Detection of ESBLs & AmpC

David Livermore

Health Protection Agency,

Colindale, London

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

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

0

10

20

30

40

50

2001 2002 2003 2004 2005 2006

E. coli Klebsiella Enterobacter

From http://www.bsacsurv.org

EARSS resistance to 3-gen cephs in E. coli

2001 2006

http://www.earss.rivm.nl

Detecting ESBL producers

2 steps:• Screen for resistance with an indicator ceph

• Do confirmatory test on those found resistant

Choice of indicator cephalosporin

Sensitivity Specificity

Cefotaxime & ceftazidime

Good Good

Cefpodoxime Good Moderate

Cefuroxime Poor Poor

Cephalexin or cephradine

Moderate Poor

Cefpirome or Cefepime

Poor Good

Detection of ESBLs: step 2

See http://www.hpa.org.uk

Seek ceph/clav synergy in ceph R isolates

•Double disc

•Combination disc

•Etest

Combination discs

Disc with cephalosporin

alone

Disc with cephalosporin + clavulanic

acid

0

10

20

30

40

50

60

-3 -1 1 3 5 7 9 11 13 15 17 19 21 23 25

Control AmpC K1 ESBL CTX-M

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

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

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

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

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

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

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

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

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 39 0.91 4.4

Cefpirome 0.57 53 2.4 10

Cefuroxime 35 >64 >64 >64

Cefoxitin 51 17 >64 51

Potz et al., JAC 2006; 58:320-6

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

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

Cefotaxime combinations vs. AmpC E. coli: London SE survey

0

5

10

15

20

25

30

<=0.

120.

25 0.5 1 2 4 8 16 32 64 >64

MIC (mg/L)

Alone

+Clavulanate, 4 mg/L

+Cloxacillin 100 mg/L

Potz et al., JAC 2006; 58:320-6

Cefotaxime combinations vs. AmpC Enterobacter: London SE survey

0

10

20

30

40

50

60

70

<=0.

120.

25 0.5 1 2 4 8 16 32 64 >64

MIC (mg/L)

Alone

+Clavulanate, 4 mg/L

+Cloxacillin 100 mg/L

Potz et al., JAC 2006; 58:320-6

Cefotaxime / cloxacillin tests for AmpC

1

10

100

1000

E. cloacae 684-con P. aeruginosa 2297-con

MIC

(m

g/L

)

Cefotaxime

Cefotaxime +cloxacillin

ARMRL- reference control data

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-1 12 128

E. coli LAT-2 12 64

Kleb DHA-1 14 128

Kleb DHA-2 13 64

E. coli ACC-1 4 4

Kleb ACT-1 13 64

ALL ESBL +ve <2 <2

Coudron JCM 2005 43 4163

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

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

Clover leaf (3 dimensional) test for AmpC

Test strainE. cloacae,

AmpC derepressed

IndicatorE. coli

NCTC10418

DiscCefoxitin 30 g

Multiplex detection of plasmid AmpC genes

Method of Perez-Perez & Hanson JCM 2002, 40, 2153

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

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

MICs (mg/L) for multi-resistant UK klebsiellas

Example 1 2 3 4

Cefotaxime >64 >64 >64 >64

Ceftazidime >64 >64 >64 >64

CTX/clav >32 >32 >32 >32

CAZ/clav >32 >32 >32 16

Cefepime >64 >64 >64 >64

Cefepime/clav 16 >32 >32 32

Ertapenem >16 >16 >16 >16

Meropenem 8 16 16 4

Imipenem 2 4 8 1

• >200 isolates; 60 centres; many strains.

• No imipenem hydrolysis with crude extract

• Carbapenem resistance not transferable

Woodford et al. IJAA 2007 ;29:456-9.

• 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

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

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

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 blaVIM; mix of clonal & non-clonal!!!

• 19 R to aztreonam--- had CTX-M ----5 susceptible

Aschbacher et al, submitted

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

Cica -Test (Mast)

No inhibitor

Mercaptoacetic acid to inhibit MBL

Clavulanate to inhibit ESBL

Boronic acid to inhibit AmpC

Cica -Test (Mast) blind testingof overnight cultures

Mechanism inferred

Reference dataMBL ESBL AmpC Mixed

OtherPen’ase

No activity

MBL (26) 20 1 0 2 3 0

ESBL (74) 3 63 2 6 0 0

AmpC (25) 2 0 18 3 2 0

K. oxytoca, K1 (10) 0 2 6 2 0 0

OXA carbapenemases (10) 0 0 0 10 0 0

P. aeruginosa OXA ESBLs (4) 1 3 0 0 0 0

KPC/SME carbapenemase (2) 0 0 2 0 0 0

Penicillinase (39) 5 3 1 0 30 0

Better but slower to use with antibiogram @ 48h

Livermore et al., JAC in press.

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