antimicrobial susceptibility test power point presentation

antimicrobial susceptibility test

1. Antimicrobial susceptibility Testing Presented by Dr Mohan Singh Dhakad PG 1 st year Department of Microbiology DLNPGMC Ratlam Moderator- Dr Gaurav Saxena Assosiate professor Department of Microbiology DLNPGMC Ratlam

2. Introduction Antimicrobial susceptibility test (AST) is the most important investigation. Guide the clinician for tailoring the empirical antibiotic therapy to pathogen-directed therapy

3. For whom AST is perform AST is performed only for pathogenic bacteria Isolated from the specimen, and not for the Commensal bacteria. For example, E. coli Isolated from urine specimen should be Subjected to AST, where as E. coli isolated from stool is a commensal ; hence AST is not performed

4. Classification of AST methods A) THE PHENOTYPIC METHODS: Disk Diffusion Method, Kirby-Bauer's disk diffusion (DD) method: Colony DD Direct DD Stokes disk diffusion methods Dilution tests: Broth dilution: Micro Broth dilution Macro Broth dilution Agar dilution methods

5. Classification of AST methods conti .. Epsilometer or E-test Automated AST, e.g. Vitek , Phoenix and Microscan systems. B) GENOTYPIC METHODS : PCR detecting drug-resistant genes

6. Kirby-Bauer's disk diffusion (DD) method Most widely used AST method. They are suitable for rapidly growing pathogenic bacteria. It is performed from colony (called colony- dd ) mostly, or Performed directly from the specimens(called direct DD)

7. Workflow of performing KB-DD test Selecting colony growth on the appropriate media ↓ Preparing the inoculum ↓ Selection of antimicrobial disks ↓ Inoculation onto the AST plates ↓

8. KBDD workflows Cont.. Applying the disks onto AST plates ↓ Incubating the AST plates ↓ Taking the reading of the AST result ↓ interpreting the AST result according to the clinical break point

9. Mueller-Hinton agar (MHA) Mueller-Hinton agar (MHA) is the best medium to used in following reason: Excellent acceptable batch-to-batch reproducibility for AST. Contains the lower amount of inhibitors that can affect the AST results of antimicrobial such as sulfonamide, trimethoprim , and tetracycline. It supports satisfactory growth of most non fastidious aerobic and facultative anaerobic bacteria. Does not alter the growth of an organism .

10. Preparation of MHA Mueller-Hinton agar is to be prepared according to the manufacturer's instructions, the following general principles need to be followed. Autoclaving: The medium is autoclaved, allowed to cool to 45-50°C in a water bath, and then poured into Petri plates with an optimum depth of 4 mm. This would correspond to 25-30 mL of medium for 100 mm plates and 60-70 mL for 150 mm plates. pH check : The pH of each MHA batch is checked after it has cooled and solidified and should have an optimum pH between 7.2 and 7.4

11. Other media Mueller-Hinton blood agar (MHBA): For certain Mueller-Hinton blood agar (MHBA) containing 5% of sheep blood is used For fastidious organisms such as S. pyogenes and S. pneumoniae Haemophilus test medium (HTM): It is a modification of MHA with addition of yeast extract, hematin and β -NAD It is recommended for H. influenzae and H. parainfluenzae .

12. Factors Influencing Zone Diameter in MHA 1. Effects of Divalent Cations : Variation in divalent cations , principally magnesium, calcium and zinc affect the zone size of certain antimicrobials 2.Effects of Thymidine or Thymines 3.Effect of pH of the Medium

13. Factors Influencing Zone Diameter in MHA 1. Effects of Divalent Cations : Magnesium and calcium : affect the AST results of aminoglycoside and tetracycline for P. aeruginosa . Excess Ca²/ Mg² reduces zone sizes (false-resistance), low Ca²/ Mg² cation content may result in large zones of inhibition (false-susceptibility). Supplemental Ca² or Mg² should not be added to MHA. Excess zinc ions may reduce carbapenem zone sizes.

14. 2 . Effects of Thymidine or Thymine MHA should have as low thymidine content as possible. Reason: excessive thymidine or thymine can reverse the inhibitory effect of sulfonamides, cotrimoxazole , and trimethoprim , drugs ineffective may result in false-resistance reports. Therefore, MHA should have as low thymidine content as possible.

