Abstract
The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.
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Introduction
Vibrio cholerae, an intestinal pathogen, is the causative agent of epidemic diarrheal cholera. Cholera epidemics caused by toxigenic V. cholerae O1 and O139 (Bengal strain) represent a major public health problem in developing countries like India and Bangladesh (Nair et al. 1994; Singh et al. 2002). Over the last few decades, V. cholerae of classical biotype has been replaced by El Tor strains (Saini et al. 1995). The resurgence of V. cholerae El Tor causing outbreaks in and around Nagpur was reported recently (Mishra et al. 2004). Many sporadic outbreaks of cholera are associated with the emergence of multiple antibiotic resistant strains. The high population density of Kerala and the proximity with the aquatic systems increase the risk of occurrence of cholera epidemics. In 2001, V. cholerae strains isolated from the Kottayam, Alleppey and Thiruvananthapuram districts in Kerala were multidrug resistant, although differences in antibiotic susceptibility patterns suggest the existence of different types of V. cholerae in different geographical locations (Sabeena et al. 2001). The resurgent strains of V. cholerae are more virulent, multi-drug resistant and have characteristic changes in surface antigenicity (Shukla and Sunil 2005). In this context, we analysed the V. cholerae strain associated with a sporadic occurrence of cholera linked to food poisoning, which resulted in the death of three persons in Ernakulam district.
Materials and methods
The present strain of V. cholerae O1 was isolated from a cholera patient during February 2006. Stool samples were collected and processed for the isolation of V. cholerae using standard bacteriological methods as followed by WHO manual (WHO 1987). The strain was stored in Tryptic soy broth with 20% glycerol at −70 °C. Initial biochemical screening was done to identify the strain followed by serological analysis. The antibiotic resistance pattern was checked by agar-disc diffusion (Bauer et al. 1966). The V. cholerae isolate in this study was examined for resistance to ampicillin (10 μg), polymyxin B (50 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), neomycin (30 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), spectinomycin (100 μg), norfloxacin (10 μg), cephataxime (30 μg) and trimethoprim (5 μg) by using commercial discs (Hi Media, Bombay, India) according to the interpretation criteria recommended by the World Health Organization. The control strain used for antibiotic resistance assay was E. coli ATCC 25922. For molecular studies, a single colony of the bacteria was inoculated into 3 mL of sterile Luria Bertani broth and incubated at 37 °C, overnight for 18 h in a shaker incubator at 180 rev/min. After 18 h of growth, cells were harvested by centrifugation (6708g). The supernatant was discarded and the cell pellet was resuspended in sterile water, boiled at 100 °C for 15 min and then snap chilled in ice and stored at −20 °C and used as template DNA for PCR reactions. Specific PCR was used to detect the presence of hemolysin (hlyA. classical and El Tor specific) (Shi et al. 1998) and Hexaplex PCR was used for the rapid detection of virulence and regulatory genes comprising cholera toxin enzymatic subunit A (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and central regulatory protein (toxR) (Singh et al. 2001).
Results and discussion
The isolated strain was identified as V. cholerae O1 biotype El Tor serotype Inaba. The hexaplex PCR assay revealed the presence of virulence and regulatory genes ctxA, zot, ace, tcpA, ompU and toxR in the strain analysed. It showed resistance to polymyxin B, trimethoprim, nalidixic acid, co-trimoxazole, ampicillin and streptomycin. Additionally it showed intermediate resistance to chloramphenicol, norfloxacin and cephataxime. It showed susceptibility to gentamycin, ciprofloxacin, ofloxacin, spectinomycin and tetracycline. The emergence of antimicrobial-resistant bacterial pathogens has become a major public health concern. The indiscriminate use of antimicrobials in disease treatment can potentially lead to widespread dissemination of antimicrobial-resistant bacteria. V. cholerae acquires antimicrobial resistance phenotypes most often via conjugative transfer of genetic material. The last reported Inaba isolate in India was observed in the northern State, Chandigarh in 2004 (Taneja et al. 2005). The reemergence of V.cholerae O1 serotype Inaba in Kerala deserves mention, as future epidemics due to this serotype can be anticipated. In this context, the antigens of Inaba should be considered in future vaccine development. A better understanding of the molecular mechanisms responsible for the emergence of multidrug resistant strains will enable us to design intervention strategies to reduce the pathogen’s progression in the community. Hence, a continued monitoring and epidemiological surveillance of V. cholerae strains has become a necessity.
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Acknowledgements
The authors are grateful to Prof. M. Radhakrishna Pillai, Director, RGCB for the facilities provided. The KSCSTE, Govt. of Kerala is gratefully acknowledged for providing the research grant to Dr. Sabu Thomas.
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Thomas, S., Ramachandran, D., Remani, B. et al. Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India. World J Microbiol Biotechnol 24, 433–434 (2008). https://doi.org/10.1007/s11274-007-9481-9
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DOI: https://doi.org/10.1007/s11274-007-9481-9