All students in the concerned school were subjected to physical examination. Children with signs and symptoms were separated and given oral antibiotics. All food and beverage supplied by the school canteen were sampled, and every food handler was screened by interview and stool culture. Drinking water and surface of cookers were also sampled for biochemical detection. An investigation of food consumed by these patients recently and the procedures of food processing and storage of the school canteen was carried out under the direction of epidemiologists to find out the origin of the epidemic.

Case definition and case ascertainment: A suspected case was defined as follows: after 1 September, 2006, any student at the school who had at least two of the following clinical manifestations: acute onset of diarrhea, >3 loose stools/day; blood and/or mucus in stool; fever; and abdominal pain. A confirmed case was a suspected case whose stool specimen was cultured positive for S. sonnei.

Plasmid analysis: 20 randomly selected isolates of S. sonnei from this outbreak were analyzed. Plasmid DNA was extracted by the Biospin Plasmid DNA Extraction Kit (Hangzhou Biotech Co, Ltd, China) according to manufacturer’s instructions, and was separated by horizontal agarose gel electrophoresis. After the electrophoresis, the gel was stained with ethidium bromide and video images were performed by a gel documentation system. The molecular mass of the unknown plasmid DNA was assessed by comparing its mobility with that of a super-coiled DNA ladder with known molecular mass.

Integron analysis: DNA was extracted from each strain. And integrons were detected by PCR with the degenerate primers hep35 (5’-TGCGGGTYAARGATBTKGATTT-3’) and hep36 (5’- CARCACATGCGTRTARAT-3’). The reaction conditions of PCR amplification were as follows: 1 cycle of 95ºC for 5 min, 30 cycles of 94ºC for 50 s, 55ºC for 90 s and 72ºC for 60 s, and 1 cycle of 72ºC for 5 min. The amplified DNA products were analyzed by conventional 1.5% (wt/vol) agarose gel electro-phoresis in 1×TBE buffer and stained with ethidium bromide. Cassette PCR products were restricted with HinfI. The sequences of amplicons were confirmed by restriction analysis with the restriction endonucleases HinfI in a water bath at 37ºC for 4 hours. Sizes of the digestion products were analyzed by electrophoresis on a 2.0% agarose gel. The digestion fragments were classified as per White Classification [5].

In the elementary school where the outbreak took place, there were 1,134 students and 140 faculty and staff members. The first reported case had abdominal pain and diarrhea 7 hours after common lunch at the school canteen. The number of reported cases peaked 2-3 days after the first case. 937 students had different manifestations (Table I). Among students with manifestations, 568 were hospitalized, including 306 boys and 262 girls. The mean age of the 568 cases was 8.4 years (range 5 to 13 years). Mean incubation period of these hospitalized patients was 2 days (ranging from 7 hours to 9 days). The first laboratory-confirmed case occurred on 3 September, and the last on 13 September. Four teachers had mild abdominal pain and diarrhea. None of them was hospitalized and they recovered well in two to three days. In the samples of food and general environments from the school canteen, the cold meat made of cold cooked pork was positive for S. sonnei. However, no evidence of origin of contamination of the meat was found.

TABLE I	Clinical Manifestations and Laboratory Evaluation in The Study Subjects

S. sonnei strains were isolated from 337 out of 937 stool samples. All the 337 S. sonnei strains had the same antimicrobial susceptibility. They were resistant to amoxicillin, trimethoprim/sulfametho-xazole (TMP-SMX), gentamicin, piperacillin, ticarcillin, cefuroxime, and cephalothin, and were susceptible to cefoxitin, imipenem, ciprofloxacin, amoxicillin/clavulantic acid, cefotaxime, cefopera-zone, fosfomycin, netilmicin, tobramycin and amikacin. The earlier isolated 13 sporadic strains of S. sonnei had different antimicrobial susceptibility as compared to the outbreak strain.

