Salmonella Typhimurium and Salmonella Enteritidis Infections in Sporadic Diarrhea in Children: Source Tracing and Resistance to Third-Generation Cephalosporins and Ciprofloxacin

Abstract

Objectives:

This study aimed to trace the transmission source of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis strains associated with enteric infections in Shanghainese children, and understand the molecular mechanism of resistance to third-generation cephalosporins and ciprofloxacin.

Materials and Methods:

The profiles of pulsed-field gel electrophoresis (PFGE) were compared among the isolates from children, animal, and environment. Antimicrobial susceptibility was determined using the minimal inhibitory concentrations and Kirby–Bauer disk diffusion method. Extended-spectrum β-lactamase (ESBL) producing isolates mediated by resistance genes were identified using polymerase chain reaction and sequencing.

Results:

Based on PFGE patterns, 49 (33.1%) of 148 human Salmonella Typhimurium isolates located in the dominant PFGE clusters were genetically related to the isolates from poultry source, environment water, aquatic products, and reptiles, whereas 97 (97.0%) of 100 human Salmonella Enteritidis isolates were genetically related to isolates from poultry and water. The rates of resistance to ceftriaxone among clinical Salmonella Typhimurium and Salmonella Enteritidis isolates were 42.0% and 14.2%, respectively. Besides, 35.1% of Salmonella Typhimurium isolates displayed resistance to ciprofloxacin; 64.9% of Salmonella Typhimurium isolates and 97.0% of Salmonella Enteritidis isolates displayed reduced susceptibility to ciprofloxacin. Of 64 ESBL/AmpC-producing strains, CTX-M, TEM, DHA, and CMY were detected at frequencies of 86.0%, 62.5%, 7.8%, 3.1%, and 3.1%, respectively.

Conclusions:

The transmission sources of Salmonella Typhimurium and Salmonella Enteritidis infections in Shanghainese children were diverse. The high prevalence of resistance to third-generation cephalosporins and ciprofloxacin mediated by multiple molecular mechanisms needs continuous monitoring and intervention.

Introduction

N ontyphoidal salmonella (NTS) is a zoonotic pathogen and infects a wide variety of animal hosts and humans (Bäumler et al., 1998). In industrialized countries, NTS is transmitted predominantly by commercially produced food of animal origin (Foley and Lynne, 2008; Hale et al., 2012; Crump et al., 2015; Antunes et al., 2016). It was estimated that NTS caused 93.8 million cases of gastroenteritis and 155,000 deaths globally each year, of which 80.3 million were foodborne (Havelaar et al., 2015; Majowicz et al., 2010). NTS is also an important cause of invasive bacteremia in infants, young children, and immunocompromised patients and in the resource-limited settings (Kirk et al., 2015; Ao et al., 2015; Wen et al., 2017). Empirical antibiotic treatment is essential for invasive NTS infections and acute invasive diarrhea (Crump et al., 2015; Wen et al., 2017; WHO, 2017). However, emergence and spread of NTS isolates resistant to extended-spectrum cephalosporins and fluoroquinolones since 1990 represent another public health concern because these clinically important antibiotics are the first-line options for the treatment of severe NTS diseases (Su et al., 2004; Arlet et al., 2006; Parry and Threlfall, 2008; Kariuki, 2015). Global priority list of antibiotic-resistant bacteria highlights antibiotic-resistant NTS as a major threat to human health (WHO, 2017).

NTS is currently recognized as one of the leading pathogens causing infectious diarrhea in China and Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis are the most common serovars (Ran et al., 2011; Li et al., 2014; Wang et al., (2015b); Yu et al., 2015; Chang et al., 2017). An estimated ∼75% (30 million) of foodborne diseases are attributed to NTS in China (Wang et al., 2007). Thus far, the exact modes of NTS transmission in sporadic and outbreak gastroenteritis are largely unknown in China. Globally, an estimated ∼20% of NTS foodborne illnesses occurred in children <5 years (Kirk et al., 2015). It is also worrisome that antimicrobial resistance among NTS clinical isolates was serious in China (Ran et al., 2011). Given the major clinical issues on NTS infections in China, we carried out a retrospective study, aiming to trace the potential transmission sources for Salmonella Typhimurium and Salmonella Enteritidis isolates causing sporadic diarrhea in children, and investigate the prevalence and mechanism of extended-spectrum β-lactamase (ESBL)-producing and ciprofloxacin-resistant isolates in these two predominant serovars.

Materials and Methods

Study setting, sample collection, and bacterial strains

During 2012–2014, Salmonella surveillance program was carried out at the enteric clinic setting of our hospital, the exclusive sentinel pediatric hospital in Shanghai. Fresh fecal samples were collected from 3143 children with probable acute bacterial diarrhea (presence of blood and mucus in the stool, and or with the presence of fecal leukocytes ≥5 cells/high-power field in the stool). Clinical information was extracted from the archived medical records.

During the same period, monitoring of S. enterica was routinely performed in foods and environmental water as one part of Salmonella surveillance program in Shanghai. In total, 1385 S. enterica isolates were recovered from raw meat (chicken, duck, pork, and cattle), cooked meat for sale, sea and freshwater fishes/shellfishes/turtles from supermarkets or farm product markets, and river/sewage water. Salmonella Typhimurium and Salmonella Enteritidis representative isolates were selected for further characterization and test.

