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Proper surveillance of Campylobacter jejuni and Campylobacter coli, major pathogens associated with human gastroenteritis, is necessary to tackle the increasing disease burden. To detect these pathogenic species, a variety of PCR assays have been developed. This study examined the sensitivity and specificity of 12 PCR assays targeting 23S rRNA, ceuE, lpxA, hipO, mapA, ask, and cdt genes of C. jejuni and C. coli. The sensitivities of PCR assays were 85.2-100%, and 97-100%, and the specificities were 90.5-100%, and 94.3-100% for the tested C. jejuni (n = 61) and C. coli (n = 33) strains, respectively. Two PCR assays, targeting cdtC and hipO genes, were found to be 100% sensitive and/or specific for all C. jejuni strains, while 3 assays, targeting cdtB, cdtA, and ask genes, were 100% sensitive and/or specific for C. coli strains. However, PCR assays for hipO and ask genes are problematic to conduct simultaneously due to the differences in PCR conditions. Overall, multiplex PCR assays targeting cdtC and cdtB genes, encoding 2 subunits of the same toxin, were concluded to be the most reliable. The results of this study would aid in proper surveillance of C. jejuni and C. coli and adopting intervention strategies in the near future.

Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.

Salmonella Gallinarum is one of the most important bacterial pathogens associated with diminished egg production in poultry. The aim of this study was to understand the occurrence, molecular traits and antimicrobial resistance patterns of Salmonella Gallinarum strains isolated from small-scale commercial layer flocks with low level biosecurity standards in Bangladesh. A total of 765 samples, including cloacal swabs (535), visceral organs (50), and droppings (180), were collected from chickens of 12 layer flocks in 11 districts. Salmonella Gallinarum was isolated and characterized through culture-based method, followed by biochemical tests, sero-grouping, PCR assays, sequencing, and antibiogram. The identity of biochemically detected isolates of Salmonella Gallinarum was confirmed via genus-specific 16S rRNA gene based PCR, followed by invA and spvC genes based PCR assays. Occurrence of Salmonella Gallinarum was detected in overall 25.75% (197/765) samples, with a significantly (p &lt, 0.05) higher incidence in visceral organs (42%) in comparison to cloacal swab (24%) and droppings (26%). Sequencing and subsequent phylogenetic analysis of invA and spvC genes in representative strains of Salmonella Gallinarum revealed a close genetic lineage, with a sequence similarity of 98.05–99.21% and 97.51–99.45%, respectively, to previously published sequences of the corresponding genes from the same serogroup strains. Remarkably, 66.5% (131/197) of the isolated strains of Salmonella Gallinarum were found to be resistant to 3 to 6 antimicrobial agents, and interpreted as multidrug resistant (MDR). The findings of this study underscore an inherent need of appropriate control measures to curb the widespread incidence of MDR Salmonella Gallinarum in small-scale commercial layer flocks, thereby, facilitating enhanced egg production and further support to the food security and safety in low resource settings.

A cross-sectional survey was conducted in selected districts of Bangladesh to estimate prevalence, risk factors, and molecular detection of Campylobacter isolates from 540 farmed cattle of 90 herds. As an individual sample, 540 feces, and as a pooled sample, 180 milk samples, 90 feed samples, 90 water samples, 90 manure samples, and 90 animal attendants’ hand-rinse water were collected and tested via culture, biochemical, and molecular assays. A pretested semi-structured questionnaire was used to collect herd-level data on risk factors with the herd owners. The herd-level data on risk factors were analyzed through univariate and multivariate analyses, and a p-value &lt, 0.05 was considered statistically significant for all analyses. Overall, farm-level prevalence of bovine Campylobacter was enumerated to be 53.3% (95% confidence interval [CI]: 42.5–63.9%). The feces sample was found to be a high level of contamination of 30.9% (95% CI: 27–35%) followed by the manure swab (pooled) at 15.6% (95% CI: 8.8–24.7%). Campylobacter jejuni was documented as an abundant species (12.6%), followed by Campylobacter coli (5.1%), and Campylobacter fetus (0.3%). Older farms (&gt, 5 years of age), no/minimum cleaning and disinfection practices, along with animal roaming outside of the farm, were documented as significant risk factors for farm-level Campylobacter occurrence. Evidence-based control measures need to be taken through stringent biosecurity and hygienic measurement to lessen the load of the Campylobacter pathogen in the farm environment and prevent further transmission to animals and humans.

Additional file 1. Primers used for PCR-based detection of the major pathogenic species of Campylobacter.

Additional file 4. Antimicrobial resistance patterns of C. jejuni and C. coli strains from poultry production and supply chain.

Additional file 2. Questionnaire used during ‘Semi Structured Interview’ of poultry farmers or managers.

Additional file 3. Representative gel images showing results of genus- and species-specific PCRs of Campylobacter isolates.

