Exploring the dynamics of toxigenic environmental Vibrio mimicus and its comparative analysis with Vibrio cholerae of the southern Gangetic delta.

Vibrio mimicus and Vibrio cholerae are closely related species. Environmental V.mimicus were comparatively analyzed with V.cholerae, for the presence of virulence genes, antibiotic susceptibility, resistance genes, in-vitro hemolysis, and biofilm formation. Phylogenetic analysis was performed depending on toxin-gene disposition and isolation area. One V.mimicus isolate harbored ctxA, tcp El-Tor, toxT and toxS, whereas several strains contained incomplete copies of virulence cassettes and associated toxin genes. V.cholerae isolates harbored ctx, tcp and toxT genes, with a higher preponderance of hlyA, rtxA and toxR genes. V.mimicus were highly sensitive to amino/carboxy-penicillins, furazolidone & gentamycin, with quinolone & tetracycline resistance genes. V.cholerae isolates were sensitive to penicillins and cephalosporins, with 29% of the strains bearing the sxt gene. Phylogenetically, the apomorphic strains of both species were unique to the inland sites. V.cholerae has embodied an enormous public health burden globally but our findings emphasize the role of V.mimicus as an emerging etiological agent with similar epidemic potential.

Seasonal influence, enteropathogenic microbial load and diarrhoeal enigma in the Gangetic Delta, India: Present scenario and health implications.

BACKGROUND: Environmental diarrheagenic enteropathogens, effect of surrounding attributes and disease dynamicity remains far from being conclusively explored. Population flux, poor sanitation and hygienic practice poses potential health threat in diarrhoea endemic tropical countries like India. We aim to identify environmental attributes, seasonality of water-borne enteropathogens and health risk assessment off the river Ganges. METHODS: A yearlong sampling data generated from three sites on either sides of the River was analysed and implications have been reported. Immediately after sample collection, physico chemical and bacterial indices were measured at the sampling site and laboratory respectively, followed by further statistical analysis of the findings. RESULTS: Annual variation of physico-chemical indices viz., temperature 18°C-36°C, pH 7.49-8.67, conductivity 215-468µS/cm and turbidity 25.6-593 NTU was recorded in the riverine water samples. High temperature and turbidity were recorded in the summer and monsoon at all sites. High bacterial dispersion has been positively correlated with turbidity and temperature variation (P<0.01; P<0.1) as we report TBC 103-105CFU/ml, TCC 103-104CFU/ml and CVC 4-212CFU/ml, with higher distribution in the monsoon and reverse in the winter. This suggests that the bacterial pool proliferates at higher temperature whereas turbidity enhances their survival providing the substratum for the bacterial pool. CVC could be positively correlated with conductivity which implies that ionic content of water augments the Vibrio load. Adaptive capability of Vibrios to sustain in very low saline riverine setting seems to be assisted by turbid water coupled with nutrient rich organic matter. CONCLUSION: Our present work establishes the interplay of seasonal variants on the dynamicity of enteropathogenic bacteria in flowing aquatic ecosystem. It also categorises the existing microbial threats in the Ganga River to help monitor the conventional as well as emerging diarrhoeal pathogens to reduce diarrheal recurrences.

Waterborne outbreaks in diarrhoea endemic foci of India: a longitudinal exploration and its implications.

Diarrhoea remains a global public health enigma raising deep concerns for the health planners since contaminated potable water often spoils the community health structure. We hereby report a 6-year odd continuing outbreak surveillance report based on potable water indices, during which 264 water samples were screened from different districts of West Bengal, India. Samples were analysed for the presence of different enteropathogenic bacterial species by conventional molecular tools and their sensitivity to antibiotics. 78.03% samples were positive for enteropathogenic bacterial organisms and 75% samples harbored Coliform. 45.45, 12.12, and 4.16% samples were positive for E.coli, V. cholerae, and V. mimicus, respectively. Diarrhoeagenic E.coli 7 EPEC, 10 ETEC, and 2 EIEC were isolated along with 2 V. cholerae O1 Ogawa (ctxA and tcpA ElTor positive), one each from tube well and pond. Interestingly, 4 V.cholerae non-O1/non-O139 also harbored hlyA gene. The detection of toxin genes among this bacterial pool of sampled water indicates the fallout of the potable water sources, thus enabling us to establish that it is none other than the contaminated potable water system which often wreaks havoc in the south Bengal diarrhoeal menace. The consequences are further complicated by the presence of drug-resistant pathogenic bacterial pool to fluoroquinolone, beta-lactams, and cephalosporins, in the accessible potable water, with threats of outbreaks exploding into an epidemic, given suitable environment, poor sanitation, and unhygienic practices. Therefore, we strongly recommend re-modelling of 'point-of-use water disinfection' measures and adequate personal hygiene for healthier community life.

