Environmental Vibrio cholerae O139 may be the progenitor of outbreak of cholera in coastal area of Orissa, eastern India, 2000: molecular evidence.

Cholera has been reported in the state of Orissa, India during the past decades. An outbreak of diarrheal disease occurred during November 1 to November 9, 2000 in Rusipada village near Puri, which was inhabited by a population of approximately 560. During the outbreak, Vibrio cholerae O139 strains were isolated from clinical specimens collected from patients with acute diarrhea admitted to the infectious diseases (ID) hospital in Puri and from environmental samples collected from multiple bodies of water in the village. The index case with acute diarrhea was a 60-year-old female resident of Rusipada who had not visited any known outbreak-related areas, including an outbreak 1 week prior to her symptom onset in a nearby village. All the isolated strains were positive for ctxA, tcpA, ace, and zot genes, produced cholera toxin, and exhibited a similar antibiogram pattern. Comparison of DNA fingerprinting analysis by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) method and dendrogram constructed from RAPD revealed that genetic homogeneity exist between the clinical and environmental O139 strains. Epidemic and endemic cholera is a major public health problem in many developing countries and continues to be an important cause of morbidity in many areas of Asia, Africa, and Latin America. Among more than 200 serogroups of V. cholerae so far identified, only O1 and recently identified O139 serogroup are capable of causing epidemic cholera. It is now widely accepted that O139 Bengal like O1 and non-1 and non-O139 Vibrio may survive better in aquatic environments and environmental water is the reservoir for infectious V. cholerae. In this report we investigated the clonal relationship between strains of V. cholerae collected both from stool specimens from persons with acute diarrhea and from environmental sources of water. During the outbreak of diarrhea, 23 non-randomly selected rectal swabs were collected from the hospitalized diarrhea patients in ID hospital, Puri in Cary-Blair Transport (CBT, DIFCO,USA) medium and transported to the Microbiology Department of Regional Medical Research Centre (RMRC), Bhubaneswar and bacteriologically analyzed following standard technique. Subsequently after the isolation of V. cholerae O139 as the etiological agent of the outbreak, 20 water samples non-randomly selected were collected from water bodies situated at various distances in that village to determine the source of contamination following a previous method. Presumptive identification of 10 and 6 V. cholerae were isolated from rectal swabs and water samples respectively and the strains were agglutinated with monoclonal O139 antiserum supplied by NICED, Kolkata, India and were confirmed to belong to V. cholerae serogroup O139. To investigate the similarities of clinical and environmental strains of V cholerae O139, drug susceptibility test, cholera toxin assay, detection of virulent genes by polymerase chain reaction (PCR) assay, RAPD finger printing assay, and PFGE were performed. A monosialoganglioside (GM1) enzyme-linked immunosorbent assay (ELISA) was used to examine cholera toxin production in V. cholerae O139 strains by the method Svennerholm and Holmgren. Drug susceptibility test was performed following the method described elsewhere, with the antibiotics (Hi-media Laboratories, Bombay, India) ampicillin (A, 10 g), chloramphenicol (C, 30 g), co-trimoxazole (Co, 25 g), ciprofloxacin (Cf, 5 g), furazolidone (fz, 100 g), gentamicin (G, 10 g), neomycin (N, 30 g), nalidixic acid (Na, 30 g), norfloxacin (Nx, 10 g), streptomycin (S, 10 g) and tetracycline (T, 30 g). Characterization of strains as susceptible or resistant was based on size of the inhibition zone around each disc according to manufacturer’s instructions, which matched interpretive criteria recommended by the WHO. Strains showing an intermediate zone of inhibition were interpreted as resistant to that drug on the basis of previous MIC studies conducted with V. cholerae. The presence of virulent genes ctxA, tcpA, zot, and ace were determined by using PCR assay as described elsewhere. The primers used for this assay and the expected amplicon sizes are listed in Table 1. V. cholerae O1 serotype Inaba biotype classical strain 569B, V. cholerae O1 serotype Ogawa biotype El Tor strain 20(VC20) and O139 isolated in 1992 in Calcutta (SG24) were used as PCR positive control for ctxA, tcpA, zot, and ace genes. RAPD was carried out following the method described previously. The pictures obtained from RAPD (1281) fingerprinting analysis of clinical and environmental strains O139 were compared with ascertaining the phylogenetic relationship among them by using Quantity–1 software. Genomic DNA of V. cholerae O139 was prepared in agarose plugs as described previously to carry out PFGE. PFGE was performed using the counter clamped homogenous electric field method on a CHEF Mapper system (BioRad, CA) with 1% PFGE grade agarose in 0.5X TBE buffer. Run condition was generated by auto algorithm mode of CHEF Mapper, PFGE system using a size range of 20–300 kb marker for V. cholerae strains. The gel was stained in 10 g/mL ethidium bromide solution for 30 minutes, de-stained in water for 15 minutes, and photographed under UV light in Alpha Imager (Alpha Infotech Corporation, USA). During 9 days of outbreak, 198 persons had acute diarrhea (1–2 members from each family). Members of the affected family came into close physical contact while handling the patients. Investigations were initiated immediately after the * Address correspondence to Guru Chhotray, C. S. Pur, Bhubaneswar, Orissa, India 751023. E-mail: [email protected] Am. J. Trop. Med. Hyg., 78(5), 2008, pp. 819–822 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene