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The rapid global spread of carbapenem-resistantEnterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-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, usingbla 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, includingbla 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 , andbla OXA-232 . Our assay identified the presence ofbla OXA-48-like β-lactamase genes and clearly distinguished betweenbla OXA-48 and its variants in control strains, including betweenbla OXA-181 andbla 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.

antimicrobial agents and chemotherapy

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