FOODBORNE PATHOGENS AND DISEASE Volume 11, Number 3, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2013.1629

Nontyphoidal Salmonella Infection in Children with Acute Gastroenteritis: Prevalence, Serotypes, and Antimicrobial Resistance in Shanghai, China Yuefang Li,1,* Xinbao Xie,2,* Xuebing Xu,3,* Xiangshi Wang,2 Hailing Chang,2 Chuanqing Wang,4 Aiming Wang,4 Yanlei He,4 Hui Yu,2 Xiaohong Wang,1 and Mei Zeng 2

Abstract

Information about nontyphoidal Salmonella (NTS) infection in children is limited in mainland China. The objective of this study was to investigate the prevalence, serotypes, and antibiotic resistance patterns of NTS infection in children in Shanghai. All cases with probable bacterial diarrhea were enrolled from the enteric clinic of a tertiary pediatric hospital between July 2010 and December 2011. Salmonella isolation, serotyping, and antimicrobial susceptibility testing were conducted by the microbiological laboratory. NTS were recovered from 316 (17.2%) of 1833 cases with isolation rate exceeding Campylobacter (7.1%) and Shigella (5.7%). NTS infection was prevalent year-round with a seasonal peak during summer and autumn. The median age of children with NTS gastroenteritis was 18 months. Fever and blood-in-stool were reported in 52.5% and 42.7% of cases, respectively. Salmonella Enteritidis (38.9%) and Salmonella Typhimurium (29.7%) were the most common serovars. Antimicrobial susceptibility showed 60.5% of isolates resistant to ‡ 1 clinically important antibiotics. Resistance to ciprofloxacin and the third-generation cephalosporins was detected in 5.5% and 7.1%–11.7% of isolates, respectively. NTS is a major enteropathogen responsible for bacterial gastroenteritis in children in Shanghai. Resistance to the current first-line antibiotics is of concern. Ongoing surveillance for NTS infection and antibiotic resistance is needed to control this pathogen in Shanghai.

Introduction

D

iarrhea continues to be a significant cause of morbidity and mortality in children in low- and middleincome countries, where there were nearly 1.7 billion episodes of diarrhea in children under 5 years old in 2010 (Fischer Walker et al., 2012). Household surveys conducted during 1988–1991 showed that the average incidence rate of diarrhea among Chinese children under 5 years old was 2.5 episodes/ child-year; enteropathogens were identified in 47%–65% of diarrheal children (Fang, 1991; Wei, 2008). Shigellosis had long been considered as one of the most important infectious diarrhea in China and has been listed among the notifiable infectious diseases since the 1950s (Wei, 2008). China has made significant infrastructural improvements regarding water supplies, sanitation, and hygiene, resulting in a remarkable decrease in shigellosis since the late 1990s (Wang et al., 2006; Ye et al., 2007; Wei et al., 2008). Health providers

have been required to report laboratory-confirmed bacterial diarrhea besides dysentery and cholera during the past years because food safety has gained growing public attention in China. Surveillance systems from developed countries have suggested that nontyphoidal Salmonella (NTS) is the most common bacterial cause of foodborne outbreaks (CDC, 2011; Gormley et al., 2011). The intensified control programs of Salmonella in animal reservoirs, particularly in poultry, and better hygiene practices throughout the food production chain have resulted in the declining trend of human salmonellosis in Europe (Lahuerta et al., 2011). Laboratory surveillance of NTS infection has been enhanced in China recently (Ran et al., 2011). A study showed that 34% of NTS diarrheal cases occurred in children < 5 years old in China (Ran et al., 2011). However, the epidemiologic profiles of NTS infection in children are far less understood in mainland China due to the considerable diagnostic gap and the reliability of reporting data.

1

Enteric Clinic and 2Department of Infectious Diseases, Children’s Hospital of Fudan University, Shanghai, China. Department of Microbiology, Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China. 4 Department of Microbiology, Children’s Hospital of Fudan University, Shanghai, China. *These authors contributed equally to this work. 3

200

NONTYPHOIDAL SALMONELLA INFECTION Given the importance of NTS infection in global foodborne illnesses and in childhood diarrhea (Marcus, 2008; Majowicz et al., 2010), knowledge of local epidemiology and antibiotic resistance patterns of the NTS serotypes causing infectious diarrhea is important to formulate appropriate therapeutic and control strategies. In this study, we retrospectively investigated NTS infection in children with bacterial gastroenteritis in Shanghai, aiming to understand the recent clinical epidemiology and antimicrobial resistance patterns of NTS isolates in Chinese children. Methods Study setting and patients This study was conducted at Children’s Hospital of Fudan University, a tertiary hospital in Shanghai with 800 beds and over 4000 outpatient visits per day. Children registered with probable acute bacterial gastroenteritis at the enteric clinic between July 2010 and December 2011 were included in this study. Probable acute bacterial gastroenteritis in our setting was based on the following presentation: diarrhea lasting < 14 days with the presence of blood and pus/mucus in the stool, or with the presence of fecal leukocytes > 5 cells/high-power field in the stool. Once a patient was referred to the enteric clinic, a stool sample was routinely subjected to bacteria isolation, and a medical record was kept on file. Patients with probable bacterial gastroenteritis were treated empirically with third-generation cephalosporins for 3–7 days, depending on the resolution of fever and bloody diarrhea and the results of stool culture.

201 identification double-tube slant, and CHROMagarTMSalmonella chromogenic agar (CAS) (Shanghai KeMaJa Biotech), and incubated for 18–24 h at 36 – 1C. Colonies that were considered as Salmonella had the following biochemical phenotypes: mannitol and glucose positive, urease enzyme, sucrose and indole negative, hydrogen sulfide positive or negative, mobility positive or negative, typical mauve colonies on CAS. Serogrouping was performed using slide agglutination with Salmonella polyvalent serum OMA, OMB, OMC, OMD (States Serum Institute, SSI Diagnostica, Denmark). The isolates showing strong positive agglutination were further tested using API 20E (bioMe´RIEUX, Marcy l’Etoile, France), and Salmonella serovars identification plate (Shanghai KeMaJa Biotech) at 36 – 1C for 18–24 h. Additionally, ‘‘O’’ and ‘‘H’’ typing sera (Tianrun, Ningbo) were used for serotyping (Grimont et al., 2007). To isolate Campylobacter, stool samples were plated onto KAMRILI agar (Shanghai KeMaJa Biotech), and kept at 42C for 48 h in an anaerobic jar (Oxoid, UK) with microaerophilic Pack (Shanghai KeMaJa Biotech). The typical Campylobacter colonies were selected for further confirmation using Gram stain, catalase, oxidase, and hippurate hydrolysis tests. Salmonella and Shigella serotypes were confirmed at the reference laboratory of Shanghai Municipal Center for Disease Control and Prevention (SCDC) using methods recommended by Denka Seiken ( Japan) and States Serum Institute SSI Diagnostica. Multiple polymerase chain reaction assay with two sets of primers targeting mapA and ceuE genes was used for identification of Campylobacter jejuni and Campylobacter coli in the SCDC reference laboratory (Denis et al., 1999).

Data collection During the 18-month period, a total of 1833 children with probable bacterial gastroenteritis were registered at the enteric clinic. Case files were reviewed regarding the following data: demographic information, date of hospital visit, disease onset, and results of stool culture. For cases with cultureconfirmed salmonellosis, additional data were retrieved including family/self-reported case clusters, clinical symptoms, microscopic examination of stool, antibiotic therapy, and treatment outcomes. For patients admitted to the wards, the results of blood culture, if any, were reviewed. This retrospective study was approved by the Hospital Ethics Committee. Microbiological methods All fecal samples were preserved in Cary-Blair transport media (Shanghai KeMaJa Biotech, China) within 24 h, and processed within 6 h for identifying different enteropathogenic bacteria at the microbiology laboratory. Fecal samples were cultured by streaking on xylose-lysine deoxycholate (XLD) agar, Sorbitol-MacConkey agar (Shanghai KeMaJa Biotech), and SSI agar (Statens Serum Institute SSI Diagnostica, Denmark), followed by incubation at 36 – 1C for 18– 24 h, for isolating Shigella, Salmonella, and enterohemorrhagic Escherichia coli, respectively. To improve the isolation of Salmonella, fecal samples were enriched in modified SBG (Selenite brilliant-green broth) at 36 – 1C for 18–24 h, followed by subculturing onto XLD agar. Three suspected colonies (characteristic black colonies reflecting H2S production) on XLD were selected, and inoculated on Columbia agar slant, intestinal biochemical

