Prevalence and Characterization of Human Shigella Infections in Henan Province, China, in 2006

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2011, p Vol. 49, No /11/$12.00 doi: /jcm Copyright 2011, American Society for Microbiology. All Rights Reserved. Prevalence and Characterization of Human Shigella Infections in Henan Province, China, in 2006 Shengli Xia, 1 Bianli Xu, 1 Lili Huang, 1 Jia-Yong Zhao, 1 Lu Ran, 2 Jin Zhang, 1 Haomin Chen, 1 Chaiwat Pulsrikarn, 3 Srirat Pornruangwong, 3 Frank M. Aarestrup, 4 and Rene S. Hendriksen 4 * Branch for Enteric Disease Control and Prevention, Institute for Infectious Disease Control and Prevention, Henan Center for Disease Control and Prevention, Zhengzhou, China 1 ; Branch for Enteric Disease Control and Prevention, Office for Disease Control and Emergency Response, Chinese Center for Disease Control and Prevention, Beijing, China 2 ; WHO National Salmonella and Shigella Center, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand 3 ; and WHO Collaborating Centre for Antimicrobial Resistance in Food borne Pathogens and European Union Reference Laboratory for Antimicrobial Resistance, National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark 4 Received 27 July 2010/Returned for modification 4 October 2010/Accepted 28 October 2010 In 2006, 3,531 fecal samples were collected from patients with diarrhea in Henan Province, China. A total of 467 (13.2%) Shigella strains were isolated and serotyped. Seventy-one Shigella flexneri strains were characterized by MIC determination, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding cephalosporin resistance. Most infections were caused by variant X [IV:(7),8] (27.6%), S. sonnei (24.2%), and 2a (20.8%). However, large regional differences were observed. Significantly higher odds (2.0) of females compared to males were infected with 2a. Untypeable ( :6) isolates were absent among males, as were untypeable [I:(7),8] isolates among females. Patient ages ranged from 2 months to 82 years, with 231 subjects (49.7%) <5 years of age. Most of the patients were male (62.1% [n 290]). Infections peaked in July; week 27 with 38 cases (8.1%). All of the 71 conferred resistance to nalidixic acid; in addition, 21% (n 15) and 79% (n 56) were high- and low-level resistant to ciprofloxacin, respectively. Six isolates {serotype 2b [II:7,(8)] and 2b [II:(3),4;7,(8)]} harbored the bla CTX-M-14 or bla CTX-M-15 gene. A total of 52 unique XbaI PFGE patterns were observed among the 71 isolates with 11 distinct PFGE clusters. This study revealed a high prevalence of shigellosis with geographical differences in the distribution of serotypes in the distribution of serotypes and also differences in comparisons by gender. A high frequency of resistance, including 100% resistance to ciprofloxacin and resistance to extended-spectrum cephalosporins, was observed. We detected several isolates exhibiting the same PFGE type and MIC profile, indicating multiple undetected outbreaks. Shigella is an important cause of acute diarrheal disease and occurs worldwide in an estimated million people per year, resulting in around 1,100,000 deaths, especially among children in developing countries (14). In China, Shigella spp. is the most frequently isolated gastrointestinal pathogen (19) accounting for up to 1.7 million episodes of bacillary dysentery annually, with up to 200,000 patients admitted to hospitals (28). Any of four species of Shigella (Shigella dysenteriae,, S. boydii, and S. sonnei) can cause shigellosis. serotypes 2a, 1b, 3a, 4a [IV:(3),4], and 6 and S. sonnei are the predominant species in developing countries, while mainly S. sonnei is predominant in industrialized countries, including S. flexneri serotype 2a and other unspecified type 2 variants (2, 14). is endemic in many developing countries and causes a higher rate of mortality than other species (3). The most common serotype is 2a, but other serotypes are also of major importance (14). In China, is the most common species, accounting for up to 80% of all Shigella spp. with * Corresponding author. Mailing address: National Food Institute, Technical University of Denmark Kemitorvet, Bygning 204, DK-2800 Kgs. Lyngby, Denmark. Phone: Fax: rshe@food.dtu.dk. Published ahead of print on 10 November serotypes 1a [I:(3),4], 2a, 3a, and 4 [IV: ], and variant X is reported as the most prevalent type (26, 28, 30). A range of different antimicrobials are effective for treatment of shigellosis, although options are becoming limited due to the emergence of multidrug resistance. Thus, sulfonamides, tetracycline, ampicillin, and cotrimoxazole are no longer recommended for empirical treatment (16). In recent years, an increase in the occurrence of antimicrobial resistance, including resistance to fluoroquinolones and extendedspectrum cephalosporins (ESC), among Shigella spp. has