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Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

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Author

  • Alma Brolund
  • Karin Tegmark Wisell
  • Petra J. Edquist
  • Lisbeth Elfstrom
  • Mats Walder
  • Christian G. Giske

Summary, in English

Introduction: Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBLM) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBLM are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. Material and methods: Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmo University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. Results and discussion: The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated. (C) 2010 Elsevier B.V. All rights reserved.

Publishing year

2010

Language

English

Pages

229-233

Publication/Series

Journal of Microbiological Methods

Volume

82

Issue

3

Document type

Journal article

Publisher

Elsevier

Topic

  • Microbiology in the medical area

Keywords

  • ESBL
  • Escherichia coli
  • Klebsiella pneumoniae
  • DHA
  • CMY

Status

Published

Research group

  • Clinical Microbiology, Malmö

ISBN/ISSN/Other

  • ISSN: 1872-8359