Antimicrobial susceptibility testing Antibiotic susceptibility was evaluated using the Kirby-Bauer disk diffusion method for 13 antimicrobial agents including ampicillin, chloramphenicol, sulfisoxazole, trimethoprim-sulfamethoxazole, tetracycline, amoxicillinclavulanate, cefepime, cefotaxime, ceftazidime, nalidixic acid, ciprofloxacin, gentamicin, and streptomycin (Oxoid, UK). Isolates were classified as resistant, intermediate, and susceptible according to the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2011). For this analysis, isolates displaying intermediate susceptibility were categorized as sensitive. According to the interpretive standards of China CDC and U.S. National Antimicrobial Resistance Monitoring System (Ran et al., 2011; Crump et al., 2011), clinically important antimicrobial resistance phenotypes are defined as resistance to one or more clinically important agents including ampicillin, ciprofloxacin, gentamicin, trimethoprimsulfamethoxazole, and third-generation cephalosporins (cefotaxime, ceftazidime, and cefepime). R-type ACSSuT is defined as resistance to ampicillin, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, and tetracycline; ACSSuTAuCx is defined as resistance to ampicillin, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, tetracycline, amoxicillin-clavulanate, and cefotaxime. Escherichia coli ATCC 25922 was used for quality control in disk diffusion test. Statistical analysis Statistical analysis was performed using SPSS (version 11.5) software. Differences between proportions were tested

202 using chi-square test or Fisher’s exact test. A p value < 0.05 was considered to be statistically significant. Results Seasonal trends of NTS infection NTS was isolated from 316 (17.2%) of 1833 children, 2 of whom had Shigella co-infection and 1 of whom had Campylobacter co-infection. Shigella spp. were isolated in 105 (5.7%) children, with 85 (81.0%) isolates being Shigella sonnei and 20 (19.0%) isolates being S. flexneri. Detection for Campylobacter spp. was performed in 1284 children since December 2010, of whom Campylobacter spp. were isolated in 91 (7.1%) children with 70 (76.9%) isolates being C. jejuni and 21 (23.1%) being C. coli. No Enterohemorrhagic E. coli O157: H7 was isolated. NTS was prevalent throughout the year (Fig. 1). Isolation rates of NTS ranged from 6.8% to 24.9% by month, with higher prevalence between May and September. The isolate rates of Campylobacter ranged from 2.7% to 17.0% by month, with higher prevalence between December and March. The isolate rates of Shigella ranged from 0% to 15% by month, with higher prevalence between August and November. Demographics The median age of 1833 enrolled children was 20 months (range: 1 month–14 years). The median ages of 316 NTS-associated diarrheal patients was 18 months (range: 1 month–12 years), which was significantly younger than the median ages of children with campylobacteriosis (44.5 months, range: 6 months–10 years, p < 0.001) and shigellosis (51.2 months, range: 1 month–10 years, p < 0.001). The age-specific isolation rates of NTS in 1833 cases were 18.3% (114/623) in children < 1 years, 22.2% (82/370) in children 1–1.9 years, 22.2% (47/ 212) in children 2–2.9 years, 16.7% (28/168) in children 3–3.9 years, 16.9% (22/130) in children 4–4.9 years, and 7.0% (23/ 330) in children ‡ 5 years, respectively ( p < 0.05). The isolation rates were 17.1% (191/1116) and 17.4% (125/717) among boys and girls, respectively ( p = 0.860). Clinical features and outcomes All 316 children with NTS-associated diarrhea had no known underlying diseases and no epidemiologic linkage.

