1. 2008 Antimicrobial Drugs ChemotherapyThe use of drugs to treat a disease Antimicrobial drugsInterfere with the growth of microbes within a host AntibioticSubstance produced by a microbe that, in small amounts, inhibits another microbe Selective toxicityA drug that kills harmful microbes without damaging the host

2. 2008 1928 – Fleming discovered penicillin, produced by Penicillium. 1940 – Howard Florey and Ernst Chain performed first clinical trials of penicillin. Figure 20.1

3. 2008 Table 20.1

4. 2008 ANTIMICROBIAL SENSITIVITY TESTS @ Used to select effective drugs for treatment. @ Not performed on commensals or contaminants @ This misleads physician and patient to receive unnecessary therapy @ Such therapy leads to side-effects & resistance of pathogens. @ Used to identify organism if it has a characteristic sensitivity pattern.

5. 2008 LIMITATIONS: 1.Measure in vitro not in vivo drug activity. 2.Selection of best drug depends on: a) Patient clinical condition b) Type and site of infection. c) History of drug hypersensitivity. 3.Drug activity : absorption, diffusion in tissues, metabolism, excretion, toxicity, effect on patient normal flora, are not known by sensitivity.

6. 2008 Table 20.2

7. 2008 The Action of Antimicrobial Drugs Broad-spectrum Superinfection Bactericidal Bacteriostatic

8. 2008 The Action of Antimicrobial Drugs Figure 20.2

9. 2008 The Action of Antimicrobial Drugs Figure 20.4

10. 2008 Penicillin Natural penicillins Semisynthetic penicillins Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

11. 2008 Penicillins Figure 20.6

12. 2008 Penicillin Penicilinase-resistant penicillins Extended-spectrum penicillins Penicillins +  -lactamase inhibitors Carbapenems Monobactam Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

13. 2008 Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis Figure 20.8

14. 2008 Cephalosporins 2 nd, 3 rd, and 4 th generations more effective against gram-negatives Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis Figure 20.9

15. 2008 Polypeptide antibiotics Bacitracin Topical application Against gram-positives Vancomycin Glycopeptide Important "last line" against antibiotic resistant S. aureus Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

16. 2008 Antimycobacterium antibiotics Isoniazid (INH) Inhibits mycolic acid synthesis Ethambutol Inhibits incorporation of mycolic acid Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

17. 2008 Chloramphenicol Broad spectrum Binds 50S subunit, inhibits peptide bond formation Aminoglycosides Streptomycin, neomycin, gentamycin Broad spectrum Changes shape of 30S subunit Antibacterial Antibiotics Inhibitors of Protein Synthesis

18. 2008 Tetracyclines Broad spectrum Interferes with tRNA attachment Macrolides Gram-positives Binds 50S, prevents translocation Erythromycin Gram-positives Binds 50S, prevents translocation Antibacterial Antibiotics Inhibitors of Protein Synthesis

19. 2008 Streptogramins Gram-positives Binds 50S subunit, inhibits translation Synercid Gram-positives Binds 50S subunit, inhibits translation Oxazolidinones Linezolid Gram-positives Binds 50S subunit, prevents formation of 70S ribosome Antibacterial Antibiotics Inhibitors of Protein Synthesis

20. 2008 Polymyxin B Topical Combined with bacitracin and neomycin in over- the-counter preparation Antibacterial Antibiotics Injury to the Plasma Membrane

21. 2008 Rifamycin Inhibits RNA synthesis Antituberculosis Quinolones and fluoroquinolones Ciprofloxacin Inhibits DNA gyrase Urinary tract infections Antibacterial Antibiotics Inhibitors of Nucleic Acid Synthesis

22. 2008 Sulfonamides (Sulfa drugs) Inhibit folic acid synthesis Broad spectrum Antibacterial Antibiotics Competitive Inhibitors Figure 5.7

23. 2008 Figure 20.13

24. 2008 Polyenes Amphotericin B Azoles Miconazole Triazoles Allylamines Antifungal Drugs Inhibition of Ergosterol Synthesis Figure 20.15

25. 2008 Echinocandins Inhibit synthesis of  -glucan Cancidas is used against Candida and Pneumocystis Antifungal Drugs Inhibition of Cell Wall Synthesis

