Phenotypic Characteristics and Genetic Diversity of Salmonella enterica Serotype Derby Isolated from Human Patients and Foods of Animal Origin

Abstract

Salmonella enterica serotype Derby is among the three most common serotypes of nontyphoidal Salmonella isolated from patients with diarrhea in China. In this study, 133 Salmonella Derby isolates from human patients (n = 74) and foods of animal origin (n = 59) in Shanghai, China, between September 2013 and December 2014, were selected to study its phenotypic characteristics and genetic diversity. The isolates were subjected to antimicrobial susceptibility testing, plasmid replicon typing, virulence profile determination, and molecular subtyping by pulsed-field gel electrophoresis (PFGE). Isolates were frequently resistant to tetracycline (87.22%), sulfisoxazole (74.44%), and streptomycin (62.41%), and a low frequency of resistance was found toward ofloxacin (3.01%), ceftazidime (2.26%), and cefepime (1.50%); in addition, 93 (69.92%) isolates were multidrug resistant. The most common plasmid incompatibility replicon types were the IncF family (FIA, 51.31%; FIC, 27.82%; and FIB, 21.80%) and IncP types (35.34%): these plasmid types may be associated with the spread of antibiotic resistance and virulence genes. All isolates were positive for the Salmonella pathogenicity island (SPI) gene avrA and the fimbrial gene bcfC from among the 10 virulence genes detected, and most of them carried ssaQ (99.25%), mgtC (97.74%), siiD (98.50%), sopB (97.74%), and sopE (96.99%). PFGE showed 68 patterns in nine main clusters at an 85% similarity threshold. Most of the isolates from different sources possessed the same fingerprints or molecular profiles in each cluster, which strongly suggests the possibility that foods of animal origin, especially pork, serve as an important source for human infection. Moreover, this diversity may suggest strains originating from multiple clones. Therefore, surveillance on this serotype should be strengthened to prevent transmission of Salmonella Derby from foods of animal origin, especially pork, to humans.

Introduction

S almonella enterica is the leading cause of human gastroenteritis worldwide, with 2637 serotypes identified on the basis of the O (lipopolysaccharide) and H (flagella) antigens (Issenhuth-Jeanjean et al., 2014). Salmonella serotypes differ in their natural reservoirs and ability to cause human infections, but only a small proportion is responsible for the majority of human infections (Jackson et al., 2013). These serotypes often have a wide range of hosts and are strongly associated with animals and agricultural products; therefore, they can easily spread through consumption of contaminated foods (Carrasco et al., 2012).

Salmonella Derby was first isolated from an outbreak of pork pie poisoning in humans in Derby, Britain (Peckham and Savage, 1923). Recently, it has been recognized as a foodborne pathogen and is mainly associated with slaughter pigs or pork products in many parts of the world (Jayarao et al., 1990; Mannion et al., 2007; Kerouanton et al., 2013). In China, Salmonella Derby isolates seem to play a more active role as they not only represent the third most common serotype isolated from patients with diarrhea after Salmonella Enteritidis and Salmonella Typhimurium (Ran et al., 2011) but also are typically one of the most commonly isolated serotypes from pork (Cai et al., 2016). In addition, Salmonella Derby was among the 10 most frequently isolated serotypes from human sources in Germany (Hauser et al., 2011), France (Kerouanton et al., 2013), and America (Valdezate et al., 2005). Outbreaks and sporadic illness caused by Salmonella Derby have spread worldwide over the past decade (Ebuchi et al., 2006; Hendriksen et al., 2011; Jackson et al., 2013; Arnedo-Pena et al., 2016). Pork is China's staple meat, and the average person in China eats more pork (intake increased from 37.1 g/d in 1992 to 64.3 g/d in 2012) than an average person in any other country (He et al., 2016). Therefore, it is important to specifically focus on the dangers of Salmonella Derby spread from pork to humans.

In recent years, the emergence of multidrug-resistant (MDR) Salmonella has grown as a global concern and has been attributed to the overuse of antibiotics in humans and livestock. Multiple studies have described MDR Salmonella Derby strains isolated from humans, retail meats, and foods of animal origin, which have become a significant public health security concern in many countries and regions (Hauser et al., 2011; Bleicher et al., 2013; Lin et al., 2015).

