Abstract

Objectives: To determine the distribution of acquired AmpC β-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance.

Methods: MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one blaAmpC amplicon was sequenced.

Results: Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum β-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative blaAmpC amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23.

Conclusions: Diverse acquired AmpC enzymes occur in E. coli and Klebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.

Introduction

The spread of resistance to third-generation cephalosporins in Escherichia coli and Klebsiella spp. is a continuing cause of public health concern, with such resistance increasingly seen in community-onset or -acquired infections. Extended-spectrum β-lactamases (ESBLs) cause most cephalosporin resistance in E. coli and Klebsiella spp., but resistance may also be mediated by AmpC cephalosporinases.1 Isolates with AmpC typically give a negative ESBL test, are cefoxitin-resistant, but are usually susceptible to cefepime and cefpirome. In E. coli an AmpC phenotype may result from overexpression of the chromosomally encoded AmpC enzyme or from acquisition of a plasmid AmpC enzyme; in Klebsiella spp. AmpC enzymes are always acquired.

Plasmidic AmpC enzymes, which fall into at least six phylogenetic groups,2 have been reported in clinical isolates of Salmonella and E. coli collected in the UK between 1993 and 2003,3–5 and have also been detected in surveys of cephalosporin-resistant isolates.1

The Health Protection Agency's Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) monitors unusual resistance among isolates referred from UK microbiology laboratories. Here, we sought to determine the distribution of genes encoding acquired AmpC β-lactamases in cephalosporin-resistant clinical isolates of E. coli and Klebsiella spp. referred since 2004.

Materials and methods

Bacterial isolates and susceptibility testing

We studied 173 isolates of E. coli (n = 135) and Klebsiella spp. (n = 38), which had been submitted to ARMRL for reference testing, mostly for confirmation of cephalosporin resistance. The isolates studied were inferred by ARMRL to have possible AmpC-mediated resistance on the basis of cephalosporin (including cefoxitin) resistance without potentiation by clavulanic acid. This working definition served to distinguish them from producers of ESBLs, which do not confer resistance to cephamycins and are inhibited by clavulanate.6 MICs were determined by agar dilution and interpreted according to BSAC guidelines.7 Antibiotics were supplied as powders of known potency by the manufacturers or were obtained from Sigma (Poole, UK).

Molecular investigation of isolates with AmpC phenotypes

Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought with a multiplex PCR assay.2,blaCTX-M alleles were sought in selected isolates,8 as was linkage of group 1 CTX-M genes and IS26.9 Selected isolates were compared by PFGE of XbaI-digested genomic DNA. Selected entire blaAmpC (CIT group) genes were amplified with primers and conditions described previously,10 and sequenced on both strands using dye terminator chemistry on a CEQ 8000 analyser (Beckman Coulter, High Wycombe, UK).

Results and discussion

Detection and susceptibilities of acquired AmpC producers

We sought genes encoding acquired AmpC enzymes in 173 clinical isolates of E. coli and Klebsiella spp. from separate patients. These had been referred to ARMRL from laboratories in the UK and Ireland since 2004, and met the selection criteria of resistance to cephalosporins, but without significant potentiation by clavulanate. Sixty-seven (49%) E. coli isolates and 21 (55%) Klebsiella spp. yielded PCR amplicons, indicating the presence of genes encoding acquired AmpC enzymes; 60 isolates had CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. No isolates producing MOX- or EBC-/ENT-type enzymes were detected. Eighty-five isolates yielded no amplicons in the multiplex assay used.

The susceptibilities of the isolates are summarized in Table 1. All producers were resistant to penicillins and third-generation cephalosporins, though cefoxitin resistance was less marked in ACC producers. There was negligible synergy upon addition of clavulanate to cefotaxime and ceftazidime. The DHA producers showed marked synergy of piperacillin by tazobactam, a characteristic of these enzymes, which have ‘escaped’ from the chromosomes of Morganella morganii.3,11 All isolates remained susceptible to carbapenems as well as to cefepime. Moreover, the AmpC-producers were less multiresistant than the majority of CTX-M ESBL producers,9 with many remaining susceptible to ciprofloxacin, amikacin and gentamicin (Table 1).