26. 2008 Flucytocine Cytosine analog interferes with RNA synthesis Pentamidine isethionate Anti-Pneumocystis; may bind DNA Antifungal Drugs Inhibition of Nucleic Acids

27. 2008 Griseofulvin Used for superficial mycoses Tolnaftate Used for athlete's foot; action unknown Antifungal Drugs Inhibition of Microtubules (Mitosis)

28. 2008 Antiviral Drugs Nucleoside and Nucleotide Analogs Figure 20.16a

29. 2008 Antiviral Drugs Nucleoside and Nucleotide Analogs Figure 20.16b, c

30. 2008 Protease inhibitors Indinavir HIV Inhibit attachment Zanamivir Influenza Inhibit uncoating Amantadine Influenza Interferons prevent spread of viruses to new cells Viral hepatitis Antiviral Drugs Enzyme Inhibitors

31. 2008 Chloroquine Inhibits DNA synthesis Malaria Diiodohydroxyquin Unknown Amoeba Metronidazole Damages DNA Entamoeba, Trichomonas Antiprotozoan Drugs

32. 2008 Niclosamide Prevents ATP generation Tapeworms Praziquantel Alters membrane permeability Flatworms Pyantel pamoate Neuromuscular block Intestinal roundworms Antihelminthic Drugs

33. 2008 Mebendazole Inhibits nutrient absorption Intestinal roundworms Ivermectin Paralyzes worm Intestinal roundworms Antihelminthic Drugs

34. 2008 Disk-Diffusion Test Figure 20.17

35. 2008 E Test Figure 20.18

36. 2008 MIC Minimal inhibitory concentration MBC Minimal bactericidal concentration

37. 2008 Broth Dilution Test Figure 20.19

38. 2008 Figure 20.20

39. 2008 A variety of mutations can lead to antibiotic resistance. Mechanisms of antibiotic resistance 1. Enzymatic destruction of drug 2. Prevention of penetration of drug 3. Alteration of drug's target site 4. Rapid ejection of the drug Resistance genes are often on plasmids or transposons that can be transferred between bacteria. Antibiotic Resistance

40. 2008 Misuse of antibiotics selects for resistance mutants. Misuse includes: Using outdated, weakened antibiotics Using antibiotics for the common cold and other inappropriate conditions Use of antibiotics in animal feed Failure to complete the prescribed regimen Using someone else's leftover prescription Antibiotic Resistance

41. 2008 Synergism occurs when the effect of two drugs together is greater than the effect of either alone. Antagonism occurs when the effect of two drugs together is less than the effect of either alone. Effects of Combinations of Drugs

42. 2008 Effects of Combinations of Drugs Figure 20.22

43. 2008 Antimicrobial peptides Broad spectrum antibiotics from plants and animals Squalamine (sharks) Protegrin (pigs) Magainin (frogs) Antisense agents Complementary DNA or peptide nucleic acids that binds to a pathogen's virulence gene(s) and prevents transcription The Future of Chemotherapeutic Agents

44. 2008 TECHNIQUES: Mainly two: 1.Diffusion technique. 2.Dilution technique.

45. 2008 DIFFUSION SENSITIVITY TECHNIQUE: @ Used in routine sensitivity testing. @ A disc of filter paper is impregnated with a known volume & concentration of a drug & placed on an agar medium inoculated with a test organism.

46. 2008 Control organisms are inoculated: @ On same plate (Stokes technique). @ On a separate plate (Kirby-Bauer technique). Drug diffuses into medium.

47. 2008 @ After an overnight incubation, culture is examined for areas of no growth (inhibition zones) around discs: 1.Sensitive bacteria are inhibited at a distance from disc. 2.Resistant bacteria grow up to the edge of disc.

48. 2008 @ In Stokes technique: inhibition zone is compared directly with that of control. @ In Kirby-Bauer technique: zone is measured & compared against a previously prepared scale that correlates zone size with MIC.

49. 2008 @ MIC is the minimum drug concentration required to inhibit bacterial multiplication under standard conditions. @ It is measured by the dilution sensitivity technique. @ Inhibition zone increases when MIC decreases.

50. 2008 Inhibition zones vary in size due to: 1.Difference in molecular structures of drugs (larger zones are obtained when drugs diffuse rapidly in medium). 2.When bacterial growth is heavy (zones are smaller, & vice versa). 3.Factors affecting the medium: (volume, moisture, pH, & constituents). 4.Factors affecting the disc: (drug concentration, storage, & application).

