During April–May 2002, an outbreak of bacillary dysentery was reported in the northern district of West Bengal, India, among tea garden workers. A team from National Institute of Cholera and Enteric Diseases investigated the episode, and S. dysenteriae 1 was found to be the sole causative agent of the outbreak (6). A similar outbreak of blood dysentery caused by S. dysenteriae 1 occurred during March–June 2002 in the southern part of West Bengal (7). Following these episodes, we intensified the surveillance of diarrheal diseases in two hospitals of Kolkata, India. Infectious Disease (I.D.) Hospital is the biggest hospital in Kolkata, if not in India, for admission and treatment of infectious disease cases and the Dr. B. C. Roy Memorial Children’s Hospital is the only referral pediatric hospital in the state of West Bengal, which usually serves an area that includes the Kolkata metropolis and suburbs. In both hospitals, patients with diarrhea were kept under continuous surveillance after admission to the Diarrhoea Treatment Unit (DTU) of the hospitals and were treated with oral rehydration solution and antimicrobial agents as advised by attending clinicians.

Rectal swabs or fresh stool samples were collected from children with acute diarrhea admitted to Dr. B. C. Roy Memorial Children’s Hospital, Kolkata from January 2001 through August 2002. The children were selected irrespective of type, duration of diarrhea, and history of antibiotic drug therapy.

During the first week of July 2002, an upsurge of acute bloody diarrhea cases was noticed in patients attending the I. D. Hospital, Kolkata, and increased numbers of patients with acute dysentery continued to be admitted to the hospital until September 2002.The patients reported bloody stools, abdominal pain, and tenesmus, with or without fever. Stool samples or rectal swabs were collected from all of these patients on admission from July 1, 2002, to August 31, 2002.

The samples were placed in Cary-Blair transport medium and processed within 2 hours of collection in the Microbiology Laboratory of the National Institute of Cholera and Enteric Diseases, Kolkata; the samples were tested for the entire gamut of enteropathogens by using standard techniques. Shigella species were confirmed with the API 20E test (Biomerieux, Marcy l’Etoile, France) and slide agglutination test with antisera specific to serotypes of Shigella species (Denka Seiken Co, Tokyo, Japan). Antimicrobial susceptibility testing of Shigella isolates was done by the disk diffusion method, and MICs were measured by using the agar dilution method.

In addition to the conventional technique, polymerase chain reaction (PCR) was performed within 4–6 hours on LB broth cultures of 77 stool samples collected from I. D. Hospital by using published primer sequences for IpaH (invasion plasmid antigen H) gene to detect further Shigella infection, which might have been missed by conventional methods (8). PCR was also performed in anticipation of rapid diagnosis and early treatment of shigellosis cases and thus prevent the development of complications.

Table 1 shows the distribution of Shigella serotypes from patients admitted in two hospitals of Kolkata. An increased isolation rate of all serotypes of shigellae was observed in patients with acute diarrhea (72/790; 9.1%) and also in patients with bloody diarrhea (72/237; 30%) in the Children’s Hospital since January 2002. Although S. flexneri continued to be the most prevalent serotype (45/72; 62.5%), followed by S. sonnei (18/72; 25%), the reemergence of S. dysenteriae serotype 1 (5/72; 7%) claimed special attention. During 2001, only one strain of S. dysenteriae 1 was isolated in July, but during January to August 2002, five S. dysenteriae 1 strains were identified:one in April and two in both July and August 2002. The other four S. dysenteriae strains isolated in 2001 were S. dysenteriae type 2 (two strains), S. dysenteriae type 3 (one strain), and S. dysenteriae type 6 (one strain).

Figure

Figure. Epidemic curve for Shigella dysenteriae 1 case-patients admitted to Infectious Diseases Hospital, Kolkata, India, during July and August 2002.

Among 77 patients with bloody diarrhea in I. D. Hospital examined through August 2002, Shigella spp. were identified as the sole pathogen from 27 (35%) patients. Of 27 Shigella strains, 24 (88%) belonged to S. dysenteriae serotype 1; other strains isolated were S. flexneri (2 strains) and S. sonnei (1 strain). PCR could detect shigellae infection in 37 (48%) patients, and 12 of these patients were not culture-positive. No other pathogen could be detected from any of the case-patients. The figure shows the epidemic curve for S. dysenteriae 1 case-patients admitted to I. D. Hospital during the period of study.