Salmonella isolation and serotyping

The microbiological method for S. enterica isolation and serotyping were described in detail as the previous study (Chang et al., 2017). In brief, fecal samples were cultured by streaking on xylose lysine deoxycholate (XLD; CHROMagar, China) agar, Sorbitol MacConkey agar (Shanghai KeMaJa Biotech), and SSI agar (Statens Serum Institute SSI Diagnostica, Denmark), followed by incubation at 36°C for 18–24 h, for isolating Salmonella. To improve the isolation of Salmonella, fecal samples were enriched in modified selenite brilliant green broth at 36°C ± 1°C for 18–24 h, followed by subculturing onto XLD agar. Presumptive colonies were screened by testing in triple sugar iron agar, motility indole urea agar, l-lysine decarboxylase, and l-galactosidase (o-nitrophenylL-d-galactopyranoside). Salmonella serovars were determined by slide agglutination with commercial Salmonella antisera (Statens Serum Institute, Denmark) and results were interpreted according to the Kauffmann–White scheme (World Health Organization, 2011).

Pulsed-field gel electrophoresis of Salmonella Typhimurium and Salmonella Enteritidis isolates

Pulsed-field gel electrophoresis (PFGE) was performed according to the international PulseNet protocol for Salmonella using a CHEFDRIII system (Bio-Rad, Hercules, CA) to investigate genetic relatedness (WHO, 2014). In brief, genomic DNA was digested with 50 U of XbaI (TaKaRa, China), and restriction fragments were separated by electrophoresis. The interpretation of the PFGE band patterns was performed with BioNumerics software (version 2.5; Applied Maths, Kortrjk, Belgium) using the Dice similarity coefficient. Isolates with a Dice similarity index ≥85% were considered to belong to the same PFGE cluster. Dendrograms were constructed on the basis of the unweighted pair group method using average linkages with a position tolerance of 1% (Tenover et al., 1995).

Antimicrobial susceptibility testing and screening test for ESBLs

Antimicrobial susceptibility testing was performed for human clinical isolates using minimum inhibitory concentrations (MICs), and nonhuman isolates using the Kirby–Bauer disk diffusion method. Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) performance standards (CLSI, 2016).

Salmonella strains exhibiting resistance to either ceftriaxone (MIC ≥4 mg/L) or ceftiofur (MIC ≥8 mg/L) were subsequently tested for resistance to additional broad-spectrum β-lactam antimicrobials (aztreonam, cefepime, cefotaxime, ceftazidime, imipenem, and piperacillin-tazobactam) to confirm ESBL/AmpC phenotypes. Quality control isolates were Escherichia coli (ATCC 25922) and Klebsiella pneumoniae (ATCC 700603).

Detection of ESBL-encoding/amoxicillin/clavulanic acid or amoxicillin–clavulanate-producing genes in Salmonella Typhimurium and Salmonella Enteritidis isolates

Isolates exhibiting ESBL phenotypes were screened by polymerase chain reaction (PCR) for the presence of blaTEM, blaSHV, blaOXA, blaDHA, blaGES, blaPER, blaVEB, blaACC, blaFOX, blaEBC, blaNDM, blaVIM, blaIMP, blaKPC, blaOKP, blaCMY, and blaCTX-M (Dallenne et al., 2010; Poirel et al., 2011). All the PCR products were directly sequenced (TaKaRa Biotechnology Cooperation, Dalian, China) for sequence analysis and aligned using the BLAST program (www.ncbi.nlm.nih.gov/BLAST/).

Statistical analysis

Statistical analysis was performed using SPSS statistics 17.0 software (IBM). Differences between proportions were tested using chi-square test or Fisher's exact test. A value of p < 0.05 was considered to be statistically significant.

Results

Serotypes of Salmonella strains isolated from children, foods, and water

The distribution of Salmonella serovars is given in Table 1. Forty Salmonella serovars were identified in 742 isolates recovered from diarrheal children, of which Salmonella Enteritidis and Salmonella Typhimurium accounted for 42.6% and 28.2%, respectively, followed by Salmonella Thompson (4.0%), Salmonella Derby (3.1%) and Salmonella Stanley (2.8%). We selected 148 Salmonella Typhimurium isolates and 100 Salmonella Enteritidis isolates for further characterization, according to the sex, age, month of isolation at a ratio of almost 1:2. The median age of 148 children infected with Salmonella Typhimurium was 12 months (range: 1–116 months), significantly younger than the median age of 100 children infected with Salmonella Enteritidis (30 months, range: 3–132 months, p = 0.000).

Table 1.

Major Serovars and Number (Frequency, %) of Salmonella enterica Recovered from Diarrheal Children, Foods, and Environmental Water

