Characterisation of the secondary effects of sub-inhibitory concentrations of 1-(1-naphthylmethyl)-piperazine (NMP) in Klebsiella pneumoniae

Efflux of antimicrobial compounds from bacterial cells is one of the important mechanisms responsible for multi-drug resistance (MDR). Inhibiting the activity of efflux pumps using chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) is currently considered as a promising strategy to overcome MDR. However, additional effects of NMP other than inhibition are rarely if ever considered. Here, using phenotypic, phenotypic microarray and transcriptomic assays we show that NMP plays a role in membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The observation of membrane destabilization was supported by RNA-seq data which showed that many up-regulated genes were either directly involved in responses to envelope stress or bacterial repair systems which are essential to maintain viability in an environment containing NMP. Membrane destabilization happens as early as 15 minutes post-NMP treatment. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition. β-Lactamase activity, membrane potential and Transmission Electron Microscopy assays were used to assay the NMP mediated membrane destabilisation in MDR Klebsiella pneumoniae MGH 78578 strain. We further used the Phenotypic Microarray (Biolog) and RNA-seq to elucidate the transcriptional and metabolic signals. Crystal violet-based assays were also used to assay the biofilm formation ability of NMP treated bacterial cells.