The Bengal delta coast harboring the famous Sundarban mangroves is extremely vulnerable to climate change. Already, salinity intrusion, increasing cyclones and anomalies in rainfall, and temperature, are causing many social and livelihood problems. However, our knowledge on the diversified climate change impacts on Sundarban ecosystems services, providing immense benefits, including foods, shelters, livelihood, and health amenities, is very limited. Therefore, this article has systematically reviewed the major functional aspects, and highlights on biodiversity, ecosystem dynamics, and services of the Sunderban mangroves, with respect to variations in climatic factors. The mangrove ecosystems are highly productive in terms of forest biomass, and nutrient contribution, especially through detritus-based food webs, to support rich biodiversity in the wetlands and adjacent estuaries. Sundarban mangroves also play vital role in atmospheric CO2 sequestration, sediment trapping and nutrient recycling. Sea level rise will engulf a huge portion of the mangroves, while the associated salinity increase is posing immense threats to biodiversity and economic losses. Climate-mediated changes in riverine discharge, tides, temperature, rainfall and evaporation will determine the wetland nutrient variations, influencing the physiological and ecological processes, thus biodiversity and productivity of Sundarban mangroves. Hydrological changes in wetland ecosystems through increased salinity and cyclones will lower the food security, and also induce human vulnerabilities to waterborne diseases. Scientific investigations producing high resolution data to identify Sundarban‟s multidimensional vulnerabilities to various climatic regimes are essential. Sustainable plans and actions are required integrating conservation and climate change adaptation strategies, including promotion of alternative livelihoods. Thus, interdisciplinary approaches are required to address the future climatic disasters, and better protection of invaluable ecosystem services of the Sunderban mangroves. Fil: Neogi, Sucharit Basu. Coastal Development Partnership; Bangladesh. Osaka Prefecture University; Japón. Leibniz Center for Tropical Marine Ecology GmbH; Alemania Fil: Dey, Mouri. University of Chittagong; Bangladesh Fil: Lutful Kabir, S. M.. Bangladesh Agricultural University; Bangladesh Fil: Masum, Syed Jahangir H.. Coastal Development Partnership; Bangladesh Fil: Kopprio, Germán Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Leibniz Center for Tropical Marine Ecology GmbH; Alemania Fil: Yamasaki, Shinji. Osaka Prefecture University; Japón Fil: Lara, Ruben Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina

Background Understanding potential risks of multi-drug resistant (MDR) pathogens from the booming poultry sector is a crucial public health concern. Campylobacter spp. are among the most important zoonotic pathogens associated with MDR infections in poultry and human. This study systematically examined potential risks and associated socio-environmental factors of MDR Campylobacter spp. in poultry farms and live bird markets (LBMs) of Bangladesh. Methods Microbial culture and PCR-based methods were applied to examine the occurrence and MDR patterns of Campylobacter spp. in potential sources (n = 224) at 7 hatcheries, 9 broiler farms and 4 LBMs in three sub-districts. Antimicrobial residues in broiler meat and liver samples (n = 50) were detected by advanced chromatographic techniques. A questionnaire based cross-sectional survey was conducted on socio-environmental factors. Results Overall, 32% (71/ 224) samples were found contaminated with Campylobacter spp. In poultry farms, Campylobacter spp. was primarily found in cloacal swab (21/49, 43%), followed by drinking water (8/24, 33%), and meat (8/28, 29%) samples of broilers. Remarkably, at LBMs, Campylobacter spp. was detected in higher prevalence (p p Campylobacter isolates, one from each of 71 positive samples, were differentiated into Campylobacter jejuni (66%) and Campylobacter coli (34%). Alarmingly, 49 and 42% strains of C. jejuni and C. coli, respectively, were observed as MDR, i.e., resistant to three or more antimicrobials, including, tetracycline, amoxicillin, streptomycin, fluoroquinolones, and macrolides. Residual antimicrobials (oxytetracycline, ciprofloxacin and enrofloxacin) were detected in majority of broiler liver (79%) and meat (62%) samples, among which 33 and 19%, respectively, had concentration above acceptable limit. Inadequate personal and environmental hygiene, unscrupulously use of antimicrobials, improper waste disposal, and lack of health surveillance were distinguishable risk factors, with local diversity and compound influences on MDR pathogens. Conclusion Potential contamination sources and anthropogenic factors associated with the alarming occurrence of MDR Campylobacter, noted in this study, would aid in developing interventions to minimize the increasing risks of poultry-associated MDR pathogens under ‘One Health’ banner that includes poultry, human and environment perspectives.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains. IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae. This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae. Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae. The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