51. 2008 DILUTION SENSITIVITY TECHNIQUE : @ Performed under conditions: 1.Patient not responding to therapy 2.Patient is immunosuppressed. @ Measures MIC. @ Measures the minimum bacterial concentration (MBC) (The minimum concentration of drug required to kill bacteria).

52. 2008 Technique: @ Dilutions of drug are added to a medium. @ A standard inoculum of organism is added. @ After overnight incubation, MIC is reported.

53. 2008 @ Clinical response is assessed by comparing MIC obtained with already known concentrations of the drug @ MBC may be determined by subculturing last tube in the dilution series to show visible growth @ Other tubes should detect no growth on subculture.

54. 2008 @ Dilution techniques require: *good standardization *good control of: inoculum, medium, drugs, incubation time, diluting techniques, reading of results. @ MIC may be determined by automated machines.

55. 2008 DRUG ASSAYS : @ Performed to: * check enough drug concentration in a body fluid to give adequate therapy (e.g.: treatment of endocarditis) * make sure that concentrations of toxic aminoglycosides remain below their toxic levels in pt. serum.

56. 2008 @ Specimens submitted for drug assays are accompanied by: drug dose, time of administration, time of collecting the specimen, and if patient is receiving other treatments.

57. 2008 STOKES DISC DIFFUSION SENSITIVITY TESTING: @ Has the following advantages: 1.Both test & control strains are inoculated on same plate. 2.Inoculum gives semi-confluent growth (neither too heavy nor too light).

58. 2008 3. The activity of each disc is controlled. 4. Inhibition zone of test organism is compared directly with that of control. 5. Errors due to too heavy or too light inocula are easily detected. 6. Different media may be used.

59. 2008 REQUIREMENTS: 1) SENSITIVITY TESTING AGAR: @ Best is Mueller - Hinton agar. @ Depth of medium is 4mm (25 ml) @ Pour plates on a level surface. @ Too thin & too thick media give false inhibition zones. @ patient not used antibiotic before 3 days. @ Plates are stored in plastic bags at 2 - 8°C for up to 2 weeks.

60. 2008 @ Before use dry plates with lids slightly open for ½ hr at 37°C. @ 5% blood is added to M-H agar to test for fastidious organisms (Neisseria, Haemophilus, Streptococcus).

61. 2008 Factors affecting antimicrobial sensitivity 1. Media containing substances inhibiting action of aminoglycosides, tetracyclines, trimethoprim, e.g: the substance thymidine. 2.pH of media: False large zones are formed if medium is acidic (tetracycline), or false small zones if medium is alkaline (aminoglycosides). Fermentable sugars are not added to medium to avoid production of acid and change of pH.

62. 2008 2) ANTIMICROBIAL DISCS : @ To select drugs for sensitivity, consult clinicians. @ Drug list must be limited & reviewed at regular intervals. @ If resistance developed, one member of each drug group is selected.

63. 2008 Other factors include: * Manufacturer instructions regarding discs: store temp., expiry date, etc. are followed * Bring discs to room-temp. one hour before use. * Do not expose discs to sunlight. * Quality control of discs essential. * Avoid dryness & heat that decrease control zone size.

64. 2008 3) CONTROL STRAINS: @ Selected according to : 1.Site of infection in patient. 2.Drug concentration at this site. 3.Strain must respond to treatment with normal doses. 4.Strain must grow at same rate as test organism.

65. 2008 Recommended control strains: 1. S.aureus – Oxford strain (NCTC 6571): Used for all except polymyxins, & for pathogens of all specimens except urine. 2. E.coli – NCTC 10418. Used for all drugs against pathogens from urine. 3. Ps.aeruginosa – NCTC 10662 : Used for controlling all drugs against Ps.

66. 2008 Precautions for control strains are : 1. Strain is cultivated on N.A. slopes. 2. Strains are stored in dark at room temp.(20-28°C). 3. Subculture is made every 3 – 6 months 4. Each week, a nutrient broth or agar culture is made & stored at 2 – 8°C. & from this culture, suspensions are prepared for daily use.

67. 2008 4) TURBIDITY (OPACITY) STANDARD @ It is a standard of barium chloride for matching turbidity of test & control strains inocula. @ Turbidity of standard is equivalent to an overnight broth culture. @ After matching, don’t incubate test or control strains for two hrs.