LI ET AL. The attendant symptoms included fever in 116 (52.5%) patients, self-reported abdominal pain in 47 (14.9%) patients, and vomiting in 37 (11.7%) patients. Stool containing visible blood was reported in 135 (42.7%) patients. The passing of > 10 stools in 24 h was reported in 42 (13.3%) patients. Fecal occult blood was present in 282 (89.2%) stool samples. Three hundred five cases received oral cefixime as outpatients and 11 received intravenous ceftriaxone or cefoperazonesulbactam as inpatients. All patients fully recovered, and no bacteremia was found in 11 inpatients. Serovars of NTS isolates The distribution of 36 Salmonella serovars is shown in Table 1. Salmonella Enteritidis and Salmonella Typhimurium were the most common serovars, representing 123 (38.9%) and 94 (29.7%) isolates, respectively. Twenty-seven serovars were detected in infants, 24 serovars were detected in children 1-4 years, and only 5 serovars in children ‡ 5 years. Salmonella Enteritidis was more commonly found in children ‡ 1 year than in infants ( p < 0.001) while Salmonella Typhimurium was more commonly found in infants than in children ‡ 1 years ( p < 0.001). Antimicrobial susceptibility in NTS isolates A total of 309 (97.8%) of 316 isolates were tested for antimicrobial susceptibility at the SCDC laboratory. As shown in Table 2, 187 (60.5%) isolates were resistant to ‡ 1 clinically important antimicrobial agents, 83 (26.9%) isolates were resistant to ‡ 3 clinically important antimicrobial agents, 62 (20.1%) isolates were R-type ACSSuT, and 7 (2.3%) isolates were ACSSuTAuCx. NTS isolates exhibited the highest frequency of resistance to the conventional first-line antibiotics (previously empirical first-line antibiotics): nalidixic acid (69.6%), ampicillin (58.6%), tetracycline (48.5%), trimethoprim-sulfamethoxazole (32.4%), and chloramphenicol (29.8%). Resistance to the current first-line antibiotics such as ciprofloxacin, cefotaxime, ceftazidime, cefepime, and amoxicillin-clavulanate was detected in 17 (5.5%), 36 (11.7%), 27 (8.7%), 22 (7.1%), and 31 (10.0%) isolates, respectively. Three Salmonella Indiana isolates and one Salmonella Enteritidis isolate were resistant to both ciprofloxacin and third-generation cephalosporins.

FIG. 1. Monthly isolation rates of nontyphoidal Salmonella, Shigella, and Campylobacter in children with probable bacterial gastroenteritis between July 2010 and December 2011 in Shanghai.

NONTYPHOIDAL SALMONELLA INFECTION

203

Table 1. Distribution of 316 Salmonella Isolates by Year and Age Groups Between July 2010 and December 2011 in Shanghai

Serotype

Rank

Enteritidis Typhimurium Paratyphi B Stanley Thompson Aberdeen Infantis Agona London Derby Stenftenberg Saintpaul Tennessee Indiana Braenderup Others

1 2 3 4 5 6 6 7 8 9 9 9 10 10 10 11–12

Total number (%) of isolates (n = 316) 123 94 11 9 8 7 7 6 5 4 4 4 3 3 3 25

(38.9) (29.7) (3.5) (2.8) (2.5) (2.2) (2.2) (1.9) (1.6) (1.3) (1.3) (1.3) (0.9) (0.9) (0.9) (7.9)

Number (%) of isolates by year 2010 (n = 89) 25 28 2 2 3 2 3 1

(28.1) (31.5) (2.2) (2.2) (3.4) (2.2) (3.4) (1.1)

1 3 2 1

(1.1) (3.4) (2.2) (1.1)

1 (1.1) 15 (16.9)

2011 (n = 227) 98 66 9 7 5 5 4 5 5 3 1 2 2 3 2 10

(43.2) (29.1) (4.0) (3.1) (2.2) (2.2) (1.8) (2.2) (2.2) (1.3) (0.4) (0.9) (0.9) (1.3) (0.9) (4.4)

< 1 (n = 114) 26 40 6 1 1 3 3 4 5 3 3 1 3

(22.8) (35.1) (5.3) (0.9) (0.9) (2.6) (2.6) (3.5) (4.4) (2.6) (2.6) (0.9) (2.6)

1 (0.9) 14 (12.3)

Number (%) of isolates by age group (years) 1-4.9 (n = 179) 84 47 5 8 7 4 3 2

(46.9) (26.1) (2.8) (4.5) (3.9) (2.2) (1.7) (1.1)

‡ 5 (n = 23) 13 (56.5) 7 (30.4)

1 (4.3)

1 (0.6) 1 (0.6) 3 (1.7) 2 (1.1) 1 (0.6) 10 (5.6)

1 (4.3) 1 (4.3) 0 (0)

Other Salmonella serotypes included Montevideo (2), Mbandaka (2), Newport (2), Virchow (2), Washington (2), Ordonez (1), Bovismorbificans (1), Pomona (1), Hvittingfoss (1), Turkey (1), Carrau (1), Cuttbus (1), Reading (1), Litchfield (1), Moualine (1), Wandsworth (1), Uganda (1), Singapore (1), Anatum (1), and Irumu (1).