This study was aimed for isolation, identification and characterization of Campylobacter species from Bangladesh Agricultural University (BAU) dairy farm during the period of January to May, 2016. A total of 80 samples (faecal samples of calves, heifers and cows; milk samples of cows) were collected from Bangladesh Agricultural University dairy farm for isolation and identification of Campylobacter species by using cultural, biochemical and molecular methods. Moreover, the isolated Campylobacter species were subjected for antimicrobial susceptibility test. Campylobacter like organisms were presumptively identified in 20 samples. Isolates were biochemically positive to catalase and oxidase tests and in hippurate hydrolysis test some of the isolates (n=6) shown negative that indicated the isolates were C. coli and some of the test isolates (n=14) shown positive that indicated the isolates were C. jejuni. Campylobacter specific 16S rRNA genes were amplified from the isolates. Out of 20 isolated Campylobacter 14 (17.5%) were detected as C. jejuni and the rest 6 (7.5%) were detected as C. coli by cdtC gene based multiplex PCR assay. C. jejuni were resistant to amoxicillin, erythromycin, azithromycin and susceptible to gentamicin, ciprofloxacin, norfloxacin and streptomycin. Furthermore, C. coli were resistant to amoxicillin and erythromycin and susceptible to gentamycin, ciproflaxacin. Out of 20 Campylobacter isolates, 57.14% C. jejuni and 33.33% C. coli were identified as multidrug resistant. To the best of our knowledge, this study has brought the first report on the occurrence of Campylobacter species with their antibiogram profiles in any dairy farm of Bangladesh.

Proper surveillance of Campylobacter jejuni and Campylobacter coli, major pathogens associated with human gastroenteritis, is necessary to tackle the increasing disease burden. To detect these pathogenic species, a variety of PCR assays have been developed. This study examined the sensitivity and specificity of 12 PCR assays targeting 23S rRNA, ceuE, lpxA, hipO, mapA, ask, and cdt genes of C. jejuni and C. coli. The sensitivities of PCR assays were 85.2-100%, and 97-100%, and the specificities were 90.5-100%, and 94.3-100% for the tested C. jejuni (n = 61) and C. coli (n = 33) strains, respectively. Two PCR assays, targeting cdtC and hipO genes, were found to be 100% sensitive and/or specific for all C. jejuni strains, while 3 assays, targeting cdtB, cdtA, and ask genes, were 100% sensitive and/or specific for C. coli strains. However, PCR assays for hipO and ask genes are problematic to conduct simultaneously due to the differences in PCR conditions. Overall, multiplex PCR assays targeting cdtC and cdtB genes, encoding 2 subunits of the same toxin, were concluded to be the most reliable. The results of this study would aid in proper surveillance of C. jejuni and C. coli and adopting intervention strategies in the near future.

Atypical El Tor strains ofVibrio choleraeO1 harboring variantctxBgenes of cholera toxin (CT) are gradually becoming a major cause of recent cholera epidemics.Vibrio mimicusoccasionally contains virulence factors associated with cholera, e.g., CT, encoded byctxABon CTXФ genome; and TCP, the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon inctx+vestrains ofV. mimicusisolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence related-genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novelctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized in an array of RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv. Additionally, a novel variant oftcpAandtoxTrespectively, showing clonal lineage to a phylogenetic clade ofV. choleraenon-O1/O139, was identified. TheV. mimicusstrains lacked the RTX and TLC elements, andVibrioseventh pandemic islands of the El Tor strains, but contained five heptamer (TTTTGAT) repeats inctxABpromoter region like some classical strains ofV. choleraeO1. PFGE analysis showed all thectx+veV. mimicusstrains were clonally related. However, theirin vitroCT production andin vivotoxigenecity were variable, which could be explained by differential transcription of virulence genes along with ToxR regulon. Taken together, our findings strongly suggest that environmentalV. mimicusstrains act as potential reservoir of atypical virulence factors, including variant CT and ToxT regulon, and may contribute to the evolution ofV. choleraehybrid strains.IMPORTANCENatural diversification of CTXФ andctxABgenes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains ofV. cholerae. This study showing the occurrence of CTXETcomprising a novel variant ofctxABinV. mimicuspoints out a previously unnoticed evolutionary event, independent to that of the El Tor strains ofV. cholerae. Identification and cluster analysis of the newly-discovered alleles oftcpAandtoxTindicates their horizontal transfer from an uncommon clone ofV. cholerae. The genomic content of ToxT regulon, and tandemly arranged multiple pre-CTXФEnvand a CTXФETinV. mimicusprobably act as salient raw materials inducing natural recombination among the hallmark virulence genes of hybridV. choleraestrains. This study will facilitate deeper understanding of the evolution of new variant CT and ToxT regulon, influencing cholera epidemiology.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicus ctx-positive (ctx(+)) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTX(ET)) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTX(Env)), and RS1 elements, which were organized as an RS1-CTX(ET)-RS1-pre-CTX(Env)-pre-CTX(Env) array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx(+) V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains. IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae. This report, showing the occurrence of CTX(ET) comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae. Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae. The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФ(Env) and of a CTXФ(ET) in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n=61) but none of the non-target strains (n=34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n=70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains.

