PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Bacterial Factors Associated with Lethal Outcome of Enteropathogenic Escherichia coli Infection: Genomic Case-Control Studies

Background

Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome.

Methods

We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality.

Results

Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains.

Conclusions

Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.     

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.     

Trends in the Epidemiology of Pandemic and Non-pandemic Strains of Vibrio parahaemolyticus Isolated from Diarrheal Patients in Kolkata,India

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.     

Organization of the CTX prophage in environmental isolates of Vibrio mimicus

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.     

Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

Schisandrin B Ameliorates ICV-Infused Amyloid β Induced Oxidative Stress and Neuronal Dysfunction through Inhibiting RAGE/NF-κB/MAPK and Up-Regulating HSP/Beclin Expression

Amyloid β (Aβ)-induced neurotoxicity is a major pathological mechanism of Alzheimer’s disease (AD). Our previous studies have demonstrated that schisandrin B (Sch B), an antioxidant lignan from Schisandra chinensis, could protect mouse brain against scopolamine- and cisplatin-induced neuronal dysfunction. In the present study, we examined the protective effect of Sch B against intracerebroventricular (ICV)-infused Aβ-induced neuronal dysfunction in rat cortex and explored the potential mechanism of its action. Our results showed that 26 days co-administration of Sch B significantly improved the behavioral performance of Aβ (1–40)-infused rats in step-through test. At the same time, Sch B attenuated Aβ-induced increases in oxidative and nitrosative stresses, inflammatory markers such as inducible nitric oxide syntheses, cyclooxygenase-2, interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and DNA damage. Several proteins such as receptor for advanced glycation end products (RAGE), nuclear factor-κB, mitogen-activated protein kinases, and apoptosis markers were over expressed in Aβ-infused rats but were significantly inhibited by Sch B treatment. Furthermore, Sch B negatively modulated the Aβ level with simultaneous up-regulation of HSP70 and beclin, autophagy markers in Aβ-infused rats. The aforementioned effects of Sch B suggest its protective role against Aβ-induced neurotoxicity through intervention in the negative cycle of RAGE-mediated Aβ accumulation during AD patho-physiology.