The antimicrobial resistance profiles of the isolated S. dysenteriae 1 strains showed multidrug resistance to at least seven or more antimicrobial agents, e.g., chloramphenicol (80%), ampicillin (100%), tetracycline (100%), cotrimoxazole (100%), furazolidone (50%), nalidixic acid (100%), ciprofloxacin (100%), norfloxacin (100%), and amoxicillin (100%). MICs of antimicrobial agents also showed higher level of resistance acquired by these strains compared to the MICs observed during 1995–2000 (5). The MICs₉₀ of antimicrobial agents tested were as follows: ampicillin (512 μg/mL), tetracycline (>256 μg/ml), chloramphenicol (<256 μg/mL), nalidixic acid (>256 μg/mL), norfloxacin (>32 μg/mL), and ciprofloxacin (>8 μg/mL). In contrast, other serotypes of shigellae were uniformly susceptible (100%) to ciprofloxacin and norfloxacin and showed partial resistance to ampicillin (60%), tetracycline (94%), cotrimoxazole (98%), and nalidixic acid (45%).

Initially, physicians advised norfloxacin and ciprofloxacin for routine treatment of shigellosis, because of poor clinical responses, subsequent patients were treated with ofloxacin to which the organism was susceptible (100%). No case fatality and no case of hemolytic uremic syndrome have been reported among the present series of patients. Median time for resolution of symptoms of the patients was 4 days from the date of admission to the hospital.

When S. dysenteriae 1 strains were screened for virulent gene profiles by PCR with published primer sequences (9), all S. dysenteriae 1 strains (100%) were found to harbor stx1 (Shiga toxin), ipaH (invasion plasmid antigen H), and ial (invasion-associated locus) gene and were negative for set (Shigella enterotoxin 1) gene. Sixteen (70%) of 23 isolates were positive for sen (Shigella enterotoxin 2) gene. Only one S. dysenteriae 1 strain showed both Shigella enterotoxin 1 and 2 (set and sen) genes.

Although the proportion of Shigella strains isolated from case-patients with acute bloody diarrhea increased (30% to 35%), the rate of S. dysenteriae 1 isolated from B.C. Roy Memorial Children’s Hospital (5/237; 2%) was not as high as that of patients at I.D. Hospital (27/77; 35%). Because I. D. Hospital was a general hospital and provided treatment to patients of all ages, including children, concerned parents of patients who had severe dysentery caused by S. dysenteriae 1 may have brought them to I.D. Hospital in anticipation that their illness would be better managed.

Isolated strains of S. dysenteriae 1 from two recently documented outbreaks in West Bengal also showed reduced susceptibility to fluoroquinolones (6,7). In recent years, the emergence of multidrug-resistant S. dysenteriae 1 strains has also been reported from Southeast Asia and Africa, although fluoroquinolone resistance was not observed among the strains (10,11).

While processing the stool samples of patients admitted to the Children’s Hospital for shigellae species by conventional method, we found a few strains that showed a biochemical reaction typical of Shigella, but were nonagglutinable by commercially available antisera. These strains were positive for ipaH gene when tested by PCR. We designated those strains as Shigella untypable strains (8). The strains were sent to the Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan, for typing, and they were found to be provisional serovers of Shigella spp. (Table 2). Table 2 shows the year of isolation and virulent gene profiles of those strains. All strains were negative for stx1 gene. They were resistant to chloramphenicol, tetracycline, cotrimoxazole, furazolidone, and amoxicillin. But all strains were susceptible to nalidixic acid, norfloxacin, ciprofloxacin, gentamicin, amikacin, and cefotaxime. This drug-resistance profile contrasted with that of recently emerged S. dysenteriae type 1 strains. Similar strains have also been identified in other studies (12–14). To our knowledge, this is the first report of the isolation and identification of provisional serovars of S. dysenteriae and S. boydii from Kolkata, India.

Our study reports increased isolation of shigellae with reemergence of S. dysenteriae 1 in and around Kolkata, India. This increase has public health importance with respect to monitoring impending outbreaks of shigellosis and implementing appropriate strategies for containment of this deadly organism.

Emergence of multidrug-resistant Shigella strains is of concern to clinicians in treating shigellosis cases. Because the recently emerged S. dysenteriae 1 strain was resistant (100%) to ampicillin, cotrimoxazole, nalidixic acid, norfloxacin, and ciprofloxacin, which were commonly used for shigellosis cases; ofloxacin is currently recommended for treatment. Concomitant search for alternate new drugs should be continued because, although newer antimicrobial drugs can offer hope for treatment of shigellosis, emergence of resistance to the new drugs is also not far in the future. Therefore, generating an effective vaccine can offer the ultimate solution to such problems. Perhaps the more effective way of reducing the impact of the disease and the risk of contracting infection lies in improving poor living condition, disseminating health education, and supplying safe drinking water. However, accomplishing those objectives and reaching the goal is not an easy task in developing countries. Laboratory detection capabilities also need to be strengthened at all levels to increase the baseline surveillance data for improved isolation of the pathogen. Identifying some strains with provisional serovars of Shigella spp. for the first time from Kolkata, India, indicates that all provisionally identified Shigella strains should be sent to a reference laboratory for typing and further characterization.

Studying plasmid profiles of isolated S. dysenteriae 1 strains and typing the strains by using various molecular tools could provide insight into the origin of these recently isolated S. dysenteriae 1 strains and the relationships among the strains.