Development of a Set of Multiplex PCR Assays for the Detection of Genes Encoding Important Beta-Lactamases in Enterobacteriaceae

J Antimicrob Chemother. 2010 Mar;65(3):490-5. doi: 10.1093/jac/dkp498. Epub 2010 Jan 12.

Abstract

Objectives: To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases.

Methods: Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes.

Results: Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1).

Conclusions: We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

Publication types

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

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • DNA Primers / genetics
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Enterobacteriaceae / enzymology*
  • Enterobacteriaceae / genetics*
  • Enterobacteriaceae / isolation & purification
  • Enterobacteriaceae Infections / microbiology
  • Humans
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • beta-Lactam Resistance
  • beta-Lactamases / biosynthesis*
  • beta-Lactamases / genetics*
  • beta-Lactams / pharmacology

Substances

  • Anti-Bacterial Agents
  • DNA Primers
  • DNA, Bacterial
  • beta-Lactams
  • beta-Lactamases