Phenotypic & genetic characterization of Bacillus cereus isolated from the acute diarrhoeal patients.

BACKGROUND & OBJECTIVES: Bacillus cereus is one of the pathogens responsible for human diarrhoea, mainly due to consumption of contaminated food. The present study was undertaken to determine the occurrence of B. cereus among diarrhoeal patients and its phenotypic and genetic characteristics that determine the virulence and clonal features. METHODS: Stool specimens were collected for two years from acute diarrhoeal patients attending the two referral hospitals in Kolkata. Presence of virulence genes in B. cereus was determined by PCR. Clonality was assessed by pulsed-field gel analysis (PFGE) by restriction digestion with SmaI and NotI enzymes. Enterotoxins were detected by haemolysin assay and using BCET-RPLA kit. Invasion assay was done on Hep-2 cell line. Antimicrobial susceptibility was tested by disc diffusion method. RESULTS: B. cereus was identified in 54 (3.5%) of the 1536 diarrhoeal cases studied. Majority of the isolates were susceptible to many antibiotics but showed resistant to amoxyclav and cephalosporins. Six genes covering the two different enterotoxic complexes determining the pathogenicity of B. cereus have been characterized by PCR. The nhe genes were detected in a higher proportion than hbl. Except in two, clonal diversity was noticed among 21 B. cereus isolates. Haemolytic enterotoxin was detected in 76 per cent of the isolates. Majority of the isolates (67%) produced in vitro enterotoxin (BCET) confirming its involvement in the infection. INTERPRETATION & CONCLUSIONS: Though the presence of B. cereus was not high in patients with diarrhoea, several virulence factors confirm their association with diarrhoea. Distinct clonality was identified in majority of the isolates indicating their origin from different sources.

Prevalence of virulence genes and cytolethal distending toxin production in Campylobacter jejuni isolates from diarrheal patients in Bangladesh.

From 300 stool samples, 58 Campylobacter strains were isolated by standard microbiological and biochemical methods. Of these, 40 strains were identified as Campylobacter jejuni and 5 as Campylobacter coli. The presence of flaA (100%), cadF (100%), racR (100%), dnaJ (100%), pldA (100%), ciaB (95%), virB11 (0%), ceuE (82.5%), cdtA (97.5%), cdtB (97.5%), cdtC (97.5%), and wlaN (7.5%) genes was detected in C. jejuni by PCR. All C. jejuni strains but one produced cytolethal distending toxin in a HeLa cell assay.

Design and Construction of Vibrio cholerae Strains That Harbor Various CTX Prophage Arrays.

Toxigenic Vibrio cholerae strains arise upon infection and integration of the lysogenic cholera toxin phage, the CTX phage, into bacterial chromosomes. The V. cholerae serogroup O1 strains identified to date can be broadly categorized into three main groups: the classical biotype strains, which harbor CTX-cla; the prototype El Tor strains (Wave 1 strains), which harbor CTX-1; and the atypical El Tor strains, which harbor CTX-2 (Wave 2 strains) or CTX-3~6 (Wave 3 strains). The efficiencies of replication and transmission of CTX phages are similar, suggesting the possibility of existence of more diverse bacterial strains harboring various CTX phages and their arrays in nature. In this study, a set of V. cholerae strains was constructed by the chromosomal integration of CTX phages into strains that already harbored CTX phages or those that did not harbor any CTX phage or RS1 element. Strains containing repeats of the same kind of CTX phage, strains containing the same kind of CTX phage in each chromosome, strains containing alternative CTX phages in one chromosome, or containing different CTX phages in each chromosome have been constructed. Thus, strains with any CTX array can be designed and constructed. Moreover, the strains described in this study contained the toxT-139F allele, which enhances the expression of TcpA and cholera toxin. These characteristics are considered to be important for cholera vaccine development. Once their capacity to provoke immunity in human against V. cholerae infection is evaluated, some of the generated strains could be developed further to yield cholera vaccine strains.

Identification of burden hotspots and risk factors for cholera in India: An observational study.

