Detection of Plasmid-Mediated AmpC β-Lactamase Genes in Clinical Isolates by Using Multiplex PCR

TLDR

Gram‑negative pathogens that carry plasmid‑mediated AmpC β‑lactamases pose a major nosocomial threat, are largely resistant to β‑lactams except for a few agents, and comprise six families that cannot be distinguished by phenotypic tests. The study aimed to develop a multiplex PCR capable of identifying the family‑specific AmpC β‑lactamase genes in gram‑negative bacteria. The assay employed six primer sets generating 190–520 bp amplicons that can be resolved by gel electrophoresis, and incorporated WAVE HPLC technology to shorten analysis time and enhance sensitivity. The multiplex PCR successfully distinguished all six AmpC families in Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella Typhimurium, with family‑specific primers showing no cross‑amplification and enabling detection of multiple genes in a single reaction.

Abstract

ABSTRACT Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-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 β-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 β-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 β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene.