title: Role of H- and D- MATE-type transporters from multidrug resistant clinical isolates of Vibrio fluvialis in conferring fluoroquinolone resistance.
abstract: BACKGROUND
  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.
  
  
  METHODS
  Two 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Increased 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.
authors.affiliation: Department of Human Health and Diseases, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India.
authors.forename: Priyabrata
authors.initials: P
authors.lastname: Mohanty
authors.forename: Arati
authors.initials: A
authors.lastname: Patel
authors.forename: Ashima
authors.initials: A
authors.lastname: Kushwaha Bhardwaj
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003141:Communicable Diseases / Q000382:microbiology; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D024841:Fluoroquinolones / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism; D014733:Vibrio / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D024841:Fluoroquinolones; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D012964:Sodium