BACKGROUND: Even though cholera has existed for centuries and many parts of the country have sporadic, endemic and epidemic cholera, it is still an under-recognized health problem in India. A Cholera Expert Group in the country was established to gather evidence and to prepare a road map for control of cholera in India. This paper identifies cholera burden hotspots and factors associated with an increased risk of the disease. METHODOLOGY/PRINCIPLE FINDINGS: We acquired district level data on cholera case reports of 2010-2015 from the Integrated Disease Surveillance Program. Socioeconomic characteristics and coverage of water and sanitation was obtained from the 2011 census. Spatial analysis was performed to identify cholera hotspots, and a zero-inflated Poisson regression was employed to identify the factors associated with cholera and predicted case count in the district. 27,615 cholera cases were reported during the 6-year period. Twenty-four of 36 states of India reported cholera during these years, and 13 states were classified as endemic. Of 641 districts, 78 districts in 15 states were identified as "hotspots" based on the reported cases. On the other hand, 111 districts in nine states were identified as "hotspots" from model-based predicted number of cases. The risk for cholera in a district was negatively associated with the coverage of literate persons, households using treated water source and owning mobile telephone, and positively associated with the coverage of poor sanitation and drainage conditions and urbanization level in the district. CONCLUSIONS/SIGNIFICANCE: The study reaffirms that cholera continues to occur throughout a large part of India and identifies the burden hotspots and risk factors. Policymakers may use the findings of the article to develop a roadmap for prevention and control of cholera in India.

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.

Mass vaccination with a new, less expensive oral cholera vaccine using public health infrastructure in India: the Odisha model.

INTRODUCTION: The substantial morbidity and mortality associated with recent cholera outbreaks in Haiti and Zimbabwe, as well as with cholera endemicity in countries throughout Asia and Africa, make a compelling case for supplementary cholera control measures in addition to existing interventions. Clinical trials conducted in Kolkata, India, have led to World Health Organization (WHO)-prequalification of Shanchol, an oral cholera vaccine (OCV) with a demonstrated 65% efficacy at 5 years post-vaccination. However, before this vaccine is widely used in endemic areas or in areas at risk of outbreaks, as recommended by the WHO, policymakers will require empirical evidence on its implementation and delivery costs in public health programs. The objective of the present report is to describe the organization, vaccine coverage, and delivery costs of mass vaccination with a new, less expensive OCV (Shanchol) using existing public health infrastructure in Odisha, India, as a model. METHODS: All healthy, non-pregnant residents aged 1 year and above residing in selected villages of the Satyabadi block (Puri district, Odisha, India) were invited to participate in a mass vaccination campaign using two doses of OCV. Prior to the campaign, a de jure census, micro-planning for vaccination and social mobilization activities were implemented. Vaccine coverage for each dose was ascertained as a percentage of the censused population. The direct vaccine delivery costs were estimated by reviewing project expenditure records and by interviewing key personnel. RESULTS: The mass vaccination was conducted during May and June, 2011, in two phases. In each phase, two vaccine doses were given 14 days apart. Sixty-two vaccination booths, staffed by 395 health workers/volunteers, were established in the community. For the censused population, 31,552 persons (61% of the target population) received the first dose and 23,751 (46%) of these completed their second dose, with a drop-out rate of 25% between the two doses. Higher coverage was observed among females and among 6-17 year-olds. Vaccine cost at market price (about US$1.85/dose) was the costliest item. The vaccine delivery cost was $0.49 per dose or $1.13 per fully vaccinated person. DISCUSSION: This is the first undertaken project to collect empirical evidence on the use of Shanchol within a mass vaccination campaign using existing public health program resources. Our findings suggest that mass vaccination is feasible but requires detailed micro-planning. The vaccine and delivery cost is affordable for resource poor countries. Given that the vaccine is now WHO pre-qualified, evidence from this study should encourage oral cholera vaccine use in countries where cholera remains a public health problem.

Impact of climate change on health and strategies for mitigation and adaptation.

