Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae
(2010) In Journal of Microbiological Methods 82(3). p.229-233- Abstract
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1695899
- author
- Brolund, Alma ; Wisell, Karin Tegmark ; Edquist, Petra J. ; Elfstrom, Lisbeth ; Walder, Mats LU and Giske, Christian G.
- organization
- publishing date
- 2010
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- ESBL, Escherichia coli, Klebsiella pneumoniae, DHA, CMY
- in
- Journal of Microbiological Methods
- volume
- 82
- issue
- 3
- pages
- 229 - 233
- publisher
- Elsevier
- external identifiers
-
- wos:000282113000006
- scopus:77955659288
- pmid:20600365
- ISSN
- 1872-8359
- DOI
- 10.1016/j.mimet.2010.06.006
- language
- English
- LU publication?
- yes
- id
- b8f19904-42c1-40fb-b989-cb578f95256d (old id 1695899)
- date added to LUP
- 2016-04-01 10:15:42
- date last changed
- 2022-04-04 08:16:27
@article{b8f19904-42c1-40fb-b989-cb578f95256d,
abstract = {{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.}},
author = {{Brolund, Alma and Wisell, Karin Tegmark and Edquist, Petra J. and Elfstrom, Lisbeth and Walder, Mats and Giske, Christian G.}},
issn = {{1872-8359}},
keywords = {{ESBL; Escherichia coli; Klebsiella pneumoniae; DHA; CMY}},
language = {{eng}},
number = {{3}},
pages = {{229--233}},
publisher = {{Elsevier}},
series = {{Journal of Microbiological Methods}},
title = {{Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae}},
url = {{http://dx.doi.org/10.1016/j.mimet.2010.06.006}},
doi = {{10.1016/j.mimet.2010.06.006}},
volume = {{82}},
year = {{2010}},
}