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Detection of ESBLs & AmpC David Livermore Health Protection Agency, Colindale, London.

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Presentation on theme: "Detection of ESBLs & AmpC David Livermore Health Protection Agency, Colindale, London."— Presentation transcript:

1 Detection of ESBLs & AmpC David Livermore Health Protection Agency, Colindale, London

2 Some premises Growing resistance to 3-gen cephs Mostly ESBLs in E. coli & Klebsiella; AmpC in Enterobacter, Citrobacter, Serratia… but not always Identification of mechanism aids –Epidemiological investigation / control –Treatment choice –Recognition of the exceptional e.g. MBLs

3 Resistance to 3 gen cephs; BSAC bacteraemia surveillance (%) From

4 EARSS resistance to 3-gen cephs in E. coli 2001 2006

5 Detecting ESBL producers 2 steps: Screen for resistance with an indicator ceph Do confirmatory test on those found resistant

6 Choice of indicator cephalosporin SensitivitySpecificity Cefotaxime & ceftazidime Good CefpodoximeGoodModerate CefuroximePoor Cephalexin or cephradine ModeratePoor Cefpirome or Cefepime PoorGood

7 Detection of ESBLs: step 2 See Seek ceph/clav synergy in ceph R isolates Double disc Combination disc Etest


9 Combination discs Disc with cephalosporin alone Disc with cephalosporin + clavulanic acid

10 Zone differences (mm), Klebs & E. coli c’pod/clav 10+1  g - c’pod 10  g

11 Etest for ESBLs Cefotaxime + clavulanate

12 Etest for ESBLs Cefotaxime + clavulanate

13 ESBL confirmatory tests ProContra Double disc CheapBest disc spacing varies with strain Combination disc Cheap, sensitive & specific Batch variation; Controls critical Etest Sensitive Internally controlled More expensive; False +ve's with K1 in K. oxytoca

14 Controls for ESBL tests +ve E. coli with TEM-3, -10 & CTX-M-15 available as NCTC 13351, 13352, 13353 No one control is perfect… and these have high levels of enzyme whilst some clinical isolate have very low levels –ve E. coli (e.g. NCTC 10418) Critical for combination discs; should give equal zones irrespective of clavulanate

15 Further investigating ESBLs Multiplex PCR for 5 bla CTX-M groups* TEM & SHV mutants require sequencing Beware…. Isolates may have >1 enzyme –e.g. Classical TEM / SHV + TEM / SHV ESBL –Many with CTX-M-15 also have OXA-1 & TEM-1 Isoelectric focusing gives fullest picture

16 ESBL tests for AmpC inducible species Methods optimised for E. coli & Klebsiella More difficult with Enterobacter –clavulanate induces AmpC; hides ESBL Advice is to do synergy test (NOT SCREEN) with 4 th gen ceph; specificity good, sensitivity moderate BSAC bacteraemia: c. 25% CephR Enterobacter have ESBL, not AmpC….. Probably an underestimate

17 Bacteria not to test for ESBLs Acinetobacters –Often S to clavulanate alone S. maltophilia –+ve result by inhibition of L-2 chromosomal  -lactamase, ubiquitous in the species

18 Suspect derepressed / plasmid AmpC if: Resistant 3-gen cephs, NOT cefepime & cefpirome Resistant to cefoxitin (but more ESBL producers R, too, nowadays) No ceph/clav synergy

19 Geometric mean MICs (mg/L): AmpC producers; 2004 London SE survey E. coli AmpC E. coli ESBL Enterobacter AmpC Enterobacter ESBL Cefotaxime122287249 Ceftazidime18393681 Cefepime0.38390.914.4 Cefpirome0.57532.410 Cefuroxime35>64 Cefoxitin5117>6451 Potz et al., JAC 2006; 58:320-6

20 Some wrinkles… AmpC-derepressed M. morganii are S to pip/tazo AmpC derepressed Serratia are S to ceftazidime Cefoxitin R an unreliable marker for Providencia, Morganella & Serratia spp. –Inducible & derepressed strains may appear I or S AmpC derepressed P. aeruginosa tend to be S to carbenicillin / efflux mutants are R

21 Confirmatory tests for AmpC Seek cefotaxime/cloxacillin synergy –Cefotaxime MIC +100 mg/L cloxacillin –Zones of cefotaxime 30  g discs on agar + 100 mg/L cloxacillin –No agreed interpretive standards Can also use phenylboronic acid as inhibitor

22 Cefotaxime combinations vs. AmpC E. coli: London SE survey Potz et al., JAC 2006; 58:320-6

23 Cefotaxime combinations vs. AmpC Enterobacter: London SE survey Potz et al., JAC 2006; 58:320-6

24 Cefotaxime / cloxacillin tests for AmpC ARMRL- reference control data

25 Phenyl boronic acid for detection of plasmid AmpC Expansion (mm) of FOX 30 zone with 400  g phenyl boronic acid Fold MIC reduction for cefoxitin + 400 mg/L phenyl boronic acid Kleb MOX-112128 E. coli LAT-21264 Kleb DHA-114128 Kleb DHA-21364 E. coli ACC-144 Kleb ACT-11364 ALL ESBL +ve<2<2<2<2 Coudron JCM 2005 43 4163

