Abstract
OXA-48-like carbapenemase gene remains a hidden threat, as different OXA-48 variants have varying presentations of susceptibility to antibiotics that might affect the treatment decisions. Rapid detection and differentiation of OXA-48-like carbapenemase genes are critical for targeted treatment and infection control. In this study, we aimed to develop high-resolution melting (HRM) analysis for the differentiation of OXA-48 variants. HRM analysis is a post-polymerase chain reaction (post-PCR) method for identification of small variations in nucleic acid sequences based on the PCR dissociation curve. A total of 82 bacterial strains, which consisted of Enterobacteriaceae and non-Enterobacteriaceae, were collected from a tertiary teaching hospital. The sensitivity and specificity of the assay were determined, and the developed assay was evaluated using the collected isolates against conventional-sequencing method. Overall, the developed assay was able to detect isolates that harboured OXA-48 and OXA232/OXA-181 by showing two distinct peaks at 81.1 ± 0.2 °C and 82.1 ± 0.2 °C, respectively. The detection limit of the assay was 1.6 x 10−6 ng/µL for OXA-48 and 1.8 × 10−7 ng/µL for OXA-232/OXA-181. This assay showed 100% specificity when evaluated on a panel of 37 isolates comprised of different species of bacteria and yeasts. When the assay with isolates collected in the year 2016 was first evaluated, the assay showed comparable results with conventional PCR-sequencing method where 34 OXA-48 and OXA-232/OXA-181 were detected. By using HRM analysis, the presence of OXA-48-like variants could be easily identified within 3 h from the pure culture.
Highlights
The prevalence of carbapenem-resistant Enterobacteriaceae (CRE), especially those harbouringOXA-48-like carbapenem hydrolysing class D β-lactamases, has increased at an alarming rate [1].OXA-48 is a plasmid borne enzyme that is only detected in Enterobacteriaceae, and is commonly associated with Klebsiella pneumoniae (K. pneumoniae) [1]
Each variant differs from OXA-48 by one to five amino acid substitution or deletion, which results in different hydrolytic profiles
In the 298-bp region amplified by the designed primers, OXA-48 differs from OXA-181 and OXA-232 by four and five amino acids substitutions, respectively, while OXA-181 and OXA-232 only differ by one nucleotide substitution at position 642 (A to T)
Summary
The prevalence of carbapenem-resistant Enterobacteriaceae (CRE), especially those harbouringOXA-48-like carbapenem hydrolysing class D β-lactamases, has increased at an alarming rate [1].OXA-48 is a plasmid borne enzyme that is only detected in Enterobacteriaceae, and is commonly associated with Klebsiella pneumoniae (K. pneumoniae) [1]. The prevalence of carbapenem-resistant Enterobacteriaceae (CRE), especially those harbouring. OXA-48-like carbapenem hydrolysing class D β-lactamases, has increased at an alarming rate [1]. OXA-48 is a plasmid borne enzyme that is only detected in Enterobacteriaceae, and is commonly associated with Klebsiella pneumoniae (K. pneumoniae) [1]. The first OXA-48 carbapenemase gene was identified in a K. pneumoniae isolate from Istanbul, Turkey in 2001 and subsequently disseminated in many countries including India, North Africa, and Europe [1,2]. Several OXA-48-like variants have been described such as OXA-48, OXA-232, OXA-181, OXA-162, OXA-163, OXA-244, and OXA-245 [3]. Each variant differs from OXA-48 by one to five amino acid substitution or deletion, which results in different hydrolytic profiles. OXA-232 exhibited five amino acid substitutions compared to OXA-48 but differed from
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