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.

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.

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.

Rise in free intracellular calcium in HeLa cells infected with aggregative Klebsiella pneumoniae strains isolated from cases of diarrhoea.

BACKGROUND & OBJECTIVES: Klebsiella pneumoniae strains occasionally cause diarrhoea in humans. This study was done to determine the involvement of calcium in the pathogenesis of aggregative K. pneumoniae strains. METHODS: A total of nine strains of K. pneumoniae were tested for adherence assay in HeLa cell line. A representative strain CO-1215 was used for [Ca2+]i study using Fura-2 fluorescence. RESULTS: Infection of cultured HeLa cells with aggregative K. pneumoniae strain resulted in five-fold elevation of intracellular level of free calcium ([Ca2+]i) with maximum Ca2+ influx at 3 h after bacterial infection. Chelation of extracellular Ca2+ with [ethylenebis(oxyethylenenitrile)] tetraacetic acid and suspension of cells in Ca2+ free buffer suggested that the rise of Ca2+ in aggregative K. pneumoniae infected HeLa cells was due to influx of Ca2+ from extracellular medium. INTERPRETATION & CONCLUSIONS: This study showed aggregative adherence in HeLa cells and this adherence leads to influx of extracellular Ca2+. The unrestricted passage of calcium ions across cell membranes could cause phosphorylation of proteins involved in ion transport across the membrane, which could result in secretory diarrhoea. Further work is in progress to study the enterotoxicity of these strains in an in vitro rabbit intestinal model.

Enteroaggregative Klebsiella pneumoniae in association with childhood diarrhoea.

A total of 19 strains of Klebsiella pneumoniae isolated as sole pathogen from children with diarrhoea were used to study their virulence mechanism using different assays. Eith strains of K. pneumoniae exhibited aggregative adherence that was distinct from the stacked brick enteroaggregative pattern shown by Escherichia coli. The study suggests the presence of a new virulence mechanism in the pathogenesis of Klebsiella-associated diarrhoea.

An outbreak of Eltor cholera in Aizwal town of Mizoram, India.

During the months of May, June and through early part of July 1994, an unusual occurrence of severe dehydrating watery diarrhoea cases and deaths were reported from Aizwal town, the capital of Mizoram, a North-Eastern state of India. Vibrio cholerae 01 biotype Eltor, the causative agent responsible for this outbreak, was isolated from 50.0% of hospitalised cases. The disease affected older children and adults more (52.9%) than younger children below five years of age. Vibrio cholerae 01 strains isolated were uniformly resistant to furazolidone and co-trimoxazole, which are commonly advocated in the treatment of cholera specially in children of developing countries. Emergence of such resistant strain is alarming and is of great public health importance.

Detection of faecal leucocytes & erythrocytes from stools of cholera patients suggesting an evidence of an inflammatory response in cholera.

BACKGROUND & OBJECTIVES: Detection of faecal leucocytes and RBCs in stool samples of cholera patients has been reported in a small number of studies. This study extends these observations by examining stool samples of cholera patients in Calcutta. METHODS: Out of 1562 diarrhoeal stool samples, Vibrio cholerae was isolated in 266 cases. Stool samples obtained were examined microscopically within two hours of collection. RBCs and faecal leucocytes were examined by normal saline and methylene blue stain. Stool culture was performed using selective and differential media for isolation of V. cholerae. RESULTS: Among 266 cholera patients, RBCs was detected in 58 per cent and faecal leucocytes in 88 per cent respectively. The extent of the changes correlated with clinical severity. INTERPRETATION & CONCLUSION: This study showed the presence of RBCs and faecal leucocytes in stools of patients of cholera caused by V. cholerae 01 and 0139 which indicates some inflammatory changes in the gut mucosa. Further study is required to elucidate the inflammatory mechanism involved in the underlying process(es).

Multi-drug resistant non-typhoidal Salmonella spp. associated with acute diarrhoeal disease.

