The Importance and Detection of AmpC and ESBL EnzymesThe β-lactamases that give cephalosporin resistance - AmpC and ESBL and how to recognise them

D69C AmpC Detection SetInterpretation of results

Confirmation of eitherchromosomal or plasmid acquiredAmpC

A CPD10+ AmpC inducer

B CPD10+ AmpC inducer + ESβL inhibitor

C CPD10+ AmpC inducer + ESβL inhibitor+ AmpC inhibitors

CPD – Cefpodoxime

ESBL Detection DiscsInterpretation of results

Compare the zones of inhibitionfor the ceftazidime, cefotaxime andcefpodoxime discs to those of theceftazidime, cefotaxime and cefpodoxime+ clavulanic acid combination discs.

An increase in zone diameter of ≥5mm in thepresence of clavulanic acid from any or all ofthe sets of MAST ID™ ESBL Detection Discsindicates the presence of ESBL in the testorganism.

MAST ID™ ESBL Detection Discs are alsocompatible with the CLSI phenotypicconfirmatory test method.

D68C AmpC & ESBL Detection SetInterpretation of results

Interpret results by comparing inhibition zonediameters in the sequence described below:-

Compare the zone of inhibition of thecefpodoxime disc (A) to the zones ofinhibition of each of the cefpodoxime plusinhibitor discs (B, C, and D) where

A. Cefpodoxime 10μg (CPD10)B. CPD10 + ESBL inhibitorC. CPD10 + Amp C inhibitorD. CPD10 + ESBL and AmpC inhibitors

Introduction

• β-Lactamases inactivate β-lactam antibiotics by hydrolysis (Figure 1),they are the main source of resistance to penicillins and cephalosporins

• Sequencing divides them into 4 families, A-D

• Ampicillin - the first broad-spectrum β-lactam – was swiftly followed by the spread of plasmidsencoding TEM-1, SHV-1 and other class A β-lactamases in Gram-negative bacteria

• Pharmaceutical chemists sought to protect β-lactams by shielding the β-lactam ring with an‘oxyimino’ group (Figure 2)

• Unfortunately, oxyimino cephalosporins have selected for further β-lactamases, able to deflectthe oxyimino and inactivate the drug.

• The most important are extended-spectrum β-lactamases (ESBLs) and AmpC types

ESBLs and AmpC: the differences

• ESBLs: Early ESBLs were mutant TEM and SHV types, with an enlarged active site.They mostly occurred in Klebsiella spp., especially from intensive care units (ICUs).

• Recently CTX-M ESBLs have become predominant (Figure 3). These:

• Evolved by new gene ‘escape’ from Kluyvera spp., an obscure genus, not bymutation of old β-lactamase types.

• Are disseminating rapidly in Escherichia coli, • Often occur outside the ICU - e.g. in urinary infections/urosepsis in older,community patients.

• Regardless of type, most ESBLs are encoded by transferable plasmids

• AmpC: Historically, AmpC β-lactamases were chromosomal and inducible in Enterobacter,Citrobacter freundii, Morganella, Providencia and Serratia spp., also P. aeruginosa. • Ampicillin and cephalexin were strong inducers and rapidly hydrolysed, and so generally lacked activity against these species

• Oxyimino cephalosporins (except cefepime and cefpirome) are hydrolysed, but induce weakly.

• They inhibit AmpC-inducible strains but not ‘derepressed’ mutants, which hyperproduce AmpC without induction.

• ampC genes are escaping to plasmids, which are spreading into other Enterobacteriaceae, e.g. E. coli and Klebsiella

Clinical impact

1) ESBLs

• ESBLs cause cephalosporin failure, even when MICs remain below CLSI breakpoints (though generally not the lower EUCAST/BSAC values)

• Many ESBL producers are resistant also to quinolones and aminoglycosides

• One E.coli clone with CTX-M-15 enzymes (ST131:O25) is distributed worldwide - it is common in the UK… but there are many diverse producers too

• Patients infected by ESBL producers often receive inadequate empirical therapy until the pathogen’s resistance is recognised.

• Consequently, patients with bacteraemias due to ESBL producers are almost twice as likely to die as those with bacteraemias due to non-producers.

2) AmpC• Oxyimino cephalosporins (except cefepime and cefpirome) select spontaneous AmpCderepressed mutants from inducible populations sometimes during therapy (Figure 4).These are resistant.

• Selection and contingent failure occurs in 20-30% of Enterobacter bacteraemia cases treated with a cephalosporin

• This increases mortality, cost and hospital stay (Table 1)

• It is important to distinguish this selection from induction

• Carbapenems and temocillin remain active against both ESBL producers and strains with high-level AmpC.

