Susceptibility Testing Susceptibility Testing AnaerobesAnaerobes

Jenny AndrewsJenny AndrewsThe BSAC Standardized Method The BSAC Standardized Method

Development CentreDevelopment CentreBirmingham UKBirmingham UK

BSAC Anaerobe StudyBSAC Anaerobe Study

Gunnar Kahlmeter & Lena Bieber Gunnar Kahlmeter & Lena Bieber SRGA SRGA

Jenny Andrews & Becky WalkerJenny Andrews & Becky Walker

SMDCSMDC

Susceptibility Testing AnaerobesSusceptibility Testing Anaerobes

Very difficult, particularly for slow growing Very difficult, particularly for slow growing organismsorganisms

CLSI have tried to give recommendations CLSI have tried to give recommendations for disc testing, but pilot studies have not for disc testing, but pilot studies have not been encouragingbeen encouraging

Now suggest MIC batch testing to see if Now suggest MIC batch testing to see if there are changes in levels of there are changes in levels of susceptibilitysusceptibility

Emergence of resistance in Emergence of resistance in B. fragilisB. fragilis

Carbapenem Carbapenem

(metallo (metallo -lactamase)-lactamase)

Metronidazole Metronidazole (nim gene)(nim gene)

NB. Resistance is rare to these agents

Is there a need for the BSAC to Is there a need for the BSAC to provide recommendations?provide recommendations?

Questionnaire sent to approximately 318 taking Questionnaire sent to approximately 318 taking part in the NEQAS schemepart in the NEQAS scheme108 replies (34%)108 replies (34%)63 DGH; 16 private laboratories; 11 Teaching 63 DGH; 16 private laboratories; 11 Teaching Hospitals; 4 Associate Teaching Hospitals; 6 Hospitals; 4 Associate Teaching Hospitals; 6 PHLS; 1 Reference Laboratory; 7 other PHLS; 1 Reference Laboratory; 7 other institutions institutions Questionnaire published in the JAC 2002 Questionnaire published in the JAC 2002 (A survey of susceptibility testing of anaerobes (A survey of susceptibility testing of anaerobes in the United Kingdom. Andrews & Wise. JAC in the United Kingdom. Andrews & Wise. JAC 2002; 2002; 5050:757):757)

Information from the QuestionnaireInformation from the Questionnaire

IdentificationIdentification28% ID anaerobes28% ID anaerobes45% ID if isolates 45% ID if isolates from blood culture, from blood culture, CSF or sterile siteCSF or sterile site27% never ID 27% never ID anaerobesanaerobes

Susceptibility testingSusceptibility testing6 never6 never26% metronidazole 26% metronidazole on the primary on the primary isolation plate. No isolation plate. No further testing unless further testing unless R to metronidazole, a R to metronidazole, a pure culture , or from pure culture , or from a blood culture, CSF a blood culture, CSF or sterile siteor sterile site

Media used for testingMedia used for testing

48% laboratories used ISA supplemented 48% laboratories used ISA supplemented with 5 % defibrinated horse blood and 20 with 5 % defibrinated horse blood and 20 mg/L NADmg/L NAD

21% Fastidious Anaerobe agar21% Fastidious Anaerobe agar

5 % Wilkins & Chalgren agar (medium 5 % Wilkins & Chalgren agar (medium recommended by the BSAC for MIC recommended by the BSAC for MIC determinations) determinations)

Method of susceptibility testingMethod of susceptibility testing

90 % disc testing90 % disc testing15 % used a combination of disc testing 15 % used a combination of disc testing and Etestand EtestAntibiotics most often tested other than Antibiotics most often tested other than metronidazole:metronidazole:penicillin 91%; clindamycin 48%; penicillin 91%; clindamycin 48%; erythromycin 44%; co-amoxiclav 44 %; a erythromycin 44%; co-amoxiclav 44 %; a carbapenem 3%; piperacillin/tazobactam carbapenem 3%; piperacillin/tazobactam 3%3%

Reasons for undertaking a Reasons for undertaking a BSAC StudyBSAC Study

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

Technical considerationsTechnical considerations

Media used for testing by diagnostic Media used for testing by diagnostic laboratories:laboratories:

Should media be stored under Should media be stored under anaerobic conditions before use?anaerobic conditions before use?

