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

Variant Salmonella Genomic Island 1 Antibiotic Resistance Gene Cluster in Salmonella enterica Serovar Albany

Salmonella genomic island 1 (SGI1) contains an antibiotic resistance gene cluster and has been previously identified in multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, and Paratyphi B. We identified a variant SGI1 antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France. In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function. Thus, this serovar Albany strain represents the fourth S. enterica serovar in which SGI1 has been identified and the first SGI1 example where gene cassette replacement took place in one of its integron structures. The antibiotic resistance gene cluster of serovar Albany strain 7205.00 constitutes a new SGI1 variant; we propose a name of SGI1-F

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

A silent mutation in human alpha-A crystallin gene in patients with age-related nuclear or cortical cataract

A cataract is a complex multifactorial disease that results from alterations in the cellular architecture, i.e. lens proteins. Genes associated with the development of lens include crystallin genes. Although crystallins are highly conserved proteins among vertebrates, a significant number of polymorphisms exist in human population. In this study, we screened for polymorphisms in crystallin alpha A (CRYAA) and alpha B (CRYAB) genes in 200 patients over 40 years of age, diagnosed with age-related cataract (ARC; nuclear and cortical cataracts). Genomic DNA was extracted from the peripheral blood. The coding regions of the CRYAA and CRYAB gene were amplified using polymerase chain reaction and subjected to restriction digestion. Restriction fragment length polymorphism (RFLP) was performed using known restriction enzymes for CRYAA and CRYAB genes. Denaturing high performance liquid chromatography and direct sequencing were performed to detect sequence variation in CRYAA gene. In silico analysis of secondary CRYAA mRNA structure was performed using CLC RNA Workbench. RFLP analysis did not show any changes in the restriction sites of CRYAA and CRYAB genes. In 6 patients (4 patients with nuclear cataract and 2 with cortical cataract), sequence analysis of the exon 1 in the CRYAA gene showed a silent single nucleotide polymorphism [D2D] (CRYAA: C to T transition). One of the patients with nuclear cataract was homozygous for this allele. The in silico analysis revealed that D2D mutation results in a compact CRYAA mRNA secondary structure, while the wild type CRYAA mRNA has a weak or loose secondary structure. D2D mutation in the CRYAA gene may be an additional risk factor for progression of ARC

Comparison of indirect cyanmethaemoglobin method and TrueHb haemometer point of care test with direct cyanmethaemoglobin (gold standard) method for strengthening anaemia detection at primary health care level

Introduction: The most reliable indicator for anaemia diagnosis at the population level is haemoglobin (Hb) estimation. The direct cyanmethaemoglobin method is considered the gold standard method for haemoglobin estimation. However, for resource constraint areas like primary health care (PHC) level, either blood samples are transported on filter paper for Hb testing (indirect cyanmethaemoglobin method) in laboratory or point of care testing is commonly used. Therefore, a comparative analysis of haemoglobin estimation of direct with indirect cyanmethaemoglobin method and also with TrueHb (Wrig Nanosystems Pvt. Ltd.) haemometer was done to strengthen anaemia diagnosis at the PHC level. Materials and Methods: This was a cross-sectional study. A total of 90 participants above 9 years of age, who visited the outpatient department (OPD) of health centre, Kheri and gave consent were included. Comparative analysis was done between Hb concentration assessed by indirect cyanmethaemoglobin method and TrueHb haemometer device against the gold standard method. Results: The mean Hb value estimated by direct method, TrueHb haemometer and indirect methods (filter paper A, B and C) was 11.42 ± 1.59 g/dl, 11.52 ± 1.54 g/dl, 10.66 ± 1.52 g/dl, 9.84 ± 1.50 g/dl and 10.19 ± 1.62 g/dl, respectively. There was no significant difference found between the mean Hb concentration estimated by the direct method and the TrueHb haemometer device. However, there was a significant difference in mean Hb values between the direct method and the indirect method. Therefore, regression analysis was done to estimate the correction factor for the indirect method. Conclusion: TrueHb metre device gave promising results in comparison to the gold standard method and can be used if resource permits in PHC centres. Indirect methods of haemoglobin estimation can be an alternative in resource-constraint settings, specifically for surveys. However, further studies are required for the validation of the indirect method

Evaluation of antioxidants and argpyrimidine in normal and cataractous lenses in north Indian population

AIM: To assess the level of glutathione, thioltransferase, and argpyrimidine in nuclear and cortical cataractous lenses as well as in the clear lenses in the north Indian population. METHODS: Human cataractous lenses were collected from the patients who underwent extracapsular cataract extraction surgery; clear lenses were collected from the freshly donated eye bank eyes. Antioxidant molecules such as glutathione and thioltransferase enzyme activity were measured; simultaneously in these lenses a blue fluorophore argpyrimidine, an advanced glycation end (AGE) product level was assessed using high performance liquid chromatography (HPLC). RESULTS: The protein concentration was found to be present at higher levels in the control lenses compared to cataract lenses. A significant decrease in the glutathione level was observed in the nuclear cataractous lenses compared to cortical cataractous (P=0.004) and clear lenses (P≤0.005), but no significant change in the level of antioxidant enzyme thioltransferase was observed. Further, argpyrimidine a blue fluorophore (AGE) was found to be significantly higher in the nuclear cataract (P=0.013) compared to cortical cataract lenses. CONCLUSION: Antioxidants such as glutathione significantly decrease in age-related nuclear and cortical cataract and an AGE, argpyrimidine are present at significantly higher levels in nuclear cataract