been observed in many countries, including China (13, 16, 18, 21, 27, 30). The emergence of antimicrobial resistance is a matter of concern and poses a major difficulty in the determination of an appropriate antimicrobial for treatment due to shifts in the prevalences of the different serogroups and changes in the resistance patterns (16). Knowledge regarding the occurrence of different serotypes in different countries and geographic regions may assist in the recognition and tracing of emerging pathogens and in the implementation of correct treatment and control strategies (14). With nearly 100 million people, Henan is the most populated province of China by residency. It borders the province of Hebei to the north, Shandong to the northeast, Anhui to the southeast, Hubei to the south, Shaanxi to the west, and Shanxi to the northwest. In the present study, we reveal the results of 232

2 VOL. 49, 2011 HUMAN SHIGELLA INFECTIONS IN HENAN PROVINCE, CHINA 233 a passive surveillance conducted in 2006 of Shigella infections from Henan Province in China. In addition, we combined various microbiological methodologies to describe the distribution of a subset of the Shigella flexneri isolates and its antimicrobial susceptibility patterns. EDTA at 6 V with an angle of 120. The running conditions consisted of one phase from 2.2 to 63.8 s at a run time of 20 h. Statistical analysis. SAS version (SAS Institute, Inc., Cary, NC) was used to assess the association of specific serotypes present in males compared to females by using a Fisher exact test. The criteria for evaluating the significance level in the model was a P value of MATERIALS AND METHODS Sample collection. A total of 3,531 cross-sectional fecal samples from diarrheal patients were investigated for the presence of Shigella during 2006 in Henan Province in China. The samples were collected during from 26 May to 31 December 2006 from 15 sentinel hospitals and 13 clinics and forwarded to six regional Center for Disease Control and Prevention (CDC) diagnostic laboratories, so all data are on a regional level. The six regional laboratories represent five different geographic regions in Henan Province. Six of the clinics were located in rural areas (Sui County and Jiyuan), whereas the remaining 15 hospitals and seven clinics were located in urban areas. Fresh fecal specimens (cotton swabs dipped with mucous or blood parts of fresh stool derived from diarrhea patients of all ages) were inoculated into Carry-Blair medium and forwarded the regional CDC laboratories within 4 h. The stool samples were streaked onto XLD agar and SS agar and then incubated at 37 C for 18 to 24 h. Colorless and transparent colonies were screened by using triple sugar iron agar and motility indole-urea agar. The presumptive positive Shigella isolates were identified by using API 20E strips (biomérieux, Marcy l Etoile, France). Based on availability, a subset of 71 isolates were selected for further analysis by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Serotyping. All presumptive positive Shigella isolates were initially serotyped by the six regional CDC diagnostic laboratories based on serological slide agglutination, using the following polyvalent antisera: A, A1, B, C, C1, C1-3, and D and monovalent antisera; S. dysenteriae type 1-12, type I-VI, and group factor (3),4,6,7,(8) antisera; S. boydii type 1-18 antiserum; and S. sonnei phase I-II antiserum from Ningbo Tianrun Bio-Pharmaceutical Co., Ltd., Zhejiang, China. From the regional CDC diagnostic laboratories, all isolates were sent to the Henan provincial CDC for confirmation of the serotype. The isolates were serotyped using the same polyvalent and monovalent antisera as initially used but from other manufacturers; Denka Seiken Co., Ltd., Tokyo, Japan, and S&A Reagents, Ltd., Bangkok, Thailand. There are no differences between the ranges of the antisera from these two companies. The serotypes were interpreted according to Manual of Clinical Microbiology criteria (4). Some isolates revealed a serologically atypical pattern that did not correspond to the official Shigella typing scheme according to the Manual of Clinical Microbiology (4). Two isolates that reacted with polyvalent antisera B were negative for type factors I to VI and reacted only with group factor antiserum 6 were considered as untypeable according to the available antisera. Similarly, 129 isolates that reacted with polyvalent antisera B that were positive for type factor IV and reacted only with group factor antiserum (7),8 were considered variant X. (N. A. Strockbine, unpublished data). Three isolates that reacted with polyvalent antisera B, were positive for type factor I, and reacted only with group factor antiserum (7),8 were considered untypeable [I:(7),8] (24). Antimicrobial susceptibility. Susceptibility to antimicrobial agents was