Exposure to Sub-inhibitory Concentrations of the Chemosensitizer 1-(1-Naphthylmethyl)-Piperazine Creates Membrane Destabilization in Multi-Drug Resistant Klebsiella pneumoniae.

João Anes,Sathesh K Sivasankaran,Shabarinath Srikumar,Séamus Fanning,Dechamma M Muthappa

doi:10.3389/fmicb.2019.00092

João Anes, Sathesh K Sivasankaran + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2019.00092

Copy DOI

Abstract

Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using β-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both β-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition.

Highlights

Bacterial multi-drug resistance (MDR) is a serious threat to infectious disease therapy (Van Boeckel et al, 2015)
We subjected K. pneumoniae MGH78578 to a β-lactamase assay in the presence of 250 μg/mL (0.5x minimum inhibitory concentration (MIC)), defined as a sub-inhibitory concentration of NMP. β-Lactamase is a periplasmic enzyme, its extracellular release correlates with disruption of the outer membrane (OM) which can be assayed by the hydrolysis of the chromogenic substrate nitrocefin (O’Callaghan et al, 1972)
Even though efflux pump inhibition is the main theme of NMP action, we propose that membrane destabilization is an additional effect of NMP on bacterial cells

Summary

Introduction

Bacterial multi-drug resistance (MDR) is a serious threat to infectious disease therapy (Van Boeckel et al, 2015). Drug resistance is recognized by the World Health Organization as one of the most significant challenges of the 21st century (World Health Organization, 2014). 25,000 people die each year in Europe alone due to multi-drug resistant (MDR) bacterial infections costing European Union € 1.5 billion annually (Davies et al, 2013). It is imperative that new technologies be developed to mitigate drug resistance in bacteria. The mechanisms used by bacteria to combat antimicrobial agents are (1) Reduced permeability of the antimicrobial compound; (2) Increased efflux of the molecule; (3) Gene mutation dependent modification of antimicrobial targets; and (4) Direct inactivation of drug molecules (Blair et al, 2015). The genes encoding proteins responsible for various antibiotic resistance mechanisms are frequently located on mobile genetic elements (MGE) such as plasmids, transposons, integrons and are disseminated via horizontal transfer (Carraro et al, 2014; Gillings, 2014)

Methods

Results

Conclusion