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

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 energydependent 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 i

Clinical isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: preponderance of SXT element and presence of Haitian ctxB variant.

BACKGROUND: Increase 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 FINDINGS: One 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. CONCLUSIONS: There 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

Antimicrobial susceptibility of IDH01572, IDH01738 and their transconjugants.

<p>The antibiotic names are as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056477#pone-0056477-g001" target="_blank">figure 1</a>.</p><p>Bold face indicates the resistance traits from recipient XL-1Blue cells.</p

Antibiotic susceptibility profile of 119 clinical isolates from Kolkata, India, in 2009.

<p>AMP, Ampicillin; CHL, Chloramphenicol; CIP, Ciprofloxacin; COT, Co-Trimoxazole; GEN, Gentamicin; KAN, Kanamycin; NAL, Nalidixic Acid; NEO, Neomycin; NOR, Norfloxacin; PB, Polymixin B; STR, Streptomycin; SUL, Sulfisoxazole; TET, Tetracycline; TRI, Trimethoprim.</p

Agarose gel (1%) analysis of PCR product of SXT integrase from IDH isolates and their transconjugants.

<p>PCR products obtained using genomic DNA templates from clinical isolates or their transconjugants have been electrophoresed in different lanes as follows: Lane M : 1 kb ladder (Fermentas); Lane 1: Positive control <i>V.cholerae</i> O139 MO10; Lane 2: Recipient <i>E. coli</i> XL-1 Blue; Lanes 3 and 4: Negative controls of no DNA template and SXT-negative IDH02095 isolate respectively; Lanes 5 and 6 : IDH01572 (SXT-positive) isolate and its transconjugant respectively; Lanes 7 and 8 : IDH01738 (SXT-positive) isolate and its transconjugant respectively.</p

Abstracts of International Conference on Innovations in Business Management

This book contains abstracts of the various research ideas of the academic community and practitioners of management presented at the International Conference on Innovations in Business Management (ICIBM 2020). The researchers have contributed toward various themes of the conference such as sustainable economy, supply chain, women-empowerment, export-import, microfinance, government policies, etc. We strongly believe that it will open up further scope for in-depth research in various disciplines of business management. Best wishes to the participants to have detailed discussions on the above-said wide range of areas. Conference Title: International Conference on Innovations in Business ManagementConference Acronym: ICIBM 2020Conference Date: 16-17 January 2020Conference Location: ICFAI University, Dehradun, IndiaConference Organizers: ICFAI Business School, ICFAI University, Dehradun, India &amp; University of Derby, United Kingdo