26 Disc tests for AmpC 60 E. coli & Klebsiella : cefoxitin MICs reduced >4-fold by 100 mg/L cloxacillin % with >5 mm zone expansion Cefoxitin + cloxacillin 100  g 86% Cefoxitin + BZB 64  g 89% Cefpodoxime + BZB 64  g 97% Cefpodoxime + clav + BZB 64  g 100% BZB: benzo(b)thiophene-2-boronic acid Brenwald et al., JAC 2005, 56, 600

27 3-D test for AmpCs Plate seeded with cefoxitin S indicator strain Cut cross in agar, heavily inoculated with test strain Cefoxitin disc Looks for distortion where cross intersects the cefoxitin zone

28 Clover leaf (3 dimensional) test for AmpC Test strain E. cloacae, AmpC derepressed Indicator E. coli NCTC10418 Disc Cefoxitin 30  g

29 Multiplex detection of plasmid AmpC genes Method of Perez-Perez & Hanson JCM 2002, 40, 2153

30 AmpC commercial tests ROSCO- ‘research only’ : high content (500  g) cloxacillin & boronic acid discs for double disc synergy tests AB Biodisk- evaluating double-ended cefotetan or cefoxitin plus cloxacillin or boronic acid ETests

31 Hyperproduction of K1 enzyme Unique to K. oxytoca, chromosomal Indole +ve Klebsiella –R cefuroxime, aztreonam, pip / tazo, ceftriaxone –Borderline (S/I/R) to cefotaxime –S to ceftazidime & carbapenems –Weak cefotaxime or cefepime/clav synergy

32 MICs (mg/L) for multi- resistant UK klebsiellas Example1234 Cefotaxime>64 Ceftazidime>64 CTX/clav>32 CAZ/clav>32 16 Cefepime>64 Cefepime/clav16>32 32 Ertapenem>16 Meropenem816 4 Imipenem2481 >200 isolates; 60 centres; many strains. No imipenem hydrolysis with crude extract Carbapenem resistance not transferable Woodford et al. IJAA 2007 ;29:456-9.

33 Mechanism is combination of porin loss & CTX-M-15 Occasionally selected during therapy Mechanisms of multi- resistant UK klebsiellas OMP Profile S R R R R Woodford et al. IJAA 2007 ;29:456-9

34 Acquired carbapenemases KPC –Class A, 4 variants –Spreading world-wide, 2 cases in UK…. so far –Often clonal, mostly Klebsiella, Enterobacter Metallo’s –Class B, VIM, IMP families, also SPM, SIM, GIM –Scattered, mostly non-fermenters –>100 UK in since 2001, mostly VIM + P. aeruginosa

35 Carbapenemase or not... KPC…. clearest R to ertapenem; no synergy in clavulanate, cloxacillin, boronic acid or EDTA tests –Easy to confuse with combination of ESBL + impermeability Metallo’s…. Suspect if isolate has reduced carbapenem susceptibility, reversed by ESBL… But –Frank carbapenem resistance not always seen –EDTA tests not specific… many false +ves –Spare aztreonam, may be affected by other mechanisms

36 A problem in Bolzano 209 Ceph R Enterobacteriaceae, most had ESBLs 24 lacked ceph / clav synergy- mix of E. coli, K. pneumoniae, K. oxytoca, Citrobacter Imipenem MICs 2- >32 mg/L, mostly 4-8 mg/L –Meropenem, ertapenem MICs lower than imipenem –Imipenem + EDTA MICs 0.12-1 mg/L All had bla VIM ; mix of clonal & non-clonal!!! 19 R to aztreonam--- had CTX-M ----5 susceptible Aschbacher et al, submitted

37 Cica  -Test (Mast) Examine hydrolysis of chromogenic oxyimino ceph, HMRZ-86- yellow to red If +ve, test inhibition IN SEQUENCE by: –Sodium mercaptoacetic acid – MBL –Clavulanic acid – Class A / ESBL –Benzo-thiophene-2-boronic acid – AmpC Count first positive result

38 Cica  -Test (Mast) No inhibitor Mercaptoacetic acid to inhibit MBL Clavulanate to inhibit ESBL Boronic acid to inhibit AmpC

39 Cica  -Test (Mast) blind testing of overnight cultures Mechanism inferred Reference data MBLESBLAmpCMixed Other Pen’ ase No activity MBL (26) 2010230 ESBL (74) 3632600 AmpC (25) 2018320 K. oxytoca, K1 (10) 026200 OXA carbapenemases (10) 0001000 P. aeruginosa OXA ESBLs (4) 130000 KPC/SME carbapenemase (2) 002000 Penicillinase (39) 5310300 Better but slower to use with antibiogram @ 48h Livermore et al., JAC in press.

40 Summary Labs should be able to recognise ESBL producers –Even among Enterobacters –Ref lab will look at difficult cases Labs should be able to recognise AmpC derepressed strains & those with plasmid AmpC Enterobacteriaceae with reduced carbapenem susceptibility need reference investigation New tests being developed

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