Avian colibacillosis is considered a major bacterial disease causing severe economic repercussions to poultry producers worldwide. Egg-laying strains of chickens, in particular, have experienced a resurgence of this disease. Most strains of Escherichia coli are not harmful to poultry; however, a few virulent strains referred to as avian pathogenic E. coli (APEC) can induce either localized or systemic infections. The APEC strains have developed resistance to many antibiotics, and they may trigger zoonotic infections. Human populations living close to poultry hubs with poor hygienic practices are especially at high risk of developing E. coli infection. The characteristics, pathologies, and invasion routes of APEC along with the host’s immune response to APEC, the virulence factors used in the counterattack by APEC, and control strategies for minimizing outbreaks of avian colibacillosis are discussed.

We evaluated the usefulness of the loop-mediated isothermal amplification (LAMP) using a portable ESE Quant tube scanner as a rapid and simple method for the detection of Vibrio parahaemolyticus, an important pathogen causing seafood-borne gastroenteritis. The real time LAMP (RT-LAMP) assay using a hemolysin gene (tlh/ldh)-specific primers was verified using V. parahaemolyticus strains (n = 91) from different countries and other non-target strains to check the utility of this method. Both the sensitivity and specificity of the RT-LAMP using 3 pairs of tlh/ldh-specific primers developed in this study were excellent (100%). The detection limit of the RT-LAMP was as low as 7 Colony forming unit per reaction and detection time was only 20 min. Comparative evaluation of the target bacterial strains with the RT-LAMP using ESE Quant tube scanner, API 20E system and conventional RT-PCR method revealed that the RT-LAMP assay developed in this study is simpler and more rapid than the latter two methods. Therefore, the RT-LAMP method using the easily portable ESE Quant tube scanner can be considered as an effective tool for the rapid screening of V. parahaemolyticus strains in environmental and clinical samples, especially, in remote areas of developing countries during epidemic periods.

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 ~ 102 to ~ 105 CFU/L) and total bacteria (TBC, from ~ 105 to ~ 109 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 ~ 102 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. Fil: Batabyal, Prasenjit. National Institute For Cholera And Enteric Diseases; India Fil: Einsporn, Marc H.. Leibniz Centre For Tropical Marine Ecology; Alemania Fil: Mookerjee, Subham. National Institute For Cholera And Enteric Diseases; India Fil: Palit, Anup. National Institute For Cholera And Enteric Diseases; India Fil: Neogi, Sucharit B.. Osaka Prefecture University; Bangladesh. International Centre for Diarrheal Disease Research; Bangladesh Fil: Nair, Gopinath B.. National Institute For Cholera And Enteric Diseases; India. Translational Health Science and Technology Institute; India Fil: Lara, Ruben Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto Argentino de Oceanografía (i); Argentina

The ecology of the most relevant Vibrio species for human health and their relation to water quality and biogeochemistrywere studied in two estuaries in Argentinian Patagonia. Vibrio cholerae and Vibrio parahaemolyticuswere reported in N29% of cases at the Río Colorado and Río Negro estuaries. Neither the pandemic serogroupsof Vibrio cholerae O1, Vibrio cholerae O139 nor the cholera toxin gene were detected in this study. However, severalstrains of V. cholerae (not O1 or O139) are able to cause human disease or acquire pathogenic genes by horizontaltransfer. Vibrio vulnificus was detected only in three instances in the microplankton fraction of the RíoNegro estuary. The higher salinity in the Río Colorado estuary and in marine stations at both estuaries favoursan abundance of culturable Vibrio. The extreme peaks for ammonium, heterotrophic bacteria and faecal coliformsin the Río Negro estuary supported a marked impact on sewage discharge. Generally, the more pathogenic strainsof Vibrio have a faecal origin. Salinity, pH, ammonium, chlorophyll a, silicate and carbon/nitrogen ratio ofsuspended organic particulates were the primary factors explaining the distribution of culturable bacteria afterdistance-based linear models. Several effects of dissolved organic carbon on bacterial distribution are inferred. Fil: Kopprio, Germán Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Leibniz Center for Tropical Marine Ecology; Alemania Fil: Streitenberger, Maria Eugenia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Okuno, Kentaro. Osaka Prefecture University; Japón Fil: Baldini, Monica Diana. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Biancalana, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina Fil: Fricke, Anna Lena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Leibniz Center for Tropical Marine Ecology; Alemania Fil: Martínez, Ana María. Universidad Nacional del Sur; Argentina Fil: Neogi, Sucharit Basu. Osaka Prefecture University; Japón Fil: Koch, Boris Peter. Alfred Wegener Institute Helmholtz Center for Polar and Marine Research; Alemania Fil: Yamasaki, Shinji. Osaka Prefecture University; Japón Fil: Lara, Ruben Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006–2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n = 175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh+ V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRSvariant, were present only in the pandemic serotype O3:K6 (n = 13) and its serovariant O4:K68 (n = 2). Temporal distribution of the tdh+ serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh+ serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh+ O3:K6 and its pandemic serovariants from the tdh+ non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh+ strains, including the observed temporal change of the predominant tdh+ serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.

