Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla _VIM, bla _OXA-48, bla _OXA-23, bla _KPC, bla _NDM, bla _OXA-40, bla _OXA-58, bla _IMP, bla _GIM, bla _GES, ISAba1-bla _OXA-51, bla _IMI, bla _FIM and bla _DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla _VIM, bla _OXA-48, bla _OXA-23, bla _KPC, bla _NDM and bla _OXA-40, while multiplex-2 included bla _OXA-58, bla _IMP, bla _GIM, bla _GES, ISAba1-bla _OXA-51 and bla _IMI.Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.