15. Effects of Thymidine or Thymine Cont.. Evaluating MHA: The thymidine content of a MHA should be evaluated-( i ) routinely for every batch of MHA before use, (ii) if the weekly QC test fails for cotrimoxazole . It is performed by testing Enterococcus faecalis ATCC 29212 or33186 with cotrimoxazole disks For a satisfactory MHA medium, a clear distinct inhibition zone of ≥20 mm is obtained. If MHA medium is unsatisfactory, it produces no zone of inhibition, growth within the zone, or a zone of <20mm.

16. Effect of pH of the Medium The MHA medium should have a pH between 7.2 and 7.4 at room temperature. Increase in pH is commonly observed when plates are incubated in a capnophilic environment. ↑pH , ↑ zone diameter of aminoglycoside (P. aeruginosa ), quinolones , clindamnycin (S. aureus ), and macrolide (S. aureus ), giving rise to false susceptible reports. Whereas it reduces the zone diameter of penicillins and tetracycline, leading to false resistant results.

17. Antimicrobial disks Storage of Disks Temperature: The stock disks -stored in a freezer (<-14°C) The working disks can be kept in refrigerator (2-8°C) for a maximum of 1 week. Certain labile disks( eg . imipenem , cefaclor , clavulanate combinations) need to be f rozen(<-l4°C) till the day of use to retain greater stability. Removal: The working disks cartridge or box should be removed from the refrigerator at least 1-2 h before use .

18. Storage of Disks Cont.. Desiccant : once opened, The cartridges should be stored in a tightly sealed desiccated contaier at 2-8°C after use.. Expiry of the disks needs to be checked regularly and the expired disks should always be discarded.

19. Preparing inoculum Fresh colony Single morphotype Method: About 3-5 fresh colonies from a non selective medium such as blood agar are touched to make a direct suspension in sterile normal saline/ pepton water. Turbidity is adjusted to 0.5 McF standard.

20. McFarland Turbidity Standard for Inoculum Preparation   A 0.5 Mcfarland mixer 0.05 ml of 1% barium chloride dihydrate (bacl 2 •2h 2 o), with 9.95 ml of 1% sulfuric acid (H 2 SO 4 ), which is equivalent to approximately 1.5 x 10^8 CFU/ml .

21. Inoculating the test plates 15-15-15 rule Inoculum preparation (i.e. turbidity adjusted) within 15 minutes ↓ Lawn culture of the inoculum on to MHA plate within 15 minutes ↓ Applying the disks on to MHA plate within 15 minutes ↓ Incubating the MHA plate

22. Lawn culture

23. Applying the disks onto MHA plate By Manually with sterile forceps or by using a disk dispenser Pressed: . Small zone disk next to the large zone : (ex erythromycin ↔ amikacin ) Disks should be placed at least 24 mm (center to center) apart on the MHA plate Ordinarily, maximum upto 6 disk on 100 mm plate and maximum upto 12 disk on 150 mm plate Place a new, rather than relocating :

24. Incubating the MHA Plates Within 15 minutes after the disks are placed, plates are inverted (with lid down) and placed inside the aerobic incubator. Temperature: The MHA plates should be incubated at 35C±2°C. When testing for oxacillin , temperature above 35°C may not reliably detect methicillin -resistant S.aureus (MRSA). Duration: For colony KB-DD, an incubation of 16-18 h.

25. Reading plates and interpreting results Growth in AST plates: A Acceptable growth, confluent lawn of growth with zones of inhibition will be uniformly circular.

26. READING PLATES AND INTERPRETING RESULTS cont.. C and D, Unacceptable growth, as individual colonies are apparent and therefore the test must be repeated.

27. The following points are to be kept in mind while measuring the zone diameter of inhibition The zone edge should be read at the point of complete inhibition, as judged by the unaided eye Reading includes the diameter of the disk (6 mm) Measurement is taken preferably by a sliding caliper ( e.g.Vernier caliper) or a ruler. Measurement is taken to the nearest whole milimeter:ex - any zones between 23.0 and 23.5 mm should be taken as 23 mm; Whereas the zones between 23.6 and 23.9 mm should be read as 24 mm.

