Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D beta-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for beta-lactams. The plasmid-borne OXA-48 beta-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla(OXA-48-like)-specific primers coupled with an unlabeled 3'-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla(KPC), bla(SME), bla(IMP), bla(NDM-1), bla(VIM), bla(OXA-48), bla(OXA-162), bla(OXA-181), bla(OXA-204), bla(OXA-244), bla(OXA-245), and bla(OXA-232). Our assay identified the presence of bla(OXA-48-like) beta-lactamase genes and clearly distinguished between bla(OXA-48) and its variants in control strains, including between bla(OXA-181) and bla(OXA-232), which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.