Outbreak of Infections Caused by Shigella ﬂexneri 2a with ESBL-Producing and Quinolone-Resistance in a Mental Healthcare Center in China

An outbreak of bacillary dysentery occurred in the Xiaoshan Mental Healthcare Center from November 8 to 16, 2012. Eight strains of Shigella flexneri 2a (S. flexneri 2a) were isolated from the same ward. The aim of the investigation was to clarify the origin of outbreak. Rectal swabs or fecal specimens from 7 doctors, 15 nurses, 2 nursing workers, 3 canteen workers, as well as 18 swabs from environmental sample in the ward were screened for Shigella. Phenotypic analysis included standard microbiological identification techniques, serotyping and antimicrobial susceptibility testing. PCR and sequencing were used to characterize drug resistance and virulence genes. Pulsed field gel electrophoresis (PFGE) was carried out for all Shigella isolates. S.flexneri 2a with multidrug resistance was isolated from eight patients and one canteen worker, which carried multiple antibiotics resistance genes and virulence determinants. The results of PFGE confirmed that nine strains of S. flexneri 2a belonged to the same clone. It was suggested that the outbreak strain spread directly from person-person or indirectly from person-food-person from the canteen worker. The outbreak was controlled after quarantine of ill residents, replacement of antibiotics and improvement of hygienic condition.

techniques, serotyping and antimicrobial susceptibility testing. PCR and sequencing were used to characterize drug resistance and virulence genes. Pulsed field gel electrophoresis (PFGE) was carried out for all Shigella isolates. S.flexneri 2a with multidrug resistance was isolated from eight patients and one canteen worker, which carried multiple antibiotics resistance genes and virulence determinants. The results of PFGE confirmed that nine strains of S. flexneri 2a belonged to the same clone. It was suggested that the outbreak strain spread directly from person-person or indirectly from person-food-person from the canteen worker. The outbreak was controlled after quarantine of ill residents, replacement of antibiotics and improvement of hygienic condition.

Background
Shigella species, which are important pathogens of the gastrointestinal tract, are responsible for causing severe diarrhea and acute gastroenteritis, especially in developing countries. It has been estimated that Shigella causes 165,000 deaths worldwide every year, of which 55,000 are children younger than 5 years of age [1,2]. In mainland China, the incidence of Shigella infections is 20.28 cases per 100,000 people per year [3].
3 Currently, third-generation cephalosporins and fluoroquinolones are the main antimicrobial agents in the treatment of Shigella infection in China; nevertheless, increasing multidrug resistant Shigella has become a serious public health threat [10].
Here, we report an outbreak of infection caused by S. flexneri 2a with ESBL-producing and

Outbreak investigation
During the outbreak, we firstly isolated all infectious patients who received treatment with antibiotics. Next, we started examining the possible reasons of the outbreak. Since all patients were mentally ill and unable to communicate effectively, all relevant medical data (frequency of diarrhea, dietary process, the patient's clinical manifestations and external contact) were provided by the patients' physicians and nurses. Next, stool sample from patients and personal (doctors, nurses, nursing workers, canteen staff), as well as environmental sample in male wards were screened for Shigella. The ethics committee of the Mental Healthcare Center approved this study.

Laboratory investigation
Stool specimens from all patients with diarrhea were cultured for the detection of enteric pathogens. Suspected isolates were identified by VITEK2 system (BioMérieux, France) and the serotype of Shigella isolates were confirmed by slide agglutination using commercial antisera (Denka Seiken Co. Ltd, Tokyo, Japan), following the manufacturer's recommendations. Antimicrobial susceptibility testing of the Shigella isolates was performed by broth microdilution method and the susceptibility was interpreted according to Clinical and Laboratory Standards Institute guidelines [21]. Polymerase Chain Reaction(PCR) and sequencing were used to characterize topoisomerases (gyrA, gyrB, parC, parE) in the quinolone-resistance determining regions (QRDRs) and the ESBL genes (bla TEM , bla OXA , bla CTX-M- 1,2,8,9 and bla SHV ) and virulence determinants (ial, iapH, set1A, set1B, sen and virA) [14,22]. Pulsed-field gel electrophoresis (PFGE) after the XbaI digestion of chromosomal DNA was carried out for all Shigella isolates [23].