Table 1

Geometric mean MICs (mg/L) for isolates of E. coli (n = 135) and Klebsiella spp. (n = 38) with AmpC phenotypes

 CIT-type ACC-type FOX-type DHA-type None detected 
 
 

 

 

 

 
Antibiotica E. coli (59 isolates; 30 centres) Klebsiella spp. (1 isolate; 1 centre) E. coli (4 isolates; 4 centres) Klebsiella spp. (10 isolates; 3 centres) E. coli (3 isolates; 2 centres) Klebsiella spp. (8 isolates; 2 centres) E. coli (1 isolate; 1 centre) Klebsiella spp. (2 isolates; 2 centres) E. coli (68 isolates; 32 centres) Klebsiella spp. (17 isolates; 9 centres) 
AMP >64 >64 >64 >64 >64 >64 >64 >64 >64 64.0 
AMC 53.0 32 32.0 27.9 40.3 32.0 64 45.3 39.9 28.1 
CTX 34.7 26.9 9.8 40.3 13.5 8.0 3.7 9.4 
CTX-CLA 15.3 16 19.0 6.5 10.1 8.0 8.0 1.6 4.3 
CAZ 28.1 16 76.1 48.5 50.8 34.9 16 16.0 5.5 5.3 
CAZ-CLA 17.2 >32 32.0 24.3 20.2 22.6 32 32.0 3.4 3.4 
FEP 1.0 ND 0.8 0.4 ND 1.3 ≤0.125 0.1 0.3 1.2 
FEP-CLA 0.2 ND 0.6 0.3 ND 0.1 ≤0.06 0.1 0.1 0.3 
FOX 55.6 >64 9.5 12.1 64.0 64.0 >64 64.0 47.4 48.1 
PIP 61.8 >64 >64 >64 64.0 >64 >64 >64 41.6 64.0 
TZP 21.2 ND 64.0 42.2 20.2 17.7 4.0 10.1 25.8 
IPM 0.3 0.5 0.2 0.3 0.2 0.3 0.5 1.0 0.2 0.4 
MEM 0.1 ≤0.06 0.1 0.1 0.1 0.1 ≤0.06 0.1 0.1 0.1 
ETP 0.2 0.1 0.1 0.1 0.1 ≤0.125 0.3 0.2 0.2 
CIP 4.6 0.5 2.8 0.8 0.2 0.5 0.5 4.0 0.5 0.7 
GEN 2.7 0.25 0.8 0.8 10.1 11.3 5.7 1.7 1.5 
AMK 3.8 2.0 2.1 2.0 1.2 1.4 2.4 2.3 
MIN 6.9 5.7 8.6 1.6 2.8 11.3 3.0 12.5 
TIG 0.5 0.5 0.3 1.1 0.3 0.4 ≤0.25 1.0 0.3 1.5 
COL 0.6 ≤0.5 0.6 0.7 0.5 0.6 1.0 0.5 1.8 
 CIT-type ACC-type FOX-type DHA-type None detected 
 
 

 

 

 

