Defining the role and control of the lpxO gene in Klebsiella pneumoniae

Abstract

Antimicrobial resistance (AMR) is fast becoming one of the greatest challenges to human mortality this century. In recent years the number of reported infections by multi-drug resistant (MDR) bacteria has seen a rapid rise. As a result, the efficacy of currently used antibiotics is decreasing, with infections by MDR bacteria increasingly difficult to treat. In more and more cases, the use of last-line drugs is becoming necessary. However, this in turn has resulted in increasing reports of last-line drug resistance, presenting a dangerous cycle that leaves us potentially short of treatment options. As a result, elucidating the intrinsic resistance mechanisms that pathogens employ, and their importance in generating an AMR phenotype, is essential to developing a comprehensive and effective antimicrobial strategy. Furthermore, identifying specific resistance mechanisms and their contribution to AMR presents potential targets for inhibitory drugs in combination with existing antimicrobials. This project looks at the role of the transcription regulator RamA in generating an AMR phenotype in the Gram-negative bacterium Klebsiella pneumoniae. K. pneumoniae is a major cause of nosocomial infections in the immunocompromised, with MDR isolates resistant to the last-line drug colistin rapidly increasing in prevalence. K. pneumoniae RamA regulates genes associated with virulence and resistance, able to modulate outer membrane permeability through altered influx and efflux. Overexpression of ramA has also been shown to upregulate the dioxygenase LpxO, a lipid A modifying enzyme associated with increased resistance to colistin, polymyxin B, and the human cationic antimicrobial peptide LL-37. Our study seeks to define the relative importance of RamA and LpxO in relation to polymyxin B, colistin and LL-37; specifically how RamA-mediated lpxO-overexpression confers this phenotype. In this regard, we have performed MIC and relative survival assay studies with wild-type K. pneumoniae, ramA- and lpxO-overexpressing strains, in order to define the role of these genes in the relevant phenotypes.