Susceptibility Testing Anaerobes

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Susceptibility Testing Anaerobes

• Jenny Andrews
• The BSAC Standardized Method Development Centre
• Birmingham UK

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

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Emergence of resistance in B. fragilis

• Carbapenem
• (metallo ?-lactamase)
• Metronidazole (nim gene)

NB. Resistance is rare to these agents
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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
  50757)

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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

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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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)

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

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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

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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-ß-lactamase) have no zone of
• inhibition

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Clindamycin 2 ?g disc

• Susceptible MIC BP 4 mg/L
• ZD BP 10 mm

B. thetaiotaomicron
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Metronidazole 5 ?g discRecommendations have been
on the website since January 2006
nim ve
MIC BP 8 mg/L ZD BP 18 mm
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QC
NB. Metronidazole ranges available on the website
since January 2006
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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

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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