Diarrheal children (N = 742)		Chicken meat (N = 158)		Cattle meat (N = 21)		Duck meat (N = 110)		Pork (N = 490)		Cooked meat (N = 61)		Freshwater fishes/shellfishes/frogs/turtles (N = 200)		Sea fishes/shellfishes (N = 78)		River/sewage water (N = 267)
Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)	Serovar	n (%)
Typhimurium	316 (42.6)	Enteritidis	77 (48.7)	Typhimurium	8 (38.1)	Typhimurium	40 (36.4)	Derby	249 (50.8)	Typhimurium	12 (19.7)	Thompson	48 (24.0)	Thompson	20 (25.6)	Derby	29 (10.9)
Enteritidis	209 (28.2)	Indiana	24 (15.2)	Derby	5 (23.8)	Enteritidis	21 (19.1)	Typhimurium	105 (21.4)	Enteritidis	11 (18.0)	Typhimurium	24 (12.0)	Aberdeen	9 (11.5)	Mbandaka	25 (9.4)
Thompson	30 (4.0)	Derby	10 (6.3)	Aberdeen	1 (4.8)	Derby	9 (8.2)	London	40 (8.2)	Indiana	8 (13.1)	Aberdeen	20 (10.0)	Agona	7 (9.0)	Senftenberg	22 (8.2)
Derby	23 (3.1)	Typhimurium	10 (6.3)	Enteritidis	1 (4.8)	Agona	4 (3.6)	Rissen	19 (3.9)	Derby	7 (11.5)	Wandsworth	20 (10.0)	Derby	5 (6.4)	Infantis	19 (7.1)
Stanley	21 (2.8)	Meleagridis	4 (2.5)	Meleagridis	1 (4.8)	Kottbus	4 (3.6)	Meleagridis	14 (2.9)	Senftenberg	3 (4.9)	Havana	9 (4.5)	Typhimurium	4 (5.1)	Typhimurium	15 (5.6)
Agona	19 (2.6)	Rissen	4 (2.5)	Livingstone	1 (4.8)	Indiana	4 (3.6)	Give	12 (2.4)	Orion	2 (3.3)	Saintpaul	9 (4.5)	Stanley	4 (5.1)	Agona	14 (5.2)
London	15 (2.0)	Thompson	4 (2.5)	Muenster	1 (4.8)	Liverpool	3 (2.7)	Enteritidis	9 (1.8)	Give	2 (3.3)	Virchow	8 (4.0)	Havana	3 (3.8)	Enteritidis	14 (5.2)
Infantis	14 (1.9)	Agona	2 (1.3)	Senftenberg	1 (4.8)	Mbandaka	3 (2.7)	Anatum	8 (1.6)	London	2 (3.3)	Give	7 (3.5)	Rissen	3 (3.8)	Thompson	14 (5.2)
Newport	12 (1.6)	London	2 (1.3)	Paratyphi B	1 (4.8)	Rissen	2 (1.8)	Agona	5 (1.0)	Rissen	2 (3.3)	Derby	7 (3.5)	Saintpaul	3 (3.8)	Meleagridis	13 (4.9)
Braenderup	7 (0.9)	Senftenberg	2 (1.3)	Indiana	1 (4.8)	Senftenberg	2 (1.8)	Thompson	4 (0.8)	Saintpaul	2 (3.3)	Livingstone	5 (2.5)	Paratyphi B	3 (3.8)	Give	12 (4.5)
		Anatum	2 (1.3)			Washington	2 (1.8)	Infantis	4 (0.8)	Thompson	2 (3.3)					Rissen	11 (4.1)
		Infantis	2 (1.3)			Anatum	2 (1.8)	Newport	3 (0.6)

Forty-three Salmonella serovars were identified in 779 Salmonella isolates from raw animal meat, of which Salmonella Typhimurium and Salmonella Enteritidis accounted for 21.9% and 13.9%, respectively. Salmonella Typhimurium was the most common in cattle and duck meat and the second most common in pork, whereas Salmonella Enteritidis was the most common in chicken meat. Nineteen serovars were identified in 61 Salmonella isolates from cooked animal meat, of which Salmonella Typhimurium and Salmonella Enteritidis accounted for 19.7% and 18.0%, respectively, being the most and the second most common serovar. Thirty-three serovars were identified in 200 Salmonella isolates from freshwater fishes/shellfishes and turtles, of which Salmonella Typhimurium accounted for 12.0%, being the second most common serovar. Twenty-five serovars were identified in 78 Salmonella isolates from sea fishes/shellfishes, of which Salmonella Typhimurium accounted for 5.1%, being the fifth most common serovar. Fifty-two serovars were identified in 267 Salmonella isolates from environmental water, of which Salmonella Typhimurium and Salmonella Enteritidis accounted for 5.6% and 5.2%, respectively, being the fourth and fifth most common serovar.

Overall, Salmonella Typhimurium was widely detected in diverse animal foods and environmental water, whereas Salmonella Enteritidis was frequently detected in chicken and duck meat and environmental water.

PFGE patterns of Salmonella Typhimurium and Salmonella Enteritidis isolates

Ninety-five PFGE patterns were generated from 148 human Salmonella Typhimurium isolates and 32 PFGE patterns were generated from 100 human Salmonella Enteritidis isolates. As given in Table 2, there were 11 and 10 separate PFGE patterns containing more than one Salmonella Typhimurium isolates and Salmonella Enteritidis isolates, respectively.

Table 2.