Resistance to clinically important antibiotics was more common in Salmonella Typhimurium (89.1%) isolates than in Salmonella Enteritidis (70%) isolates. The frequency of resistance to ‡ 3 clinically important antimicrobial agents was high in Salmonella Typhimurium (58.7%), Salmonella Indiana (100%), and Salmonella London (60%) isolates. R-type ACSSuT was more common in Salmonella Typhimurium (58.7%) and Salmonella London (60%) isolates. Discussion This study confirmed that NTS has become a leading bacterial pathogen causing childhood acute enteritis in Shanghai, with an isolation frequency of 17.2%, surpassing Campylobacter spp. (7.1%) and Shigella spp. (5.7%). Our findings highlight the importance of NTS infection in Chinese children. The data from FoodNet showed that NTS caused most bacterial enteric illness among American children < 5 years old, followed by Campylobacter and Shigella (Scallan et al., 2013). However, in the United Kingdom Campylobacter was the most common bacterial pathogens among cases with acute gastroenteritis presenting to health care (Tam et al., 2012). Our findings are similar to those of the American report. A study from our hospital reported that NTS was isolated in only 0.5% of outpatients with bacterial gastroenteritis in 2007 (Wang et al., 2009). Previous domestic studies conducted in Wuhan and Beijing since 2007 indicated that NTS was isolated in 3.4%–5.9% of young children with diarrhea (Cui et al., 2009; Qu et al., 2012). The significantly higher prevalence of NTS infection in this study might be due to an effective SBG enrichment in combination with XLD/CAS inoculation method being used in our microbiology laboratory for Salmonella isolation since July 2010. This method was shown to greatly improve the efficiency of isolation of Salmonella (Zhao et al., 2010).

Consistent with the previous studies in China and North America (Koehler et al., 2006; Ran et al., 2011; Ravel et al., 2010), this study observed that NTS infection was common year-round with higher activity in summer and autumn, distinct from the seasonal patterns of campylobacteriosis and shigellosis. It is documented that salmonellosis cases are subject to seasonal variation with higher activity in warmer months (Ravel et al., 2010; Lal et al., 2012). The seasonal distribution pattern might be related to environmental change, human activity, dietary differences, and/or food consumption (Lal et al., 2012; Ravel et al., 2010). Understanding diseasespecific seasonal pattern is also important for generating appropriate prevention strategies and proposing probable microorganisms causing acute diarrhea. Our results support the previous studies indicating that young children, especially infants, are more susceptible to salmonellosis (Lee et al., 1998; Koehler et al., 2006; Marcus, 2008; Deng et al., 2012). Furthermore, the greatest diversity of Salmonella serovars was found in infants who generally are restricted to a limited diet. Salmonella contamination in raw food of animal origin such as chicken, pork, beef, and lamb is relatively common in China (Shao et al., 2011); however, Chinese young children usually eat well-cooked food. Therefore, crosscontamination between raw and cooked food of origin at home might be a primary risk factor of NTS infection for Chinese infants. Also, environmental contamination and exposure might be associated with NTS infection in infants, an hypothesis suggested for American infants ( Jones et al., 2006). Salmonella Enteritidis and Salmonella Typhimurium were the most frequently isolated serovars, representing 68.6% of isolates. Both serovars were also predominant among diarrheal patients in Shanghai (50%) during 2006–2009 and other regions in China (58.7%) (Ran et al., 2011; Zhang et al., 2013). However, the serovars of Salmonella Derby, Salmonella Agona, and Salmonella Stanley were quite prevalent in diarrheal

204

0 (0) 0 (0)

0 0 0 0

0 (0)

0 (0) 0 (0)

0 (0)

0 (0)

(42.9) (0)

(0) (60.0) (0) (0)

(25)

(100) (0)

(0)

(12)

(0) (80) (50) (0)

3 (12)

0 (0)

3 (100) 0 (0)

0 (0)

0 4 2 0

1 (14.3) 0 (0)

0 (0) 2 (25)

(0) (0) (0) (0)

0 (0)

0 (0)

3 (100) 0 (0)

0 (0)

0 0 0 0

0 (0) 0 (0) (0) (0) (0) (0)

1 (4)

0 (0)

3 (100) 0 (0)

1 (25)

0 0 0 0

0 (0) 0 (0)

0 (0) 1 (12.5)

0 (0)

8 (8.7)

8 (6.7)

CEP 8 (6.7)

CAZ

(0) (0) (0) (0)

(0) (0) (0) (0)

2 (8)

0 (0)

1 (33.3) 0 (0)

0 (0)

0 0 0 0

0 (0) 0 (0)

0 (0) 3 (37.5)

1 (10)

36 (11.7) 27 (8.7)

2 (8)

0 (0)

3 (100) 0 (0)

1 (25)

