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 antimicro-bial 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 carbapen-emase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and