Susceptibility Testing Anaerobes Jenny Andrews The BSAC Standardized Method Development Centre Birmingham UK

BSAC Anaerobe Study Gunnar Kahlmeter & Lena Bieber SRGA Jenny Andrews & Becky Walker SMDC

Susceptibility Testing Anaerobes Very difficult, particularly for slow growing organisms CLSI have tried to give recommendations for disc testing, but pilot studies have not been encouraging Now suggest MIC batch testing to see if there are changes in levels of susceptibility

Emergence of resistance in B. fragilis Carbapenem (metallo -lactamase) Metronidazole (nim gene) NB. Resistance is rare to these agents

Is there a need for the BSAC to provide recommendations? Questionnaire sent to approximately 318 taking part in the NEQAS scheme 108 replies (34%) 63 DGH; 16 private laboratories; 11 Teaching Hospitals; 4 Associate Teaching Hospitals; 6 PHLS; 1 Reference Laboratory; 7 other institutions Questionnaire published in the JAC 2002 (A survey of susceptibility testing of anaerobes in the United Kingdom. Andrews & Wise. JAC 2002; 50:757)

Information from the Questionnaire Identification 28% ID anaerobes 45% ID if isolates from blood culture, CSF or sterile site 27% never ID anaerobes Susceptibility testing 6 never 26% metronidazole on the primary isolation plate. No further testing unless R to metronidazole, a pure culture , or from a blood culture, CSF or sterile site

Media used for testing 48% laboratories used ISA supplemented with 5 % defibrinated horse blood and 20 mg/L NAD 21% Fastidious Anaerobe agar 5 % Wilkins & Chalgren agar (medium recommended by the BSAC for MIC determinations)

Method of susceptibility testing 90 % disc testing 15 % used a combination of disc testing and Etest Antibiotics most often tested other than metronidazole: penicillin 91%; clindamycin 48%; erythromycin 44%; co-amoxiclav 44 %; a carbapenem 3%; piperacillin/tazobactam 3%

Reasons for undertaking a BSAC Study Working parties concern over emerging resistance (carbapenems & metronidazole) Laboratories were routinely undertaking disc testing, so the BSAC should try to provide recommendations The Working Party chose 6 antibiotics to be tested based on those generally used for treatment of anaerobic infections Pragmatic decision to use ISA + 5% defibrinated horse blood and 20 mg/L NAD for testing because this was the medium used most often by diagnostic laboratories (data from the questionnaire) Only fast growing anaerobes B. fragilis, B. thetaiotaomicron and C. perfringens to be studied

Technical considerations Media used for testing by diagnostic laboratories: Should media be stored under anaerobic conditions before use? Were results comparable between the two centres undertaking the study: This is important when combining data for analysis

Initial studies by the SRGA & the SMDC Disc testing control strains on media prepared on the day and used immediately; prepared and stored anaerobically until use; media stored at 4-80C for up to 14 days (SRGA) No significant difference Disc testing control strains SRGA & SMDC Within ± 2mm

Materials for disc testing Discs Co-amoxiclav 30 g Clindamycin 2 g Meropenem 10 g Metronidazole 5 g Pip/tazobactam 85 g Penicillin 1 unit Incubation for 18-20 hours 75 B. fragilis 25 B. thetaiotaomicron 50 C. perfringens Controls: ATCC 29741 B. thetaiotaomicron NCTC 9343/ATCC 25285 B. fragilis NCTC 8359/ATCC 12915 C. perfringens ISA + 5% blood + 20 mg/L NAD Prepared in-house bioMérieux pre-poured Oxoid pre-poured

Disc testing Clinical isolates Sweden & SMDC Acceptable ranges for control strains SMDC Media depth of 3.5, 4 and 4.5 mm Pre-poured Oxoid & bioMérieux Tested 5 times on 6 separate occasions

MIC testing SMDC ISA + 5% blood + 20 mg/L NAD Same antibiotics as those chosen for disc testing (antibiotic powders obtained from the pharmaceutical company or Sigma) Tests read after 18-20 h incubation

General comments about disc testing Unable to supply plates to both centres from the same batches Organisms grew less well on the pre-poured plates from Oxoid & bioMérieux Zones for metronidazole and clindamycin difficult to measure

Data analysis Identification of the `wild sensitive’ population used to suggest zone diameter BPs EUCAST clinical MIC BPs (where available) or BSAC MIC BPs If available clinical response data (to be discussed by Robin Howe)

Combined SRGA & SMDC data for C. perfringens: penicillin 1 unit disc NB B. fragilis and thetaiotaomicron resistant to penicillin

Co-amoxiclav 30 g disc Susceptible MIC BP 8 mg/L ZD BP 28 mm for B. fragilis & C. perfringens only. For B. thetaiotaomicron a poor relationship between MIC and zone diameter

Piperacillin/tazobactam 75/10 g disc Susceptible MIC BP 16 mg/L ZD BP of 27 mm for C. perfringens & B. Fragilis only For B. thetaiotaomicron , unacceptable merging of S & R populations

Meropenem 10 g disc I I EUCAST MIC BP 2/8 mg/L 26 mm = S; 19-25 = I; 18 = R Carbapenem resistant B. fragilis (due to the presence of metallo-β-lactamase) have no zone of inhibition

Clindamycin 2 g disc Susceptible MIC BP 4 mg/L ZD BP 10 mm B. thetaiotaomicron

Metronidazole 5 g disc Recommendations have been on the website since January 2006 nim+ ve MIC BP 8 mg/L ZD BP 18 mm

QC Acceptable range (mm) Antibiotic Disc content (g unless stated) B. fragilis NCTC 9343 B. thetaiotaomicron ATCC 29741 C. Perfringens NCTC 8359 Clindamycin 2 13-27 11-25 23-28 Co-amoxiclav 30 43-49 40-45 Meropenem 10 42-50 36-43 39-45 Penicillin 1 unit 6 26-30 Piperacillin/tazobactam 75/10 41-48 29-34 37-43 NB. Metronidazole ranges available on the website since January 2006

Comments 1 From these data it would appear that supplemented ISA media can be used for testing fast growing anaerobes Media does not have to be stored under anaerobic conditions before use, but can be stored in the refrigerator at 4-80C Difficult group of organisms to test Only fast growing anaerobes B. fragilis, B. thetaiotaomicron & C. perfringens studied Zones for clindamycin and metronidazole are more difficult to measure than for the other antibiotics tested

Comments 2 Unable to give recommendations for B. thetaiotaomicron when testing inhibitor combinations because of the poor relationship between MIC and zone of inhibition Organisms resistant by disc testing should have resistance confirmed by an MIC For debate: Should organisms considered resistant be sent to the Anaerobe Reference Laboratory for further investigation? Look forward to comments from users