Climate change and its negative impacts on health are now globally recognized. A wide variety of diseases and health conditions - ranging from heat and radiation-related illnesses to water and vector-borne diseases, under-nutrition, respiratory and cardiac problems, drowning, injuries and mental stress arising from extreme and sudden weather events and their resultant population displacements - all have been associated with various components of changing climate. However, the exact nature and extent of such impacts are yet to be firmly established since many other non-climate factors also produce or affect similar outcomes. This calls for more research specially from the underdeveloped countries, where such impacts are disproportionately more but reliable data are remarkably less. Recognizing the importance of human influences on global warming, almost all countries in the world have undertaken some kind of policies and measures to mitigate adverse climatic changes. Unfortunately, even without further addition of greenhouse gases (GHGs) in our climate, the amount of GHGs already released has the potential to continue the damages for many more decades to come. Thus, all countries should also place priorities in assessing their own vulnerabilities from climate change and take adaptive measures accordingly. As climate change exerts its impact simultaneously in many non-health sectors as well, this would require strong intersectoral cooperation at various levels.

Role of Shrimp Chitin in the Ecology of Toxigenic Vibrio cholerae and Cholera Transmission.

Seasonal plankton blooms correlate with occurrence of cholera in Bangladesh, although the mechanism of how dormant Vibrio cholerae, enduring interepidemic period in biofilms and plankton, initiates seasonal cholera is not fully understood. In this study, laboratory microcosms prepared with estuarine Mathbaria water (MW) samples supported active growth of toxigenic V. cholerae O1 up to 7 weeks as opposed to 6 months when microcosms were supplemented with dehydrated shrimp chitin chips (CC) as the single source of nutrient. Bacterial counting and detection of wbe and ctxA genes were done employing culture, direct fluorescent antibody (DFA) assay, and multiplex-polymerase chain reaction methods. In MW microcosm, the aqueous phase became clear as the non-culturable cells settled, whereas the aqueous phase of the MW-CC microcosm became turbid from bacterial growth stimulated by chitin. Bacterial chitin degradation and biofilm formation proceeded from an initial steady state to a gradually declining bacterial culturable count. V. cholerae within the microenvironments of chitin and chitin-associated biofilms remained metabolically active even in a high acidic environment without losing either viability or virulence. It is concluded that the abundance of chitin that occurs during blooms plays an important role in the aquatic life cycle of V. cholerae and, ultimately, in the seasonal transmission of cholera.

Are probiotics a feasible intervention for prevention of diarrhoea in the developing world?

With more than 1.4 million of the 9 million child deaths being attributed to diarrhoea in 2008 and 49% of them occurring in five countries namely, India, Nigeria, Democratic Republic of the Congo, Pakistan and China, there is an urgent need for intervention to prevent and control diarrhoeal diseases. Of the various interventions, probiotics offer immense potential. The past decade has witnessed the validation of their utility for the prevention, treatment and management of a variety of infective and non infective disorders. The most investigated field continues to remain infectious diarrhoea and compelling evidence comes from randomized placebo controlled trials. While results from these studies are encouraging most of them reflect the outcomes of the developed world. Developing countries like India continue to struggle with nutritional and health challenges and bear the greatest burden of diarrhoea. A paucity of data from the developing countries limits the definite recommendation of probiotics. In these countries curd, often confused for a probiotic, is practiced as an integral part of the culture. While the nutritional benefits of these products cannot be understated, it is still uncertain whether these products can be classified as a probiotic. The emergence of probiotic foods which are scientifically validated for their efficacy and impart defined health benefits offer an excellent opportunity to improve public health. A recent randomized controlled trial conducted by the National Institute of Cholera and Enteric Diseases in Kolkata, India demonstrated a protective efficacy of 14% in preventing diarrhoea among children who received a probiotic. For the developing world however the vision for probiotics would mean a fundamental change in perception and developing a well planned strategy to allow interventions like probiotics to permeate to impoverished settings, where the assault of micro organisms is on a daily basis. This would mean that probiotics are ingrained into the public health system without being seen as a medicine.

Genetic analysis of CTX prophages with special reference to ctxB and rstR alleles of Vibrio cholerae O139 strains isolated from Kolkata over a decade.

