BSAC Susceptibility Testing Residential Workshop

Transcription

1 BSAC Susceptibility Testing Residential Workshop Susceptibility Testing Methodology Insert name of presentation on Master Slide Mandy Wootton

2 How to determine susceptibility/resistance Minimum Inhibitory Concentration Macro broth dilution Micro broth dilution Agar dilution Gradient Strips Automated systems Category (S/I/R) Disc Breakpoint method

3 Macrobroth dilution (tube dilution)

4 Macrobroth dilution (tube dilution) Broth media (1-2mL) Antimicrobial dilution: log2 Control tube Inoculum; direct or growth touching 2 to 5 morphologically similar colonies 0.5 McFarland (10 8 ); dilute to achieve 10 5 Control organisms Incubation Reading Minimum Inhibitory Concentration

5 Microbroth dilution Gold Standard

6 Standardisation Critical population of bacterial cells Known inoculum Defined media Defined media Batch variation ph Gaseous environment Drug Streptomycin Gentamicin Tobramycin Amikacin Increased - Acidic Increased - Alkaline Decreased - Acidic Amoxicillin Amikacin Azithromycin Ampicillin Clindamycin Clindamycin Cloxacillin Metronidazole Metronidazole Doxycycline Minocycline Nitrofurantoin Piperacillin Tetracycline Clarithromycin Erythromycin Nalidixic acid Quinolones Tobramycin

7 Standardisation Defined media cont. Cation concentration (Mg 2+ & Ca 2+) tetracycline, aminoglycoside, daptomycin Osmolarity (NaCl) - aminoglycoside Thymine/thymidine - Trim/Co-trim Incubation temperature / time Enterococci & glycopeptides Staphylococci & cefoxitin Standardised methods need to be performed without alteration or modifications

8 Agar dilution Agar media (20mL) Antimicrobial dilution: log2 Range 0.008mg/L to 128mg/L Control plate Inoculum; direct or growth touching 2 to 5 morphologically similar colonies 0.5 McFarland (10 8 ); dilute to achieve 10 7 Control organism

9 Agar dilution Inoculating plates Inoculate 1uL onto plates (equivalent to 10 4 ) Reading Growth on control plate Check control organisms MIC values: 1= >128 mg/l, 2 = 0.5 mg/l, 3 = 0.25 mg/l, 4 = 0.12 mg/l

10 Gradient strips Validated against MBD Easy to use Variety of manufacturers & antimicrobials Always use media recommended by manufacturer QC - more difficult than AD/MBD Reading

11 Automated AST systems More reliable/consistent results Closer to standard Reduced scope for error Reproducible Easy to use Speed of results hrs for ID, 4-7hrs for AST Gram neg, Gram pos, Yeast panels Large range of antimicrobials

12 Automated AST systems ID linked to AST EUCAST/BSAC/CLSI Algorithms for resistance Expert rules Linked to LIMS Staff mix & lab workflow Costly Other methods to fill the gaps Fastidious organisms Large labs, put all isolates through? Quick AST results not always useful BPs updated promptly

13 Agar incorporation Breakpoint method Agar incorporation method Antimicrobials: in-house or commercial tabs Plates either side of breakpoints Inoculum 10 4 per spot Control plate + 2 concentrations Reading: manual or reader Comparison of growth on plates Cheap & easy to perform No MIC generated Not widely used now Amikacin vs. Enterobactericeae EUCAST BP: S R> mg/L 32mg/L

14 Disc diffusion method Most used method Cheap & easy to perform No MIC, just category Correlated against MIC method Zone diameter governed by three dynamics Zone of inhibition formation: Critical concentration: conc n just capable of inhibiting growth & conc n at zone edge at critical time Critical time: time it takes for critical conc n to be reached Critical population: Number of bacterial cells found at the critical time at the ultimate zone edge

15 Disc diffusion method - Dynamics Critical concentration Antimicrobial diffuses in a decreasing gradient Diffusion rate Depth of agar Osmolarity of agar Initial concentration Molecular size Shape of drug Charge of drug Growth of bacteria Diffusion of drug Zone of inhibition formation: Critical concentration: conc n just capable of inhibiting growth & conc n at zone edge at critical time Critical time: time it takes for critical conc n to be reached Critical population: Number of bacterial cells found at the critical time at the ultimate zone edge