28. KBDD reading and interpretation Cont.. Holding the plate : The plate is held about 30 cm from the eye and against a black, non reflecting background the plate at a 45° angle to the work bench may facilitate reading when zone edges are difficult to define. The sliding caliper is held on the back of the inverted Petri plate, with lid closéd . Reflected light : Reading of MHA plate is mostly taken under reflected light few drugs/organism for which transmitted light is needed Ex (LZ/ S.aureus.,penicillin / S.aureus )

29. General Rule While Reading KB-DD Plates Colonies within zone : If discrete visible colony within a clear zone of inhibition, then the colony is subjected to identification to check for the purity. If identification is different, then the outer zone should be measured. If identification is found to be same, the test should be repeated with a pure culture. If repeat AST yields similar discrete colonies within the zone of inhibition, then the colony-free inner zone should be measured.

30. General Rule While Reading KB-DD Plates Cont.. Fuzzy zone : If smearing of the colonies is observed by naked eyes inside the zone edge, then only the inner colony-free zone should be measured.

31. COTRIMOXAZOLE For cotrimoxazole : Presence of antagonists in MHA medium may allow some growth within the inhibition Zone surrounding the cotrimoxazole disk. Sight growth (20% of the lawn of growth) should be disregarded( ignored), Ex Enterobacterales , Acinetobacter

32. D -formation Inducible clindamycin resistance : Detected by antagonism of clindamycin activity by a macrolide agent Erythromycin and clindamycin disks are placed adjacent D-shaped flattening of clindamycin zone or colonies inside clindamycin zone of inhibition on the side adjacent to erythromycin is suggestive of a positive test.

33. Quality control The components of a QC program are: Monitoring of reproducibility and accuracy of susceptibility test procedures. Monitoring of the performance of reagents and equipment used in the susceptibility testing. Proficiency testing: Evaluating the performance of persons who carry out the tests and report the results. Taking corrective actions if necessary. Record keeping and maintenance.

34. Quality control cont.. Shared QC Responsibilities Manufacturer and The laboratory

35. The advantages of the Kirby-Bauer method  Easy to perform, Little need for specialized equipment, and The different drugs used in testing can be changed readily.

36. Disadvantage Some bacteria grow poorly or not at all on the media, Minimum inhibitory concentration (MIC) cannot be determined disk diffusion testing is not reliable for daptomycin . . insufficient calcium content reduces zone sizes, whereas high calcium content may increase zone sizes;

37. AST PART II COMING SOON…

38. Penicillin/S. aureus (Zone-Edge test) The interpretation is dependent on the edge of the zone, whether sharp or fuzzy. The test is considered as positive if the zone edge is sharp or like a "cliff" indicating Beta- lactamase production and reported as ‘R’ The test is considered as negative if the zone edge is fuzzy or like a "beach" indicating no B- lactamase production and reported as ‘S’

39. Nitrocefin based test QC Testing for β - Lactamase For both DD and MIC: Positive control: S. aureus ATCC 29213 Negative control: S. aureus ATCC 25923

40. Enterococcus spp. S. aureus ATCC 25923 E. faecalis ATCC 29212 for HLG Haemophilus influernzae and Haemophilus parainfluenzae H. influenzae ATCC 49247 and/or H. influenzae ATCC 49766E. coli ATCC 35218 (when testing amoxicillin- clavulanate ) Streptococcus pneumoniae S. pneumoniae ATCC 49619 Oxacillin disk- S. aureus ATCC 25923 β - Hemoolytic Streptococcus S. pneumoniae ATCC 49619 Viridans Streptococcus Group S. pneumoniae ATCC 49619

41. Routine quality control (QC) strains to be used in daily/weekly plan Organism/Family Recommended quality control strains Enterobacterales E. coli ATCC 25922 P. aeruginosa ATCC 27853 (for carbapenems ) Pseudomonas aeruginosa P aeruginosa ATCC 27853 Acinetobacter spp P. aeruginosa ATCC 27853 E. coli ATCC 25922 (for tetracyclines and cotrimoxazole ) Staphylococcus spp . S. Aureus ATCC 25923