Were results comparable between the two Were results comparable between the two centres undertaking the study:centres undertaking the study:

This is important when combining data This is important when combining data for analysisfor analysis

Initial studies by the Initial studies by the SRGA & the SMDCSRGA & the SMDC

Disc testing control strains on media Disc testing control strains on media prepared on the day and used prepared on the day and used immediately; prepared and stored immediately; prepared and stored anaerobically until use; media stored at anaerobically until use; media stored at 4-84-800C for up to 14 days (SRGA)C for up to 14 days (SRGA)

No significant differenceNo significant differenceDisc testing control strains SRGA & SMDCDisc testing control strains SRGA & SMDC

Within Within ± 2mm± 2mm

Materials for disc testingMaterials for disc testing

75 75 B. fragilisB. fragilis

25 25 B. thetaiotaomicronB. thetaiotaomicron

50 50 C. perfringensC. perfringens

Controls:Controls:ATCC 29741ATCC 29741 B. thetaiotaomicronB. thetaiotaomicron

NCTC 9343/ATCC 25285NCTC 9343/ATCC 25285 B. fragilisB. fragilis

NCTC 8359/ATCC 12915NCTC 8359/ATCC 12915 C. perfringensC. perfringens

ISA + 5% blood + 20 mg/L NADISA + 5% blood + 20 mg/L NAD

Prepared in-housePrepared in-house

bioMérieux pre-pouredbioMérieux pre-poured

Oxoid pre-pouredOxoid pre-poured

DiscsDiscsCo-amoxiclav Co-amoxiclav 30 30 gg

Clindamycin Clindamycin 2 2 gg

Meropenem Meropenem 10 10 gg

Metronidazole Metronidazole 5 5 gg

Pip/tazobactam 85 Pip/tazobactam 85 gg

PenicillinPenicillin 1 unit1 unit

Incubation for 18-20 hoursIncubation for 18-20 hours

Disc testingDisc testing

Clinical isolatesClinical isolates

Sweden & SMDC Sweden & SMDC

Acceptable ranges for control strainsAcceptable ranges for control strains

SMDCSMDC

Media depth of 3.5, 4 and 4.5 mmMedia depth of 3.5, 4 and 4.5 mm

Pre-poured Oxoid & bioMérieuxPre-poured Oxoid & bioMérieux

Tested 5 times on 6 separate occasionsTested 5 times on 6 separate occasions

MIC testingMIC testing

SMDCSMDC

ISA + 5% blood + 20 mg/L NADISA + 5% blood + 20 mg/L NAD

Same antibiotics as those chosen for Same antibiotics as those chosen for disc testing (antibiotic powders disc testing (antibiotic powders obtained from the pharmaceutical obtained from the pharmaceutical company or Sigma)company or Sigma)

Tests read after 18-20 h incubationTests read after 18-20 h incubation

General comments about disc General comments about disc testingtesting

Unable to supply plates to both centres Unable to supply plates to both centres from the same batchesfrom the same batches

Organisms grew less well on the pre-Organisms grew less well on the pre-poured plates from Oxoid & bioMérieuxpoured plates from Oxoid & bioMérieux

Zones for metronidazole and Zones for metronidazole and clindamycin difficult to measureclindamycin difficult to measure

Data analysisData analysis

Identification of the `wild sensitive’ Identification of the `wild sensitive’ population used to suggest zone diameter population used to suggest zone diameter BPsBPs

EUCAST clinical MIC BPs (where EUCAST clinical MIC BPs (where available) or BSAC MIC BPs available) or BSAC MIC BPs

If available clinical response data If available clinical response data (to be discussed by Robin Howe) (to be discussed by Robin Howe)

Combined SRGA & SMDC data for Combined SRGA & SMDC data for C. perfringensC. perfringens: : penicillin 1 unit discpenicillin 1 unit disc

0

5

10

15

20

25

30

35

40

45

50

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Zone diameter (mm)