0 0 0 0

0 (0) 0 (0)

0 (0) 2 (25)

1 (10)

NAL

(0) (60) (0) (0)

(0) (0) (25) (0)

5 (20)

0 (0)

3 (100) 2 (66.7)

0 (0)

0 0 1 0

3 (42.9) 0 (0)

3 (33.3) 1 (12.5)

1 (10)

(16.7) (100) (25) (0)

9 (36)

1 (33.3)

3 (100) 0 (0)

2 (50)

1 5 1 0

3 (42.9) 4 (66.7)

2 (22.2) 5 (62.5)

9 (90)

82 (89.1)

81 (67.5)

SSS

(0) (60) (25) (0)

3 (12)

0 (0)

3 (100) 0 (0)

1 (25)

0 3 1 0

1 (14.3) 0 (0)

0 (0) 2 (25)

0 (0)

69 (75)

17 (14.2)

SXT

(0) (80) (100) (0)

4 (16)

0 (0)

0 (0) 0 (0)

1 (25)

0 4 4 0

3 (42.9) 1 (16.7)

0 (0) 3 (37.5)

2 (20)

80 (87.0)

48 (40.0)

TET

(16.7) (100) (75) (25)

6 (24)

1 (33.3)

3 (100) 0 (0)

3 (75)

1 5 3 1

1 (14.3) 3 (50)

6 (66.7) 3 (37.5)

4 (40)

77 (83.7)

71 (59.2)

STR

(66.7) (0) (0) (75)

10 (40)

1 (33.3)

0 (100) 1 (33.3)

0 (0)

4 0 0 3

1 (14.3) 0 (0)

1 (11.1) 3 (37.5)

1 (10)

4 (4.3)

0 (0)

Fully susceptible

76 (24.6) 215 (69.6) 208 (67.3) 100 (32.4) 150 (48.5) 188 (60.8) 29 (9.4)

1 (4)

0 (0)

3 (100) 0 (0)

1 (25)

0 3 0 0

1 (14.3) 0 (0)

0 (0) 0 (0)

0 (0)

77 (83.7)

14 (11.7) 119 (99.2)

GEN

14 (15.2) 12 (13.0) 53 (57.6)

13 (10.8)

CTX

Number (%) of isolates by antimicrobial resistance patterns

(0) (60) (0) (0)

187 (60.5)

5 (20)

0 (0)

3 (100) 0 (0)

2 (50)

0 3 0 0

3 (42.9) 0 (0)

0 (0) 3 (37.5)

2 (20)

82 (89.1)

84 (70)

(0) (60) (0) (0)

83 (26.9)

3 (12)

0 (0)

3 (100) 0 (0)

1 (25.0)

0 3 0 0

0 (0) 0 (0)

0 (0) 3 (37.5)

1 (10)

54 (58.7)

15 (12.5)

(0) (60) (0) (0)

62 (20.1)

2 (8)

0 (0)

0 (0) 0 (0)

0 (0)

0 3 0 0

0 (0) 0 (0)

0 (0) 2 (25)

0 (0)

51 (55.4)

4 (3.3)

(0) (0) (0) (0)

7 (2.3)

0 (0)

0 (0)

0 (0) 0 (0)

0 (0)

0 0 0 0

0 (0) 0 (0)

0 (0) 1 (12.5)

1 (10)

4 (4.3)

1 (0.8)

‡ 1 Clinically ‡ 3 Clinically important R-type important agents ACSSuT ACSSuTAuCx agent

Antimicrobial agents used for susceptibility testing of Salmonella species (disk concentration): AMP, ampicillin (10 lg/mL); AMC, amoxicillin-clavulanate (20/10 lg/mL); CHL, chloramphenicol (30 lg/mL); CIP, ciprofloxacin (5 lg/mL); CEP, cefepime (30 lg/mL); CTX, cefotaxime (30 lg/mL); CAZ, ceftazidime (30 lg/mL); GEN, gentamicin (10 lg/mL); NAL, nalidixic acid (30 lg/mL); SSS, sulfisoxazole (30 lg/mL); SXT, trimethoprim-sulfamethoxazole (23.75/1.25 lg/mL); TET, tetracycline (30 lg/mL); STR, streptomycin (10 lg/mL). Clinically important antibiotics included the following agents: ampicilin, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole and third-generation cephalosporins such as cefotaxime, ceftazidime and cefepime. Fully susceptible, susceptible to all antimicrobial agents tested; ‡ 1 agent, resistant to one or more antimicrobial agent tested; ‡ 3 clinically important agents, resistant to ampicillin, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole and or third-generation cephalosporins (cefotaxime, ceftazidime, cefepime); R-type ACSSuT, resistance to ampicillin, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, and tetracycline; ACSSuTAuCx, resistance to ampicillin, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, tetracycline, amoxicillin-clavulanate, and cefotaxime.

