Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR

J Clin Microbiol. 2002 Jun;40(6):2153-62. doi: 10.1128/JCM.40.6.2153-2162.2002.

Abstract

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC beta-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC beta-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins*
  • Chromatography, High Pressure Liquid
  • DNA Primers
  • Gram-Negative Bacteria / enzymology*
  • Humans
  • Molecular Sequence Data
  • Plasmids / genetics
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Species Specificity
  • beta-Lactam Resistance
  • beta-Lactamases / genetics*
  • beta-Lactamases / metabolism

Substances

  • Bacterial Proteins
  • DNA Primers
  • AmpC beta-lactamases
  • beta-Lactamases