The Predominant XbaI-Pulsed-Field Gel Electrophoresis Patterns of Salmonella Typhimurium and Salmonella Enteritidis

Salmonella. Typhimurium (N = 148)		Salmonella Enteritidis (N = 100)
XbaI-PFGE pattern	Isolates, n (%)	XbaI-PFGE pattern	Isolates, n (%)
JPXX01.SH0002	10 (6.8)	JEGX01.SH0003	34 (34.0)
JPXX01.SH0003	10 (6.8)	JEGX01.SH0001	13 (13.0)
JPXX01.SH0004	6 (4.1)	JEGX01.SH0002	9 (9.0)
JPXX01.SH0006	6 (4.1)	JEGX01.SH0092	4 (4.0)
JPXX01.SH0005	4 (2.7)	JEGX01.SH0007	3 (3.0)
JPXX01.SH0008	4 (2.7)	JEGX01.SH0010	3 (3.0)
JPXX01.SH0124	4 (2.7)	JEGX01.SH0011	3 (3.0)
JPXX01.SH0007	3 (2.0)	JEGX01.SH0019	3 (3.0)
JPXX01.SH0009	3 (2.0)	JEGX01.SH0050	3 (3.0)
JPXX01.SH0010	3 (2.0)	JEGX01.SH0005	2 (2.0)
JPXX01.SH0012	3 (2.0)

PFGE, pulsed-field gel electrophoresis.

We obtained the PFGE patterns of 21 Salmonella Enteritidis isolates and 37 Salmonella Typhimurium isolates recovered from foods and environmental water during 2012–2014 through Shanghai PulseNet database. By comparative analysis of the PFGE patterns of human and nonhuman isolates, 10 Salmonella Typhimurium clusters contained both human and nonhuman isolates, whereas all human and nonhuman Salmonella Enteritidis isolates were located in a single cluster. As given in Figures 1 and 2, 33.1% of clinical Salmonella Typhimurium isolates belonged to the dominant PFGE cluster 3, which shared high similarity (85–100%) in PFGE banding patterns to the same serovar isolates from poultry source, animal products, aquatic products, and water; 97.0% of clinical Salmonella Enteritidis isolates shared extremely high similarity (85–100%) in PFGE banding patterns to the same serovar isolates from poultry source and environmental water.

FIG. 1.

XbaI PFGE patterns and antimicrobial resistance profiles of Salmonella Typhimurium isolates in this study. An 85% similarity threshold defines 10 major cluster (cluster1 to cluster10) among 126 Salmonella Typhimurium human isolates and 37 nonhuman Salmonella Typhimurium isolates. AMC, amoxicillin/clavulanic acid or amoxicillin–clavulanate; AXO, ceftriaxone; COT, trimethoprim/sulfamethoxazole; TET, tetracycline; TIO, ceftiofur; FOX, cefoxitin; GEN, gentamicin; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; FIS, sulfisoxazole; NAL, nalidixic acid; PFGE, pulsed-field gel electrophoresis; STR, streptomycin; AZI, azithromycin; IPM, imipenem; OFX, ofloxacin; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; W, trimethoprim; S3, sulfonamides; NONE, isolates are susceptible to all of the test agents.

FIG. 2.

XbaI PFGE patterns and antimicrobial resistance profiles of Salmonella Enteritidis isolates in this study. An 85% similarity threshold defines 1 major cluster (cluster1) among 56 human Salmonella Enteritidis isolates and 21 nonhuman Salmonella Enteritidis isolates.AMC: amoxicillin/clavulanic acid or amoxicillin–clavulanate; AXO, ceftriaxone; COT, trimethoprim/sulfamethoxazole; TET, tetracycline; TIO, ceftiofur, FOX, cefoxitin; GEN, gentamicin; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; FIS, sulfisoxazole; NAL, nalidixic acid; PFGE, pulsed-field gel electrophoresis; STR, streptomycin; AZI, azithromycin; IPM, imipenem; OFX, ofloxacin; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; W, trimethoprim; S3, sulfonamides; NONE, isolates are susceptible to all of the test agents.

Antimicrobial resistance phenotypes and patterns

As given in Table 3, the frequencies of multidrug resistance among human Salmonella Typhimurium and Salmonella Enteritidis isolates were 83.8% and 79.0%, respectively. The frequencies of resistance to ceftriaxone (MIC ≥4 μg/mL) among Salmonella Enteritidis and Salmonella Typhimurium isolates were 42.0% and 14.2%, respectively (p = 0.000). Based on the ESBL phenotype confirmation test, 15.5% of Salmonella Typhimurium isolates and 41% of Salmonella Enteritidis isolates were ESBL/AmpC producing. However, 35.1% of Salmonella Typhimurium isolates displayed resistance to ciprofloxacin (MIC ≥1 μg/mL); 64.9% of Salmonella Typhimurium isolates and 97.0% of Salmonella Enteritidis isolates displayed reduced susceptibility to ciprofloxacin (MIC ≥0.12 μg/mL). The concurrent resistance to both ciprofloxacin and ceftriaxone was detected in 6.8% of Salmonella Typhimurium isolates but was not found in Salmonella Enteritidis isolates. Resistance to azithromycin was identified in 3.4% of Salmonella Typhimurium isolates but not found in Salmonella Enteritidis.

Table 3.

