Optimisation of a multiplex PCR assay for the detection of carbapenem resistance genes in Klebsiella pneumoniae

Abstract

Carbapenem-resistant Enterobacterales have been classified by the World Health Organisation as one of the most critical bacterial threats to public health, including Klebsiella pneumoniae. This study aimed to design and optimise two multiplex PCR assays for the simultaneous detection of five carbapenem resistance genes in K. pneumoniae, including mPCR1 (bla_KPC, bla_IMP, bla_VIM) and mPCR2 (bla_NDM, bla_{OXA-48- like}). DNA extracted from bacterial colonies using the heat-shock method was used, while positive controls consisted of bacterial strains and plasmids carrying the target genes confirmed by DNA sequencing. The optimised conditions were successfully established for mPCR1, with an annealing temperature of 54ºC and primer concentrations of 0.4, 0.2, and 0.3 µM for bla_KPC, bla_IMP, bla_VIM, respectively; and for mPCR2, with an annealing temperature of 58ºC and primer concentrations of 0.5 µM for bla_NDM and 0.3 µM for bla_OXA-48-like. Evaluation of the optimised assays on 30 clinical K. pneumoniae isolates from Military Hospital 103 showed that 26.7% (8/30) harbored carbapenemase genes, predominantly single-gene carriers (75.0%, 6/8), while 25.0% (2/8) carried two carbapenemase genes. No isolates carrying blaIMP or blaVIM were detected. The optimised mPCR protocol provides a rapid tool for screening of selected carbapenemase-encoding genes in clinical K. pneumoniae isolates, thereby supporting treatment selection and infection control.