detection of esbls & ampc david livermore health protection agency, colindale, london
TRANSCRIPT
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Detection of ESBLs & AmpC
David Livermore
Health Protection Agency,
Colindale, London
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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
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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
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EARSS resistance to 3-gen cephs in E. coli
2001 2006
http://www.earss.rivm.nl
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Detecting ESBL producers
2 steps:• Screen for resistance with an indicator ceph
• Do confirmatory test on those found resistant
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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
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Detection of ESBLs: step 2
See http://www.hpa.org.uk
Seek ceph/clav synergy in ceph R isolates
•Double disc
•Combination disc
•Etest
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Combination discs
Disc with cephalosporin
alone
Disc with cephalosporin + clavulanic
acid
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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
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Etest for ESBLs
Cefotaxime
Cefotaxime+
clavulanate
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Etest for ESBLs
Cefotaxime
Cefotaxime+
clavulanate
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Clover leaf (3 dimensional) test for AmpC
Test strainE. cloacae,
AmpC derepressed
IndicatorE. coli
NCTC10418
DiscCefoxitin 30 g
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Multiplex detection of plasmid AmpC genes
Method of Perez-Perez & Hanson JCM 2002, 40, 2153
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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
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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
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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.
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• 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
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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
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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
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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
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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
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Cica -Test (Mast)
No inhibitor
Mercaptoacetic acid to inhibit MBL
Clavulanate to inhibit ESBL
Boronic acid to inhibit AmpC
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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.
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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