Cholera is water-borne infectious disease caused by Vibrio cholerae O1 and O139. Currently a proportion of people living in rural area like Matlab, Bangladesh are dependent on surface water for various purposes to surface water. People of the area are affected by cholera particularly in rainy seasons. We measured in situ the physicochemical parameters of water using portable meters and counted the Vibrio sp. from water, phytoplankton and zooplankton samples of river and pond water of Matlab, Bangladesh. The culturable Vibrios were counted thiosulfate-citrate-bile salts-sucrose agar (TCBS) and taurocholate tellurite gelatin agar (TTGA) plates and viable but nonculturable (VBNC) by direct fluorescent microscopy. To confirm, we further did PCR of ompW gene and serology for V. cholerae O1/O139. Strong correlation of Vibrios cultural counts with pH, total coliform and faecal coliform heterotropic plate counts and viable but nonculturable (VBNC) counts with only total dissolved solids (TDS) noticed from the results. We identified 25 V. cholerae non-O1 and five V. cholerae O1 serotype which is main cause of cholera from water, phytoplankton and zooplankton samples of pond and rivers. Therefore, the present study indicated that the surface water sources are the main causes of cholera and reestablished the Matlab as epidemic area of Bangladesh.

Little is known about bacterial dynamics in the oligotrophic ocean, particularly about cultivable bacteria. We examined the abundance of total and cultivable bacteria in relation to changes in biogeochemical conditions in the eastern Atlantic Ocean with special regard to Vibrio spp., a group of bacteria that can cause diseases in human and aquatic organisms. Surface, deep water and plankton (55 µm) samples were collected between 50◦ N and 24◦ S. Chlorophyll-a was very low (99 %) were found in the nanoplankton fraction (

Laribacter hongkongensis is a recently discovered bacterium associated with gastroenteritis. In this study, a total of 199 isolates of this species obtained from aquatic products (n=462) in Guangzhou City, China, were examined for their susceptibility to 19 antimicrobial agents and the presence of antimicrobial resistance integrons. The genetic relatedness of the isolates with integrons was also evaluated. A PCR-based method was used to screen integrons and found that 13 (6.5%) of the isolates harbored class 1 integrons. The antimicrobial resistance rates of integron-positive isolates were significantly higher than integron-negative ones for cefepime, ciprofloxacin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole and rifampicin. Genetic sequence analysis revealed that these integrons contained various antimicrobial-resistance genes (dfrA1, dfrA14, dfrA17, dfrA32, aadA1, aadA2, aadA5, cmlA5, arr2, ereA and orfC) organized into different gene cassettes arrangements including a novel array of dfrA14-arr2-cmlA5. Pulsed-field gel electrophoresis (PFGE) yielded 13 different patterns among 13 integron-positive isolates, which could be grouped into four clusters. These indicate the dispersal of multi-resistant integrons among different molecular types. To the best of our knowledge, this is the first report showing distribution and characterization of class 1 integrons among L. hongkongensis isolates.

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.

Aims: To develop simple and rapid PCR-fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. Methods and Results: PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat-amplified fragment length polymorphism (VCR-AFLP) assay was also developed. In the PCR-RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR-AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed-field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). Conclusions: The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. Significance and Impact of Study: This newly developed method is more discriminatory, simple, rapid and cost-effective in comparison with PFGE, and thus can be widely applicable.

The use of natural compounds as inhibitory agents for virulence factor production is a new approach to overcome increased antimicrobial resistance in pathogenic bacteria. In this study, we examined whether red chilli (Capsicum annuum) contains any such compound(s) that can repress the cholera toxin (CT) production in Vibrio cholerae. We found that the methanol extract of red chilli could inhibit CT production in recently emerged V. cholerae O1 El Tor variant strains without affecting their viability. Interestingly, capsaicin, a well-studied active component of red chilli, also drastically inhibited CT production in V. cholerae strains belonging to various serogroups including variants. Real-time quantitative reverse transcription-PCR assay revealed that capsaicin effectively repressed the transcription of ctxA, tcpA and toxT genes, but not of toxR and toxS genes. On the contrary, capsaicin significantly enhanced the transcription of the hns gene, the product of which is known to regulate negatively the transcription of ctxAB, tcpA and toxT genes. These results suggest that capsaicin might act as a potent repressor for CT production possibly by enhancing the transcription of hns.