Epidemiologic investigation
Eight strains of S. flexneri 2a were isolated from fresh fecal specimens of 12 patients (average age 48.9±18.9 years old; youngest patient was seventeen and the oldest one was seventy-four years old) with diarrhea from the same ward of Xiaoshan Mental Healthcare Center in Hangzhou, China. The Mental Healthcare Centre is a fully enclosed medical and rehabilitation institution, which is divided into male ward, female ward and intensive care area. The outbreak occurred in the male ward with twenty-five patients; twelve patients had different degrees of diarrhea accompanied by high temperature (>38°C ) with blood-free mucus feces. Finally, a total of eight Shigella strains were isolated from twelve samples and the serotype of all isolates were flexneri 2a.
The investigation showed that no doctors, nurses or nursing workers were infected with Shigella. Interestingly, S. flexneri 2a was detected in stool sample of a canteen staff worker (his job was to distribute food to patients in the male wards). The staff member suffered from diarrhea and abdominal pain. Those symptoms accrued before the outbreak in patients, which suggested the worker was likely to be a source of infection. Moreover, the serotype, drug sensitivity, resistance and virulence genes of the strain were the same as that in the Shigella presented in the outbreak.
Furthermore, in March 2013, we isolated the S. flexneri 2a from feces of two patients who have been infected with Shigella. In February 2014, one S. flexneri 2a was isolated from patient with initial infection. Then we confirmed that these isolates belong to the same clone as the outbreak strains. After that, we continued the monitoring for three years, except for four patients who had been discharged from the hospital. The investigation ended in December 2016. This phenomenon suggests that patients infected with Shigella do not have long-term immunity; it may turn into a latent infection under the intervention of antibiotics, and relapse when the body's immune system decreases.

Laboratory results
All 8 S. flexneri 2a isolates were resistant to ampicillin, trimethoprim/sulfamethoxazole, tetracycline, cefotaxime, ceftazidime, cefepime, ciprofloxacin, but were sensitive to piperacillin/tazobactam, imipenem, and the MIC of azithromycin was 2 μg/mL (no interpretation standard) ( Table 1). Moreover, all strains produced extended-spectrum βlactamase (ESBL). In addition, the PCR and sequencing results showed that all outbreak isolates carried bla CTX-M-57 , bla OXA-30 , bla TEM-1 resistant genes and ia1, ipaH, setlB, sen and virA virulence genes. Meanwhile, the gyrA (Ser83Leu) and parC (Ser80Ile) amino acid mutations were discovered in quinolone resistance determining regions (QRDRs) ( Table   2). Also, the PFGE results showed that all 9 S. flexneri 2a isolates (including the strain isolated from canteen staff) had the same band patterns, which indicated that they belong to the same clone (Figure 1).

Control measures
During the outbreak of the epidemic, the hospital took emergency preventive and control Fortunately, no patients died following episodes of shigellosis.
Laboratory results showed that these outbreak isolates carry multiple antibiotics resistance genes, including CTX-M gene. So far, at least 109 variants of CTX-M enzymes (CTX-M-1 to 124) have been described. Among these enzymes, some exhibit increased hydrolysis activity against ceftazidime, while others display a significantly higher rate of hydrolysis of cefotaxime than ceftazidime [15]. The presence of D240C mutations in bla  in this study resulted in the increased hydrolysis activity against ceftazidime [16]. Besides, the outbreak S. flexneri 2a isolates carried a bla OXA-30 , bla TEM-1 genes and conferred resistance to cefotaxime, ceftazidime and cefepime. The main mechanism of quinolone resistance in the Shigella spp. was the mutation of gyrA, such as at codon 83 or 87 and of parC at codon 80 [17,22]. So, the resistance of ciprofloxacin and levofloxacin may be caused by the mutation of gyrA (Ser83Leu) and parC (Ser80Ile) in the outbreak isolates.
The ability of Shigella spp. to cause shigellosis is attributed to the expression of arrays of virulence genes associated with colonization, invasion/penetration and toxin-mediated disease, such as the invasion-associated locus (ial), the invasion plasmid antigen H gene (ipaH), Shigella enterotoxin 1 (ShET-1) gene (set1A and set1B), Shigella enterotoxin 2 (ShET-2) gene (sen) and virA gene [18][19][20]. Estimating the existence of virulence determinants in Shigella would help us to further understand its pathogenicity. The S. flexneri 2a isolates harbored virA, ial, ipaH, set1B, sen virulence genes, which indicated that the strain possesses a strong virulence, which in turn can cause diarrhea and abdominal pain.
This study has several limitations. First, although it has been determined that the outbreak was transmitted by a canteen staff personnel who distributed food to patients in 8 the male ward, the source of infection for the staff remains unclear due to limited information. Second, due to the lack of in-depth investigation, it is not clear whether workers introduced Shigella into the community before isolation thus causing infection among residents.

Conclusions
This is the first report on the outbreak of bacillary dysentery in a psychiatric ward in China. Our data suggested that the outbreak was caused by an ESBL-Producing and

Availability of data and materials
All data generated or analyzed during this study are included in this published article   Figure 1 The pulsed-field gel electrophoresis patterns of 9 isolates. Lane M1, M2, molecular mass markers. Line 1-8, patient. Line 9, canteen staff