Environmental Vibrio cholerae non O1/ non O139 from the Gangetic delta: a diarrhoeal disease purview.

Diarrhoea still remains an unsolved enigma in developing countries, a major concern for the health planners. We targeted the abundance and toxicity of Vibrio cholerae non-O1/non-O139 (NOVC) in Gangetic riverine-estuarine ecosystem. A total of 74 V. cholerae were isolated from 120 water samples (68 NOVC, 6 V. cholerae O1) from two sampling sites off river Ganges. V. cholerae showed distinct seasonality, with steady increase from summer to monsoon, steep ascent in post-monsoon and an abrupt decline in winter. Highest number of NOVC was isolated form Howrah, attributed to low salinity and high anthropogenic influence. Environmental NOVC harboured hlyA (94.0 %), rtxA (81.0 %) and toxR (28.0 %) genes. About 23.4 % of the hlyA harbouring NOVC showed haemolytic activity. Accessory toxin genes (tlcR, toxT, RJ and LJ and aldA), among 3-5 % of the NOVC carry significant health implications. Haemolytic activity and biofilm formation in NOVC, during unfavourable conditions, facilitates gene transfer and emphasises the role of environmental NOVC in diarrhoeal incidence in South Bengal, India.

Seasonal Prevalence of Enteropathogenic Vibrio and Their Phages in the Riverine Estuarine Ecosystem of South Bengal.

Diarrheal disease remains an unsolved problem in developing countries. The emergence of new etiological agents (non-cholera vibrios) is a major cause of concern for health planners. We attempted to unveil the seasonal dynamics of entero-pathogenic Vibrios in Gangetic riverine-estuarine ecosystem. 120 surface water samples were collected for a period of one year from 3 sampling sites on the Hooghly river. Five enteropathogenic Vibrio species, V. cholerae (35%), V. parahaemolyticus (22.5%), V. mimicus (19.1%), V. alginolyticus (15.8%) and V. vulnificus (11.6%), were present in the water samples. The vibriophages, V. vulnificus ɸ (17.5%), V. alginolyticus ɸ (17.5%), V. parahaemolyticus ɸ (10%), V. cholerae non-O1/O139 ɸ (26.6%) and V. mimicus ɸ (9.1%), were also detected in these samples. The highest number of Vibrios were noted in the monsoon (20-34°C), and to a lesser extent, in the summer (24-36°C) seasons. Samples positive for phages for any of the identified Vibrio species were mostly devoid of that particular bacterial organism and vice versa. The detection of toxin genes and resistance to ß-lactam antibiotics in some environmental enteropathogenic Vibrio species in the aquatic niches is a significant outcome. This finding is instrumental in the south Bengal diarrhoeal incidence.

Specificity of coliphages in evaluating marker efficacy: a new insight for water quality indicators.

Conventional procedures for qualitative assessment of coliphage are time consuming multiple step approach for achieving results. A modified and rapid technique has been introduced for determination of coliphage contamination among potable water sources during water borne outbreaks. During December 2013, 40 water samples from different potable water sources, were received for water quality analyses, from a jaundice affected Municipality of West Bengal, India. Altogether, 30% water samples were contaminated with coliform (1-20 cfu/ml) and 5% with E. coli (2-5 cfu/ml). Among post-outbreak samples, preponderance of coliform has decreased (1-4 cfu/ml) with total absence of E. coli. While standard technique has detected 55% outbreak samples with coliphage contamination, modified technique revealed that 80%, double than that of bacteriological identification rate, were contaminated with coliphages (4-20 pfu/10 ml). However, post-outbreak samples were detected with 1-5 pfu/10 ml coliphages among 20% samples. Coliphage detection rate through modified technique was nearly double (50%) than that of standard technique (27.5%). In few samples (with coliform load of 10-100 cfu/ml), while modified technique could detect coliphages among six samples (10-20 pfu/10 ml), standard protocol failed to detect coliphage in any of them. An easy, rapid and accurate modified technique has thereby been implemented for coliphage assessment from water samples. Coliform free water does not always signify pathogen free potable water and it is demonstrated that coliphage is a more reliable 'biomarker' to ascertain contamination level in potable water.

