1. Lecture 3 Antimicrobials and Susceptibility tests Dr. Abdelraouf A. Elmanama Islamic University-Gaza Medical Technology Department

2. Lecture outlines Kirby-Bauer susceptibility test Antimicrobial profiles selection Reporting susceptibility test

3. What Does the Laboratory Need to Know about Antimicrobial Susceptibility Testing (AST) ? Which organisms to test? What methods to use? What antibiotics to test? How to report results?

4. What Does a Laboratory Need to Know about AST? (con’t) How to determine the clinical significance of results? How to ensure accuracy of results? –Quality control / quality assurance When to call the MD, infection control, public health?

5. What Does a Laboratory Need to Know about AST? (con’t) When to ask for help? Where to go for help?

6. Brief Review of Routine AST Methods

7. Routine Susceptibility Tests Disk diffusion (Kirby Bauer) Broth micro-dilution MIC –NCCLS reference method Etest

8. Disk Diffusion Test

9. Select colonies Prepare inoculum suspension

10. Mix well Standardize inoculum suspension

11. Swab plate Remove sample

12. Add disks Incubate overnight

13. Measure Zones Transmitted LightReflected Light

14. Zone Interpretive Criteria (mm) Drug Disk content (ug) ResIntSusc Cefazolin30  14 15-17  18 Gentamicin10  12 13-14  15 Flash presentation for summary

15. Qualitative results –Susceptible –Intermediate – may respond if infection is at body site where drug concentrates (e.g. urine) or if higher than normal dose can be safely given –Resistant Disk Diffusion Test

16. Modify methods for fastidious bacteria

17. Clinical Conditions when MICs are Useful Endocarditis Meningitis Septicemia Osteomyelitis Immunosuppressed patients (HIV, cancer, etc.) Prosthetic devices Patients not responding despite “Sensitive results”

18. MIC Minimal inhibitory concentration The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium Interpret: –Susceptible –Intermediate –Resistant

19. Inoculum Preparation MIC Testing (NCCLS Reference Method) Standardize inoculum suspension Final inoculum concentration –3 – 5 x 10 5 CFU/ml –(3 – 5 x 10 4 CFU/well)

20. Microdilution MIC tray Prepare inoculum suspension

21. Dilute & mix inoculum suspension

22. Pour inoculum into reservoir and inoculate MIC tray

23. Inoculate purity plate Incubate overnight

24. Examining purity plate Reflected light Transmitted light

25. Read MICs

26. -+ 64 32 16 8 4 2 1 >64 0.5 MICs >64

27. MIC on a strip

29. S. pneumoniae Penicillin MIC = 3  g/ml

30. MIC Interpretive Criteria (  g/ml) DrugSuscIntRes cefazolin  8 16  32 gentamicin  4 4 8  16

31. Empirical Treatment Infants 1-3 mos Ampicillin + cefotaxime or ceftriaxone Immunocompetent children > 3 mos and adults <55 Cefotaxime or ceftriaxone + vancomycin Adults > 55 and adults of any age with alcoholism or other debilitating illnesses Ampicillin + cefotaxime or ceftriaxone + vancomycin Hospital-acquired meningitis, posttraumatic or postneurosurgery meningitis, neutropenic patients, or patients with impaired cell-mediated immunity Ampicillin + ceftazidime + vancomycin

32. Ceftazidime should be substituted for ceftriaxone or cefotaxime in neurosurgical patients and in neutropenic patients

33. Specific treatment N. meningitidis –Penicillin sensitive  Penicillin G or Ampicillin –Penicillin-resistant  Ceftriaxone or cefotaxime

34. Chemoprophylaxis for N. meningitidis Rifampin 600 mg every 12 h for 2 days in adults and 10 mg/kg every 12 h for 2 days in children >1 year Or One dose of ciprofloxacin (750 mg) One dose of azithromycin (500 mg) One intramuscular dose of ceftriaxone (250 mg) Rifampicin is not recommended in pregnant women.

35. Pneumococci –Penicillin-sensitive  Penicillin G –Penicillin-intermediate  Ceftriaxone or cefotaxime –Penicillin-resistant  (Ceftriaxone or cefotaxime) + vancomycin

36. Gram-negative bacilli (except Pseudomonas spp.)  Ceftriaxone or cefotaxime Pseudomonas aeruginosa  Ceftazidime

37. Staphylococci spp. –Methicillin-sensitive  Nafcillin –Methicillin-resistant  Vancomycin Listeria monocytogenes  Ampicillin + gentamicin Haemophilus influenzae  Ceftriaxone or cefotaxime Streptococcus agalactiae  Penicillin G or ampicillin Bacteroides fragilis  Metronidazole Fusobacterium spp.  Metronidazole

38. Local Data and protocols should be observed and reviewed periodically Thank you