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
Sensitivity
Specificity
Cefotaxime & ceftazidime
Good
Cefpodoxime
Moderate
Cefuroxime
Poor
Cephalexin or cephradine
Cefpirome or Cefepime

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

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

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

11. Etest for ESBLs
Cefotaxime
Cefotaxime +
clavulanate

12. Etest for ESBLs
Cefotaxime
Cefotaxime +
clavulanate

13. ESBL confirmatory tests
Pro
Contra
Double disc
Cheap
Best 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 blaCTX-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 4th 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 b-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
Cefotaxime 12
228 72 49
Ceftazidime 18 39 36 81
Cefepime
0.38
0.91
4.4
Cefpirome
0.57 53
2.4 10
Cefuroxime 35
>64
Cefoxitin 51 17
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 mg discs on agar 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 mg phenyl boronic acid
Fold MIC reduction for cefoxitin mg/L phenyl boronic acid
Kleb MOX-1 12
128
E. coli LAT-2 64
Kleb DHA-1 14
Kleb DHA-2 13
E. coli ACC-1 4
Kleb ACT-1
ALL ESBL +ve
<2
Coudron JCM

26. BZB: benzo(b)thiophene-2-boronic acid
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 mg
86%
Cefoxitin + BZB 64 mg
89%
Cefpodoxime + BZB 64 mg
97%
Cefpodoxime + clav + BZB 64 mg
100%
BZB: benzo(b)thiophene-2-boronic acid
Brenwald et al., JAC 2005, 56, 600

27. Looks for distortion where cross intersects the cefoxitin zone
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 mg

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 mg) 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
Example 1 2 3 4
Cefotaxime
>64
Ceftazidime
CTX/clav
>32
CAZ/clav 16
Cefepime
Cefepime/clav 32
Ertapenem
>16
Meropenem 8
Imipenem
>200 isolates; 60 centres; many strains.
No imipenem hydrolysis with crude extract
Carbapenem resistance not transferable
In the last year we have had 21 isolates from 12 centres referred to us with a characteristic antibiogram. They were referred on the basis of meropenem resistance for carbapenemase investigation. MIC’s showed a pattern whereby the ertapenem MIC was greater than the meropenem MIC which was greater than the imipenem MIC which tended to be around the breakpoint. The isolates were resistant to all antibioticss tested, including the beta-lactamase inhibitors with the exception of colistin and in some cases tigecycline.
We found the isolates to be mostly unrelated by PFGE with small strain groups from single centres. No imipenem hydrolysis was evident using crude cell extract and carbapenem resistance was not transferable. We found the isolates to be negative for every carbapenemase gene tested.
Woodford et al. IJAA 2007 ;29:456-9.

33. Mechanisms of multi-resistant UK klebsiellas
OMP Profile
S R R R R
Mechanism is combination of porin loss & CTX-M-15
Occasionally selected during therapy
Further characterisation showed that the isolates carried CTX-M and OXA-1-like genes and showed loss of at least one major outer membrane protein.
Strains all carry at least one plasmid of >100Kb.
IEF showed bands corresponding to CTX-M-15 and OXA-1-like with some also having a TEM enzyme, these results were confirmed by PCR.
PCR mapping of the OmpK genes showed disruption of one or more of OmpK35, 36 and 37 and sequenced examples showed the disruption to be caused by insertion of an IS element into the gene. Other examples showed truncation due to a point mutation
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 mg/L
All had blaVIM; mix of clonal & non-clonal!!!
19 R to aztreonam--- had CTX-M susceptible
Aschbacher et al, submitted

37. Cica b-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 b-Test (Mast) No inhibitor Mercaptoacetic acid to inhibit MBL
Clavulanate to inhibit ESBL
Boronic acid to inhibit AmpC

39. Cica b-Test (Mast) blind testing of overnight cultures
Mechanism inferred
Reference data
MBL
ESBL
AmpC
Mixed Other
Pen’
ase
No activity
MBL (26) 20 1 2 3
ESBL (74) 63 6
AmpC (25) 18
K. oxytoca, K1 (10)
OXA carbapenemases (10) 10
P. aeruginosa OXA ESBLs (4)
KPC/SME carbapenemase (2)
Penicillinase (39) 5 30
Better but slower to use with 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