Detection of ESBLs

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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 ()
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
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Detection of ESBLs step 2

• Seek ceph/clav synergy in ceph R isolates
• Double disc
• Combination disc
• Etest

See http//www.hpa.org.uk
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Combination discs
Disc with cephalosporin clavulanic acid
Disc with cephalosporin alone
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Zone differences (mm), Klebs E. colicpod/clav
101 mg - cpod 10 mg
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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
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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 gt1 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
  b-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
Potz et al., JAC 2006 58320-6
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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 mg 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
Potz et al., JAC 2006 58320-6
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Cefotaxime combinations vs. AmpC Enterobacter
London SE survey
Potz et al., JAC 2006 58320-6
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Cefotaxime / cloxacillin tests for AmpC
ARMRL- reference control data
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Phenyl boronic acid for detection of plasmid AmpC
Coudron JCM 2005 43 4163
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Disc tests for AmpC
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 strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefoxitin 30 mg
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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 mg)
  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

• gt200 isolates 60 centres many strains.
• No imipenem hydrolysis with crude extract
• Carbapenem resistance not transferable

Woodford et al. IJAA 2007 29456-9.
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Mechanisms of multi-resistant UK klebsiellas

• Mechanism is combination of porin loss CTX-M-15
• Occasionally selected during therapy

Woodford et al. IJAA 2007 29456-9
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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
• Metallos
• Class B, VIM, IMP families, also SPM, SIM, GIM
• Scattered, mostly non-fermenters
• gt100 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
• Metallos. 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- gt32 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 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

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Cica b-Test (Mast)
No inhibitor
Mercaptoacetic acid to inhibit MBL
Clavulanate to inhibit ESBL
Boronic acid to inhibit AmpC
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Cica b-Test (Mast) blind testingof overnight
cultures
Better but slower to use with antibiogram _at_ 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