Table 1. Consequences of selecting AmpC-derepressed Enterobactermutants during therapy.

Case ControlSelection occurred No selection

Mortality (%) 26 13Hospital stay (days) 29.5 19Cost (US$) 79000 40000

Why it’s important to distinguish ESBL and AmpC producers• It’s epidemiologically important

• ESBL producers are increasing rapidly• Some belong to internationally successful clones• Some ESBL plasmids transfer readily among strains

• It’s clinically important

• Cefepime and cefpirome are active vs. many AmpC-derepressed strains, not ESBLproducers

• β-Lactamase inhibitor combinations are active vs. some ESBL producers…. But only a minority in the UK

How to distinguish ESBL and AmpC producers

• ESBLs are inhibited by clavulanate, AmpC by cloxacillin. These traits can be exploited insimple diagnostic disc tests (see below).

• A zone ≥5 mm larger to cefpodoxime 10 μg + clavulanate 1 μg than to cefpodoxime10 μg indicates an ESBL in E. coli, Klebsiella or P. mirabilis

• Limitation: Induction of AmpC by clavulanate may mask the ESBL inhibition ine.g. Enterobacter or C. freundii

• A zone ≥5 mm larger to cefpodoxime 10 μg + cloxacillin 500 μg than to cefpodoxime10 μg indicates derepressed or plasmidic AmpC

• Limitation: Cloxacillin does not enter P. aeruginosa; the method does not work for this species

• A zone ≥5 mm larger to cefpodoxime 10 μg + cloxacillin 500 μg + clavulanate 1 μg disc than to cefpodoxime plus either inhibitor suggests both an ESBL and AmpC

• Alternatively, rapid detection can be performed with the Cica-Beta-Test, which uses the chromogenic cephalosporin HMRZ-86 together with specific inhibitors of AmpC and ESBLs

• Enterobacteriaceae remaining resistant to cefpodoxime irrespective of clavulanate and cloxacillin (i) may have an ESBL or AmpC plus impermeability or (ii) may have acarbapenemase. They deserve reference investigation e.g. by the HPA’s AntibioticResistance Monitoring & Reference Laboratory, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ.

N

S

COOH O

Active penicillin Inactive penicilloate

HN

S

COOH O OH

H2O

S

N

CONH

O

C

COOH

Oxyimino group protects β-lactam ring

R

S N

H2N

N OR

e.g. cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime, cefpirome

24

20

16

12

8

4

002 03 04 05 06

E. coli

K1 Other/Undefined AmpC non-CTX-M ESBL CTX-M

24

20

16

12

8

4

002 03 04 05 06

Klebsiella

Mutant emergesrandomly

Sensitive cellskilled

Mutant progenyoverrun

Most Enterobacter cells are inducible forAmpC but 1 per 107 is spontaneouslyderepressed

Oxyimino-cephalosphorin is labile buta weaker inducer; kills inducible cells

Derepressed mutant survives andmultiplies

Selection can cause RX failure

by David M. Livermore

Figure 4 Selection of AmpC-derepression

Figure 3 Cephalosporin resistance % Prevalence BSACsurveillance

http://www.bsacsurv.org.uk

Figure 1 β-Lactamase action

Figure 2‘β-Lactamase-stable’oxyimino cephalosporins

If all zones indicate suseptibilityand are within 2mm of each

other, record theorganism as demonstratingneither ESBL nor AmpC

activity.

Step 1

InterpretationNeither ESBL norAmpC producer

Subtract A from Band C from D

If each of B – Aand D – C ≥ 5mm

AND the differences of eachof B and D and discs A and C

are <4mm

Organism has ESBLactivity alone.

Step 2

InterpretationESBL alone

Subtract B from Dand A from C

If each of D – Band C – A ≥ 5mm

AND the differences of eachof A and B and C and D

are <4mm

Organism has AmpCactivity alone.

Step 3

InterpretationAmpC alone

Subtract C from D

If D – C ≥ 5mm

AND the difference betweendiscs A and B is <4mm

Organism has both ESBLand AmpC activity.

Step 4

InterpretationESBL & AmpC combined

Alternatively a calculation program is available atwww.mastgrp.com

www.mastgrp.com Mast Group Ltd.Mast House, Derby Road, Bootle Merseyside L20 1EA UK

Tel: 44 (0)151 933 7277 Fax: 44 (0)151 944 1332

V2.0 CEV 07/12

ZA

ZB ZC

ZA

ZB ZC

ZC-ZA and ZC-ZB ≥ 5mm All zones differ by ≤ 3mm

AmpC Positive AmpC Negative