The objectives of this study were (1) to provide a detailed analysis of the antimicrobial resistance, virulence, and plasmid replicon types of Salmonella Derby from human clinical specimens and foods of animal origin, (2) to compare the genetic diversity or similarity by using a combination of different molecular profiles, and (3) to assess the epidemiologic relatedness of isolates.

Materials and Methods

Bacterial isolates

From September 2013 to December 2014, a total of 1968 S. enterica isolates were isolated from human patients (n = 1585) and foods of animal origin (n = 383). Of the 1968 Salmonella isolates, 133 belonged to Salmonella Derby and were selected for this study; among the samples, 74 were of human origin, and 59 were from foods of animal origin (44 pork, 4 duck meat, 4 chicken meat, 3 beef, 2 chicken egg, 1 mutton, and 1 aquatic product). The human origin samples were from feces of diarrhea patients from 26 sentinel hospitals and eight regional Shanghai Center for Disease Control and Prevention diagnostic laboratories spread across 10 administrative regions of Shanghai. The foods of animal origin were sampled by the Global Foodborne Infections Network Surveillance System from supermarkets and retailers in Shanghai over the same period.

Antimicrobial susceptibility testing

All isolates of Salmonella Derby used in this study were tested for their antimicrobial susceptibility to 16 antimicrobial agents by the Kirby–Bauer disk diffusion method (CLSI, 2009). The following antibiotic disks (Oxoid, United Kingdom) were used (content in μg): ampicillin (AMP, 10), amoxicillin/clavulanate acid (AMC, 20/10), cefotaxime (CTX, 30), cefepime (FEP, 30), ceftazidime (CAZ, 30), imipenem (IPM, 10), streptomycin (STR, 10), gentamicin (GEN, 10), tetracycline (TET, 30), chloramphenicol (CHL, 30), nalidixic acid (NAL, 30), ciprofloxacin (CIP, 5), ofloxacin (OFX, 5), trimethoprim (TMP, 5), sulfisoxazole (SUL, 300), and trimethoprim/sulfamethoxazole (STX, 1.25/23.75). Escherichia coli strain ATCC 25922 was used as a quality control organism. The classes of resistance levels were defined as described by the Clinical and Laboratory Standards Institute (CLSI, 2010).

Polymerase chain reaction detection of plasmid replicon typing

All 133 Salmonella Derby isolates were examined for the presence of 18 plasmid replicons by a simplex polymerase chain reaction (PCR) method (Carattoli et al., 2005). Genomic DNA was extracted using a DNA extraction kit (Qiagen, Germany) according to the manufacturer's instructions. The PCR reaction was set up in a final volume of 20 μL that contained 10 μL Premix ExTaq mixture (Takara, China), 7 μL ddH₂O, 1 μL template DNA (15–50 ng), and 1 μL of each primer (10 μM; forward and reverse primers). Reactions were carried out under the following cycling conditions: an initial denaturation step at 95°C for 5 min, followed by 30 cycles at 95°C for 30 s, 55°C for 45 s, and 72°C for 90 s; and a final extension of 5 min at 72°C.

PCR detection of virulence genes

Ten virulence genes (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE, spvC, and bcfC) were detected by PCR assays as previously described (Ren et al., 2016). The PCR cycling conditions were as follows: 5 min at 95°C; 30 cycles of 40 s at 94°C, 60 s at 55°C, and 90 s at 72°C; and a final extension of 10 min at 72°C.

Pulsed-field gel electrophoresis analysis

Pulsed-field gel electrophoresis (PFGE) of the 133 Salmonella Derby isolates was performed according to the PulseNet standardized protocol (Ribot et al., 2006). Digested DNA was separated using a Chef Mapper (Bio-Rad, Hercules, CA), and the gels were stained with Lonza GelStar Nucleic Acid Gel Stain (Cambrex Bio Science, Walkersville, MD) and analyzed using BioNumerics software (Applied Maths, Kortrijk, Belgium). Briefly, agarose-embedded DNA was digested with 50 U of XbaI (TaKaRa, Dalian, China) for 1.5–2 h in a water bath at 37°C. Restriction fragments were separated by electrophoresis in a 0.5 × TBE buffer at 14°C for 18 h using a Chef Mapper electrophoresis system, with a pulse time of 2.16–63.8 s. The Salmonella Braenderup H9812 was used as the molecular weight size standard. The unweighted-pair group method with arithmetic means, using 1.00% tolerance limit and 1.00% optimization, was used to obtain the dendrogram.

