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

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:italic>ampC</jats:italic>-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis.<jats:italic>ampC</jats:italic>multiplex PCR differentiated the six plasmid-mediated<jats:italic>ampC</jats:italic>-specific families in organisms such as<jats:italic>Klebsiella pneumoniae</jats:italic>,<jats:italic>Escherichia coli</jats:italic>,<jats:italic>Proteus mirabilis</jats:italic>, and<jats:italic>Salmonella enterica</jats:italic>serovar Typhimurium. Family-specific primers did not amplify genes from the other families of<jats:italic>ampC</jats:italic>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<jats:italic>ampC</jats:italic>genes responsible for AmpC β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene.</jats:p>