. What is a growth factor analog? 9. How does sulfanilamide inhibit the growth of some bacteria? 10. Know the names and targets of the antibiotics presented in lecture. 11. Know the following antiviral agents:rifampin, azidothymidine, interferon. Azidothymidine inhibits reverse transcriptase. What viruses are affected this? 12. Know the names and target of the antifungal agents (polyenes, azoles). 13. Why is selective toxicity more difficult to obtain with antivirals and antifungals?

49. 980 Study objectives 14. Give 3 examples of antibiotic-resistant bacteria. What are the major causes of antibiotic resistance? 15. What are the mechanisms by which bacteria require antibiotic resistance? What is the role of R-plasmids in resistance? Understand how antibiotic-inactivating enzymes work. 16. Explain why alcohol should not be consumed while taking some antibiotics. Why do disulfiram-like antibiotics cause symptoms when consumed with alcohol?