Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae.
J Microbiol Methods
; 82(3): 229-33, 2010 Sep.
Article
en En
| MEDLINE
| ID: mdl-20600365
ABSTRACT
INTRODUCTION:
Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBL(M)) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL(M) 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 ANDMETHODS:
Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmö 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 ANDDISCUSSION:
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 3h, 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.
Texto completo:
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Bases de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
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Beta-Lactamasas
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Reacción en Cadena de la Polimerasa
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Enterobacteriaceae
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Infecciones por Enterobacteriaceae
Tipo de estudio:
Diagnostic_studies
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Evaluation_studies
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Guideline
Límite:
Humans
Idioma:
En
Revista:
J Microbiol Methods
Año:
2010
Tipo del documento:
Article
País de afiliación:
Suecia