The aim of this study was to investigate the prevalence and genetic diversity of Staphylococcus aureus and staphylococcal enterotoxins (SEs) in ready-to-eat foods marketed in Hanoi, Vietnam. Out of 212 samples tested, 45 were contaminated with this bacterium and 18 contained strains having classical SEs. Among different food products, the bacterial occurrence ranged between 12.5% and 35.4% and milk samples had the highest prevalence. The production of SEs varied according to the food types and 26 different ribotype patterns were observed among the 45 strains. Our dendogram analysis showed the existence of many clones without clear-cut affiliation to any particular food type indicating both diverse primary and secondary contamination sources. The SEs also occurred in a wide variety of genetically different S. aureus strains which might facilitate the bacterium with better ecological fitness to cause epidemics. It can be concluded that Vietnamese ready-to-eat foods pose a high risk of contamination by toxigenic S. aureus population urging the need of implementing proper hygienic practices.

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 103 MPN 100 mL−1 seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20–24 °C, 24–28 p.p.t. and 7.95–8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.

Gastroenteritis caused by Vibrio parahaemolyticus has recently been associated with foods prepared with seaweeds, but little is known about the bacterium's abundance and diversity among seaweeds in coastal environment. Therefore, we determined its phenotypic and genotypic diversity in relation to its seasonal abundance in seawater and seaweed samples from three areas of Kii Channel, Japan during June 2003 to May 2004. Isolates were obtained by selective enrichment of samples and detection of V. parahaemolyticus by colony hybridization with a species-specific probe. A total of 128 isolates comprising 16 from each source in each season were characterized by serotyping and ribotyping. V. parahaemolyticus was more abundant in seaweeds (3,762 isolates) than in water samples (2,238 isolates). Twenty and 17 serotypes were found among the selected seaweed and seawater isolates, respectively. Cluster analysis revealed 19, 11, 7 and 9 ribotypes during summer, autumn, winter and spring, respectively. Seaweeds supported a diverse V. parahaemolyticus population throughout the year and thus seaweeds are a reservoir for the organism. However, V. parahaemolyticus occurrence had positive correlation with water temperature and its abundance in seaweeds was at least 50 times higher during summer than in winter.

The increasing loads of anthropogenic pollutants, compounded with climate change events, are likely to induce environmental changes in many wetlands with impacts on the native microinvertebrates and pathogens causing increased occurrence of water-borne diseases, which affect millions of people each year. In wetlands bacterial pathogens are actively preyed on by many protozoa and filter-feeding organisms but this predation can be compensated by the nourishment and protection offered by certain microinvertebrates, acting as hosts, e.g., chitinous rotifers, copepods and cladocerans. The complex interactions of ecological, biological, and genetic components mediate disease-causing organisms to exploit microinvertebrate hosts to occupy diverse niches, obtain nutrition, and withstand physico-chemical stresses. The persistence of the human pathogens in wetlands is often enabled by their association with microinvertebrates and also depends on their quorum sensing mediated colonization, biofilm formation, switching into dormant stage, and horizontal transfer of adaptive genes. The symbiosis with microinvertebrates is facilitated by the pathogen’s immune evasion and fitness factors, e.g., Type-IV pili, capsular-polysaccharides, nutrient transportation, virulence and binding proteins, proteases, chitinases, and secretion systems. Spatio-temporal variation in the population of copepods and aquatic eggs/larvae of mosquitoes and midge flies, which act as vectors, can influence the outbreaks of cholera, diarrhea, malaria, dengue, filariasis and drucunculiasis. Changes in climatic factors (temperature, salinity, cyclones, rainfall, etc.) and anthropogenic pollutions (sewage, fertilizer and insecticide) may modify the abundance and biodiversity of microinvertebrates, and thus possibly exacerbate the persistence and dispersal of water-borne pathogens. Thus there is a need to adopt ecohydrological and eco-friendly interventions for managing wetlands while conserving them. Fil: Neogi, Sucharit Basu. International Centre For Diarrhoeal Disease Research; Bangladesh. Osaka Prefecture University; Japón Fil: Yamasaki, Shinji. Osaka Prefecture University; Japón Fil: Alam, Munirul. International Centre For Diarrhoeal Disease Research; Bangladesh Fil: Lara, Ruben Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto Argentino de Oceanografía (i); Argentina. Leibniz Centre for Tropical Marine Ecology; Alemania

