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 (%)
50
E. coli
Klebsiella
Enterobacter
40
30
20
10
0
2001
2002
2003
2004
2005
2006
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
Cefpodoxime
Good
Good
Good
Moderate
Cefuroxime
Poor
Poor
Cephalexin or
cephradine
Cefpirome or
Cefepime
Moderate
Poor
Poor
Good
Detection of ESBLs: step 2
Seek ceph/clav synergy in ceph R isolates
• Double disc
• Combination disc
• Etest
See http://www.hpa.org.uk
Combination discs
Disc with
cephalosporin
+ clavulanic
acid
Disc with
cephalosporin
alone
Zone differences (mm), Klebs & E. coli
c’pod/clav 10+1 mg - c’pod 10 mg
60
Control
AmpC
K1
ESBL
CTX-M
50
40
30
20
10
25
23
21
19
17
15
13
11
9
7
5
3
1
-1
-3
0
Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
ESBL confirmatory tests
Double disc
Combination
disc
Etest
Pro
Contra
Cheap
Best disc spacing
varies with strain
Cheap, sensitive &
specific
Batch variation;
Sensitive
More expensive;
False +ve's with
K1 in K. oxytoca
Internally controlled
Controls critical
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
b-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 Enterobacter
AmpC
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 mg 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
30
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
25
20
15
10
5
4
>6
64
32
16
8
4
2
1
5
0.
25
0.
<=
0.
12
0
MIC (mg/L)
Potz et al., JAC 2006; 58:320-6
Cefotaxime combinations vs. AmpC
Enterobacter: London SE survey
70
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
60
50
40
30
20
10
4
>6
64
32
16
8
4
2
1
5
0.
25
0.
<=
0.
12
0
MIC (mg/L)
Potz et al., JAC 2006; 58:320-6
Cefotaxime / cloxacillin
tests for AmpC
1000
MIC (mg/L)
Cefotaxime
100
Cefotaxime +
cloxacillin
10
1
E. cloacae 684-con
P. aeruginosa 2297-con
ARMRL- reference control data
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 + 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 % with >5 mm
reduced >4-fold by 100 mg/L cloxacillin 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
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 strain
E. cloacae,
AmpC
derepressed
Indicator
E. coli
NCTC10418
Disc
Cefoxitin 30 mg
Multiplex detection of
plasmid AmpC genes
Method of Perez-Perez & Hanson JCM 2002, 40, 2153
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
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 multiresistant 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.
Mechanisms of multiresistant UK klebsiellas
OMP Profile
S R
R
R
R
• Mechanism is
combination of porin
loss & CTX-M-15
• Occasionally selected
during therapy
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 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
Cica b-Test (Mast)
No inhibitor
Mercaptoacetic acid to inhibit MBL
Clavulanate to inhibit ESBL
Boronic acid to inhibit AmpC
Cica b-Test (Mast) blind testing
of overnight cultures
Mechanism inferred
Pen’
ase
No
activity
2
3
0
2
6
0
0
0
18
3
2
0
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
MBL
ESBL
MBL (26)
20
1
0
ESBL (74)
3
63
AmpC (25)
2
K. oxytoca, K1 (10)
Reference data
AmpC Mixed
Other
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