Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates

Abstract

Background: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE).

Methods: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes.

Results: Among the carbapenem-resistant isolates the genes bla _KPC, bla _VIM, bla _NDM or bla _OXA were detected. Both RT-PCR assays detected all bla _KPC, bla _VIM and bla _NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials.

Conclusion: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Keywords: Acinetobacter baumannii; Carbapenemases; MDR gram-negative bacteria; RT-PCR.

Fig. 1

Comparison of the in-house PCR assay and the Check-Direct CPE assay for the identification of carbapenemase-producing isolates (n = 116 isolates)

Fig. 2

Melting-curve analysis of OXA-23-like and OXA-48-like gene variants. The carbapenemase genes were amplified by SYBR green multiplex PCR mix with OXA-23-like and OXA-48-like primer followed by melt curve analysis. The melting-curve was visualised at the wavelength channel of 475/520 nm. The melting peak for OXA-23-like was observed between 80.6 and 80.9 °C and for OXA-48-like between 83.2 and 83.3 °C