 
Antibiotica E. coli (59 isolates; 30 centres) Klebsiella spp. (1 isolate; 1 centre) E. coli (4 isolates; 4 centres) Klebsiella spp. (10 isolates; 3 centres) E. coli (3 isolates; 2 centres) Klebsiella spp. (8 isolates; 2 centres) E. coli (1 isolate; 1 centre) Klebsiella spp. (2 isolates; 2 centres) E. coli (68 isolates; 32 centres) Klebsiella spp. (17 isolates; 9 centres) 
AMP >64 >64 >64 >64 >64 >64 >64 >64 >64 64.0 
AMC 53.0 32 32.0 27.9 40.3 32.0 64 45.3 39.9 28.1 
CTX 34.7 26.9 9.8 40.3 13.5 8.0 3.7 9.4 
CTX-CLA 15.3 16 19.0 6.5 10.1 8.0 8.0 1.6 4.3 
CAZ 28.1 16 76.1 48.5 50.8 34.9 16 16.0 5.5 5.3 
CAZ-CLA 17.2 >32 32.0 24.3 20.2 22.6 32 32.0 3.4 3.4 
FEP 1.0 ND 0.8 0.4 ND 1.3 ≤0.125 0.1 0.3 1.2 
FEP-CLA 0.2 ND 0.6 0.3 ND 0.1 ≤0.06 0.1 0.1 0.3 
FOX 55.6 >64 9.5 12.1 64.0 64.0 >64 64.0 47.4 48.1 
PIP 61.8 >64 >64 >64 64.0 >64 >64 >64 41.6 64.0 
TZP 21.2 ND 64.0 42.2 20.2 17.7 4.0 10.1 25.8 
IPM 0.3 0.5 0.2 0.3 0.2 0.3 0.5 1.0 0.2 0.4 
MEM 0.1 ≤0.06 0.1 0.1 0.1 0.1 ≤0.06 0.1 0.1 0.1 
ETP 0.2 0.1 0.1 0.1 0.1 ≤0.125 0.3 0.2 0.2 
CIP 4.6 0.5 2.8 0.8 0.2 0.5 0.5 4.0 0.5 0.7 
GEN 2.7 0.25 0.8 0.8 10.1 11.3 5.7 1.7 1.5 
AMK 3.8 2.0 2.1 2.0 1.2 1.4 2.4 2.3 
MIN 6.9 5.7 8.6 1.6 2.8 11.3 3.0 12.5 
TIG 0.5 0.5 0.3 1.1 0.3 0.4 ≤0.25 1.0 0.3 1.5 
COL 0.6 ≤0.5 0.6 0.7 0.5 0.6 1.0 0.5 1.8 

aAMP, ampicillin; AMC, co-amoxiclav; CTX, cefotaxime; CLA, clavulanate; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; PIP, piperacillin; TZP, piperacillin/tazobactam; IPM, imipenem; MEM, meropenem; ETP, ertapenem; CIP, ciprofloxacin; GEN, gentamicin; AMK, amikacin; MIN, minocycline; TIG, tigecycline; COL, colistin.

Isolates without a detectable acquired blaAmpC gene tended to be less resistant to third-generation cephalosporins (all geometric mean MICs ≤5.5 mg/L). Sixty-eight (80%) of these 85 isolates were E. coli, and resistance likely reflected up-regulation of the chromosomal ampC gene through mutations in the promoter region,5 though this requires formal investigation. Klebsiella spp. do not have a chromosomally encoded AmpC enzyme, and the basis of low-level cephalosporin resistance in 17 isolates may reflect permeability changes; again, this needs further study. It is also possible that some of the 85 isolates had novel AmpC types, which were not detected by primers in the multiplex assay used.2

Distribution in the UK and Ireland

Producers of CIT enzymes (59 E. coli and one Klebsiella) were received from 30 centres scattered across the UK. The three producers of DHA types comprised single Klebsiella spp. isolates from laboratories in Ireland and Scotland and an E. coli from England, also indicating wide scatter; all three isolates had inducible AmpC activity (data not shown). DHA-1 enzyme was also reported in 2004 in two clinical isolates of Salmonella enterica serotype Senftenberg from London.3 Other AmpC types were more clustered, suggesting that their underlying epidemiology deserves further investigation. ACC enzymes, which were first reported in Germany in 1999 and have been reported from a growing number of European countries,12 were detected only in isolates from Ireland (in E. coli and Klebsiella spp. from four referring laboratories).13 Moreover, one centre in Wales referred 8 of the 11 producers of FOX-type enzymes detected in this study, which is consistent with local spread of the producing Klebsiella oxytoca strain.