(58.6) 31 (10.0) 92 (29.8) 17 (5.5) 22 (7.1)

(0) (0) (0) (0)

0 (0) 4 (37.5)

(0) (37.5)

0 (0) 0 (0)

0 (0)

1 (10)

1 (20)

5 (4.2)

CIP

(20)

11 (9.2)

CHL

9 (9.8)

8 (6.7)

AMC

(87.0) 18 (19.6) 65 (70.7)

(69.2)

AMP

Enteritidis 83 (n = 120) Typhimurium 80 (n = 92) Paratyphoid B 2 (n = 10) Stanley (n = 9) 0 Thompson 3 (n = 8) Infantis (n = 7) 3 Aberdeen 0 (n = 6) Agona (n = 6) 0 London (n = 5) 3 Derby (n = 4) 0 Senftenberg 0 (n = 4) Saintpaul 1 (n = 4) Indiana (n = 3) 3 Braenderup 0 (n = 3) Tennessee 0 (n = 3) All other s 3 erotypes (n = 25) Total (n = 309) 181

Salmonella serotypes (number of isolates)

Table 2. Antimicrobial Resistance by Serotype of Salmonella Isolates from Diarrheal Children in Shanghai, from July 2010 to December 2011

NONTYPHOIDAL SALMONELLA INFECTION children in Henan, Wuhan, and Guangdong provinces (Cui et al., 2009; Xia et al., 2009; Deng et al., 2012). Furthermore, we found that the distribution of these two dominant serovars differed within age groups. Salmonella Typhimurium was the most commonly isolated serovar in infants while Salmonella Enteritidis was the most commonly isolated serovar in children ‡ 1 years. Certain kind of foods such as undercooked eggs and broiler chickens have been documented as the main sources of Salmonella Enteritidis infection in the United States (Chai et al., 2012). Thus far, the potential risk factors such as food consumption, animal contact, environmental exposure, and hygiene habit associated with Salmonella Enteritidis and Salmonella Typhimurium infections in Chinese children are poorly understand and need additional investigation. Comprehensive epidemiologic surveillance programs for human, food, and animals and pulsed-field gel electrophoresis–based surveillance for foodborne diseases will help to explain the regional variation in Salmonella serovars, identify the important reservoirs, and trace back to the possible source of infection. Antimicrobial resistance in NTS is a global problem (Su et al., 2004; Arlet et al., 2006). The antibiotic resistance patterns of Salmonella in Shanghai are similar to the recent national trend (Ran et al., 2011). Nearly 61% of isolates were resistant to at least one clinically important antibiotic and 27% of isolates were resistant to multiple important antibiotics. We observed that the prevalence of NTS resistance against the conventional first-line antibiotics remains high (30%–59%). It is of clinical concern that NTS isolates displayed the increased level of resistance to the current first-line antibiotics for treating salmonellosis such as ciprofloxacin (5%) and third-generation cephalosporins (7%–12%). Of note, a few of the isolates displayed resistance to both ciprofloxacin and third-generation cephalosporins. High prevalence of ciprofloxacin resistance (MIC ‡ 4 lg/mL) among NTS isolates recovered from diarrheal children was reported in Wuhan (12%), Jiangxi (19.4%), and Henan (25%) in China (Cui et al., 2009; Xia et al., 2009; Yu et al., 2011). However, the overall prevalence of resistance to third-generation cephalosporins in this study is higher than that (2.1%–4%) in the previous domestic reports (Cui et al., 2009; Ran et al., 2011; Xia et al., 2009). Of course, the difference in antibiotic resistance between this study and previous studies could be related to the method determining the antimicrobial susceptibility. However, these available data refect Salmonella resistance being a serious fact in China. Particularly, Salmonella Typhimurium was more resistant compared to the other major Salmonella serovars (Cui et al., 2009; Xia et al., 2009; Yu et al., 2011; Jabeen et al., 2012). Recently, a monophasic variant of Salmonella Typhimurium has been emerging associated frequently with R-type ACSSuT in Europe and other countries (Hopkins et al., 2010). Given that R-type ACSSuT Salmonella Typhimurium represents over 50% of isolates in Shanghai, the monophasic variant should be included in the ongoing surveillance. Studies have associated antimicrobial-resistant NTS with more invasive disease and hospitalization (Varma et al., 2005). Empirical antimicrobial therapy of severe NTS infection is challenging with the increasing resistance to extended-spectrum cephalosporins and ciprofloxacin in China and other countries (Keddy, 2008; Yu et al., 2011; Jabeen et al., 2012). Therefore, continued monitoring of circulating Salmonella serovars and antibiotic resistance patterns and invasive NTS infection is imperative for guiding the initial empiric choice of antibiotics in the clinical setting.

