Abstract
BackgroundThe study seeks to understand the role of efflux pumps in multidrug resistance displayed by the clinical isolates of Vibrio fluvialis, a pathogen known to cause cholera-like diarrhoea.MethodologyTwo putative MATE family efflux pumps (H- and D-type) were PCR amplified from clinical isolates of V. fluvialis obtained from Kolkata, India, in 2006 and sequenced. Bioinformatic analysis of these proteins was done to predict protein structures. Subsequently, the genes were cloned and expressed in a drug hypersusceptible Escherichia coli strain KAM32 using the vector pBR322. The recombinant clones were tested for the functionality of the efflux pump proteins by MIC determination and drug transport assays using fluorimeter.ResultsThe sequences of the genes were found to be around 99% identical to their counterparts in V. cholerae. Protein structure predicting servers TMHMM and I-TASSER depicted ten-twelve membrane helical structures for both type of pumps. Real time PCR showed that these genes were expressed in the native V. fluvialis isolates. In the drug transport assays, the V. fluvialis clinical isolates as well as recombinant E. coli harbouring the efflux pump genes showed the energy-dependent and sodium ion-dependent drug transport activity. KAM32 cells harbouring the recombinant plasmids showed elevated MIC to the fluoroquinolones, norfloxacin and ciprofloxacin but H-type pumps VCH and VFH from V. cholerae and V. fluvialis respectively, showed decreased MIC to aminoglycosides like gentamicin, kanamycin and streptomycin. Decrease in MIC was also observed for acriflavin, ethidium bromide, safranin and nalidixic acid.SignificanceIncreased resistance towards fluoroquinolones exhibited due to these efflux pumps from multidrug resistant clinical isolates of V. fluvialis implies that treatment procedure may become more elaborate for this simple but highly infectious disease. To the best of our knowledge, this is the first report of cloning and characterization of efflux pumps from multidrug resistant clinical isolates of V. fluvialis.
Highlights
Vibrio fluvialis causes sporadic cases of diarrhoea clinically indistinguishable from cholera
Efflux pumps are categorized into five major superfamilies i.e the major facilitator superfamily (MFS), the ATP-binding cassette family (ABC), the resistance-nodulation-division family (RND), the small multidrug resistance protein family (SMR) and multidrug and toxic compound extrusion family (MATE), the latter being the newest member of antimicrobial transporters [8,9]
The present study was aimed at detailed characterization of the efflux pump proteins from the not-so-well studied pathogen Vibrio fluvialis responsible for sporadic cases of diarrhoea and known to be associated with pediatric diarrhoea [1,2,3]
Summary
Vibrio fluvialis causes sporadic cases of diarrhoea clinically indistinguishable from cholera. Efflux pumps are one of the major determinants of the accumulation of various compounds including antibacterials inside a bacterial cell and are thought to work in synergy with other mechanisms like target gene mutations and quinolone resistance genes (qnr) to acquire clinical breakpoints for resistance [5,6]. MATE family efflux pumps utilize Na+/H+ gradient for transport and has been reported to contain three branches: the NorM branch, a branch containing several eukaryotic proteins and a branch containing E. coli DinF [15]. The study seeks to understand the role of efflux pumps in multidrug resistance displayed by the clinical isolates of Vibrio fluvialis, a pathogen known to cause cholera-like diarrhoea
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