Seasonal dynamics of Vibrio cholerae and its phages in riverine ecosystem of Gangetic West Bengal: cholera paradigm.

The Gangetic delta is a century-old cholera endemic belt where the role of riverine-estuarine ecosystem in cholera transmission has never been elucidated. Seasonality, distribution, and abundance of environmental Vibrio cholerae O1/O139 and vibriophage in Hooghly riverine-estuarine environment and their correlation with cholera incidence pattern in West Bengal, India, have been analyzed for the first time across summer, monsoon, and winter months. A total of 146 water samples collected from two sites of the Hooghly River (Howrah and Diamond Harbour) were analyzed physicochemically along with cultivable Vibrio count (CVC), V. cholerae O1/O139, and vibriophages. V. cholerae O1 was detected in 56 (38.3%) samples, while 66 (45.2%) were positive for V. cholerae O1 phages. Flood tide, water temperature (31 ± 1.6 °C), and turbidity (≥250 nephelometric turbidity unit (NTU)) significantly stimulated V. cholerae and vibriophage abundance in riverine ecosystem. Solitary existence of V. cholerae O1 and phages (p < 0.0001) in aquatic environment divulges the dominance of either of the entity (V. cholerae O1 or V. cholerae O1 Φ) on the other. Significant association (p < 0.05) between Kolkata cholera cases and V. cholerae O1 in aquatic environment implies the role of riverine-estuarine ecosystem in cholera transmission. A "biomonitoring tool" of physicochemical stimulants, tidal, and climatic variants has been proposed collating V. cholerae and phage dynamics that can forewarn any impending cholera outbreak.

Influence of hydrologic and anthropogenic factors on the abundance variability of enteropathogens in the Ganges estuary, a cholera endemic region.

This study deals with the influence of water physico-chemical properties, tides, rainfall and fecal pollution on the abundance of enteropathogens in a main distributary of the Ganges, in the endemic cholera belt of West Bengal. Between January and June 2011, water and sediments were sampled from two sites of the Hooghly River by Kolkata and Diamond Harbour. Counts of cultivable Vibrio (CVC, from~10(2) to~10(5)CFU/L) and total bacteria (TBC, from~10(5) to~10(9)CFU/L) increased with water temperature (17°C to 37°C). A combination of variations in tidal height, salinity and turbidity had a distinct influence on CVC, TBC and coliform counts. At Diamond Harbour, a salinity increase from 0.6 to 7.9 was accompanied by a 1000-fold amplification of initial CVC~10(2)CFU/L, whereas higher prevalence of coliforms in Kolkata was related to greater disposal of untreated sewage into the river. Turbidity-dependent variation of CVC was noteworthy, particularly at Diamond Harbour, where CVC in intertidal surface sediments showed an analogous trend as in surface waters, suggesting bentho-pelagic coupling of Vibrio dynamics. Besides the influence of salinity variation with tidal cycles, sediment re-suspension from tidal flats can play a role on Vibrio abundance in aquatic ecosystems.

Diarrheogenic Escherechia coli in potable water sources of West Bengal, India.

Diarrheal diseases are a major cause of morbidity and mortality in developing countries. Among the bacterial pathogens, diarrheagenic Escherichia coli are most frequently connected in cases with epidemic and endemic diarrhea worldwide. Environmental surveillance for monitoring of E.coli is very rare. In the present study, we have applied a modified technique to quantify coliform and E. coli in different potable water sources and their subsequent characterization (in relation to diarrheal pathogenicity) in the diarrhea endemic foci of West Bengal. More than one-fifth of the targeted sources (21.4%) have been identified harboring E. coli. Serotyping and molecular analysis reveals multidrug resistant EPEC, EIEC and ETEC among 9% of positive sources. Rainy season seems to be the most conducive period for E. coli induced diarrhea. While non-diarrheogenic E.coli were sensitive to most of the drugs, diarrheogenic E. coli, possessing toxicity, showed resistance against tetracycline, kanamycin, furazolidone, amoxicillin, ampicillin, norfloxacin, ciprofloxacin. Presence of multidrug resistant diarrheogenic E.coli justifies the potentiality of potable water sources as its vehicle and as a potent diarrheal inducer in diarrhea prone area along with increasing concern of drug resistance. Presence of diarrheogenic E. coli stresses the urgent need of its environmental surveillance like diarrheogenic Vibrios.