16 Disc diffusion method - Dynamics Critical time 3 to 4 hours under standard conditions Standard: observable growth at 18hrs Longer for some bug:drug combinations Standardisation Apply discs within 15-30mins after inoculation Incubate 15-30mins after application of discs Zone of inhibition formation: Critical concentration: conc n just capable of inhibiting growth & conc n at zone edge at critical time Critical time: time it takes for critical conc n to be reached Critical population: Number of bacterial cells found at the critical time at the ultimate zone edge Growth of bacteria Diffusion of drug

17 Disc diffusion method - Dynamics Critical population Known inoculum Too high: quicker to critical population, overwhelms antimicrobial so smaller zone (FALSE RESISTANCE) Too low: slower to critical population, antimicrobial overwhelms bacteria (FALSE SUSCEPTIBILITY) Known lag phase & generation times Most antimicrobial work on dividing cells Growth of bacteria Diffusion of drug Zone of inhibition formation: Critical concentration: conc n just capable of inhibiting growth & conc n at zone edge at critical time Critical time: time it takes for critical conc n to be reached Critical population: Number of bacterial cells found at the critical time at the ultimate zone edge

18 Disc diffusion method - standardisation STANDARDISATION is IMPORTANT Growth of bacteria Diffusion of drug Zone of inhibition formation: Critical concentration: conc n just capable of inhibiting growth & conc n at zone edge at critical time Critical time: time it takes for critical conc n to be reached Critical population: Number of bacterial cells found at the critical time at the ultimate zone edge

19 Disc diffusion method correlation with MIC

20 Disc diffusion method correlation with MIC

21 Disc diffusion method correlation with MIC

22 Disc diffusion method correlation with MIC

23 Disc diffusion method correlation with MIC

24 Disc diffusion method correlation with MIC

25 Disc diffusion method (BSAC) Antimicrobial in paper disc IsoSensitest agar or ISA + 5% sheep blood + NAD Semi confluent inoculum by direct method Touching 2-5 morphologically similar colonies Dilute Dilute Dilute 0.5 McFarland Application of discs 15 minute rule No dilution 1:100 1:10 -Haemolytic streptococci Staphylococci Neisseria gonorrhoeae Enterococci Serratia spp. Campylobacter spp. Enterobacteriaceae Streptococcus pneumoniae Pseudomonas spp. Neisseria meningitidis Stenotrophomonas maltophilia Moraxella catarrhalis Acinetobacter spp. -haemolytic streptococci Haemophilus spp. Clostridium perfringens Pasteurella multocida Coryneform organisms Bacteroides fragilis Bacteroides thetaiotaomicron

26 Disc diffusion method (BSAC) 18-20hr at C in air VAN/TEIC resistance in enterococci: 24hr FOX resistance in Staphylococci: 35±1 C Campylobacter: 42 C microaerophilic for 24hr Fastidious orgs: 4-6% CO 2 Anaerobes: 10% hydrogen/10%co 2 /80% Nitrogen Reading

27 Disc diffusion method (EUCAST) Antimicrobial in paper disc Some conc n are different: Mueller Hinton agar (Enterobacteriaeae, Pseudomonas, staphylococci & enterococci) MH-F (5% defibrinated horse blood + NAD) (S. pneumoniae, streptococci, Haemophilus, Moraxella, Pasteurella, Listeria, Campylobacter & Corynebacterium)

28 Disc diffusion method (EUCAST) Confluent inoculum by direct method Touching 2-5 morphologically similar colonies Suspension equivalent to 0.5 McFarland in 0.85% saline Inoculation should result in confluent growth without being too heavy Application of discs 15 minute rule (use inoculum within 15m & always within 60m. Apply discs within 15m, incubate plates within 15m) Incubation 16-20h at 35±1 C MH plates in air MH-F plates in 5% CO 2

29 Disc diffusion method (EUCAST) Plates should look like this....and NOT like this!

30 EUCAST - reading zones MH plates: Read zones from the back of the plate against a dark background and illuminated with reflected light MH-F plates Read zones from the front with lid removed and illuminated with reflected light