Chronological analysis of 125 Vibrio cholerae O139 strains isolated during 1993-2005 in Kolkata revealed the prevalence of two new genotypes of cholera toxin (CT) and novel combinations of ctxB and rstR alleles resulting in variant CTX prophages. One of the new genotypes of ctxB, which first appeared in 1996 with the re-emerged V. cholerae O139 strains that had CTX Calcutta phage, was designated as genotype 4. In 1998, another new genotype, designated as genotype 5, was detected that prevailed mostly in CTX phages with El Tor rstR. The prototype El Tor CTX phage with genotype 3 gradually disappeared in O139, and since 2002 the predominant CTX prophages in O139 are Calcutta phages with genotype 4 and El Tor phages with genotype 5. Results showed that V. cholerae O139 strains of Kolkata, isolated over a decade, harboured CTX prophages in the large chromosome having no RS1 downstream of CTX prophage. During the course of its intermittent incidence over a decade, five types of O139 strains were detected based on CT genotypes. Such abrupt genetic changes in O139 strains might not favour its continued prevalence in human cases in Kolkata, India.

Influence of catastrophic climatic events and human waste on Vibrio distribution in the Karnaphuli estuary, Bangladesh.

Vibrios are bacteria of marine and estuarine origin that can cause human diseases, such as cholera, and also affect aquatic organisms. The impact of storm-driven changes in salinity and suspended particulate matter (SPM) on cultivable Vibrio counts (CVC) and distribution in Karnaphuli estuary, Bangladesh, was compared before and after a strong cyclone in mid May 2007 and after a monsoon landslide a month later. CVC were higher (approximately 10(3) colony forming units-cfu/ml) at estuary's mouth (salinity 20-15 parts per thousand, ppt) and steeply declined landwards. CVC and their proportion of total aerobic bacteria were highest after the cyclone and also increased after the landslide, likely due to higher SPM loads. The cyclone did not significantly change previous fecal coliform abundance, contrasting with the ten times increase after the landslide. Sewage input enhanced CVC near the point sources. CVC and salinity correlated highly significantly at salinities <10 ppt; however, at higher values dispersion increased, probably due to the effect of sediment resuspension on CVC. Cyclone or heavy rainfall-mediated turbidity changes jointly with salinity gradients can significantly influence abundance and distribution of estuarine vibrios. Extended salt intrusion and higher turbidities in tropical estuaries by stronger and more frequent storms and deforestation-derived erosion could favor Vibrio growth, with increasing risks for aquatic resources and human health in the coastal zone.

Surveillance of vibriophages reveals their role as biomonitoring agents in Kolkata.

Cholera is a public health threat in all developing countries. Kolkata, a city in eastern India, is an endemic zone for cholera. During the course of a comprehensive investigation on the distribution of phages of Vibrio cholerae O1 and O139 in freshwater bodies in Kolkata, we were able to isolate the phages of V. cholerae O1 and O139. Vibrio cholerae O1 phages were found at all the sites and exhibited a distinct seasonal cycle, with a primary peak (13.6-17.2 PFU mL(-1)) during monsoon (June to August) in both 2006 and 2007. Vibrio cholerae O139 phages were present in the environment and were predominant during monsoon in the year 2006, except for late winter and early summer from February to April. In contrast, in the year 2007, the O139 phages could be isolated only during July to December, with the highest counts of 12.0 PFU mL(-1) determined in August. The multiplex PCR results showed that 90 samples were positive for wbe of V. cholerae O1, 32 samples for O139 (wbf) and 18 samples for both. This study shows that surveillance of vibriophages indicates the presence of V. cholerae O1 and O139 in water bodies in and around Kolkata and could therefore serve as a powerful biomonitoring agent.

Rapid culture-free identification and molecular typing of Shiga toxin-producing Escherichia coli by PCR-RFLP.

Using culture-independent technology, PCR-RFLP were used to identify and type STEC in the stool of a patient with HUS. Fecal PCR-RFLP patterns were identical to those of the STEC O157:H7 isolated from the patient.

Fluoroquinolone resistance linked to both gyrA and parC mutations in the quinolone resistance-determining region of Shigella dysenteriae type 1.

We examined the quinolone resistance-determining region (QRDR) of gyrA, gyrB, and parC of recently isolated fluoroquinolone-resistant S. dysenteriae type 1 strains from south Asia and compared data with fluoroquinolone-susceptible strains associated with previous epidemics of 1978, 1984, and 1994. In fluoroquinolone-resistant strains, double mutations (Ser83-->Leu, Asp87-->Asn or Gly) and a single mutation (Ser80-->Ile) were detected in the QRDRs of gyrA and parC, respectively.

Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004.

Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.