68. 2008 Preparation of Turbidity Standard: 1. Add I ml of H 2 SO4 to 99 ml water to make 1% H 2 SO4. 2. Dissolve 2.35g barium chloride in 200 ml water.

69. 2008 3. Mix 0.5 ml barium chloride solution to 99.5 ml H 2 SO4 solution 4. Transfer turbid solution to screw-cap bottle of same type as that used to prepare test & control strain suspension 5. Store turbidity standard sealed in dark at room temp. for 6 months.

70. 2008 INDIRECT SENSITIVITY TESTING: @ Indirect (Secondary) test: Inoculum is a pure culture. @ Direct (primary) test: Inoculum is a specimen.

71. 2008 METHODS OF INDIRECT TESTING: @ Apply Stokes technique as follows: 1. Emulsify colonies of organism in Muller- Hinton broth. 2. Match turbidity developed against standard turbidity. * To match, view against a printed card * No incubation.

72. 2008 3. Using a sterile a 4mm loop, apply the organism suspension to center of sensitivity plate. @ Using a sterile swab, spread inoculum across the center third of the plate.

73. 2008 Control Test

74. 2008 @ Do not use same swab for both application and spreading. @ Judge your turbidity with turbidity standard, not by naked eye.

75. 2008 4. Similarly, inoculate the broth culture of control strain across the upper and lower thirds of plate, leaving 5 mm. on each side of test organism. @ Control suspension must be standardized against the standard. 5. Allow the inocula to dry for few minutes, with the Petri dish closed.

76. 2008 6. Place antibiotic discs (after warming to room temp.) between test and control inocula. Press disc down a little, and do not move once it is placed. 7. After ½ hr. incubate at 37°C. @ For methicillin, incubate at 35°C.

77. 2008 8. Read test when : @ Growth of both test and control strains is not too heavy or too light. @ Control inhibition zones measure 8-15 mm. radius. * Measurement is from edge of disc to edge of zone.

78. 2008 @ Using an electric rotary inoculator, test strain may be inoculated in a ring around the control strain. @ If growth of test & control strains is not semi confluent, sensitivity test is repeated.

79. 2008 INTERPRETATION OF RESULTS: Test is reported: sensitive, intermediate, & resistant: a) Sensitive: Test zone is: @ wider than control zone @ or equal to control zone @ or not than 3 mm smaller of control zone.

80. 2008 b)Intermediate:Test zone is: @ more than 3 mm smaller of control zone but not less than 3 mm in diameter. c) Resistant: Test zone is: @ 2 mm or less. @ No zone of inhibition

81. 2008 1. Intermediate zone drugs must be prescribed in high doses to cure infection, or when drug is concentrated at site of infection, e.g.: UTI. 2. With sulphonamides & trimethoprim, slight growth may occur within inhibition zone. This is due to presence of inhibitors (thymidine), and it must be ignored.

82. 2008 3. Strains are considered resistant if: @ Growth is heaped-up at zone edge without gradual fading up towards disc (penicillin-resistant Staph.) @ Large colonies are seen growing within inhibition zone (Staph.aureus) 4. To test for sensitivity of co- trimoxazole, test for sensitivity of sulphamoxazole & trimethoprim separated, using individual discs each.

83. 2008 5. Colistin & polymyxin give smaller zones of inhibition because of their large molecular size. Hence control zone must be at least 3-4mm.. 6. If Proteus swarms across its inhibition zone, no problem, the zone is clear. 7. Check your discs daily for any decrease in zone size resulting from drug deterioration.

84. 2008 DIRECT SENSITIVITY TESTING: @ Performed when : 1. Gram smear stain showed large number of one type of organism. 2. To obtain a presumptive result for serious cases 3. For urine, pus, pos. blood cultures, 4. To isolate & identify a pathogen or to detect a resistant strain.

85. 2008 @ Sensitivity plate must not replace routine culture plate. @ Blood is added to Muller Hinton agar to be used for direct sensitivity @ Procedure for direct sensitivity is same as for indirect sensitivity. @ Result of direct sensitivity must be confirmed by indirect sensitivity.

86. 2008 @ Do not report direct sensitivity result if: * Growth of bacteria is too heavy or too light. * Zone size is smaller than that of the control.