Short Communication
The differential importance of mutations within AmpD in cephalosporin resistance of Enterobacter aerogenes and Enterobacter cloacae

https://doi.org/10.1016/j.ijantimicag.2016.07.021Get rights and content

Highlights

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    In contrast to E. cloacae, cephalosporin resistance in E. aerogenes does not seem to be associated with lesions in AmpD.

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    Loss of Omp35, primarily due to decreased transcription (up to 250×), was observed in ertapenem-resistant E. aerogenes.

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    Meropenem resistance was observed in E. aerogenes isolates also showing decreased or no production of Omp36.

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    Presence of Ala294Thr in H-10 helix of AmpC refers to a possible extended-spectrum AmpC variant.

Abstract

Mechanisms leading to carbapenem and cephalosporin resistance were sought in Enterobacter aerogenes isolates that were highly resistant to carbapenems but had no known carbapenemase. Results were compared with recent work examining carbapenem-resistant Enterobacter cloacae. Eighteen carbapenem-resistant E. aerogenes were screened for known β-lactamase and carbapenemase genes, and novel carbapenemases were sought in whole-genome sequencing (WGS) data of the three most resistant isolates. For all isolates, ampC, ampR, ampD and the porin genes omp35 and omp36 were investigated by Sanger sequencing or from available WGS data. Expression of ampC and porin genes was measured in comparison with cephalosporin- and carbapenem-susceptible control strains by reverse transcriptase PCR, with porin translation also detected by SDS-PAGE. Loss of Omp35, primarily due to decreased transcription (up to 250×), was observed in ertapenem-resistant isolates (MICs ≥ 2 mg/L), whereas meropenem resistance (MICs ≥ 4 mg/L) was observed in those isolates also showing decreased or no production of Omp36. Loss of Omp36 was due to combinations of premature translation termination or reduced transcription. In contrast to E. cloacae, cephalosporin resistance in E. aerogenes was not associated with lesions in AmpD. High-level cefepime resistance (MIC = 32 mg/L) was caused by a novel modification in the H-10 helix of AmpC in one isolate. The differential importance of AmpD lesions in cephalosporin resistance in E. cloacae and E. aerogenes underlines the differences between these contrasting members of the Enterobacter genus. Porin loss resulted in high-level carbapenem resistance with gradual loss of Omp36, which led to high-level meropenem resistance.

Introduction

As a common hospital-associated pathogen, Enterobacter aerogenes can exhibit resistance to a variety of antimicrobials, including emerging resistance to carbapenems. Besides the acquisition of carbapenemases, changes in outer membrane permeability and overexpression of AmpC have been described as the main contributors to cephalosporin and carbapenem resistance [1], [2], [3], [4]. We recently reported highly carbapenem-resistant Enterobacter cloacae with no known carbapenemase, in which resistance was mediated by lesions in ampD and hyperproduction of AmpC [5]. In this study, we sought to define the contributions of these resistance mechanisms in a collection of carbapenem-resistant E. aerogenes isolates.

Section snippets

Clinical isolates and antimicrobial susceptibility testing

Twenty-three E. aerogenes isolates were tested. These included 18 ertapenem-resistant [minimum inhibitory concentrations (MICs) ≥ 2 mg/L] clinical isolates [15 submitted from different UK laboratories to Public Health England's Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit (London, UK) during 2012 and 2013 and 3 isolates from Basel, Switzerland (CH isolates)] and 5 isolates susceptible to carbapenems and third-generation cephalosporins that were included

Results

The basic characteristics of the 18 carbapenem-resistant E. aerogenes isolates were that they were genetically distinct by PFGE and were uniformly resistant to third-generation cephalosporins and ertapenem (median ertapenem MIC = 16 mg/L). Most were also non-susceptible, defined by clinical breakpoints, to meropenem and imipenem (15 and 17 isolates, respectively), with median MICs of 8 mg/L for both compounds. Six isolates were also resistant to cefepime (MICs > 4 mg/L) (Supplementary Fig. S1).

Discussion

In this diverse collection of ertapenem-resistant E. aerogenes isolates, we found that loss of Omp36, due to a combination of premature translation termination or reduced transcription, led to high-level meropenem resistance (MIC ≥ 16 mg/L), with disruption of the Omp36 coding sequence being observed in one-half of the carbapenem-resistant isolates.

All tested isolates lacked Omp35, suggesting that loss of Omp35 is primarily responsible for the ertapenem-resistant phenotype of E. aerogenes. With

Conclusion

Loss of Omp35 due to reduced transcription primarily led to an ertapenem-resistant phenotype in E. aerogenes, whereas loss of Omp36 led also to high-level meropenem resistance. Cephalosporin resistance was not dependent on mutations within AmpD, which contrasts with findings in E. cloacae.

Nucleotide sequence accession numbers

The accession numbers (for EMBL/GenBank/DDBJ) for the nucleotide sequence data in this study are as follows: KT894102 for ampC; KT894103 for ampR; KT894104 for ampD; KT894105 for omp35; and KT894106 for omp36 from NCTC 10336.

Acknowledgments

The authors would like to thank Dr Reno Frei and Dr Vladimira Hinic for providing the Swiss isolates. The authors would also like to thank Jayesh Shah, Claire Perry, Marina Warner, Ayisha Chaudry, Rachael Adkin, Jacqueline Findlay, Tabassum Noorie, Rachel Pike and Shazad Mushtaq for technical advice and assistance.

Funding: This study was supported by an award to BBF from Prof. Dr Max Cloëtta and Uniscienta Foundation and the Margarete and Walter Lichtenstein Foundation, University of Basel

References (20)

  • F. Fernandez-Cuenca et al.

    In vivo selection of Enterobacter aerogenes with reduced susceptibility to cefepime and carbapenems associated with decreased expression of a 40 kDa outer membrane protein and hyperproduction of AmpC β-lactamase

    Int J Antimicrob Agents

    (2006)
  • J.F. Turton et al.

    A prevalent, multiresistant clone of Acinetobacter baumannii in Southeast England

    J Hosp Infect

    (2004)
  • H. Yigit et al.

    Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs

    Antimicrob Agents Chemother

    (2002)
  • A. Thiolas et al.

    Successive emergence of Enterobacter aerogenes strains resistant to imipenem and colistin in a patient

    Antimicrob Agents Chemother

    (2005)
  • L.J. Piddock et al.

    β-Lactamase expression and outer membrane protein changes in cefpirome-resistant and ceftazidime-resistant Gram-negative bacteria

    J Antimicrob Chemother

    (1991)
  • B. Babouee Flury et al.

    Association of novel nonsynonymous single nucleotide polymorphisms in ampD with cephalosporin resistance and phylogenetic variations in ampC, ampR, ompF and ompC in Enterobacter cloacae isolates that are highly resistant to carbapenems

    Antimicrob Agents Chemother

    (2016)
  • J.M. Andrews

    Determination of minimum inhibitory concentrations

    J Antimicrob Chemother

    (2001)
  • R.A. Walker et al.

    Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype Typhimurium phage-type U302 (MR U302) from humans, animals, and foods

    Microb Drug Resist

    (2001)
  • N. Woodford et al.

    Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases

    J Antimicrob Chemother

    (2006)
  • F.J. Perez-Perez et al.

    Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR

    J Clin Microbiol

    (2002)
There are more references available in the full text version of this article.

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