CMY-23, a novel AmpC enzyme associated with an epidemic E. coli strain

The CIT-producers represented numerous PFGE types (not shown), with little evidence for spread of strains between centres. However, 24 E. coli isolates referred from one hospital all produced a CIT-type enzyme. In 20 of these we also detected a gene for a group 1 CTX-M ESBL, linked immediately upstream to a copy of IS26; four isolates lacked a blaCTX-M allele. The IS26-blaCTX-M arrangement is typical of UK epidemic CTX-M-15-producing strain A,9 and PFGE of 12 representative isolates confirmed that they clustered with strain A.9 The presence of the IS26 element causes reduced expression of blaCTX-M, with the result that isolates of strain A typically have lower MICs of cephalosporins than many other CTX-M producers.9 Isolates of E. coli strain A with CTX-M-15, but without an acquired AmpC enzyme have been referred to ARMRL from >45 UK laboratories (N. Woodford, unpublished data), and are locally dominant in some locales, e.g. Shropshire and Hampshire. Given the epidemiological success of this strain and its propensity to spread by as yet undefined routes, acquisition of an AmpC in addition to CTX-M-15 gives cause for concern.

Differences in the antibiograms of isolates of this strain that produced AmpC alone versus producers of both AmpC and CTX-M-15 were surprisingly subtle, with the co-resident ESBL suggested only by slight (typically 4-fold) potentiation of cefepime MICs by clavulanate (Table 2). Sequencing the blaAmpC amplicon from a representative of this strain identified a novel allele; the predicted enzyme differed from CMY-2 by a Glu239Gly amino acid substitution and has been designated CMY-23. The sequence of blaCMY-23 has been deposited with GenBank under accession number DQ438952.

Table 2

MICs (mg/L) of β-lactams for three representative isolates of PFGE-defined E. coli strain A9 isolated at a single centre

 Genotype 
 
 
Antibiotic blaCTX-M only blaCMY-23 only blaCTX-M + blaCMY-23 
AMP >64 >64 >64 
AMC 16 64 64 
PIP >64 >64 >64 
TZP 16 64 64 
CTX 64 64 64 
CTX-CLA ≤0.06 >32 >32 
CAZ 64 32 
CAZ-CLA 0.25 32 32 
FOX 16 >64 >64 
FEP 0.5 
FEP-CLA ≤0.06 0.5 0.25 
IPM 0.125 0.25 0.25 
MEM ≤0.06 ≤0.06 ≤0.06 
ETP ≤0.125 0.25 0.25 
 Genotype 
 
 
Antibiotic blaCTX-M only blaCMY-23 only blaCTX-M + blaCMY-23 
AMP >64 >64 >64 
AMC 16 64 64 
PIP >64 >64 >64 
TZP 16 64 64 
CTX 64 64 64 
CTX-CLA ≤0.06 >32 >32 
CAZ 64 32 
CAZ-CLA 0.25 32 32 
FOX 16 >64 >64 
FEP 0.5 
FEP-CLA ≤0.06 0.5 0.25 
IPM 0.125 0.25 0.25 
MEM ≤0.06 ≤0.06 ≤0.06 
ETP ≤0.125 0.25 0.25 

AMP, ampicillin; AMC, co-amoxiclav; PIP, piperacillin; TZP, piperacillin/tazobactam; CTX, cefotaxime; CLA, clavulanate; CAZ, ceftazidime; FOX, cefoxitin; FEP, cefepime; IPM, imipenem; MEM, meropenem; ETP, ertapenem.

In conclusion, the broad resistance profiles of AmpC enzymes compromises patient management; they spare only carbapenems, cefepime, cefpirome and temocillin. As with ESBL producers, isolation of multiresistant AmpC producers drives choice for serious infections towards increased use of carbapenems, with consequent concerns of further resistance development. The variety of enzymes detected, the diversity of producing strains, and especially the acquisition of a CMY-2-like enzyme by epidemic E. coli strain A, suggest that acquired AmpC enzymes may be poised to become an important public health issue in the UK and Ireland.

Transparency declarations

None to declare.

We would like to thank the microbiologists in the UK and Ireland who submitted isolates that were included in this analysis. Parts of this work were presented at the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy (Washington, DC, USA, 2005; Poster C2-776), FIS (Cardiff, UK, 2005; Oral presentation O10) and the Sixteenth European Congress of Clinical Microbiology and Infectious Diseases (Nice, France, 2006; Poster P1622).

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Abstract C2-776