The prevalence of different serotypes of non-typhoidal Salmonella spp. among patients suffering from acute diarrhoea admitted to the Infectious Diseases Hospital, Calcutta was investigated. The predominant serogroup was C and Salmonella infantis was the major serotype isolated followed by S. worthington, S. enteritidis, S. typhimurium, S. weltevereden and S. newport. All the Salmonella strains were isolated from adults. Multidrug resistance to various antimicrobial agents was observed in 37.5 per cent of the strains. All the strains were sensitive to ciprofloxacin, norfloxacin and gentamycin.

Distribution of phage type of Vibrio cholerae O1 biotype ElTor in Indian scenario (1991-98).

During the period 1991-98, distribution of biotype, serotype and phage type of V. cholerae O1 strains isolated from different parts of the country and referred to the National Institute of Cholera and Enteric Diseases, Calcutta were studied. Of the 8101 strains received, 5613 (69.2%) were subjected to phage typing. All these strains belonged to the biotype ElTor and Ogawa was the predominant serotype (96.08%). The strains were clustered into only two types--types 2 and 4 and around 10 per cent strains remained untypable. However, using the new scheme, all these strains were found to be typable and 8 major types were recognized of which type number 27 was the predominant type (66.12%). The distribution of a common type throughout the country suggests that a particular clone of V. cholerae O1 is probably circulating all over India. A constant monitoring through phage typing is necessary to observe the emergence of any new clone of V. cholerae O1 in India.

Comparison between the multiplex PCR, sensitivity to biotype specific phages & polymyxin B for biotyping of Vibrio cholerae O1.

A total of 196 Vibrio cholerae O1 strains isolated between 1970 and 1996 were biotyped by multiplex PCR, susceptibility to polymyxin B and sensitivity to biotype specific phages. We modified the multiplex PCR by increasing the primer concentration of tcpA to improve the results. Comparison of the results of modified multiplex PCR and sensitivity to biotype specific phages and to polymyxin B showed that multiplex PCR was as efficient as phage typing for biotyping of V. cholerae O1. All the strains of V. cholerae O1 could be accurately distinguished based on polymyxin B sensitivity. Thus our results show that susceptibility of strains of V. cholerae O1 to polymyxin B is the easiest method to biotype V. cholerae O1 and is feasible in most laboratories when compared with multiplex PCR and sensitivity to biotype specific phages.

Frequency of isolation of toxigenic Clostridium difficile from healthy adults.

An attempt was made to isolate Clostridium difficile from 129 faecal specimens from 87 young and 42 elderly healthy adults. C.difficile was isolated using bacterial culture from 7 (5.4%) specimens and the frequency of isolation of toxigenic C. difficile as detected by cytotoxin (tissue culture) assay was 1.1 per cent and 2.4 per cent in healthy young and elderly adults respectively. The carrier rate of toxigenic C. difficile amongst healthy adults is relatively low in this part of our country.

Isolation of a fluoroquinolone resistant Shigella dysenteriae 1 strain from Calcutta.

Fourteen strains of Shigella spp. were isolated from patients aged 5-12 yr, hospitalized with dysentery during 1995-1996. The strains isolated were Shigella dysenteriae 1, S. flexneri and S.sonnei. All but one of the 14 strains were sensitive to ciprofloxacin and norfloxacin. The MIC of both the fluoroquinolones was 15 micrograms/ml for the resistant S.dysenteriae 1 strain (NRM4). Therefore, the careful use of antimicrobials is required for therapy of Shigellosis.

Association of enterotoxigenic Bacteroides fragilis with childhood diarrhoea.

This study was conducted in a hospital setting to determine whether enterotoxigenic strains of Bacteroides fragilis (ETBF) were associated with childhood diarrhoea. ETBF was isolated from 6 (2.6%) of 226 patients and 3 (1.7%) of 172 controls and was found mostly in children between 1-5 yr of age. The syndrome associated with ETBF was secretory in nature with watery diarrhoea and of mild severity. ETBF may be associated with diarrhoeal illness in children but is not a major problem in this part of the country.

Outbreak of cholera due to Vibrio cholerae 01 in Orissa state.

During May-June 1993, an outbreak of acute diarrhoea resulting in deaths primarily in adults was reported in two districts of Orissa state. Epidemiological and microbiological investigations revealed that this outbreak was caused by V. cholerae 01 biotype EITor. V. cholerae 01 strains were uniformly resistant to furazolidone.