Extensive analyses of particulate lipids and lipid classes were conducted to gain insight into lipid production and related factors along the biogeochemical provinces of the Eastern Atlantic Ocean. Data are supported by particulate organic carbon (POC), chlorophyll a (Chl a), phaeopigments, Chl a concentrations and carbon content of eukaryotic micro-, nano- and picophytoplankton, including cell abundances for the latter two and for cyanobacteria and prokaryotic heterotrophs. We focused on the productive ocean surface (2 m depth and deep Chl a maximum (DCM)). Samples from the deep ocean provided information about the relative reactivity and preservation potential of particular lipid classes. Surface and DCM particulate lipid concentrations (3.5–29.4 g L-1) were higher than in samples from deep waters (3.2-9.3 g L-1) where an increased contribution to the POC pool was observed. The highest lipid concentrations were measured in high latitude temperate waters and in the North Atlantic Tropical Gyral Province (13-25°N). Factors responsible for the enhanced lipid synthesis in the eastern Atlantic appeared to be phytoplankton size (micro, nano, pico) and the low nutrient status with microphytoplankton having the most expressed influence in the surface and eukaryotic nano- and picophytoplankton in the DCM layer. Higher lipid to Chl a ratios suggest enhanced lipid biosynthesis in the nutrient poorer regions. The various lipid classes pointed to possible mechanisms of phytoplankton adaptation to the nutritional conditions. Thus, it is likely that adaptation comprises the replacement of membrane phospholipids by non-phosphorus containing glycolipids under low phosphorus conditions. The qualitative and quantitative lipid compositions revealed that phospholipids were the most degradable lipids, and their occurrence decreased with increasing depth. In contrast, wax esters, possibly originating from zooplankton, survived downward transport probably due to the fast sinking rate of particles (fecal pellets). The important contribution of glycolipids in deep waters reflected their relatively stable nature and degradation resistance. A lipid-based proxy for the lipid degradative state (Lipolysis Index) suggests that many lipid classes were quite resistant to degradation even in the deep ocean.

Polyphosphate provides a substitute for ATP and energy source when phosphorus is a limiting resource in nature. The present study focuses on the role ofpolyphosphate for the survival of Vibrio cholerae in the aquatic habitats as an autochthonous bacterium. The survival advantages of polyphosphate of V. cholerae O1 having (wild type) and lacking (mutant) polyphosphate kinase (ppk) gene in surface water and with Anabaena variabilis were compared by cultural, Direct Fluorescent Antibody (DFA) and polymerase chain reaction methods in natural water microcosms. The microcosm's water was prepared by filtering and physicochemical parameters were also investigated by standard methods. The results revealed that both fresh and saline water, the wild type strain enhanced survival in cultural conditioned than ppk mutant strain. However, Fluorescent Antibody Direct Viable Counts (FADVC) and Polymerase Chain Reaction (PCR) results noted both strains have the equal survival strategy in viable but nonculturable state (VNC). In conclusion, it could be hypothesized that the polyphosphate inclusion body might keep cultivable and survivable at low phosphate natural environment of the aquatic bacterium.

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

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae . These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number ( n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 ( n = 485) or O139 ( n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae , which may be associated with extraintestinal infections rather than enterotoxicity.

Cholera epidemics are thought to be influenced by changes in populations of estuarine Vibrio cholerae. We investigated the abundance and distribution of this bacterium, as ‘‘free-living’’ (\20 lm fraction) and associated with microphytoplankton ([20 lm) or zooplankton ([60 lm), in the Karnaphuli estuary of Bangladesh during pre- and post-monsoon seasons. Cultivable Vibrio populations were *102 –104 colony forming units (CFU) ml-1 in the high saline zone (19–23 practical salinity unit, PSU) and declined in freshwater (\101 CFU ml-1 ). Culture independent detection of toxigenic V. cholerae O1 and O139 serogroups revealed a higher abundance of ‘‘free-living’’ (104 –105 cells l-1 ) than those attached to plankton (101 –103 cells l-1 ). However, ‘‘free-living’’ O1 and O139 cells were sometimes absent in the medium saline and freshwater areas (0.0–11 practical salinity unit [PSU]). In contrast, plankton samples always harbored these serogroups despite changes in salinity and other physico-chemical properties. Microphytoplankton and zooplankton were dominated by diatoms and blue-green algae, and copepods and rotifers, respectively. Toxigenic V. cholerae abundance did not correlate with plankton abundance or species but had a positive correlation with chitin in the\20 lm fraction, where suspended particulate matter (SPM), V. cholerae and chitin concentrations were highest. C:N ratios indicated that organic matter in SPM originated predominantly from plankton. The differential occurrence of ‘‘free-living’’ and attached V. cholerae suggests a pivotal function of plankton in V. cholerae spreading into freshwater areas. The probable association of this pathogen with organisms and particles in the nanoplankton (\20 lm) fraction requires validation of the concept of the ‘‘free living’’ state of V. cholerae in aquatic habitats. Fil: Neogi, Sucharit Basu. Osaka Prefecture University; Japón. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Islam, Mohammad Sirajul. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Nair, Balakrish Gopinath. National Institute of Cholera and Enteric Diseases; India Fil: Yamasaki, Shinji. Osaka Prefecture University; Japón Fil: Lara, Ruben Jose. Leibniz Centre for Tropical Marine Ecology GmbH; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 lM) and nitrogen (9.1 ± 3.3 lM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1–2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with *98% of the total in the fraction \20 lm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes [20 lm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes \20 lm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5–10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant ([22 lM N and 100 lM Si). However, nitrite increased *9 times in the outer sector, reaching *1.2 lM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality. Fil: Lara, Ruben Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Leibniz Centre for Tropical Marine Ecology; Alemania Fil: Neogi, Sucharit B.. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Islam, Mohammad Sirajul. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Mahmud, Zahid H.. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Islam, Shafiqul. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Paul, Debasish. International Centre for Diarrhoeal Disease Research; Bangladesh Fil: Demoz, Biniam. University of Hohenheim; Alemania Fil: Yamasaki, Shinji. Osaka Prefecture University; Japón Fil: Nair, Gopinath B.. National Institute of Cholera and Enteric Diseases; India Fil: Kattner, Gerhard. Alfred Wegener Institute for Polar and Marine Research; Alemania

The rise in multi-drug resistant Vibrio cholerae strains is a big problem in treatment of patients suffering from severe cholera. Only a few studies have evaluated the potential of natural compounds against V. cholerae. Extracts from plants like ‘neem’, ‘guazuma’, ‘daio’, apple, hop, green tea and elephant garlic have been shown to inhibit bacterial growth or the secreted cholera toxin (CT). However, inhibiting bacterial growth like common antimicrobial agents may also impose selective pressure facilitating development of resistant strains. A natural compound that can inhibit virulence in V. cholerae is an alternative choice for remedy. Recently, some common spices were examined to check their inhibitory capacity against virulence expression of V. cholerae. Among them methanol extracts of red chili, sweet fennel and white pepper could substantially inhibit CT production. Fractionation of red chili methanol extracts indicated a hydrophobic nature of the inhibitory compound(s), and the n-hexane and 90 per cent methanol fractions could inhibit >90 per cent of CT production. Purification and further fractionation revealed that capsaicin is one of the major components among these red chili fractions. Indeed, capsaicin inhibited the production of CT in various V. cholerae strains regardless of serogroups and biotypes. The quantitative reverse transcription real-time PCR assay revealed that capsaicin dramatically reduced the expression of major virulence-related genes such as ctxA, tcpA and toxT but enhanced the expression of hns gene that transcribes a global prokaryotic gene regulator (H-NS). This indicates that the repression of CT production by capsaicin or red chili might be due to the repression of virulence genes transcription by H-NS. Regular intake of spices like red chili might be a good approach to fight against devastating cholera.

This chapter treats two main topics: the relationship between human health, aquatic ecosystems, and water use; and the necessity of interdisciplinary approaches for the development of water management policies and disease control. Main waterborne diseases, mostly affecting developing countries and relevant in terms of water management and changes in land use, such as malaria, schistosomiasis, or cholera, are discussed stressing links to the global water crisis. Also, the role of artificial and natural wetlands in influenza epidemics is treated. The effects of increasing water use and scarcity on human health are discussed considering historical and contemporary incidence of diarrheal diseases in European and South Asian megacities, relationships between dams and on waterborne diseases in Asia and Africa, and intensive agri- and aquaculture resulting in man-made ecotones, fragmented aquatic ecosystems, and pathogen mutations. It is emphasized that the comprehension of the multiple interactions among changes in environmental settings, land use, and human health requires a new synthesis of ecohydrology, biomedical sciences, and water management for surveillance and control of waterborne diseases in basin-based, transboundary health systems. Surveillance systems should monitor changes in water management, ecotones, and hydrological cycles and shifts in, for example, the outbreak timing of strongly seasonal diseases. These indicators would provide criteria for the development of innovative water management policies, combining methods of vector control and the safe creation of water reservoirs, irrigation systems, and wetland habitats.

A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), as well as PCR screening to identify genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples representing various food products. The pathogen was mainly isolated from frozen food made of wheat flour or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323) of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance was most frequently observed for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than five antimicrobials. Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes, and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.

Vibrio cholerae and campylobacters are recognized as important water-borne and food-borne pathogens, respectively, associated with acute gastroenteritis. In this article, we describe the recent scenario of cholera on the basis of active surveillance conducted at the National Institute of Cholera and Enteric diseases, Kolkata, India with special reference to a newly emerged V. cholerae O1 El Tor variant and the recent molecular epidemiologic investigation related with Campylobacter and related organisms in Japan, Thailand and the People’s Republic of China.

Aim: To develop a haemolysin (hly) gene-based species-specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species-specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost-effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.