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We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae. Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as ‘no known aetiology’. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower Ct values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.

To study the prevalence pattern and trends in the phenotypic and genetic characteristics of shigellae, we tested 212 isolates isolated from diarrhoeal patients admitted to the Infectious Diseases Hospital, Kolkata, India, from November 2007 to October 2010. Prevalence of Shigella spp. was higher in the >5 years age group (69 %) than in children in the

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region., Author Summary Vibrio parahaemolyticus has been associated with several epidemics of foodborne diarrheal infection. Recent observations in several counties have shown the emergence of pandemic strains of V. parahaemolyticus with unique genetic features and their role in diarrheal outbreaks. Unlike other enteric pathogens, the appearance of pandemic strains of V. parahaemolyticus has not been associated with the economic/hygiene status of the population. The pandemic strains of V. parahaemolyticus continue to prevail in Kolkata, India since its appearance during 1996. The present communication describes not only the prevalence of pandemic serovars of V. parahaemolyticus, but also the appearance of novel serovars under the pandemic strain category. In addition, the trh gene was detected in some of the pandemic strains for the first time. In the newly emerged serovars genetic changes have occurred, as evidenced from the PFGE analysis. Overall, the antimicrobial susceptibility of pandemic strains remains unchanged for the past 20 years. The observations made in this study re-emphasize the importance of this pathogen and shows the recent genetic and serovar changes in the epidemiology of V. parahaemolyticus-mediated diarrhea.

This study examined established enteric pathogens, Arcobacter species and enterotoxigenic Bacteroides fragilis (ETBF), in 201 U.S. and European travelers with acute diarrhea acquired in Mexico, Guatemala, and India. Arcobacter butzleri and ETBF were detected in 8% and 7% of diarrhea cases, respectively.

Background This study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens. Methods Active surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes. Results Of the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5–10 years age group (33%). Shigellosis was more in children from 2–5 years of age (19%), whereas children

BACKGROUND: To analyse the trends in the prevalence of different pathogroups of diarrheagenic Escherichia coli (DEC) among hospitalized acute diarrheal patients. METHODOLOGY/PRINCIPAL FINDINGS: From the active surveillance of diarrheal disease at the Infectious Diseases Hospital, Kolkata, 3826 stool specimens collected during 2008-2011 were screened for DEC and other enteric pathogens. PCR was used in the detection of enterotoxigenic, enteropathogenic and enteroaggregative E. coli and 10 major colonization factor antigens (CFs) of enterotoxigenic E. coli. The relationship between DEC infected patient's age group and clinical symptoms were also investigated. Multiplex PCR assay showed that the prevalence of EAEC was most common (5.7%) followed by ETEC (4.2%) and EPEC (1.8%). In diarrheal children >2 year of age, EAEC and EPEC were detected significantly (p = 0.000 and 0.007, respectively). In children >2 to 5 and >5 to 14 years, ETEC was significantly associated with diarrhea (p = 0.000 each). EAEC was significantly associated with diarrheal patients with age groups >14 to 30 and >30 to 50 years (p = 0.001, and p = 0.009, respectively). Clinical symptoms such as vomiting, abdominal pain, watery diarrhea, were recorded in patients infected with ETEC. Dehydration status was severe among patients infected by ST-ETEC (19%) and EPEC (15%). CS6 was frequently detected (37%) among ETEC. CONCLUSIONS/SIGNIFICANCE: Hospital based surveillance reviled that specific pathogroups of DEC are important to certain age groups and among ETEC, CS6 was predominant.