Efficacy of cocktail phage therapy in treating Vibrio cholerae infection in rabbit model.

Ability of a cocktail of five lytic vibriophages to combatting Vibrio cholerae O1 infection in rabbit model was examined. In one group, rabbits were administered 1 × 10(8) plaque forming unit of phage cocktail 6 and 12 h prior to the administration of V. cholerae O1, while in the other group, same procedure was applied 6 and 12 h post infection. It was observed that oral administration of the phage cocktail after oral bacterial challenge lowered the shedding of bacteria significantly (p < 0.01). In contrast phage treatment prior to bacterial challenge had no such effect (p > 0.05). Results suggest that oral administration of phage subsequent to V. cholerae challenge could provide a possible means of combatting V. cholerae infection.

In-house contamination of potable water in urban slum of Kolkata, India: a possible transmission route of diarrhea.

We have investigated and determined the potentiality of different water sources, both for drinking and domestic purposes, in diarrheal disease transmission in diarrhea endemic foci of urban slums in Kolkata, India in a one and half year prospective study. Out of 517 water samples, collected from different sources, stored water (washing) showed higher prevalence of fecal coliforms (58%) (p < 0.0001) in comparison with stored (drinking) samples (28%) and tap/tubewell water (8%) respectively. Among different sources, stored water (washing) samples had the highest non-permissible range of physico-chemical parameters. Fecal coliform levels in household water containers (washing) were comparatively high and almost 2/3 of these samples failed to reach the satisfactory level of residual chlorine. Interestingly, 7% stored water (washing) samples were found to be harboring Vibrio cholerae Improper usage of stored water and unsafe/poor sanitation practices such as hand washing etc. are highlighted as contributory factors for sustained diarrheal episodes. Vulnerability of stored water for domestic usage, a hitherto unexplored source, at domiciliary level in an urban slum where enteric infections are endemic, is reported for the first time. This attempt highlights the impact of quality of stored water at domiciliary level for fecal-oral contamination vis-à-vis disease transmission.

Increasing prevalence of Salmonella enterica serotype Paratyphi-A in patients with enteric fever in a periurban slum setting of Kolkata, India.

A sudden rise in the occurrence of Salmonella enterica serotype Paratyphi-A (SPTA) was noted in a longitudinal community-based study in Kolkata, India, during 2004 - 2005. We compared the incidence rate of Salmonella enterica serotype Typhi (ST) and SPTA and their antimicrobial susceptibility pattern. Rate of isolation of SPTA was 1.5 times higher than that of ST, a trend detected for the first time in that particular focus. Almost all the isolates were sensitive to Gentamycin and Norfloxacin. Most of the strains (90%) were sensitive to Ciprofloxacin. Two thirds of the strains were also sensitive to Chloramphenicol. The high occurrence of SPTA in the present study could be the signal of the emergence of SPTA as a pathogen in India. Quinolone derivatives (namely, Ciprofloxacin, Norfloxacin and Chloramphenicol) can be suggested as drugs of choice for treatment of enteric fever caused by SPTA. Future vaccination strategies should include bivalent vaccines with protection capacities against both ST as well as SPTA.

Asymptomatic cryptosporiosis in a periurban slum setting in Kolkata, India--a pilot study.

We report the results of a prospective study conducted among asymptomatic children of slum dwellers in Kolkata, eastern India, to assess the prevalence of Cryptosporidium parvum during the period of October 2002 to July 2003. Of the 609 fecal samples examined, C. parvum was detected in 14 (2.3%). The study indicated that poor sanitary conditions may be cofactors for the cryptosporidial disease burden among slum dwellers. The implications of asymptomatic infections have been discussed.