Rates of Resistance by 14 Antimicrobial Agents Among Salmonella Typhimurium Isolates and Salmonella Enteritidis Isolates Recovered from Diarrhea Children Using the Minimum Inhibitory Concentrations

Diarrheal children	Isolates n (%)
	Salmonella Typhimurium (N = 148)		Salmonella Enteritidis (N = 100)
	R	I	R	I
Ampicillin	118 (79.7))	1 (0.7)	81 (81.0)	1 (1.0)
Amoxicillin–clavulanic acid	17 (11.5)	58 (39.2)	11 (11.0)	27 (27.0)
Ceftiofur	21 (14.2)	2 (1.4)	43 (43.0)	3 (3.0)
Cefoxitin	8 (5.4)	6 (4.1)	4 (4.0)	3 (3.0)
Ceftriaxone	21 (14.2)	0 (0)	42 (42.0)	0 (0)
Tetracycline	118 (79.7)	1 (0.7)	21 (21.0)	0 (0)
Chloramphenicol	71 (48.0)	3 (2.0)	9 (9.0)	3 (3.0)
Azithromycin	5 (3.4)	0 (0)	0 (0)	0 (0)
Nalidixic acid	75 (50.7)	0 (0)	97 (97.0)	0 (0)
Ciprofloxacin	52 (35.1)	44 (29.7)	0 (0)	97 (97.0)
Sulfisoxazole	127 (85.8)	0 (0)	51 (51.0)	0 (0)
Trimethoprim–sulfamethoxazole	67 (45.3)	0 (0)	2 (2.0)	0 (0)
Streptomycin	81 (54.7)	0 (0)	48 (48.0)	0 (0)
Gentamicin	40 (27.0)	4 (2.7)	5 (5.0)	0 (0)
MDR patterns
MDR (≥3 CLSI classes)	124 (83.8)		79 (79.0)
ACSSuT	32 (21.6)		7 (7.0)
ACSSuTAuCx	2 (1.4)		2 (2.0)
DSC	96 (64.9)		97 (97.0)
At least DSC and ceftriaxone	15 (10.1)		42 (42.0)
ESBLs/AmpC phenotype	23 (15.5)		41 (41.0)

MDR, multidrug resistance; R, resistant; I, intermediate; CLSI, Clinical and Laboratory Standards Institute; ACSSuT, resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole/sulfisoxazole, and tetracycline; ACSSuTAuCx, resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole/sulfisoxazole, tetracycline, amoxicillin–clavulanic acid, and ceftriaxone; DSC, decreased susceptibility to ciprofloxacin, NARMS categorized Salmonella isolates with intermediate or resistant MICs (≥0.12 μg/mL) for ciprofloxacin as having DSC; NARMS, National Antimicrobial Resistance Monitoring System; ESBLs, extended-spectrum β-lactamase; AmpC, AmpC beta-lactamase; MICs, minimum inhibitory concentrations.

During the same period, 208 nonhuman Salmonella Typhimurium isolates and 130 nonhuman Salmonella Enteritidis isolates were tested for antimicrobial susceptibility. As given in Tables 4 and 5, the overall frequency of resistance to ciprofloxacin was 26.0% in Salmonella Typhimurium and 2.3% in Salmonella Enteritidis; however, Salmonella Typhimurium isolates recovered from freshwater food animals and Salmonella Enteritidis isolates recovered from pork showed a higher frequency of resistance to ciprofloxacin, being 69.6% and 9.1%, respectively (shown in Tables 4 and 5). The overall frequency of resistance to cefotaxime was 2.4% in Salmonella Typhimurium isolates and 6.9% in Salmonella Enteritidis isolates; however, Salmonella Typhimurium isolates recovered from freshwater food animals and Salmonella Enteritidis isolates recovered from pork showed a higher frequency of resistance to cefotaxime, being 13.0% and 18.2%, respectively (given in Tables 4 and 5). Only 1.4% of Salmonella Typhimurium isolates and 0.8% of Salmonella Enteritidis isolates were co-resistant to cefotaxime and ciprofloxacin.

Table 4.

Rates of Resistance by 16 Antimicrobial Agents Among Nonhuman Salmonella Typhimurium Isolates Using the Kirby–Bauer Disk-Diffusion Method