evaluated at the National Food Institute, Kgs. Lyngby, Denmark, for the subset of 71 isolates as MIC determinations according to a previously described method (8). Reduced susceptibility (low-level resistance) to ciprofloxacin was defined as an MIC of g/ml, and high-level resistance was defined as an MIC of 4 g/ml. Extended-spectrum -lactamase production was confirmed by the Kirby-Bauer disc diffusion method. Detection of resistance genes. Of the subset of 71 isolates, six strains resistant to ceftiofur were examined for the presence of genes encoding bla TEM, bla CTX, and bla SHV as previously described (9). All PCR amplifications were conducted with the buffer supplied by the manufacturers using 20 pmol of each primer, 0.5 U of Super-Taq (HT Biotechnology, Cambridge, United Kingdom), and the reaction mixtures (50 l). PCR products were purified by using a GFX PCR DNA kit (GE Healthcare, Chalfont St. Giles, United Kingdom), and submitted to Macrogen, Inc., Seoul, Korea, for sequencing. Sequence analysis and alignment was performed by using the Vecton NTI suite 9 (InforMax, Inc., Bethesda, MD). The resulting nucleotide sequences were compared to sequences obtained from GenBank ( PFGE. The subset of 71 isolates were analyzed for genetic relatedness by PFGE using XbaI according to the U.S. CDC PulseNet protocol (20). Electrophoresis was performed with a CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA) using 1% SeaKem Gold agarose in 0.5 Tris-borate- RESULTS Prevalence. The prevalences for human Shigella infections based on patient data from six surveillance points, including Sui County (Sui County is located in the eastern plain belonging to the rural area of Shangqiu City and includes 20 villages and towns, with a total area of 926 square kilometers and 800,000 inhabitants), Zhengzhou (Zhengzhou is the capital [an urban area] and is located in the center of Henan Province, consisting of 12 counties or cities with a total of 7,356,000 inhabitants), Jiyuan (Jiyuan is located in a rural area in the northwest of Henan Province consisting of eight towns and four townships and covers a total area of 1,931 square kilometers and 680,000 inhabitants), Zhoukou (Zhoukou is located in an urban area southeast of Henan Province and consists of one city and 10 towns, covering a total area of 11,959 square kilometers; its population is 10,860,000), Shangqiu (Shangqiu is located in an urban area east of Henan Province and consists of one city, three districts, and six towns; it covers a total area of 10,704 square kilometers, with a total population of 8,243,700), and Anyang (Anyang is located in the north of Henan Province and consists of five districts and five counties, with a total area of 7,413 square kilometers and a population of 5,420,000), in Henan Province, China, are presented in Table 1 and Fig. 1. Of 3,531 collected stool samples, 467 samples were positive for Shigella, resulting in an overall prevalence of 13.2%. The prevalence differed between the different surveillance points from 3.1% in Shangqiu (urban area) to 27.9% in the Sui County region (a rural area) (Table 1). Interestingly, a low prevalence of 4.1% was observed in rural Jiyuan compared to the high prevalence of 27.9% in rural Sui county. In addition, the low prevalence in rural Jiyuan was almost the same as in urban Shangqiu. Moreover, a proportionally larger number of samples per inhabitants were investigated from rural areas (Sui County and Jiyuan) compared to the urban areas. Serotypes of and S. sonnei. The passive surveillance of human Shigella infections in Henan Province, China, showed that among the 467 strains, 354 (75.8%) isolates belonged to the species and 113 isolates (24.2%) belonged to the species S. sonnei (Table 1). Most infections were caused by serotype variant X [IV:(7),8] (27.6%), followed by serotype 2a and serotype 1a [I:(3),4]. Large regional differences in the geographical distribution of the serotypes were observed (Table 1). serotype 1a [I:(3),4] was most frequently observed in the Northwestern region of Jiyuan (40.0%) and to a lesser degree in the central, northern, and eastern regions of Zhengzhou (16.7%), Anyang (13.8%), and Zhoukou (12.2%). serotype 2a was associated with rural, since most cases of this serotype were reported from Sui County (25.2%) and Jiyuan (24.0%). However, the serotype was also more frequently observed in the eastern part of Henan Province, with 23.5% in Zhoukou region and 20.7% in Shangqiu. In comparison to serotype

3 234 XIA ET AL. J. CLIN. MICROBIOL. TABLE 1. Prevalence data and the number and percentages of the different Shigella species and serotypes isolated from Henan Province of China in 2006 Prevalence No. (percentage) of the different Shigella species and serotypes S. sonnei untypable serotype [ :6] variant Y variant X variant X IV:(7),8 serotype 4a IV:(3),4 serotype 4a [IV: ] serotype 2b II:(3),4; (7),8 serotype 2b II:(7),8 serotype 2a untypable serotype I:(7),8 serotype 1a I:(3),4 serotype 1a [I: ] No. (%) of isolates No. of samples Surveillance point (area type), population (27.9) 2 (1.0) 13 (6.4) 3 (1.5) 51 (25.2) 7 (3.5) 10 (5.0) 4 (2.0) 3 (1.5) 33 (16.3) 13 (6.4) 6 (3.0) 2 (1.0) 55 (27.2) Sui County (rural area), 800, (8.5) 0 8 (16.7) 0 7 (14.6) 1 (2.1) 2 (4.2) (37.5) 1 (2.1) (22.9) Zhengzhou (urban area), 7,356, (4.1) 0 10 (40.0) 0 6 (24.0) 1 (4.0) (24.0) 2 (8.0) (3.1) 0 2 (6.9) 1 (3.4) 6 (20.7) (3.4) 0 13 (44.8) 1 (3.4) 1 (3.4) 0 4 (13.8) Jiyuan (rural area), 680,000 Shangqiu (urban area), 8,243, (25.1) 0 12 (12.2) 0 23 (23.5) 2 (2.0) 2 (2.0) 2 (2.0) 0 27 (27.6) 1 (1.0) (30.0) Zhoukou (urban area), 10,860, (21.7) 0 9 (13.8) 0 4 (6.2) 0 3 (4.6) 1 (1.5) 0 32 (32.7) 2 (3.1) (21.5) Anyang (urban area), 5,420,000 Total no. (%) (13.2) 2 (0.4) 54 (11.6) 4 (0.9) 97 (20.8) 11 (2.4) 17 (3.6) 8 (1.7) 3 (0.6) 129 (27.6) 20 (4.3) 7 (1.5) 2 (0.4) 113 (24.2) FIG. 1. Location of the six surveillance points in Henan Province, China. 2a, serotype variant X [IV:(7),8] was not associated with rural areas, since fewer cases were reported from Sui County (16.3%) and Jiyuan (24.0%) compared to urban areas such as Shangqiu with 44.8% cases. S. sonnei predominated in the eastern regions, with 30.0% cases in Zhoukou and 27.2% in Sui County, whereas no cases were observed in the northwestern region of Jiyuan. Age distribution. Information about the age of the patient was known for 465 of the 467 patients included in the surveillance project. Patient ages ranged from 2 months to 82 years, with a median of 25 years; 49.7% (n 231) were of positive cases occurred in patients between 0 and 5 years of age (Table 2). The frequency of infections among the patients rapidly decreased after the fifth year to a level between 7.3% (n 34) for the 6- to 11-year-old age group to 0.2% (n 1) for patients aged 81 to 85 years (Fig. 2). No specific species and serotype patterns were observed among the different age groups. However, serotype 2a, variant X [IV:(7),8], and S. sonnei were found in almost all patients of all ages (Table 2). Gender. Data regarding the patient gender were available for all of the 467 Shigella isolates. Almost two-thirds of the patients infected were males, accounting for 62.1% (n 290) (Fig. 2). The predominant Shigella species and serotypes among males were serotype variant X [IV:(7),8] (28.3%, n 82), S. sonnei (26.6%, n 77), and serotype 2a (16.2%, n 47), respectively. Among females, S. flexneri serotype 2a were the most common isolates (28.2%, n 50), followed by serotype variant X [IV:(7),8] (26.6%, n 47) and S. sonnei (20.3%, n 36), respectively. Infection with serotype 2a was seen in a significantly (P ) higher number of female patients compared to males (odds ratio 2.0; 95% confidence interval 1.29 to 3.20). In addition, differences in the distributions of serotypes were observed between the genders with the absence

4 VOL. 49, 2011 HUMAN SHIGELLA INFECTIONS IN HENAN PROVINCE, CHINA 235 a The column subheadings indicate the age groups in years. Total 465 (100.0) 231 (49.7) 34 (7.3) 26 (5.6) 19 (4.1) 22 (4.7) 16 (3.4) 19 (4.1) 20 (4.3) 12 (2.6) 13 (2.8) 9 (1.9) 14 (3.0) 9 (1.9) 12 (2.6) 4 (0.9) 4 (0.9) 1 (0.2) S. sonnei 112 (24.1) 66 (28.6) 8 (23.5) 7 (26.9) 2 (10.6) 6 (27.3) 2 (12.5) 5 (26.3) 1 (5.0) 2 (16.7) 5 (38.5) 2 (22.2) 1 (7.1) 2 (22.2) 2 (16.7) 1 (25.0) untypeable serotype [ :6] 2 (0.4) 2 (0.9) variant X 20 (4.3) 11 (4.8) 1 (5.3) 1 (4.5) 1 (5.3) 2 (16.7) 1 (2.7) 3 (25.0) variant Y 7 (1.5) 3 (1.3) 1 (2.9) 1 (5.3) 1 (5.0) 1 (2.7) variant X IV:(7),8 129 (27.7) 70 (30.0) 9 (26.5) 5 (19.2) 5 (26.3) 4 (18.2) 5 (31.3) 6 (31.6) 6 (30.0) 4 (33.3) 4 (30.8) 3 (33.3) 2 (14.2) 3 (33.3) 2 (16.7) 1 (25.0) serotype 4a IV:(3),4 3 (0.6) 2 (0.9) 1 (5.3) serotype 4a [IV: ] 8 (1.7) 4 (1.7) 1 (2.9) 1 (3.8) 1 (7.1) 1 (25.0) serotype 2b II:(3),4;(7),8 17 (3.7) 7 (3.0) 1 (2.9) 1 (5.3) 1 (4.5) 2 (10.5) 1 (5.0) 1 (8.3) 1 (2.7) 1 (7.1) 1 (11.1) untypeable 4 (0.7) 3 (0.1) 1 (3.8) serotype I:(7),8 serotype 2a 96 (20.6) 43 (18.6) 11 (32.4) 6 (23.1) 1 (5.3) 5 (22.7) 4 (25.0) 2 (10.5) 7 (35.0) 2 (16.7) 1 (2.7) 1 (11.1) 5 (35.7) 2 (22.2) 4 (33.3) 1 (25.0) 1 (25.0) 11 (2.4) 3 (1.3) 1 (3.8) 1 (6.3) 1 (5.3) 1 (5.0) 1 (11.1) 1 (11.1) 2 (50.0) serotype 2b II:(7),8 serotype 1a I:(3),4 54 (11.6) 17 (7.4) 3 (8.8) 5 (19.2) 8 (42.1) 5 (22.7) 4 (25.0) 1 (5.3) 2 (10.0) 2 (22.2) 4 (28.6) 1 (8.3) 1 (25.0) 1 (100.0) serotype 1a [I: ] 2 (0.2) 1 (5.0) 1 (8.3) Total Species and serotype No. (%) of isolates per age group a TABLE 2. Distribution of age groups per numbers among Chinese patients with shigellosis from 2006 in Henan Province, China FIG. 2. Distribution of the serotypes between genders among Chinese patients with shigellosis from 2006 in Henan Province, China. of untypeable [ :6] isolates among males and the absence of untypeable [I:(7),8] among females (Fig. 2). Seasonal trends. Data on onset of infection were available for all of the 467 Shigella isolates. Infections increased from May, week 21 (n 6, 1.3%), and reached the highest number

5 236 XIA ET AL. J. CLIN. MICROBIOL. Downloaded from FIG. 3. Seasonal variation in the numbers of Shigella infections among Chinese patients during 2006 in Henan Province, China. of infections in July, week 27, with 38 cases (8.1%) (Fig. 3). The serotypes variant X [IV:(7),8] and 2a were primarily responsible for the increased level of infections peaking in July, week 27 (Fig. 3). The infection rate slowly declined to a level of 13 cases (2.8%) in August, week 35. From this week on to October, week 40, the levels of infection were relatively consistent, with between 12 and 17 cases per week. However, in October, week 41, the number of infections dramatically increased to 28 cases (6.0%); this was caused mainly by 15 cases of S. sonnei. This high level gradually decreased until the overall infection rate in November reached 8 cases, including 1 caused by S. sonnei. The infection rate leveled out with only two cases (0.4%) at the end of the year in December, week 52. Throughout the weeks included in this study, the infection rate seemed to fluctuate among several Shigella species and serotypes without a profound pattern. However, specific Shigella species and serotypes peaked randomly throughout the survey period (Fig. 3). Antimicrobial resistance. The subset of the 71 isolates were multidrug resistant (i.e., resistant to more than three antimicrobial drug classes) and exhibited resistance to up to 14 of the 18 antimicrobials tested (Table 3 and Fig. 4). One hundred percent (n 71) of all of the tested isolates were resistant to nalidixic acid; of these, 21% (n 15) were highly resistant to ciprofloxacin (MIC 4 g/ml). The remainder of the isolates were resistant to nalidixic acid conferred low-level resistance (MIC to 2 g/ml) to ciprofloxacin (79%, n 56). Only, serotype 2b [II:(7),8], serotype 2b [II:(3),4; (7),8], and serotype variant X [IV:(7),8] demonstrated resistance to cefpodoxime (n 8; 11%) (serotype 2b [II:(7),8], serotype 2b [II:(3),4 (7),8], and serotype variant X [IV:(7),8] and ceftiofur (an extended-spectrum cephalosporin for veterinary use) (n 6; 8%) (serotype 2b [II:(7),8] and serotype 2b [II:(3),4;(7),8]) (Table 3 and Fig. 4). Resistance to apramycin (only approved for veterinary use) on April 7, 2019 by guest

6 VOL. 49, 2011 HUMAN SHIGELLA INFECTIONS IN HENAN PROVINCE, CHINA 237 TABLE 3. Frequency of resistance among Shigella serotypes from Chinese patients during 2006 in Henan Province, China No. (%) of isolates resistant to various antimicrobial agents at the indicated breakpoints a Species and serotype No of isolates AUG, 4 AMP, 4 APR, 16 CEF, 16 POD, 1 XNL, 2 CHL, 16 CIP COL, 8 FFN, 2 GEN, 2 NAL, 16 NEO, 8 SPE, 64 STR, 16 SMX, 256 TET, 8 TMP, 2 serotype 1a [I: ] serotype 1a I:(3),4 S. flexner untypeable serotype I:(7), (100) 0 1 (100) (100) 0 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 4 4 (100) 4 (100) (100) 2 (50) 2 (50) (100) 0 4 (100) 4 (100) 2 (50) 4 (100) 4 (100) 3 3 (100) 3 (100) (100) 0 3 (100) (100) 0 3 (100) 3 (100) 0 3 (100) 3 (100) serotype 2a 5 4 (80) 5 (100) (100) 5 (100) (100) 0 5 (100) 5 (100) 3 (60) 5 (100) 5 (100) 7 6 (86) 6 (86) 0 4 (57) 4 (57) 3 (43) 6 (86) 1 (14) 6 (86) (100) 0 7 (100) 7 (100) 5 (71) 7 (100) 7 (100) serotype 2b II:(7),8 serotype 2b II:(3),4;(7),8 serotype 4a [IV: ] serotype 4a IV:(3),4 variant X IV:(7), (93) 14 (100) 0 3 (21) 3 (21) 3 (21) 14 (100) 0 14 (100) (100) 0 14 (100) 14 (100) 12 (86) 14 (100) 14 (100) 7 6 (86) 7 (100) 0 2 (29) (71) 3 (43) 4 (57) (14) 7 (100) 0 5 (71) 6 (86) 5 (71) 6 (86) 7 (100) 4 4 (100) 4 (100) (100) 0 4 (100) (25) 4 (100) 0 4 (100) 4 (100) 2 (50) 4 (100) 4 (100) (100) 10 (100) 0 4 (40) 1 (10) 0 10 (100) 3 (30) 7 (70) (40) 10 (100) 0 10 (100) 10 (100) 9 (90) 10 (100) 10 (100) variant X 9 9 (100) 9 (100) 0 1 (11) (100) 0 9 (100) (100) 0 9 (100) 9 (100) 3 (33) 9 (100) 9 (100) untypeable 2 2 (100) 2 (100) (100) 0 2 (100) (100) 0 2 (100) 2 (100) 2 (100) 2 (100) 2 (100) serotype [ :6] variant Y 5 5 (100) 5 (100) (100) 1 (20) 4 (80) (100) 0 5 (100) 5 (100) 2 (63) 5 (100) 5 (100) Total (93) 69 (97) 0 14 (20) 8 (11) 6 (8) 68 (96) 15 (21) 56 (79) (8) 71 (100) 0 69 (97) 70 (99) 46 (65) 70 (99) 71 (100) a Antimicrobial agent breakpoints in the table subheadings are given in micrograms per milliliter. Abbreviations: AMP, ampicillin; AUG, amoxicillin plus clavulanic acid; APR, apramycin; CEF, cefalothin; POD, cefpodoxime; XNL, ceftiofur; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; FFN, florfenicol; GEN, gentamicin, NAL, nalidixic acid; NEO, neomycin; SPE, spectinomycin; STR, streptomycin; SMX, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim.

7 238 XIA ET AL. J. CLIN. MICROBIOL.

8 VOL. 49, 2011 HUMAN SHIGELLA INFECTIONS IN HENAN PROVINCE, CHINA 239 colistin, florfenicol (only approved for veterinary use), and neomycin was not detected among any of the isolates. Frequent resistance ranging from 93 to 100% was also observed among all serotypes against amoxicillin plus clavulanic acid (93%), ampicillin (97%), chloramphenicol (96%), spectinomycin (97%), streptomycin (99%), tetracycline (99%), and trimethoprim (100%) (Table 3 and Fig. 4). Moderate resistance was observed in all of the serotypes with the exception of untypeable [I:(7),8] to sulfamethoxazole ranging from 33 to 100%. In contrast, all of the untypeable [I: (7),8] isolates were susceptible to sulfamethoxazole. Resistance to gentamicin was observed in serotypes 4 [IV: ], 4a [(IV:(3),4], and variant X [IV:(7),8], ranging from 14 to 40%. Identification of resistance genes in the ESC-producing isolates. Based on MIC determination, six (8%) serotype 2b [II:(7),8] and 2b [II:(3),4;(7),8] conferred resistance to extended-spectrum -lactamase genes (Table 3). These isolates were confirmed by PCR and subsequently sequenced and showed 100% similarity to strains in the GenBank encoding for bla CTX-M-14 ( , serotype 2b [II:(3),4;(7),8]; , serotype 2b [II:(7),8]; , serotype 2b [II:(7),8]; and , serotype 2b [II:(7),8]) and bla CTX-M-15 ( , serotype 2b [II:(3),4;(7),8]; , serotype 2b [II:(3),4; (7),8]) (Fig. 4). PFGE. Fifty unique XbaI PFGE patterns were observed among a subset of 71 isolates (Fig. 4). There were 11 distinct PFGE clusters (a to k), five of which (clusters a, f, h, i, and k) shared isolates with the same serotype or antigenic formula. Cluster b contained six isolates with different antigenic formulas: five of serotype variant X [IV:(7),8] and one of serotype variant X. Similar observations were made in clusters j, c, d, e, and g. Cluster j consisted of three isolates, one of which was serotype 1a [I:(3),4] and two of which were untypeable [I:(7),8]. Cluster c included four isolates, three of which exhibited antigenic formula 2b [II:(7),8] and one of which exhibited the antigenic formula 2b [II:(3),4;(7),8]. Clusters d and e included two isolates of different antigenic formulas: variant X [IV:(7),8] and variant X. Finally, cluster g consisted of four isolates, three belonging to serotype 2b [II: (3),4;(7),8] and one belonging to serotype 1a [I:(3),4] (Fig. 4). All of the isolates in clusters a, c, and e to k conferred resistance to the same antimicrobials. In addition, all isolates in clusters c and f harbored the same extended-spectrum -lactamase genes, bla CTX-M-14 and bla CTX-M-15, respectively (Fig. 4). The isolation dates ranged within the clusters between 1 day (cluster e) up to 2 months (cluster j), with a median of 11 days. All of the clusters consisted of isolates belonging to S. flexneri serotype 1a [I:(3),4], untypeable [I:(7),8], 2a, 2b [II:(7),8], 2b [II:(3),4;(7),8], 4 [IV: ], variant X [IV: (7),8], untypeable [ 16], and variant X (Fig. 4). DISCUSSION In this study, the ratios between and S. sonnei were approximately 75% and 25%, respectively, indicating that Henan Province exhibits the same serotype distribution as developing countries (14). We observed the same distribution of serotypes compared to what had been reported earlier. Ye et al. (30) reported the distribution of serotypes to have fluctuated greatly over the years, with serovars 1a [I:(3),4], 2a, and variant X variant [IV:(7),8] (not an officially recognized serotype) being the most common serotypes in Henan Province between 2000 and 2008 (30). In the present study, we found that the top three most common serotypes consisted of serotype variants X [IV:(7),8], 2a, and 1a [I:(3),4]. Our data support the finding that, in 2006, serotype variant X [IV:(7),8] may have played a more significant role in the ranking of the top serotypes in Henan Province compared to 2008 (30). It is difficult to explain the observed regional differences, since these most likely are associated with social economical related issues, geography, and population density. It is unknown why S. sonnei was predominant in the eastern regions of Zhoukou and Sui County with no cases in the northwestern region of Jiyuan and why serotype 1a [I:(3),4] was predominant in the northwestern region of Jiyuan. However, correlating serotype 2a (predominating in rural areas) with gender and age, we noticed that females were significantly more affected by this serotype than males in contrast to serotype variant X [IV:(7),8] (dominating urban areas), which is evenly distributed among the genders. We have no explanation for these findings but feel they should be further explored. The present study also indicates an interesting absence of S. dysenteriae and S. boydii among the relatively large number of collected isolates. In the last 30 years, the number of clinical cases caused by these two species has declined in Henan Province. However, we do not know what has caused this decline. We have also examined serotypes with serologically atypical antigenic formulas: variant X [IV:(7),8], 2b [II:(3),4;(7),8], untypeable [ :6], and untypeable [I:(7),8], the last two of which were reported for the first time from Henan Province. In this respect, it is noteworthy to mention that untypeable [I:(7),8]) has been increasingly observed in Asia (7, 22 24) and was initially described by Talukder et al. (24). The serotype has not yet been formally included the official scheme but is will be included in the future under the name serotype 1c. In the present study, we chose to use the antigenic formula [I:(7),8] when referring to this serotype. Similarly, in variant X [IV:(7),8], the serological reaction with type IV monovalent antisera is believed to be due to the presence of a confounding E1037 antigen among immu- FIG. 4. PFGE (XbaI) analysis of 71 serotypes from Chinese patients during 2006 in Henan Province, China. Black spots in the dendrogram indicate the isolates are resistant. Abbreviations: AMP, ampicillin; AUG2, amoxicillin plus clavulanic acid; APR, apramycin; CEF, cefalothin; POD, cefpodoxime; XNL, ceftiofur; CHL, chloramphenicol; CIP ciprofloxacin; COL colistin; FFN, florfenicol; GEN, gentamicin; NAL, nalidixic acid; NEO, neomycin; SPE, spectinomycin; STR, streptomycin; SMX, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim. *, High-level resistance to ciprofloxacin with an MIC of 4 g/ml.

9 240 XIA ET AL. J. CLIN. MICROBIOL. nization strains and the lack of a standardized panel of strains for use by antisera producers (Strockbine, unpublished). The isolates producing this atypical serological antigenic pattern are considered to belong to variant X strains. In 1988, isolates producing this unusual antigenic pattern were proposed by Pryamukhina and Khomenko to be classified as serotype 4c (17). This classification was proposed to the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Enterobacteriaceae for its incorporation into the scheme; however, it has not been officially recognized. Age also seemed to influence serotype 2a compared to serotype variant X [IV:(7),8], since the infection with serotype 2a seemed to fluctuate per age group, with most infections affecting patients between 0 and 15, 21 and 30, and 56 and 80 years of age. In contrast, serotype variant X [IV:(7),8] affected mostly patients 0 to 10, 16 to 20, 26 to 55, and 61 to 65 years old. The behavior and differences between the various serotypes are important for the implementation of vaccine strategies and highlight the need for continuous monitoring in the future. In 1997, a bivalent S. flexneri serotype 2a and S. sonnei vaccine was produced by Lanzhou Institute of Biological products and licensed in China. The vaccine was tested in Henan Province on 26,230 volunteers and was 61 to 65% effective against serotype 2a and 57 to 72% effective against S. sonnei but was never evaluated outside of China (15). From 1999 to 2007, only 1,000 doses were sold in Henan Province. From 2007, the production of the vaccine was stopped according to the Immune Program Department of Henan, Center for Disease Control and Prevention. The reason why only a limited amount of the vaccines were sold in Henan Province and why the production stopped after its trial is unknown. However, we speculate that the overall prevalence of Shigella in Henan Province would be different today if the vaccine had been more used more extensively in previous years, since serotype 2a and S. sonnei are two of the most common serotypes in Henan Province today. Interestingly, in the present study, the gender distribution seemed to be skewed, with 62.1% of the infected patients being male. The skewed distribution might be associated with China s one-child-per-family policy. The policy has been criticized for discriminating against females, who may be aborted, abandoned, or unregistered and thereby may not receive the proper medical care (12). This hypothesis may be the reason for the skewed distribution among males and female patients infected with Shigella. In addition, we found a significant difference in the infection patterns caused by serotype 2a, which seemed more commonly associated with infections in females than in males. We also observed nonsignificant differences in serotype variant X, 2b [II:(7),8], 4a [IV: (3),4], untypeable isolates [ :6], and 1a [I: ], which were present in both males or females. The reasons for these discrepancies are unknown and remain to be further investigated. Not surprisingly, we found, as in many other developing countries, that primarily children of up to 5 years of age were infected (26). We did not observe any particular distribution of Shigella species and serotype among the different age groups. We provide here the first description of bla CTX-M-14 or bla CTX-M-15 in isolates originating from China. To date, only a few publications have described the presence of extended spectrum -lactamases in. The first observation was reported from Argentina, where a survey revealed six human isolates from 1997 to All of these belonged to the bla CTX-M-2 group (1). In 2004, a bla TEM-116 isolate from Zhengzhou, Henan Province, China, was obtained from a chicken (10), and human bla CTX strains were isolated in 2005 from Anhui Province, China (29). In 2006, four cases of shigellosis due to isolates harboring bla CTX-M-15 were described in New Zealand (25). We describe here the worrisome fact that all of the 71 S. flexneri isolates conferred resistance to nalidixic acid and, additionally, either high-level resistance or reduced susceptibility to ciprofloxacin. The same high level of resistance to ciprofloxacin and nalidixic acid has also previously been described in the Anhui and Henan provinces in China (11, 18, 30, 31). The two provinces are situated in the eastern part of mainland China and share borders. Thus, nalidixic acid- and ciprofloxacin-resistant isolates might have become endemic for this area in China and perhaps even beyond these provinces. In addition, treatment with fluoroquinolones when isolates show reduced susceptibility to ciprofloxacin is not advised, since such treatment has been associated with treatment failures. The highly resistant nature of the isolates in Henan Province makes antimicrobial treatment difficult. Most of the isolates were multidrug resistant and conferred resistance to up to 14 antimicrobial agents, including antimicrobial agents commonly used to treat Shigella infections in children. This development of resistance to extended-spectrum cephalosporins and fluoroquinolones is a major concern since these antimicrobials are categorized by the World Health Organization as critically important antimicrobials for the treatment of human infections (5). The reason for the high level of antimicrobial resistance in China is most likely the overuse of antimicrobial agents in hospitals and in the agricultural area due to a lack of legislative guidelines and limitation in usage. A recent article evaluated the antibiotic use in the five largest children hospitals in four large cities in China from 2002 to 2006 (32). They found that 35 to 56 different antibiotics were used, with penicillins, cephalosporins, and macrolides accounting for the largest total volume and with extended-spectrum cephalosporins being the most used antibiotic among the group of cephalosporins. In addition, the overall consumption of antibiotics ranged from 49.9 to 68.2 defined daily doses (DDD)/100 bed-days among the five hospitals (32), whereas, in comparison, the overall consumption of antibiotics in Danish hospitals in 2008 was 74.9 DDD/100 bed-days (6). In Denmark, legislation enforcing a prescription-only policy has been implemented. In contrast to Denmark, antimicrobials are still easily obtainable at pharmacies in China, causing a huge selection pressure for antimicrobial resistance in Chinese society. In the present study, we report the insufficiently discriminatory power of the PFGE protocol utilizing the XbaI enzyme, which resulted in clusters with mixed serotypes or antigenic formulas. Thus, more discriminatory protocols have to be developed for tracking the epidemiology of. We combined various microbiological typing techniques that often serve as epidemiological markers to determine the sources of infections in combination with epidemiological data. This approach is unique, and in this case we detected several PFGE clusters, e.g., clusters f and k, that shared the same

10 VOL. 49, 2011 HUMAN SHIGELLA INFECTIONS IN HENAN PROVINCE, CHINA 241 serotypes, antimicrobial susceptibility patterns, and genes and were isolated within the range from 2 days to 1 month. Based on these data, we speculate that these groups represent Shigella outbreaks in Henan Province. This hypothesis is strongly supported by the weekly incident rates of the specific serotypes, e.g., serotype 2a in week 27, serotype variant X [IV:(7),8] in weeks 26 and 27, and S. sonnei in week 41. The low number of cases (n 467) examined here may be biased due to underreporting. Shigellosis has previously been described as the most common cause of diarrheal diseases in China; thus, 467 cases of shigellosis is rather few compared to a population of 93.8 million people in Henan Province, China. Wang et al. (28) also described an underreporting rate of 71% in this region. Therefore, we expect there to be many more Shigella infections in Henan Province than were included the present study (28). The underestimation may be due to the fact that some of those infected do not consult a doctor, not every doctor orders a test, and not every test is positive. Moreover, we do not know which pathogen is responsible for the remaining 3,064 diarrheal cases since the present study has focused entirely on Shigella. The differences in the Shigella species and serotypes isolated among the surveillance points, differed in proportion between Shigella flexneri serotype 2a, variant X [IV:(7),8], and 1a [I:(3),4] and Shigella sonnei, are worth mentioning. Interestingly, serotype 1a [I:(3),4], ranking overall as the fourth most common serotype, was ranked first in Jiyuan. We do not have any information that could explain this ranking, but it might be a result of a low prevalence or the northern geographical location of the surveillance point. We demonstrated here a high prevalence of shigellosis with geographical differences, as well as differences between the genders, in the distribution of serotypes. We observed a worrisome level of multidrug resistance in serotypes conferring resistance to ciprofloxacin and expanded-spectrum cephalosporins in serotypes 2b [II:(7),8] and 2b [II:(3),4;(7),8], providing for the first time a description of bla CTX-M-14 and bla CTX-M-15 in in Henan Province, China. We report a low discriminating power in testing the isolates using PFGE and XbaI. However, we found several isolates exhibiting the same PFGE type, MIC profile, and isolated in the same period, indicating multiple undetected outbreaks. The majority of infections were observed in patients younger than 5 years and in male patients. We encourage general practitioners to avoid empirical treatment and conduct susceptibility testing in order to select the most appropriate antimicrobial for treatment. The authorities in Henan Province should implement timely surveillance of Shigella to facilitate control and prevention of outbreaks for the benefit of the Chinese people. In addition, the authorities should implement a prescription-only policy for antimicrobials, thereby forcing the population to seek professional medical care rather than self-medication of antimicrobials in order to lower the selection pressure for resistance in Shigella spp. ACKNOWLEDGMENTS We are grateful to Christina Aaby Svendsen and Jacob Dyring Jensen (DTU-Food) for outstanding technical assistance. We also thank Nancy A. Strockbine and Matthew Mikoleit at the U.S. Centers for Disease Control and Prevention for assisting the interpretation of the serotypes. This study was supported by the World Health Organization Global Food-Borne Infections Network (WHO GFN) ( REFERENCES 1. Andres, P., A. Petroni, D. Faccone, F. Pasteran, R. Melano, M. Rapoport, M. Martinez, C. Culasso, B. A. Di, B. Irigoyen, J. Mulki, A. 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