title: 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.
abstract: The rapid global spread of carbapenem-resistant Enterobacteriaceae (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, 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 β-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.
authors.affiliation: Pathology and Laboratory Medicine, UCLA, Los Angeles, California, USA.
authors.forename: Peera
authors.initials: P
authors.lastname: Hemarajata
authors.affiliation: Pathology and Laboratory Medicine, UCLA, Los Angeles, California, USA.
authors.forename: Shangxin
authors.initials: S
authors.lastname: Yang
authors.affiliation: Pathology and Laboratory Medicine, UCLA, Los Angeles, California, USA.
authors.forename: Janet A
authors.initials: JA
authors.lastname: Hindler
authors.affiliation: Pathology and Laboratory Medicine, UCLA, Los Angeles, California, USA rhumphries@mednet.ucla.edu.
authors.forename: Romney M
authors.initials: RM
authors.lastname: Humphries
mesh_terms: D015780:Carbapenems / Q000494:pharmacology*; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D015780:Carbapenems; D001618:beta-Lactamases