Salmonella Typhimurium	Freshwater fishes/shellfishes/frogs/turtles (N = 23)		Seafishes/shellfishes (N = 5)		Chicken meat (N = 11)		Duck meat (N = 41)		Cattle meat (N = 8)		Pork (N = 105)		River/sewage water (N = 15)		Total (N = 208)
Salmonella Typhimurium	R	I	R	I	R	I	R	I	R	I	R	I	R	I	R	I
Tetracycline	22 (95.7)	0 (0)	3 (60.0)	0 (0)	5 (45.5)	0 (0)	4 (9.8)	0 (0)	4 (50.0)	0 (0)	89 (84.8)	2 (1.9)	14 (93.3)	0 (0)	141 (67.8)	2 (1.0)
Imipenem	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Amoxicillin–clavulanate	13 (56.5)	7 (30.4)	1 (20.0)	0 (0)	2 (18.2)	1 (9.1)	1 (2.4)	2 (4.9)	0 (0)	1 (12.5)	13 (12.4)	32 (30.5)	3 (20.0)	4 (26.7)	33 (15.9)	47 (22.6)
Ampicillin	20 (87.0)	0 (0)	3 (60.0)	0 (0)	6 (54.5)	0 (0)	4 (9.8)	0 (0)	3 (37.5)	0 (0)	86 (81.9)	0 (0)	12 (80.0)	0 (0)	134 (64.4)	0 (0)
Trimethoprim-sulfamethoxazole	13 (56.5)	0 (0)	3 (60.0)	0 (0)	3 (27.3)	0 (0)	2 (4.9)	0 (0)	1 (12.5)	0 (0)	68 (64.8)	0 (0)	9 (60.0)	0 (0)	99 (47.6)	0 (0)
Ciprofloxacin	16 (69.6)	6 (26.1)	1 (20.0)	3 (60.0)	2 (18.2)	7 (63.6)	2 (4.9)	38 (92.7)	0 (0)	6 (75.0)	28 (26.7)	72 (68.6)	5 (33.3)	6 (40.0)	54 (26.0)	138 (66.3)
Chloramphenicol	14 (60.9)	1 (4.3)	2 (40.0)	1 (20.0)	4 (36.4)	0 (0)	3 (7.3)	0 (0)	1 (12.5)	0 (0)	69 (65.7)	0 (0)	8 (53.3)	0 (0)	101 (48.6)	2 (1.0)
Ofloxacin	4 (17.4)	1 (4.3)	0 (0)	0 (0)	1 (9.1)	0 (0)	0 (0)	2 (4.9)	0 (0)	0 (0)	1 (1.0)	8 (7.6)	0 (0)	0 (0)	6 (2.9)	11 (5.3)
Nalidixic acid	14 (60.9)	4 (17.4)	4 (80.0)	0 (0)	8 (72.7)	1 (9.1)	34 (82.9)	0 (0)	5 (62.5)	2 (25.0)	59 (56.2)	14 (13.3)	7 (46.7)	2 (13.3)	131 (63.0)	23 (11.1)
Cefepime	0 (0)	1 (4.3)	0 (0)	0 (0)	0 (0)	0 (0)	1 (2.4)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0.5)	1 (0.5)
Cefotaxime	3 (13.0)	6 (26.1)	0 (0)	0 (0)	0 (0)	0 (0)	1 (2.4)	1 (2.4)	1 (12.5)	1 (12.5)	0 (0)	8 (7.6)	0 (0)	0 (0)	5 (2.4)	16 (7.7)
Trimethoprim	13 (56.5)	0 (0)	1 (20.0)	0 (0)	2 (18.2)	0 (0)	2 (4.9)	0 (0)	1 (12.5)	0 (0)	66 (62.9)	1 (1.0)	9 (60.0)	0 (0)	94 (45.2)	1 (0.5)
Ceftazidime	0 (0)	2 (8.7)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (1.0)
Gentamicin	7 (30.4)	0 (0)	1 (20.0)	0 (0)	3 (27.3)	0 (0)	2 (4.9)	0 (0)	0 (0)	0 (0)	37 (35.2)	1 (1.0)	5 (33.3)	0 (0)	55 (26.4)	1 (0.5)
Sulfonamides	22 (95.7)	0 (0)	4 (80.0)	0 (0)	6 (54.5)	0 (0)	6 (14.6)	4 (9.8)	1 (75.0)	1 (12.5)	92 (87.6)	3 (2.9)	14 (93.3)	1 (6.7)	150 (72.1)	9 (4.3)
Streptomycin	18 (78.3)	5 (21.7)	4 (80.0)	1 (20.0)	8 (72.7)	0 (0)	25 (61.0)	15 (36.6)	7 (87.5)	1 (12.5)	92 (87.6)	12 (11.4)	14 (93.3)	1 (6.7)	168 (80.8)	35 (16.8)
Cefotaxime and ciprofloxacin	3 (13.0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	3 (1.4)	0 (0)

Values are given as n (%). R, resistant; I, intermediate.

Table 5.

Rates of Resistance by 16 Antimicrobial Agents Among Nonhuman Salmonella Enteritidis Isolates Using the Kirby-Bauer Disk-Diffusion Method