205 Conclusion NTS is a major enteric bacteria causing childhood infectious diarrhea in Shanghai. NTS resistance to the current first-line antibiotics such as ciprofloxacin and third-generation cephalosporins is an important concern when clinicians treat severe salmonellosis. Further epidemiologic studies are needed to understand the potential risk factors of the source of NTS infection, NTS asymptomatic carriage in children in China, and identify risk factors for severe invasive salmonellosis secondary to gastroenteritis in the clinical setting. Ongoing surveillance of antibiotic resistance in NTS and control measures for containment of antimicrobial resistance such as avoiding unnecessary antibiotic therapy in non-severe NTS gut infections are essential in our country. Acknowledgments We thank Professor John Klena (Centers for Disease Control and Prevention [U.S. CDC] at the U.S. Embassy, Beijing, China) for his critical comments on our manuscript and English language editing. This work was funded by the Mega-projects of Science and Technology Research of China (No. 2012ZX 10004215-003), the Subproject 6 of the China-US EID (emerging infectious diseases) cooperation project, and Shanghai Municipal Natural Science Foundation (No. 13ZR1404100). Disclosure Statement No competing financial interests exist. References Arlet G, Barrett TJ, Butaye P, Cloeckaert A, Mulvey MR, White DG. Salmonella resistant to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes Infect 2006;8:1945–1954. [CDC] Prevention. Vital signs: incidence and trends of infection with pathogens transmitted commonly through food: Foodborne diseases active surveillance network, 10 U.S. sites, 1996-2010. MMWR Morb Mortal Wkly Rep 2011;60:749–755. Chai SJ, White PL, Lathrop SL, Solghan SM, Medus C, McGlinchey BM, et al. Salmonella enterica serotype Enteritidis: increasing incidence of domestically acquired infections. Clin Infect Dis 2012;54(Suppl 5):S488–S497. [CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: 20th Informational Supplement. CLSI Document M100-S20. Wayne, PA: CLSI, 2010. Crump JA, Medalla FM, Joyce KW, Krueger AL, Hoekstra RM, Whichard JM, Barzilay EJ. Emerging Infections Program NARMS Working Group. Antimicrobial resistance among invasive nontyphoidal Salmonella enterica isolates in the United States: National Antimicrobial Resistance Monitoring System, 1996 to 2007. Antimicrob Agents Chemother 2011;55:1148–1154. Cui S, Li J, Sun Z, Hu C, Jin S, Li F, Guo Y, Ran L, Ma Y. Characterization of Salmonella enterica isolates from infants and toddlers in Wuhan, China. J Antimicrob Chemother 2009;63:87–94. Deng X, Ran L, Wu S, Ke B, He D, Yang X, Zhang Y, Ke C, Klena JD, Yan M, Feng Z, Kan B, Liu X, Mikoleit M, Varma JK. Laboratory-based surveillance of non-typhoidal Salmonella infections in Guangdong Province, China. Foodborne Pathog Dis 2012;9:305–312. Denis M, Soumet C, Rivoal K, Ermel G, Blivet D, Salvat G, Colin P. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli. Lett Appl Microbiol 1999;29:406–410.

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Address correspondence to: Mei Zeng, MD, PhD Department of Infectious Diseases Children’s Hospital of Fudan University 399 Wanyuan Road Shanghai 201102, China E-mail: [email protected] Xiaohong Wang, MD Enteric Clinic Children’s Hospital of Fudan University 399 Wanyuan Road Shanghai 201102, China E-mail: [email protected]

Nontyphoidal salmonella infection in children with acute gastroenteritis: prevalence, serotypes, and antimicrobial resistance in Shanghai, China.

Information about nontyphoidal Salmonella (NTS) infection in children is limited in mainland China. The objective of this study was to investigate the...
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