Class I Integrons and SXT Elements in El Tor Strains Isolated before and after 1992 Vibrio cholerae O139 Outbreak, Calcutta, India

We examined the distribution of class I integrons and SXT elements in Vibrio cholerae O1 El Tor strains, isolated in Calcutta, India, before and after the V. cholerae O139 outbreak in 1992. Class I integrons, with aadA1 gene cassette, were detected primarily in the pre-O139 strains; the SXT element was found mainly in the post-O139 strains

The value of and challenges for cholera vaccines in Africa.

The 21st century saw a shift in the cholera burden from Asia to Africa. The risk factors for cholera outbreaks in Africa are incompletely understood, and the traditional emphasis on providing safe drinking water and improving sanitation and hygiene has proven remarkably insufficient to contain outbreaks. Current killed whole-cell oral cholera vaccines (OCVs) are safe and guarantee a high level of protection for several years. OCVs have been licensed for >20 years, but their potential for preventing and control cholera outbreaks in Africa has not been realized. Although each item in the long list of technical reasons why cholera vaccination campaigns have been deferred is plausible, we believe that the biggest barrier is that populations affected by cholera outbreaks are underprivileged and lack a strong political voice. The evaluation and use of OCVs as a tool for cholera control will require a new, more compassionate, less risk-averse generation of decision makers

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

Distribution of Class 1 Integrons with IS26-Mediated Deletions in Their 3′-Conserved Segments in Escherichia coli of Human and Animal Origin

Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer

Resveratrol regulates body weight in healthy and ovariectomized rats

Abstract Objective The elevated body weight in post-menopausal state attributes to the reduced estrogen levels which is alleviated by resveratrol (RES) but its role in control rats is not well understood. The main objective of the study was to explore the effects of RES on the body weight of ovariectomized (OVX) female rats with controls and to relate their biochemical parameters. Methods Female Wistar rats weighing 200–300 g underwent bilateral ovariectomy (OVX) and were fed soya free diet (n = 8 rats per group). In all groups: (Control, Control + Resveratrol, OVX and OVX + Resveratrol) resveratrol was administered orally at a dose of 5 mg/kg/day for 1 month. Glucose and other biochemical parameters were examined. Results Significant reduction in the gain of body weight was observed in the control rats treated with resveratrol. Ovariectomy caused an escalation in gain of body weight due to loss of estrogen which was brought down with resveratrol. There was a slight dip in the blood glucose levels after resveratrol treatment. Conclusion Resveratrol significantly reduced the gain of body weight in the control rats and in OVX rats showing its antiobesogenic effects

Strategies for the production of a potential candidate vaccine for cholera

First attempt at cholera vaccination was made by Jaime Ferran in 1884. Since then, a variety of strategies and methods have been evolved to create a safe, efficacious vaccine against cholera. For the first few years emphasis was on the development of parenteral vaccines. However, as a result of accumulation of a tremendous amount of knowledge, not only on Vibrio cholerae-the causative agent, but also on its interaction with the host, emphasis has shifted towards the development of oral vaccines. Two such vaccines, one killed, a whole cell/B subunit combination vaccine and the other a live attenuated one, have shown promise. The combination vaccine in its present state of development confers only a transient protection in young children, while the live attenuated one produces adverse reaction. To combat these, various strategies are being evolved. In one attempt, a potential candidate vaccine strain has been constructed from a non-reactogenic clinical isolate of V. cholerae, which is devoid of all known major virulence genes and is also a good colonizer. In animal studies this construct has shown considerable promise. This review discusses the various strategies that have been employed so far in the quest for an ideal cholera vaccine

Occurrence of Antibiotic Resistance Gene Cassettes aac(6′)-Ib, dfrA5, dfrA12, and ereA2 in Class I Integrons in Non-O1, Non-O139 Vibrio cholerae Strains in India

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6′)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6′)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance

Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype inaba biotype El Tor and assessment of its reactogenicity and immunogenicity in the rabbit model

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae El Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the 'B' subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and El Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity