Genetic characteristics and changing antimicrobial resistance among Shigella spp. isolated from hospitalized diarrhoeal patients in Kolkata, India.

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 <5 years age group (31 %). Serotypes 2a, 3a and untypable isolates of Shigella flexneri were frequently detected. An increase in the isolation of Shigella sonnei (15 %) is a novel trend in this region. Fluoroquinolone resistance among S. flexneri serotypes 2a, 3a and other serogroups of shigellae is another evolving trend. The set gene was exclusively present in S. flexneri 2a, and the sen gene was detected in all serogroups. PFGE revealed the grouping of S. flexneri isolates according to their serotypes with approximately 80-100 % similarity, whilst Shigella dysenteriae type 2 and S. sonnei were clonal in nature. There was no demarcation in the prevalence of serotypes, antimicrobial resistance or clonality between the two age groups.

Role of probiotic in preventing acute diarrhoea in children: a community-based, randomized, double-blind placebo-controlled field trial in an urban slum.

Acute diarrhoea remains a major public health challenge in developing countries. We examined the role of a probiotic in the prevention of acute diarrhoea to discover if there was an effect directed towards a specific aetiology. A double-blind, randomized, controlled field trial involving 3758 children aged 1-5 years was conducted in an urban slum community in Kolkata, India. Participants were given either a probiotic drink containing Lactobacillus casei strain Shirota or a nutrient drink daily for 12 weeks. They were followed up for another 12 weeks. The primary outcome of this study was the occurrence of first episodes of diarrhoea. We assessed this during 12 weeks of intake of study agent and also for 12 weeks of follow-up. There were 608 subjects with diarrhoea in the probiotic group and 674 subjects in the nutrient group during the study period of 24 weeks. The level of protective efficacy for the probiotic was 14% (95% confidence interval 4-23, P<0·01 in adjusted model). The reduced occurrence of acute diarrhoea in the probiotic group compared to nutrient group was not associated with any specific aetiology. No adverse event was observed in children of either probiotic or nutrient groups. The study suggests that daily intake of a probiotic drink can play a role in prevention of acute diarrhoea in young children in a community setting of a developing country.

Multilocus variable-number tandem repeat analysis of Vibrio cholerae O1 El Tor strains harbouring classical toxin B.

Atypical Vibrio cholerae O1 strains - hybrid strains (strains that cannot be classified either as El Tor or classical biotype) and altered strains (El Tor biotype strains that produce classical cholera toxin) - are currently prevalent in Asia and Africa. A total of 74 hybrid and altered strains that harboured classical cholera toxin were investigated by multilocus variable-number tandem repeat analysis (MLVA). The results showed that the hybrid/altered strains could be categorized into three groups and that they were distant from the El Tor strain responsible for the seventh cholera pandemic. Hybrid/altered strains with a tandem repeat of the classical CTX prophage on the small chromosome were divided into two MLVA groups (group I: Mozambique/Bangladesh group; group III: Vietnam group), and altered strains with the RS1-CTX prophage containing the El Tor type rstR and classical ctxB on the large chromosome were placed in two MLVA groups (group II: India/Bangladesh group; group III: India/Vietnam group).

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure.

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.

Increasing antimicrobial resistance--an emerging problem in the treatment of shigellosis.

Shigellosis is a major cause of diarrhoea-related morbidity and mortality, especially in developing countries. Effective antibiotic treatment reduces the average duration of illness by reducing faecal excretion of the bacterium and preventing further transmission and potentially lethal complications. Treatment of shigellosis is currently limited by the high prevalence of multidrug-resistant strains of Shigella. Although fluoroquinolones are currently effective in treating adults, resistance to fluoroquinolones among Shigella spp. is emerging, and their use in children is subject to limitations. Azithromycin and third-generation cephalosporins are also effective in the treatment of shigellosis, but monitoring of Shigella isolates to detect the emergence of resistance is essential.

Phage typing, biotyping & antimicrobial resistance profile of Salmonella enterica serotype Typhi from Kolkata.

BACKGROUND & OBJECTIVE: Kolkata and its suburbs in eastern India are known to be endemic for typhoid fever. The objective of this study was to determine phage types, biotypes and antimicrobial resistance patterns of Salmonella enterica serotype Typhi isolated during the period 2003-2005 from a prospective surveillance for typhoid fever in two urban slums in Kolkata. METHODS: A total of 195 Salmonella enterica serotype Typhi isolated from blood cultures were phage typed, biotyped and tested for their antimicrobial susceptibility profile. RESULTS: Phage type E1 was the most common (60.3%) followed by phage type A among five phage types identified. Biotype I (95%) was predominant, 28 isolates were multidrug resistant (MDR) and most of the MDR strains belonged to phage type E1 and biotype I. INTERPRETATION & CONCLUSION: A single phage type and biotype were prevalent among the Salmonella enterica serotype Typhi isolates studied from Kolkata, India.

Molecular epidemiology of human astrovirus infections in Kolkata, India.

UNLABELLED: The study is aimed to determine the seasonal distribution and clinical characteristics of astroviruses associated with acute watery diarrhoea among children in Kolkata and characterize them at the molecular level. METHOD OF STUDY: Faecal specimens of acute watery diarrhoea cases (n=857) and non-diarrhoeic samples (n=211) from the hospitals and a nearby field community were screened with IDEIA Astrovirus detection kit; astrovirus co-infections with rotavirus and/or picobirnavirus were detected by RNA-PAGE and silver staining. Further RT-PCR was carried out using specific primers, viz. Mon340 (+) and Mon348 (-) targeting a highly conserved domain of ORF1a (289 bp) of human astroviruses. RESULTS: Astrovirus infection was detected in 50 cases (50/857); astroviruses were detected mostly in children aged 6-12 months (50%); all non-diarrhoeic samples (n=211) were negative for astrovirus. In 52% of astrovirus positive cases, the virus was detected as the sole agent; mixed infections were also detected with other diarrhoeic pathogens such as rotavirus (32%), picobirnavirus (2%), rotavirus and picobirnavirus (2%), picobirnavirus and Enterotoxigenic E. coli (ETEC) (2%), rotavirus and ETEC (2%), rotavirus and Enteroaggregative E. coli (EAEC) (2%), Enteropathogenic E. coli (EPEC) (2%), Shigella flexneri type 3a (2%) and Ascaris (2%). RT-PCR and sequencing of amplicons of astroviruses from Kolkata, with specific primers targeted to the conserved domain of ORF1a (289 bp) of the astrovirus genome, showed maximum homology to the astrovirus strain ("5-158") from Seoul (98%). RESULTS AND CONCLUSIONS: Clinical characteristics of the diarrhoeic children in Kolkata indicated that astrovirus infections were detected throughout the year and were associated with varying degree of dehydration and acute watery diarrhoea. In-depth molecular epidemiological surveillance of astroviruses in Kolkata is essential for better understanding of their overall genetic nature.

Epidemiological, microbiological & electron microscopic study of a cholera outbreak in a Kolkata slum community.

BACKGROUND & OBJECTIVE: Epidemics of cholera caused by Vibrio cholerae O1 or O139 have been reported from different parts of India. Factors such as unsafe water supply, poor environmental sanitation, indiscriminate defaecation and lack of personal hygiene are mainly responsible for continued transmission of this disease. We report here epidemiological and microbiological findings of a localized outbreak of cholera, which occurred during March and April 2004 in the eastern part of Kolkata city. METHODS: The affected slum area has a population of 4409, predominantly muslims. Patients suffering from acute watery diarrhoea attended the health outposts organized by National Institute of Cholera and Enteric Diseases, Kolkata and International Vaccine Institute, South Korea as part of a routine surveillance programme at the locality as well as the emergency medical camp organized by the Kolkata Municipal Corporation. Stool and water samples were collected and tested for diarrhoeagenic pathogens in the laboratory. Bacteriophages specific for V. cholerae were isolates and studied electron microscopically for morphology. RESULTS: A total of 89 diarrhoea cases were reported giving an attack rate of 2 per cent. V. cholerae O1 biotype ElTor, serotype Ogawa was isolated as a sole pathogen from 15 (15.8%) of 89 stool samples screened. Water samples (2 from tube wells, 3 from municipal taps and 1 from well) showed presence of coliform bacilli with high MPN (Most Probable Number) count. Bacteriophages specific to V. cholerae were isolated from 2 of 6 water samples examined. A leakage was detected in the main pipeline supplying drinking water to that area. INTERPRETATION & CONCLUSION: The outbreak was caused by V. cholerae O1 (Ogawa) biotype ElTor. The presence of phages in the water samples was an additional indicator for V. cholerae contamination in this community. Occurrences of such outbreaks support vaccination against cholera as an alternative strategy.

Epidemiology of HIV infection among brothel-based sex workers in Kolkata, India.

A cross-sectional study was carried out during January-June 2004 to assess the status of HIV infection among brothel-based sex workers in Kolkata city, India. Six hundred and twenty-two sex workers, selected from six brothels, were included in the study to assess their HIV status, and 362 sex workers, a subset of the above population, were interviewed to study their risk behaviour and practices. Blood samples were collected from each sex worker for testing HIV by an unlinked anonymous method. The prevalence of HIV infection was 9.6%, but was much higher among younger sex workers aged 20 years or less (27.7%) compared to the older age group (8.4%). This difference was statistically significant, indicating an association of younger ages with HIV infection [p = 0.006 and odds ratio (OR) = 4.18; (95% confidence interval (CI) 1.2-13.8)]. HIV was not associated with duration of sex work, average number of clients entertained per day, condom use, practice of sex during menstruation, pre-coital examination of penis for visible ulcer/discharge, suffering from sexually transmitted infections, and entertaining clients outside the brothel. There was a big gap between the reported and the evaluated condom use by sex workers. The results suggest that there is a need to develop suitable HIV intervention strategies, considering the socioeconomic and cultural aspects of city sex workers, with a provision for continuous monitoring and evaluation.

Species diversity and antimicrobial resistance of Shigella spp. isolated between 2001 and 2004 from hospitalized children with diarrhoea in Kolkata (Calcutta), India.

