JAC Advance Access originally published online on December 7, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):457-458; doi:10.1093/jac/dkm472
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Research letters |
Plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: prevalence at a Swiss university hospital and occurrence of the different molecular types in Switzerland
Microbiology Laboratory, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland
* Corresponding author. Tel: +41-61-2654248; Fax: +41-61-2655355; E-mail: hadler{at}uhbs.ch
Keywords: resistance genes , antibiotic resistance , plasmids
Since 1989, plasmid-mediated AmpC β-lactamases have been known to exist in various species lacking inducible chromosomal ampC genes such as Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella. They descend from chromosomal ampC genes and fall into six phylogenetic groups. Origins are the ampC genes of Hafnia alvei, Morganella morganii, Citrobacter freundii, Enterobacter cloacae and two unknown organisms.1 Organisms producing plasmid-mediated AmpC β-lactamases raise special concerns because of the high rate of clinical failure among infected patients.2 We report here on the prevalence of plasmid-mediated AmpC β-lactamases in isolates of Enterobacteriaceae lacking an inducible chromosomal ampC gene at University Hospital Basel and the occurrence of different molecular types of plasmid-mediated AmpC β-lactamases in Switzerland.
Between 27 January 2006 and 27 January 2007, a total of 3217 consecutive clinical isolates of various species of Enterobacteriaceae lacking inducible chromosomal ampC genes (i.e. 2434 E. coli, 174 Klebsiella oxytoca, 459 Klebsiella pneumoniae, 8 Klebsiella spp., 134 P. mirabilis, 7 Salmonella enterica ssp. enterica and 1 Shigella flexneri) were screened for resistance to cefoxitin with the disc diffusion test according to CLSI guidelines.3 Isolates with an inhibition zone diameter of <18 mm were considered putative AmpC producers and were stored at –70°C for further investigation. Additionally, 45 clinical isolates suspected to harbour a plasmid-mediated AmpC β-lactamase were collected from 5 laboratories situated in Switzerland. ampC genes were identified by a ampC multiplex PCR with primers specific for the genes of six different phylogenetic groups.1 For sequencing, the ampC genes were amplified by PCR using primer pairs as described previously for blaCMY and blaDHA, respectively.4,5 In addition, blaCMY-31 was amplified using a second pair of primers.6 PCR products were purified using Montage PCR Units (Millipore, Zug, Switzerland) and sequencing reactions were carried out using a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Rotkreuz, Switzerland) as described by the manufacturer. Sequencing products were purified with Dye Ex 2.0 Spin Kit (Qiagen, Hombrechtikon, Switzerland) and electrophoresis was performed with the 3130 Genetic Analyzer (Applied Biosystems). The isoelectric point (pI) of the new β-lactamase was determined by isoelectric focusing, applying the supernatants of crude sonic cell extracts to Phast gels (GE HealthCare, Fairfield, CT, USA) with a pH gradient of 3–9 in a Phast system (GE HealthCare). Extended-spectrum β-lactamases (ESBLs) with known pI values (TEM-1, TEM-12, SHV-12 and CTX-M15) were included as pI markers. A filter paper containing 2.5% flucloxacillin (GlaxoSmithKline, Munchenbuchsee, Switzerland) as an inhibitor of the AmpC β-lactamase was applied for 2 min to one of the gels before staining with nitrocefin (Oxoid, Basel, Switzerland).
Of 3217 consecutive clinical isolates that were obtained at our hospital, 124 (3.8%; 103 E. coli, 3 K. oxytoca and 18 K. pneumoniae) were resistant to cefoxitin and were thus considered putative AmpC producers. Among these, five isolates (all of them E. coli) carrying an ampC gene known to be plasmid-encoded were found by ampC multiplex PCR. Thus, the prevalence of plasmid-mediated AmpC β-lactamases at University Hospital Basel was 0.16% for Enterobacteriaceae lacking an inducible chromosomal ampC gene (0.2% for E. coli).
Overall, plasmid-mediated AmpC β-lactamases were found in 17 isolates obtained from five Swiss laboratories. Details are given in Table 1. Fourteen of the isolates were E. coli, two were K. pneumoniae and one was P. mirabilis. Fifteen of the plasmid-mediated AmpC β-lactamases were CIT enzymes, a phylogenetic group that has its origin in the chromosomal ampC gene of C. freundii. Fourteen of them were CMY-2. For one isolate of K. pneumoniae, sequencing of the full gene revealed a unique sequence that has been designated CMY-31 (GenBank accession no. EF622224). The derived amino acid sequence differed from CMY-2 by one amino acid (Q235R). Isoelectric focusing revealed a β-lactamase with a pI between 8.8 and 9.0. Enzyme activity of CMY-31 was inhibited by flucloxacillin which is indicative of an AmpC β-lactamase. Enzyme activity of the ESBLs that served as pI markers was retained. This is the first report of the isolation and characterization of CMY-31, a new β-lactamase which is closely related to CMY-2. DHA-1, a plasmid-mediated AmpC β-lactamase that has its origin in the chromosomal ampC gene of M. morganii, was detected in two isolates of E. coli. DHA-1 is an inducible plasmid-mediated AmpC β-lactamase whose emergence raises concerns because the mortality of patients infected with organisms that produce DHA-1 has been shown to be higher than that of patients infected with organisms that produce CMY-1.2
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In conclusion, we have demonstrated that plasmid-mediated AmpC β-lactamases have emerged in Switzerland. The prevalence of 0.16% is still low. However, the occurrence of DHA-1, an inducible type of enzyme, raises clinical concerns. Additionally, a novel plasmid-mediated AmpC β-lactamase, which was designated CMY-31, was found.
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This study was funded by the Department of Laboratory Medicine, University Hospital Basel.
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None to declare.
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1 Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol (2002) 40:2153–62.
2
Pai H, Kang CI, Byeon JH, et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-β-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother (2004) 48:3720–8.
3 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16 (2006) Wayne, PA, USA: CLSI.
4
Winokur PL, Brueggemann A, DeSalvo DL, et al. Animal and human multidrug-resistant, cephalosporin-resistant salmonella isolates expressing a plasmid-mediated CMY-2 AmpC β-lactamase. Antimicrob Agents Chemother (2000) 44:2777–83.
5 Muratani T, Kobayashi T, Matsumoto T. Emergence and prevalence of β-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan. Int J Antimicrob Agents (2006) 27:491–9.[CrossRef][Web of Science][Medline]
6 Liebana E, Gibbs M, Clouting C, et al. Characterization of β-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom. Microb Drug Resist (2004) 10:1–9.[CrossRef][Web of Science][Medline]
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