Susceptible MIC BP of 0.12 mg/L used to analyse the dataAll MICs less than 0.12 mg/L

NB B. fragilis and thetaiotaomicron resistant to penicillin

Co-amoxiclav 30 Co-amoxiclav 30 g discg disc

0

10

20

30

40

50

60

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Zone diameter (mm)

C. perfringens

0

10

20

30

40

50

60

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Zone diameter (mm)

B. fragilis

0

2

4

6

8

10

12

14

16

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Zone diameter (mm)

Nu

mb

er

B. thetaiotaomicron

•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 Piperacillin/tazobactam 75/10 75/10 g discg disc

0

10

20

30

40

50

60

70

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Zone diameter (mm)

Nu

mb

er

B. fragilis

0

2

4

6

8

10

12

14

16

18

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Zone diameter (mm)

Nu

mb

er

B. thetaiotaomicron

0

10

20

30

40

50

60

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Zone diameter (mm)

C. perfringens

•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 Meropenem 10 g discg disc

0

10

20

30

40

50

60

70

80

90

100

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Zone diameter (mm)

Nu

mb

er

B. fragilis

I

0

2

4

6

8

10

12

14

16

18

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Zone diameter (mm)

Nu

mb

er

B. thetaiotaomicron

0

10

20

30

40

50

60

70

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Zone diameter (mm)

C. perfringens

•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

I

Clindamycin 2 Clindamycin 2 g discg disc

0

5

10

15

20

25

30

35

40

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Zone diameter (mm)

Nu

mb

er

B. fragilis

0

5

10

15

20

25

30

35

40

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Zone diameter (mm)

C. perfringens

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

0

5

10

15

20

25

30

35

40

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Zone diameter (mm)

Nu

mb

er

B. thetaiotaomicron

Metronidazole 5 Metronidazole 5 g discg discRecommendations have been on the website since January 2006Recommendations have been on the website since January 2006

0

10

20

30

40

50

60

70

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Zone diameter (mm)

Nu

mb

er

B. fragilis

nim+ ve

0

5

10

15

20

25

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Zone diameter (mm)

Nu

mb

er

B. thetaiotaomicron

0

5

10

15

20

25

30

35

40

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Zone diameter (mm)

C. perfringens

MIC BP 8 mg/LZD BP 18 mm

QC QC Acceptable range (mm)Acceptable range (mm)

AntibioticAntibiotic Disc contentDisc content

((g unless g unless stated)stated)

B. fragilisB. fragilis

NCTC 9343NCTC 9343

B. thetaiotaomicronB. thetaiotaomicron

ATCC 29741ATCC 29741

C. PerfringensC. Perfringens

NCTC 8359NCTC 8359

ClindamycinClindamycin 22 13-2713-27 11-2511-25 23-2823-28

Co-amoxiclavCo-amoxiclav 3030 43-4943-49 40-4540-45 40-4540-45

MeropenemMeropenem 1010 42-5042-50 36-4336-43 39-4539-45

PenicillinPenicillin 1 unit1 unit 66 66 26-3026-30

Piperacillin/Piperacillin/tazobactamtazobactam

75/1075/10 41-4841-48 29-3429-34 37-4337-43

NB. Metronidazole ranges available on the website since January 2006

Comments 1Comments 1

From these data it would appear that From these data it would appear that supplemented ISA media can be used for testing supplemented ISA media can be used for testing fast growing anaerobesfast growing anaerobesMedia does not have to be stored under Media does not have to be stored under anaerobic conditions before use, but can be anaerobic conditions before use, but can be stored in the refrigerator at 4-8stored in the refrigerator at 4-800C C Difficult group of organisms to testDifficult group of organisms to testOnly fast growing anaerobes Only fast growing anaerobes B. fragilisB. fragilis, , B. thetaiotaomicronB. thetaiotaomicron & & C. perfringensC. perfringens studied studiedZones for clindamycin and metronidazole are Zones for clindamycin and metronidazole are more difficult to measure than for the other more difficult to measure than for the other antibiotics testedantibiotics tested

Comments 2Comments 2

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