In vitro susceptibility of Vibrio cholerae O1 biotype El Tor strains associated with an outbreak of cholera in Kerala, Southern India

F. Sabeenaa, G. Thirivikramjib, G. Radhakuttyc, P. Indud and D. V. Singha

a Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuram; b Department of Microbiology, Medical College, Kottayam; c Department of Microbiology, TD Medical College, Alleppey; d Department of Microbiology, Medical College, Thiruvananthapuram, India

Sir,

Cholera is endemic in many parts of India, and marked variation in the in vitro susceptibility to antibiotics of Vibrio cholerae O1 has been observed, with emerging resistance to nalidixic acid, co-trimoxazole, furazolidone and streptomycin.1 The emergence of multidrug-resistant strains of V. cholerae O1 has been a matter of concern, as tetracycline is not recommended for use in children, and quinolones are also not advocated for use in children and pregnant women.2 The state of Kerala has recently experienced outbreaks of cholera, and it was decided to screen the strains of V. cholerae O1 biotype El Tor serotype Ogawa for antibiotic susceptibility.

A total of 25 distinct clinical strains of Vibrio cholerae O1 were included in the study, eight isolates from cholera patients admitted to the Medical College Hospital, Kottayam, District Hospital, Kottayam, and Vaikom Taluk Hospital, Kottayam, between 28 December 1999 and 21 January 2000, and six strains from patients admitted to TD Medical College Hospital, Alleppey, between 3 May and 26 June 2000 and 11 strains from cases admitted to the Medical College Hospital, Thiruvananthapuram, from 3 to 24 July 2000. Faecal samples were collected and processed for isolation of V. cholerae using standard bacteriological methods, as recommended by the WHO.3 The strains of V. cholerae were maintained in 20% glycerol Luria–Bertani broth at –70°C.

Susceptibility to antimicrobial agents was examined by an agar disc diffusion method4 on Mueller–Hinton agar (MHA), using antibiotic-impregnated discs (Hi-Media Laboratories, Bombay, India). The following antibiotic discs with concentration of the drug per disc as stated in parentheses were used, ampicillin (10 µg), cefotaxime (30 µg), cephalexin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), co-trimoxazole (25 µg), furazolidone (100 µg), gentamicin (10 µg), neomycin (30 µg), nalidixic acid (30 µg), norfloxacin (10 µg), streptomycin (10 µg) and tetracycline (30 µg). After 18 h incubation at 37°C, strains, were characterized as susceptible or resistant based on inhibition zone sizes. A control strain of Escherichia coli ATCC 25922 was used.

Of the 25 isolates tested, all showed resistance to two or more drugs. All V. cholerae strains from Kottayam were uniformly resistant to cephalexin, co-trimoxazole, furazolidone, nalidixic acid (except for one strain), neomycin and streptomycin. Five strains showed additional resistance to ampicillin whereas three strains each, either in combination or separately, also showed resistance to cefotaxime, chloramphenicol, ciprofloxacin and norfloxacin, respectively (TableGo). Moreover, one strain also showed resistance to tetracycline. However, all isolates were sensitive to the other drugs tested.


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Table. Distribution of drug-resistant V. cholerae O1 biotype El Tor strains from hospitalized patients in different locations in Kerala, India
 
Among the V. cholerae strains from Alleppey, three strains showed resistance to five drugs, namely ampicillin, cephalexin, co-trimoxazole, furazolidone and nalidixic acid. Two isolates were resistant to four drugs, namely ampicillin, cephalexin, furazolidone and nalidixic acid. However, one isolate was resistant to cephalexin, furazolidone and nalidixic acid only (TableGo). All these strains, however, were uniformly susceptible to the other drugs tested.

Of the 11 V. cholerae strains from Thiruvananthapuram, the majority of strains (10) showed resistance to five drugs, namely cephalexin (except for one strain), co-trimoxazole, furazolidone, nalidixic acid (except for one strain) and streptomycin; however, two strains showed additional resistance to ampicillin. Moreover, one strain was resistant to all drugs tested (TableGo).

V. cholerae strains from Kottayam, Alleppey and Thiruvananthapuram, were multidrug resistant, although differences in antibiotic susceptibility patterns suggest the existence of different R-types of V. cholerae in different geographical locations in Kerala, India. Only co-trimoxazole and furazolidone are probably safe for use in children. However, from this study, it is clear that until a safe and effective alternative drug is available for use in children as well as adults, the problems in treating diarrhoeal patients remain serious. The transfer of genetic elements of V. cholerae O1 also necessitates continuous surveillance of antimicrobial resistance.

Acknowledgments

The Department of Biotechnology, Government of India and Rajiv Gandhi Centre for Biotechnology are gratefully acknowledged for funds contributed to this study and for providing a Research Fellowship to F. S.

Notes

J Antimicrob Chemother 2001;47: 361–362

*Corresponding author. Tel: +91-471-345-899; Fax: +91-471-329-472; E-mail: durg-singh{at}mailcity.com

References

1 . Mukhopadhyay, A. K., Garg, S., Nair, G. B., Kar, S., Ghosh, R. K., Pajni, S. et al. (1995). Biotype traits and antibiotic susceptibility of Vibrio cholerae serogroup O1 before, during and after the emergence of the O139 serogroup. Epidemiology and Infection 115, 427–34.[ISI][Medline]

2 . Hooper, D. C. & Wolfson, J. S. (1985). The fluoroquinolones: pharmacology, clinical uses, and toxicities in humans. Antimicrobial Agents and Chemotherapy 28, 716–21.[ISI][Medline]

3 . World Health Organization. (1987). Manual for Laboratory Investigations of Acute Enteric Infections, pp. 15–28. WHO, Geneva.

4 . Bauer, A. W., Kirby, W. M., Sherris, J. C. & Truck, M. (1966). Antibiotic susceptibility testing by standardized single disc method. American Journal of Clinical Pathology 45, 493–6.[ISI][Medline]





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