Antibiotic Susceptibility Testing and Data Interpretation

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1 Antibiotic Susceptibility Testing and Data Interpretation Dr Shabbir Simjee Microbiologist Co-Chair CLSI VAST Basingstoke England Bangkok, 7-8 October 2014 For clarity, these are solely my personal views/opinions and do not necessarily represent those of any organization to which I am affiliated.

2 House keeping If I m going too fast.. tell me to slow down If you need me to explain it again.. just ask If you have questions don t wait till the end

3 Agenda 1. Antibiotic Susceptibility Testing Methodology 2. Quality Control and Interpretive Criteria - Does it Matter? 3. Q&A

4 Agenda 1. Antibiotic Susceptibility Testing Methodology 2. Quality Control and Interpretive Criteria - Does it Matter? 3. Q&A

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6 What is AST Data Used For? Reporting S I R for the intention of treatment Will my antibiotic work or not? Looking for resistance trends over time AST data is used as part of the Risk Assessment process Continued AST is needed to ensure implemented Risk Management processes are adequate

7 AST Methods British Society of Antimicrobial Chemotherapy (BSAC) - UK Only European Committee on Antimicrobial Susceptibility Testing (EuCAST) - Human Only Clinical and Laboratory Standards Institute (CLSI) - Human (AST Sub-Committee) - Fungal (AFST Sub-Committee) - Veterinary (VAST Sub-Committee)

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9 CLSI Approved AST Methods 1. Agar Dilution 2. Broth micro/macro dilution 3. Disk diffusion (Not going to cover today)

10 What Are You Measuring? Minimal Inhibitory Concentration (MIC) The lowest concentration of an antimicrobial agent that prevents visible growth (to the naked eye) of a microorganism in an agar or broth dilution susceptibility test.

11 The basic for each method

12 Preparing a standard Turbidity of 0.5 McFarland Isolate bacterium in pure culture Inoculate broth Standardize turbidity 0.5 McFarland

13 0.5 McFarland Water Bacteria OD McFarland Water Bacteria OD You can make the Wickerham card in any way you like with a pattern, or text, for instance. Remember that the number of the McFarland standard unit does not correspond to the OD 600 value. A McFarland standard of 0.5 should have an OD 600 between about 0.08 and 0.1 using a spectrophotometer.

14 Preparing Antibiotic Stock Solutions Either of the following formulas below may be used to determine the amount of powder of diluent needed for a standard solution: or Weight (mg) = Volume (ml) x Concentration ( g/ml) Potency ( g/mg) Volume (ml) = Weight (mg) x Potency ( g/mg) Concentration ( g/ml)

15 Example: To prepare a stock solution containing 1280 g/ml of antimicrobial agent with antimicrobial powder that has a potency of 750 g/mg then 170 to 200 mg of the antimicrobial powder should be accurately weighed. If the actual weight is mg, the volume of solvent needed is then as follows: mg x 750 g/mg Volume (ml) = (Actual Weight) x (Potency) = ml 1280 g/ml (Desired Concentration) Therefore, dissolve the mg of antimicrobial powder in ml of appropriate solvent.

16 Agar Dilution Susceptibility Test An in vitro antimicrobial susceptibility test method conducted using serial concentration of an antimicrobial agent incorporated into an agar growth medium in separate Petri dishes that are inoculated with a bacterial suspension to determine the minimal inhibitory concentration. The method described is as specified by CLSI. Agar is poured to give an approximate depth of 4mm. This corresponds to approximately 60-70ml of medium for a 150mm plate and approximately 25-30ml for a 100mm plate

17 Assuming a stock solution of 5120µg/ml Solution Method of Preparation Antibiotic Concentration (µg/ml) A 2.0 ml stock solution ml SDW 1280 B 3.0 ml Solution A ml SDW 640 C 3.0 ml Solution B ml SDW 320 D 3.0 ml Solution C ml SDW 160 E 3.0 ml Solution D ml SDW 80 F 3.0 ml Solution E ml SDW 40 G 3.0 ml Solution F ml SDW 20 H 3.0 ml Solution G ml SDW 10 I 3.0 ml Solution H ml SDW 5.0 J 3.0 ml Solution I ml SDW 2.5 K 3.0 ml Solution J ml SDW 1.25 L 3.0 ml Solution K ml SDW 0.62

18 2ml sol n. A (1280µg/ml) 2ml sol n. B (640µg/ml) 2ml sol n. C (320µg/ml) 2ml sol n. D (160µg/ml) 2ml sol n. E (80µg/ml) ml agar 18ml agar 18ml agar 18ml agar 18ml agar 128 µg/ml 64 µg/ml 32 µg/ml 16 µg/ml 8 µg/ml

19 Check the ph of each batch of agar when the medium is prepared. The exact method used will depend largely on the type of equipment available in the laboratory. The agar medium should have a ph between 7.2 and 7.4 at room temperature after gelling. If the ph is less than 7.2, certain drugs will appear to lose potency (e.g., aminoglycosides and quinolones), while other agents may appear to have excessive activity (e.g., tetracyclines). If the ph is greater than 7.4, the opposite effects can be expected. Check the ph by one of the following means: Macerate enough agar to submerge the tip of a ph electrode. Allow a small amount of agar to solidify around the tip of a ph electrode in a beaker or cup. Use a surface electrode. ph Check

20 Moisture Check If, just before use, excess surface moisture is present on the plates, place them in an incubator (35 C) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually ten to 30 minutes). The surface of the plate should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the petri dish covers when the plates are inoculated.

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22 Incubate and record MIC

23 Inoculate a control agar plate (no antimicro bial agent) first Then, starting with the lowest concentration, inoculate the plates con taining the different anti micro bial concentra tions Inocu late a second control agar plate last to ensure there was no contam ination or antimi crobial agent carryover during the inocula tion Let the inoculated agar plates remain at room tem perature until the moisture in the inoculum spots is absorbed into the agar; that is, until the spots are dry. Invert the plates and incu bate them at 35 C for 16 to 20 hours.

24 2 µg/ml 4 µg/ml 8 µg/ml

25 Broth Dilution Susceptibility Test An in vitro antimicrobial susceptibility test conducted using serial concentrations of an antimicrobial agent incorporated in liquid nutrient media that are inoculated with a bacterial suspension to determine the minimal inhibitory concentration of an antimicrobial agent. NOTE: When this procedure is carried out in test tubes, it is referred to as broth macrodilution; when performed in microdilution plates, it is called broth microdilution.

26 Assuming a stock solution of 5120µg/ml Solution Method of Preparation Antibiotic Concentration (µg/ml) A 2.0 ml stock solution ml SDW 1280 B 3.0 ml Solution A ml SDW 640 C 3.0 ml Solution B ml SDW 320 D 3.0 ml Solution C ml SDW 160 E 3.0 ml Solution D ml SDW 80 F 3.0 ml Solution E ml SDW 40 G 3.0 ml Solution F ml SDW 20 H 3.0 ml Solution G ml SDW 10 I 3.0 ml Solution H ml SDW 5.0 J 3.0 ml Solution I ml SDW 2.5 K 3.0 ml Solution J ml SDW 1.25 L 3.0 ml Solution K ml SDW 0.62

27 Commercial: - Frozen - Freeze dried 0.1ml sol n. A (1280µg/ml) 0.4ml broth 0.1ml sol n. B (640µg/ml) 0.1ml sol n. C (320µg/ml) 0.1ml sol n. D (160µg/ml) 0.1ml sol n. E (80µg/ml) 0.1ml sol n. F (40µg/ml) 0.1ml sol n. G (20µg/ml) 0.1ml sol n. H (10µg/ml) ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 128µg/ml 64µg/ml 32µg/ml 16µg/ml 8µg/ml 4µg/ml 2µg/ml 1µg/ml

28 Broth Dilution autoinoculator

29 Broth Dilution

30 Drug Broth Dilution Concentration MIC A B C D E F G. 2 Repeat (?) Repeat >64 Repeat H Controls 0.06

31 Agenda 1. Antibiotic Susceptibility Testing Methodology 2. Quality Control and Interpretive Criteria - Does it Matter? 3. Q&A

32 Why use QC strains? QC = Quality Control strains, these can be considered positive controls QC are bacterial isolates that have undergone rigorous testing to ensure that under a standard test system they will always give the same MIC range with a given antibiotic If a QC is out of range it invalidates the AST and indicates there are problems in the method e.g. ph, ion concentrations, temperature etc

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34 What does QC tell us. As long as our QC strains are in range we have a valid test system It does NOT tell us if test bacteria are susceptible or resistant

35 What are interpretive criteria? These are commonly known as breakpoints; S, I, R (Susceptible, Intermediate, Resistant) Susceptible This category implies an infection due to the isolate may be appropriately treated with the dosage regimen of an antimicrobial agent recommended for that type of infection and infecting species, unless otherwise indicated. Intermediate This category implies an infection due to the isolate may be appropriately treated in body sites where the drug are physiologically concentrated or when a high dosage of drug can be used; also indicates a buffer zone that should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretation. Resistant Resistant isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or fall in the range where specific microbial resistance mechanisms are likely, and clinical efficacy has not been reliable in treatment studies.

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38 Questions?