Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Abstract

Background: Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods: In this study, a 15-µL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla_OXA-51 gene by PCR, qPCR and 15 µL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 µL-RPA data for detecting the carbapenem-resistance gene bla_OXA-23 were comparatively assessed. Results: The detection limit of the bla_OXA-51 gene by 15 µL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 µL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 µL RPA data for detecting bla_OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. Conclusion: We demonstrated that the new 15 µL RPA assay for detecting bla_OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

Keywords

Acinetobacter baumannii / Recombinase polymerase amplification / Rapid detection / Carbapenem-resistance gene / blaOXA-51 / blaOXA-23

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