Rapid detection of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases from Acinetobacter species by real-time PCR

Background: Carbapenemase-producing Acinetobacter species, especially A. baumannii, are frequently associated with treatment failures and hospital outbreaks; thus, rapid and reliable detection of specific resistance markers is paramount. The most common carbapenemases found in A. baumannii, namely OXA-23-like, OXA-24-like, and OXA-58-like, belong to the oxacillinase group (class D β-lactamases) which is notoriously difficult to identify phenotypically due to the lack of specific inhibitors. Aim: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay to detect and differentiate the above three oxacillinases. Methods: All available variants of the above three oxacillinase subfamilies were downloaded (as of November 2019) from the Beta-Lactamase DataBase (http://bldb.eu/) aligned with Clustal Omega and oligonucleotides designed using Primer-BLAST. A multiplex real-time PCR assay that included an internal control to discount inhibition was optimized on the Rotor-Gene Q (Qiagen) using the Rotor-Gene Multiplex PCR Kit (Qiagen) and validated using a panel of 122 previously characterized strains carrying a wide range of β-lactamases, often in combination. Findings: The in-silico approach enabled the design of oligonucleotides in conserved regions of the OXA-24-like and OXA-58-like alignments. Among the 42 described OXA-23-like variants, a single nucleotide polymorphism (SNP) was present in one of the oligonucleotide binding sites of OXA-27, OXA-166, OXA-811, OXA-812, and OXA-816. The assay was 100% sensitive and highly specific. Inhibition was not observed. Conclusion: The assay is easy to perform with results available in about 70 min. It enables unequivocal detection and differentiation of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases even when more than one is simultaneously present.