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J Clin Microbiol. Dec 2003; 41(12): 5833–5834.
PMCID: PMC308999

Newly Emerged Multiple-Antibiotic-Resistant Shigella dysenteriae Type 1 Strains in and around Kolkata, India, Are Clonal

Shanta Dutta,* Ayantika Ghosh, and Kaushik Ghosh
Division of Microbiology
Dharitri Dutta and Sujit K. Bhattacharya
Clinical Medicine
National Institute of Cholera  and Enteric Diseases
Kolkata, India
G. Balakrish Nair
International Centre for Diarrheal  Disease Research Bangladesh
Dhaka, Bangladesh

Several parts of India, including Kolkata, witnessed a devastating epidemic of shigellosis with increased (35.6%) isolation of Shigella dysenteriae type 1 strains during 1984 (10). In the postepidemic period, shigellosis became endemic in Kolkata and S. flexneri (58%) was the most prevalent species, followed by S. sonnei (28%), S. boydii (9%), and S. dysenteriae (5%) (5, 7). In the year 2002, a sudden resurgence in the isolation of S. dysenteriae type 1 strain was noticed, with the occurrence of several dysentery outbreaks, which swept from northern to southern districts of West Bengal, including Kolkata (4, 11; K. Sarkar, S. Ghosh, S. K. Niyogi, and S. K. Bhattacharya, Letter, Lancet 361:785, 2003). These newly emerged strains of S. dysenteriae type 1 were multidrug resistant, with resistance to commonly used antimicrobials such as ampicillin, tetracycline, cotrimoxazole, amoxicillin, nalidixic acid, and fluoroquinolones (such as ciprofloxacin and norfloxacin), which had high MICs (4, 6). Plasmid analysis and pulsed-field gel electrophoresis (PFGE) have long been used as molecular tools for subtyping a group of similar strains and for studying their genetic relatedness (1, 2, 8, 9). This suddenly increased rate of isolation of S. dysenteriae type 1 (36%) from dysentery patients in Kolkata after a gap of almost 18 years was noteworthy (4). Hence the present study was undertaken to monitor the antibiotic resistance profile of recently emerged S. dysenteriae type 1 strains and to determine the molecular subtyping of the strains by plasmid DNA analysis and PFGE.

A cluster of 36 strains of S. dysenteriae type 1 was included in this study, of which 35 strains were isolated from July 2002 to October 2002 as the sole causative agents of dysentery cases admitted to Infectious Disease Hospital and Dr. B. C. Roy Memorial Children's Hospital, two referral hospitals in Kolkata (4, 6). The remaining strain was isolated from a patient in a dysentery outbreak (Sarkar et al., letter) affecting the northern part of West Bengal. The study strains were further characterized for antimicrobial resistance profiles and subjected to plasmid DNA analysis and PFGE by standard techniques (8, 9).

The commonest antimicrobial resistance profile, observed in 97% (35 of 36) strains, was resistance to seven antimicrobials: ampicillin, tetracycline, nalidixic acid, amoxicillin, cotrimoxazole, ciprofloxacin, and norfloxacin. Plasmid profile analysis of 34 strains of S. dysenteriae type 1 revealed four different profiles (types I to IV), of which the type I profile was the most predominant and was found in 88% (30 of 34) strains. All strains had a heavy plasmid of 210 kb. Other types (types II to IV) showed gain or loss of two or three smaller plasmids.

PFGE patterns of all isolates were compared, and Dice coefficients were calculated for each pattern (3). XbaI restriction enzyme (New England Biolabs) digests of genomic DNA of S. dysenteriae type 1 strains produced a PFGE pattern with 14 to 17 DNA bands between 380 and 40 kb (Fig. (Fig.1).1). When analyzed according to the methods of Chiou et al. (2), the PFGE patterns of the isolates were classified into a single pulsotype (X1) with four subtypes (X11, X12, X13, and X14). If PFGE patterns of two phenotypically similar strains were different from each other by four or more bands, the strains were considered to be genetically different and to belong to different pulsotypes. On the other hand, in one pulsotype, if two strains had three or fewer band differences or shifts with a Dice coefficient of ≥80, they were designated subtypes, which were genetically related to the main type (12). The phenomenon could be explained by simple insertion or deletions or gain or loss of restriction sites.

FIG. 1.
PFGE patterns of S. dysenteriae type 1 strains isolated from Kolkata after digesting genomic DNA with the XbaI restriction endonuclease enzyme. Lanes M, 48.5-kb concatemers of bacteriophage λ DNA (Bio-Rad, Hercules, Calif.) used as molecular size ...

Of 36 strains of S. dysenteriae type 1, 32 (88%) belonged to the PFGE main type (X1) (Fig. (Fig.1;1; lanes 1 to 4, 6 to 8, and 10 to 12) and were considered genetically identical (clonal). Each of the remaining four strains belonged to one of four different subtypes (X11, X12, X13, or X14) (Fig. (Fig.1,1, lanes 5 [X11] and 9 [X12]). Subtypes X13 and X14 are not shown in Fig. Fig.11.

The present study revealed that most (27 of 36; 75%) of the recently emerged multidrug-resistant S. dysenteriae type 1 strains were indistinguishable by their antimicrobial resistance patterns, plasmid DNA profiles, and PFGE profiles, indicating their genetic similarity. The emergence of one predominantly circulating multidrug-resistant clone of S. dysenteriae type 1 during a specified period was alarming and might be responsible for significant number of the recently reported dysentery outbreaks in eastern India. Therefore, urgent preventive intervention is necessary to contain further spread of this deadly organism.

Acknowledgments

A part of this work was supported by funds from the Indian Council of Medical Research and from the US-Japan Cooperative Medical Science Programme (USJCMSP) 2001 for the USJCMSP Asian Region Collaboration Research Project. S. Dutta is a recipient of RONPAKU Fellowship 2001 (DST-10114) from the Japan Society for Promotion of Science.

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