Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae

TLDR

The study aimed to develop a rapid multiplex PCR assay set for detecting the most common β‑lactamase genes in Enterobacteriaceae. The authors designed and optimized six multiplex PCRs plus one simplex PCR, validated on 31 clinical isolates, and enabled single‑run detection and sequencing of β‑lactamase genes. The assay proved specific, identifying ESBLs in 22 of 31 isolates (mostly CTX‑M‑15), plasmid‑mediated AmpC in 6, and both in 3, and allowed direct sequencing of PCR products.

Abstract

To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread β-lactamase genes encoding the OXA-1-like broad-spectrum β-lactamases, extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases and class A, B and D carbapenemases. 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 β-lactamase genes. 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 β-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 β-lactamase (DHA-1). 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 β-lactamases. This method allowed direct sequencing from the PCR products.