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Development of a re...
Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae
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- Brolund, Alma (author)
- Karolinska Institutet
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Wisell, Karin Tegmark (author)
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Edquist, Petra J. (author)
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Elfstrom, Lisbeth (author)
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- Walder, Mats (author)
- Lund University,Lunds universitet,Klinisk mikrobiologi, Malmö,Forskargrupper vid Lunds universitet,Clinical Microbiology, Malmö,Lund University Research Groups
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- Giske, Christian G. (author)
- Karolinska Institutet
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(creator_code:org_t)
- Elsevier BV, 2010
- 2010
- English.
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In: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 82:3, s. 229-233
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Abstract
Subject headings
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- 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.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Microbiology in the medical area (hsv//eng)
Keyword
- ESBL
- Escherichia coli
- Klebsiella pneumoniae
- DHA
- CMY
Publication and Content Type
- art (subject category)
- ref (subject category)
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