原创力文档role of h- and d- mate-type transporters from multidrug resistant clinical isolates of vibrio fluvialis in conferring fluoroquinolone resistanceh - d mate-type转运蛋白的作用从耐药的临床分离株弧菌fluvialis赋予氟喹诺酮类耐药性.pdfVIP
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role of h- and d- mate-type transporters from multidrug resistant clinical isolates of vibrio fluvialis in conferring fluoroquinolone resistanceh - d mate-type转运蛋白的作用从耐药的临床分离株弧菌fluvialis赋予氟喹诺酮类耐药性
Role of H- and D- MATE-Type Transporters from
Multidrug Resistant Clinical Isolates of Vibrio fluvialis in
Conferring Fluoroquinolone Resistance
Priyabrata Mohanty, Arati Patel, Ashima Kushwaha Bhardwaj*
Department of Human Health and Diseases, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India
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.
Methodology: 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
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