Salmonella Enteritidis	Freshwater fishes/shellfishes/frogs/turtles (N = 2)		Chicken meat (N = 81)		Duck meat (N = 21)		Cattle meat (N = 1)		Pork (N = 11)		River/sewage water (N = 14)		Total (N = 130)
Salmonella Enteritidis	R	I	R	I	R	I	R	I	R	I	R	I	R	I
Tetracycline	0 (0)	0 (0)	20 (24.7)	0 (0)	5 (23.8)	0 (0)	1 (100.0)	0 (0)	1 (9.1)	0 (0)	3 (21.4)	0 (0)	30 (23.1)	0 (0)
Imipenem	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Amoxicillin–clavulanate	0 (0)	0 (0)	4 (4.9)	30 (37.0)	0 (0)	3 (14.3)	0 (0)	0 (0)	1 (9.1)	5 (45.5)	1 (7.1)	2 (14.3)	6 (4.6)	40 (30.8)
Ampicillin	1 (50.0)	0 (0)	52 (64.2)	0 (0)	16 (76.2)	0 (0)	1 (100.0)	0 (0)	8 (72.7)	0 (0)	10 (71.4)	0 (0)	88 (67.7)	0 (0)
Trimethoprim–sulfamethoxazole	0 (0)	0 (0)	7 (8.6)	2 (2.5)	1 (4.8)	0 (0)	0 (0)	0 (0)	3 (27.3)	0 (0)	0 (0)	0 (0)	11 (8.5)	2 (1.5)
Ciprofloxacin	0 (0)	2 (100.0)	2 (2.5)	78 (96.3)	0 (0)	20 (95.2)	0 (0)	1 (100.0)	1 (9.1)	9 (81.8)	0 (0)	14 (100.0)	3 (2.3)	124 (11.3)
Chloramphenicol	0 (0)	0 (0)	4 (4.9)	1 (1.2)	1 (4.8)	0 (0)	0 (0)	0 (0)	1 (9.1)	1 (9.1)	1 (7.1)	0 (0)	7 (5.4)	2 (1.5)
Ofloxacin	0 (0)	0 (0)	1 (1.2)	1 (1.2)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0.8)	1 (0.8)
Nalidixic acid	2 (100.0)	0 (0)	80 (98.8)	0 (0)	21 (100.0)	0 (0)	1 (100.0)	0 (0)	11 (100.0)	0 (0)	14 (100.0)	0 (0)	129 (99.2)	0 (0)
Cefepime	0 (0)	0 (0)	2 (2.5)	1 (1.2)	1 (4.8)	0 (0)	0 (0)	0 (0)	1 (9.1)	0 (0)	0 (0)	0 (0)	4 (3.1)	1 (0.8)
Cefotaxime	0 (0)	0 (0)	5 (6.2)	2 (2.5)	1 (4.8)	0 (0)	0 (0)	0 (0)	2 (18.2)	0 (0)	1 (7.1)	0 (0)	9 (6.9)	2 (1.5)
Trimethoprim	0 (0)	0 (0)	3 (3.7)	1 (1.2)	0 (0)	0 (0)	0 (0)	0 (0)	3 (27.3)	0 (0)	0 (0)	0 (0)	6 (4.6)	1 (0.8)
Ceftazidime	0 (0)	0 (0)	2 (2.5)	1 (1.2)	1 (4.8)	0 (0)	0 (0)	0 (0)	1 (9.1)	0 (0)	1 (7.1)	0 (0)	5 (3.8)	1 (0.8)
Gentamicin	0 (0)	0 (0)	2 (2.5)	0 (0)	4 (19.0)	0 (0)	0 (0)	0 (0)	1 (9.1)	0 (0)	0 (0)	0 (0)	7 (5.4)	0 (0)
Sulfonamides	1 (50.0)	0 (0)	52 (64.2)	2 (2.5)	13 (61.9)	1 (4.8)	1 (100.0)	0 (0)	5 (45.5)	0 (0)	10 (71.4)	1 (7.1)	82 (63.1)	4 (3.1)
Streptomycin	1 (50.0)	1 (50.0)	52 (64.2)	15 (18.5)	13 (61.9)	3 (14.3)	1 (100.0)	0 (0)	6 (54.5)	4 (36.4)	10 (71.4)	3 (21.4)	83 (63.8)	26 (20.0)
Cefotaxime and ciprofloxacin	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (9.1)	0 (0)	0 (0)	0 (0)	1 (0.8%)	0 (0)

Values are given as n (%). R, resistant; I, intermediate.

Prevalence of β-lactamase resistance-encoding genes among Salmonella Typhimurium and Salmonella Enteritidis isolates

Of 64 ESBL/AmpC-producing isolates, 55 (85.9%) contained blaCTX-M, 40 (62.5%) contained blaTEM-1, 5 (7.8%) contained blaDHA-1, 2 (3.1%) contained blaCMY-2 and 2 (3.1%) contained blaSHV-12. Thirty-four (53.1%) of 64 isolates co-harbored blaCTX-M and blaTEM-1 genes, 1 (1.6%) co-harbored blaDHA-1 and blaTEM-1 genes, 2 (3.1%) co-harbored blaDHA-1, blaSHV-12, and blaTEM-1 genes, and 1 (1.6%) co-harbored blaTEM-1 and blaCMY-2. Of 55 blaCTX-M-containing isolates, CTX-M-55, CTX-M-14, CTX-M-64 were identified in 43 (78.2%), 11 (20.0%) and 1 (1.8%) isolates, respectively.

Discussion

This study revealed the epidemiological link between sporadic Salmonella Enteritidis and Salmonella Typhimurium infections in Shanghainese children and possible exposure to foods of animal origin and environmental water based on the similar distribution of common serovars and PFGE profiles. The high prevalence of ESBL/AmpC-producing Salmonella Enteritidis and ciprofloxacin-resistant Salmonella Typhimurium was a serious concern in Shanghai. These findings are significant for designing the intervention measures to control the transmission of these two predominant NTS serovars and recommending appropriate antibiotic for the empirical treatment of serovar-specific NTS infection.

A recent case–control study showed exposure to diarrheal patient is not a risk factor for sporadic enteric NTS-associated diarrhea in Shanghainese children (Chang et al., 2017). In this study, we observed Salmonella Enteritidis and Salmonella Typhimurium serovars were commonly recovered from diarrheal children, animal foods, and river/sewage water. Furthermore, comparative analysis of PFGE profiles suggests that ingestion of exposure to contaminant poultry meat or environmental water is linked with a major transmission route of Salmonella Enteritidis in children. Although Salmonella Typhimurium was widely detected in poultry and livestock meat, freshwater and sea products, and environmental water, only 33.1% of clinical isolates within a dominant cluster showed high similarity in PFGE patterns with nonhuman isolates, suggesting the diverse transmission sources of Salmonella Typhimurium in children. Further food-specific analysis revealed Salmonella Typhimurium from pork, sea fishes, and environmental water shared 100% similarity with clinical isolates in PFGE patterns, highly suggesting ingestion of or exposure to contaminant pork or sea fishes or environmental water being linked to the major transmission route of Salmonella Typhimurium in children. Obviously, the main transmission vehicles of Salmonella Typhimurium and Salmonella Enteritidis are different and the transmission sources of Salmonella Typhimurium are more diverse. The difference in the main transmission vehicles of Salmonella Typhimurium and Salmonella Enteritidis could explain why infants and young children were more susceptible to Salmonella Typhimurium than to Salmonella Enteritidis that usually affected older children.

At present, the spread of ciprofloxacin-resistant and third-generation cephalosporins-resistant S. enterica is one of the major public health concerns, especially in developing countries (Hur et al., 2012). This situation was serious in human and animal foods in China (Yu et al., 2011; Cao et al., 2017; Wang et al., 2017). In European countries, at present, the proportions of Salmonella isolates resistant to either of ciprofloxacin (0.6%) and cefotaxime (0.5%) were overall relatively low (EFSA and ECDC 2018). In this study, both the prevalence of resistance to ciprofloxacin in Salmonella Typhimurium and the prevalence of resistance to ceftriaxone in Salmonella Enteritidis were significantly higher than that reported in South Korea and European countries (Kiliç et al., 2001; Maraki and Papadakis, 2014; Wang et al., 2015a; Petrov et al., 2017; EFSA and ECDC 2018). Of particular note, 6.8% of human Salmonella Typhimurium isolates were co-resistant to cefotaxime and ciprofloxacin in this study. The current status of resistance patterns makes the empirical antibiotic treatment for severe NTS infections complicated in Shanghai. Although the proportions of nonhuman Salmonella Typhimurium and Salmonella Enteritidis isolates resistant to cefotaxime and ciprofloxacin were overall low, these two serovars recovered from freshwater food animals and pork displayed relatively high frequency of resistance to cefotaxime and ciprofloxacin, which were similar to that in human isolates. These findings indirectly indicated exposure to contaminated freshwater products and pork being a major source of transmission for children to acquire NTS infections in Shanghai. A latest study from 13 provinces of China showed that a high prevalence (68.2%) of resistance to ciprofloxacin among Salmonella isolates was likely correlated to the common use of these antimicrobials in poultry farms in China (Wang et al., 2017). The distinct resistance patterns between human and nonhuman isolates and by locations could be related to the different antibiotic consumption and use for human and animals, resulting in the selected high-level resistance to these two clinically important antibiotics (Chiu et al., 2004).

BlaTEM, blaCMY, blaPSE, blaSHV, blaOXA, blaDHA, blaACC, and blaCTX-M genes have been detected in NTS isolates recovered from both poultry and human in some provinces of China (Yang et al., 2014). In Shanxi provinces, >50% of ceftriaxone-resistant and/or cefoperazone-resistant Salmonella isolates from retail meat harbored blaTEM or blaCMY-2 (Yang et al., 2010). In Wuhan, Beijing, and Chongqing blaCTX-M-14, blaCTX-M-15, and blaTEM-1 genes were commonly detected in clinical Salmonella isolates displaying high-level resistance to third-generation cephalosporins (MIC ≥32 mg/L) (Wong et al., 2015; Tian et al., 2016). In this study, blaCTX-M-55, blaCTX-M-14, blaCTX-M-64, blaTEM-1, blaSHV-12, blaDHA-1, and blaCMY-2 were detected in clinical Salmonella isolates. The result is similar to the previous reports that CTX-M-group ESBLs are now by far the most common ESBLs globally (D'Andrea et al., 2013). Unlike the reports from the United Kingdom, Italy, and other countries, where blaCTX-M-14 genes and blaCTX-M-15 genes are most commonly found in humans and blaCTX-M-1 genes are commonly found in animals, blaCTX-M-55 gene was most commonly found in Shanghai clinical isolates (Doi et al., 2017). In addition, blaCTX-M-64 gene was detected among Salmonella Enteritidis isolates in this study that had been well documented in E. coli isolates or Shigella sonnei isolates within animals and patients all over the world (Nagano et al., 2009; Sun et al., 2010; Gu et al., 2015; Pfeifer et al., 2018). Thus far, blaCTX-M-64 has rarely been described in human Salmonella isolates in China. This finding may suggest blaCTX-M-64 dissemination through horizontal transfer. Meanwhile, we also noticed that 53.1% of isolates co-harbored two kinds of ESBL-encoding genes and coexistence of ESBLs and AmpC β-lactamases were detected in 1.6% of Salmonella Enteritidis isolates and 4.7% of Salmonella Typhimurium isolates. Of special note, the majority of ESBL/AmpC β-lactamase-producing Salmonella isolates showed a high level of resistance to ceftriaxone (MIC ≥64 mg/L). Taken together, the prevalence of β-lactamase-encoding genes in S. enterica varies by Salmonella serovars, sample sources, geographical regions, and antibiotic consumption patterns. It is worthy of further investigating the correlation between the level of resistance and the different groups of β-lactamases and their alleles conferring resistance to third-generation cephalosporins.

Conclusion

The transmission sources of Salmonella Enteritidis in diarrheal children mainly originated from animal foods and environmental water. Of particular concern, the transmission sources and routes of the most predominant serovar Salmonella Typhimurium were diverse; furthermore, Salmonella Typhimurium displayed the high prevalence of resistance to third-generation cephalosporins and ciprofloxacin. Ongoing surveillance of NTS infections and antimicrobial resistance, especially Salmonella Enteritidis and Salmonella Typhimurium, is important to identify and prioritize successful food safety interventions and appropriately treat NTS illnesses in Shanghai.

Footnotes

Acknowledgments

We sincerely thank physicians and nurses from infectious disease clinic for their routinely collecting the fecal specimen and recording the medical history.

Disclosure Statement

No competing financial interests exist.

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