The incidence, phenotypic characteristics and antimicrobial resistance patterns of 193 Shigella strains isolated from 2489 hospitalized children with acute diarrhoea were studied during January 2001 to August 2004. S. flexneri (60%) was the most prevalent serogroup, followed by S. sonnei (23.8%), S. dysenteriae (9.8%) and S. boydii (5.7%). Since 2002, S. flexneri 2a was the most dominant serotype. Almost all S. flexneri strains exhibited resistance to ampicillin, co-trimoxazole, tetracycline, nalidixic acid and fluoroquinolones. After a lapse of almost 14 years, S. dysenteriae type 1 strains reemerged for the first time during 2002 and these strains were resistant to more than two antibiotics (multidrug resistance), including fluoroquinolones. An upsurge of similar resistance patterns was also noted among S. flexneri type 2a since December 2003. Resistance to fluoroquinolone increased year on year among S. dysenteriae type 1 and S. flexneri, but not in S. boydii or S. sonnei. Monitoring of antimicrobial susceptibility through a surveillance programme is recommended to select appropriate antibiotics for the effective treatment of shigellosis in this region.

The burden of cholera in the slums of Kolkata, India: data from a prospective, community based study.

AIMS: To conduct a prospective, community based study in an impoverished urban site in Kolkata (formerly Calcutta) in order to measure the burden of cholera, describe its epidemiology, and search for potential risk factors that could be addressed by public health strategies. METHODS: The study population was enumerated at the beginning and end of the study period. Surveillance through five field outposts and two referral hospitals for acute, watery, non-bloody diarrhoea was conducted from 1 May 2003 to 30 April 2004. Data and a stool sample for culture of Vibrio cholerae were collected from each patient. Treatment was provided in accordance with national guidelines. RESULTS: From 62 329 individuals under surveillance, 3284 diarrhoea episodes were detected, of which 3276 (99%) had a stool sample collected and 126 (4%) were culture confirmed cholera. Nineteen (15%) were children less than 2 years of age, 29 (23%) had severe dehydration, and 48 (38%) were hospitalised. Risk factors for cholera included a household member with cholera during the period of surveillance, young age, and lower educational level. CONCLUSIONS: There was a substantial burden of cholera in Kolkata with risk factors not easily amenable to intervention. Young children bear the brunt not only of diarrhoeal diseases in general, but of cholera as well. Mass vaccination could be a potentially useful tool to prevent and control seasonal cholera in this community.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

OBJECTIVES: The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. METHODS: The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. RESULTS: Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. CONCLUSIONS: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Prevalence of the sat, set and sen genes among diverse serotypes of Shigella flexneri strains isolated from patients with acute diarrhoea.

New virulence factors associated with the genus Shigella have been described recently. These comprise enterotoxins ShET-1 and ShET-2, encoded by the set and sen genes, respectively, and the secreted autotransporter toxin Sat, encoded by the sat gene. The present study aimed to determine the prevalence of genes encoding these toxins among a collection of Shigella flexneri clinical isolates belonging to different serotypes. While the set gene was detected only in S. flexneri serotypes 2a and 2b, the sen and sat genes were present in diverse serotypes of S. flexneri.

Clonal multidrug-resistant Shigella dysenteriae type 1 strains associated with epidemic and sporadic dysenteries in eastern India.

Multidrug-resistant strains of Shigella dysenteriae type 1 were implicated in three outbreaks and sporadic cases of dysentery in eastern India in 2002 and 2003. After a hiatus of 14 years, this pathogen reemerged with an altered antibiotic resistance pattern. In addition to ampicillin, co-trimoxazole, tetracycline, chloramphenicol, and nalidixic acid, all the recent strains were resistant to norfloxacin, lomefloxacin, pefloxacin, and ofloxacin and showed reduced susceptibility to ciprofloxacin. Pulsed-field gel electrophoresis identified a new clone of S. dysenteriae type 1 that was associated with the recent outbreaks and sporadic cases. Based on the spatial and temporal spread of multidrug-resistant S. dysenteriae type 1, we predict that this clonal type may spread further in this region.

Decreasing trend in the occurrence of Salmonella enterica serotype Typhi amongst hospitalised children in Kolkata, India during 1990-2000.

In a prospective hospital based surveillance, 1454 children clinically diagnosed as typhoid fever were enrolled during the period between 1990 to 2000. Of them 336 (23.1%) children were positive for Salmonella enterica serotype Typhi by blood culture. A declining trend of hospitalization and identification of the pathogen was observed from 1992 to 2000 as compared to 1990-1991. A declining trend of resistance to the commonly used anti-typhoid drugs was seen in the S. enterica serotype Typhi isolates. Recently in 2000, nine strains were detected as ciprofloxacin resistant. Misuse and overuse of ciprofloxacin for the treatment of typhoid fever influenced the development of ciprofloxacin resistant strains of S. enterica serotype Typhi in and around Kolkata.

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation.

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781-788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross-linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild-type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A-activated receptors can stimulate mitogen-activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S-phase are both decreased relative to 1 nM wild-type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild-type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waal's weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival.