Clinical isolates of Vibrio fluvialis from Kolkata, India, obtained during 2006: plasmids, the qnr gene and a mutation in gyrase A as mechanisms of multidrug resistance

Singh, Rochika; Rajpara, Neha; Tak, Jyoti; Patel, Arati; Mohanty, Priyabrata; Vinothkumar, Kittappa; Chowdhury, Goutam; Ramamurthy, Thandavarayan; Ghosh, Amit; Bhardwaj, Ashima Kushwaha

doi:10.1099/jmm.0.037226-0

Cited by 25 publications

(33 citation statements)

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“…MATE pumps utilize energy from the proton motive force (PMF) for transport of different substrates [12]. Altering the generation of PMF using reserpine blocks pump activity resulting in the enhanced accumulation of substrates [9], [17]. Same effect was observed when the V. fluvialis cells were treated with reserpine that aborted the PMF and increased the ethidium bromide concentration inside the cells (Fig.…”

Section: Resultsmentioning

confidence: 86%

“…Recent studies from our laboratory have suggested the role of various mobile genetic elements as well as chromosome-borne factors in multiple drug resistance of various clinical isolates of V. fluvialis [3], [17]. Though most of the studies of efflux pumps have been done in V. cholerae O1 or non-O1/non-139 strains, studies have not been envisaged in a lesser known organism like V. fluvialis .…”

Section: Introductionmentioning

confidence: 99%

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Role of H- and D- MATE-Type Transporters from Multidrug Resistant Clinical Isolates of Vibrio fluvialis in Conferring Fluoroquinolone Resistance

2012

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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.

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Section: Resultsmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Role of H- and D- MATE-Type Transporters from Multidrug Resistant Clinical Isolates of Vibrio fluvialis in Conferring Fluoroquinolone Resistance

2012

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“…DMAMA-PCR was carried out to screen for the type of ctxB gene present using the primers ctxB-F3, ctxB-F4, Fw-con, Rv-El Tor and Rv-Cla as described recently [23]. PCR amplifications for four topoisomerase genes ( gyrA , gyrB , parC , parE ) were carried out as described earlier [18]. PCR reactions were performed using a PTC-225 DNA Engine Tetrad™ Cycler (MJ Research Inc., MA, USA) and Pfu (Fermentas International Inc., Ontario, Canada) or Taq DNA polymerases (Bangalore Genei, Bangalore, India).…”

Section: Methodsmentioning

confidence: 99%

Clinical Isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: Preponderance of SXT Element and Presence of Haitian ctxB Variant

et al. 2013

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BackgroundIncrease in the number of multidrug resistant pathogens and the accompanied rise in case fatality rates has hampered the treatment of many infectious diseases including cholera. Unraveling the mechanisms responsible for multidrug resistance in the clinical isolates of Vibrio cholerae would help in understanding evolution of these pathogenic bacteria and their epidemic potential. This study was carried out to identify genetic factors responsible for multiple drug resistance in clinical isolates of Vibrio cholerae O1, serotype Ogawa, biotype El Tor isolated from the patients admitted to the Infectious Diseases Hospital, Kolkata, India, in 2009.Methodology/Principal FindingsOne hundred and nineteen clinical isolates of V. cholerae were analysed for their antibiotic resistance phenotypes. Antibiogram analysis revealed that majority of the isolates showed resistance to co-trimoxazole, nalidixic acid, polymixin B and streptomycin. In PCR, SXT integrase was detected in 117 isolates and its sequence showed 99% identity notably to ICEVchInd5 from Sevagram, India, ICEVchBan5 from Bangladesh and VC1786ICE sequence from Haiti outbreak among others. Antibiotic resistance traits corresponding to SXT element were transferred from the parent Vibrio isolate to the recipient E. coli XL-1 Blue cells during conjugation. Double-mismatch-amplification mutation assay (DMAMA) revealed the presence of Haitian type ctxB allele of genotype 7 in 55 isolates and the classical ctxB allele of genotype 1 in 59 isolates. Analysis of topoisomerase sequences revealed the presence of mutation Ser83 → Ile in gyrA and Ser85→ Leu in parC. This clearly showed the circulation of SXT-containing V. cholerae as causative agent for cholera in Kolkata.ConclusionsThere was predominance of SXT element in these clinical isolates from Kolkata region which also accounted for their antibiotic resistance phenotype typical of this element. DMAMA PCR showed them to be a mixture of isolates with different ctxB alleles like classical, El Tor and Haitian variants.

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“…qnrVC is so far the only qnr gene located in a cassette with a linked attC site (92). qnrVC genes have been found on plasmids in A. punctata (30), and V. fluvialis (32), within integrons in A. baumannii (93) and P. aeruginosa (94), and within the transmissible SXT integrating element of V. cholerae (34, 95). …”

Section: Qnr Plasmidsmentioning

confidence: 99%

Plasmid-Mediated Quinolone Resistance

2014

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Summary Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.

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Clinical isolates of Vibrio fluvialis from Kolkata, India, obtained during 2006: plasmids, the qnr gene and a mutation in gyrase A as mechanisms of multidrug resistance

Cited by 25 publications

References 22 publications

Role of H- and D- MATE-Type Transporters from Multidrug Resistant Clinical Isolates of Vibrio fluvialis in Conferring Fluoroquinolone Resistance

Role of H- and D- MATE-Type Transporters from Multidrug Resistant Clinical Isolates of Vibrio fluvialis in Conferring Fluoroquinolone Resistance

Clinical Isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: Preponderance of SXT Element and Presence of Haitian ctxB Variant

Plasmid-Mediated Quinolone Resistance

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