At the early stage of the outbreak, empirical antibiotic treatment was given to the patients at the discretion of the attending physicians. As soon as the S. sonnei strains were isolated and antimicrobial susceptibility was obtained, a 7-day intravenous antibiotic treatment plan of ceftazidime (50 mg/kg every 12 h) or cefotaxime (50 mg/kg every 12 h) was administered to the patients, and the follow-up stool cultures were carried out on the fifth day after treatment. The next course of treatment was given if the follow-up stool cultures were still positive. Hospitalized patients whose two consecutive stool cultures were negative for S. sonnei were allowed to go home. Patients who were not hospitalized received oral antibiotic treatment with cefixime (3 mg/kg every 12 h for 3 days). After treatment, manifestations had disappeared within 2-3 days in 58.8% children. No mortality was reported and there were no serious complications.

Plasmid analysis revealed that all the 20 S. sonnei strains had the same plasmid profile. Plasmid profiles of the outbreak strains were different from those of the sporadic strains. All S. sonnei isolates except one sporadic strain were positive for the integrons, and class 1 and class 2 integrons were detected. The results of gel electrophoresis showed that 3 strains (including 1 sporadic strain and 2 outbreak strains) contained class 1 integrons, and class 2 integrons were found in 31 strains (including 11 sporadic strains and 20 outbreak strains). One sporadic isolate which was resistant to ciprofloxacin contained only class 1 integron. No class 3 integron was detected.

S. sonnei is an important cause of acute gastroenteritis in both developing and developed countries. It is the second most common serotype in China where S. flexneri is the most common serotype [3]. In this outbreak, 82.6% of all students who had lunch at school had manifestations. To our knowledge, this high attack rate has been rarely reported before.

Diarrhea, fever, and abdominal pain were the three most common clinical manifestations observed in the patients infected with S. sonnei in this study. Compared with a previous report about outbreak of S. flexneri infections [6], the incidence of diarrhea, fever, abdominal pain and vomiting of S. sonnei infections is much lower.

High-level antibiotic resistance to many antibiotics recommended in the treatment of shigellosis was observed among the epidemic and sporadic S. sonnei strains. This is in accordance with other studies [7-9]. Antimicrobial resistance among Shigella organisms is an emerging problem. Resistance to ampicillin and TMP-SMX is common [10]. S. flexneri strains are much more resistant to most antibiotics presently used in the treatment of shigellosis than S. sonnei strains. Multi-resistance of Shigella strains is also striking [7, 8,11].

In this outbreak, children with severe shigellosis showed good clinical response to the third generation cephalosporin such as cefotaxime and ceftazidime. However, resistance of S. sonnei to the third generation cephalosporin has also been reported [7,9]. Although all outbreak S. sonnei strains were sensitive to ciprofloxacin in our study, fluoroquinolones are not recommended in children in China because there remains doubt about the potential damage of fluoroquinolones to growing cartilage [2,10].

In our study, plasmid profile analysis indicated that these outbreak isolates came from the same source. However, because it relies on the detection of extra-chromosomal genetic elements which can be lost [12], plasmid profile analysis technique has its limitations, which are not seen with Pulsed-Field Gel Electrophoresis (PFGE) [13]. PFGE could not be done due to limited resources.

High prevalence of class 2 integrons in S. sonnei isolates has been observed in our study, which is com-parable with previous reports [8,14]. Resis-tance of S. sonnei strains to ciprofloxacin has also been observed in other reports [15, 16]. Not all the isolates were examined for plasmid profiles and integrons, which may limit the strength of our conclusion in this regard.

In conclusion, high rate of antimicrobial resistance as well as high prevalence of class 2 integrons among S. sonnei species was observed in this study. It is mandatory to continuously monitor the local antibiotic resistant patterns of Shigella species to help to formulate appropriate treatment plans.

Contributors: All authors contributed to data acquisition and drafting the paper. GGX and JF analyzed and interpreted data. Concept and design were provided by JJD, CHC and CMW.

Funding: None.

Competing interests: None stated.

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