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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. 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 like gentamicin, kanamycin and streptomycin. Decrease in MIC was also observed for acriflavin, ethidium bromide, safranin and nalidixic acid. SIGNIFICANCE: 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.

SUBMITTER: Mohanty P

PROVIDER: S-EPMC3335099 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Role of H- and D- MATE-type transporters from multidrug resistant clinical isolates of Vibrio fluvialis in conferring fluoroquinolone resistance.

Mohanty Priyabrata P Patel Arati A Kushwaha Bhardwaj Ashima A

PloS one 20120423 4

<h4>Background</h4>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.<h4>Methodology</h4>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 cl ...[more]

PMID: 22540001

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Project description:BACKGROUND:Nearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait. FINDINGS:Direct DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence. CONCLUSIONS:Although fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.

Project description:The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region.Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates.Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94-32 and 100-32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region.In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Dataset Information

Role of H- and D- MATE-type transporters from multidrug resistant clinical isolates of Vibrio fluvialis in conferring fluoroquinolone resistance.

Publications

Role of H- and D- MATE-type transporters from multidrug resistant clinical isolates of Vibrio fluvialis in conferring fluoroquinolone resistance.

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