Statistical analyses

Differences between PFGE, antibiotic resistance patterns (ARPs), and plasmid replicon types were compared using the chi-square test and Fisher's exact test, with odds ratios and 95% confidence intervals. Analyses were performed using SAS 9.2. For all analyses, p < 0.05 was considered statistically significant.

Results

Antimicrobial susceptibility

Of the 133 Salmonella Derby isolates, only 3 isolates were susceptible to all antimicrobials tested, whereas 130 (97.74%) isolates were resistant to one or more of the antimicrobial agents. The strains tested were most often resistant to tetracycline (87.22%), sulfisoxazole (74.44%), and streptomycin (62.41%). All isolates were susceptible to imipenem, and a low frequency of resistance was found toward ofloxacin (3.01%), ceftazidime (2.26%), and cefepime (1.50%) (Table 1).

Table 1.

Antimicrobial Susceptibility Testing and Resistance Phenotypes of Salmonella Derby Isolates

	% Intermediate or resistant isolates (No. of isolates)
Antimicrobial agent	Human (74)		Foods of animal origin (59)		Total (133)
Ampicillin	0	43.24	0	38.98	0	41.35
Amoxicillin/clavulanate acid	35.14	4.05	27.12	5.08	31.58	4.51
Cefotaxime	24.32	4.05	23.73	1.69	24.06	3.01
Cefepime	0	2.70	1.50	0.00	0.75	1.50
Ceftazidime	2.70	2.70	0	1.69	1.50	2.26
Imipenem	0	0	0	0	0	0
Streptomycin	24.32	75.68	54.24	45.76	37.59	62.41
Gentamicin	5.41	29.73	1.69	28.81	3.76	29.32
Tetracycline	0	87.84	0	86.44	0	87.22
Chloramphenicol	16.22	41.89	32.20	32.20	23.31	37.59
Nalidixic acid	24.32	31.08	30.15	42.37	27.07	36.09
Ciprofloxacin	28.38	5.41	22.03	8.47	25.56	6.77
Ofloxacin	24.32	2.70	23.73	3.39	24.06	3.01
Trimethoprim	0	40.54	0	28.81	0	35.34
Sulfisoxazole	10.81	79.73	13.56	67.80	12.03	74.44
Trimethoprim/sulfamethoxazole	1.35	43.24	3.39	37.29	2.26	40.60

Overall, 93 (69.92%) isolates were resistant to three or more antimicrobials (defined as MDR), contributing to 37 distinct MDR profiles. A total of 58 (78.38%) human origin MDR isolates displayed 24 different MDR profiles, and the most common MDR profile was to SIZ-STR-TET (n = 19), followed by AMP-SXT-CHL-NAL-TMP-GEN-SIZ-STR-TET (n = 12). The percentage of MDR strains was slightly higher from human origin than from foods of animal origin. Only 35 (59.32%) of the foods of animal origin displayed MDR, which exhibited 20 distinct MDR profiles, and the pattern most frequently observed was to SIZ-STR-TET (n = 8), followed by AMP-SXT-CHL-NAL-TMP-GEN-SIZ-TET (n = 6).

Plasmid replicon types

The incompatibility (Inc) groups of plasmids were determined by PCR-based replicon typing, which showed that more than half the strains carried an FIA (51.31%) replicon-type plasmid, followed by P (35.34%), FIC (27.82%), FIB (21.80%), I1 (8.27%), HI2 (7.52%), HI1 (6.77%), F_repB (4.51%), T (2.26%), and FIIs (0.75%). Replicon types A/C, B/O, K/B, L/M, N, T, X, and W were not detected from these strains. One or more replicon types were detected in a majority (84.96%) of the isolates. Plasmid replicon typing revealed that all isolates exhibited 21 different replicon combinations. The most frequent combination was FIA-P in 13 isolates.

Virulence genotyping and gene profiles

All the Salmonella Derby isolates were PCR positive for the Salmonella pathogenicity island (SPI) gene avrA and the fimbrial gene bcfC, and most of them carried the ssaQ (99.25%), mgtC (97.74%), siiD (98.50%), sopB (97.74%), and sopE (96.99%) genes. Lower prevalence was observed for the prophage gene gipA (5.26%) and plasmid gene spvC (0.75%). A total of 11 virulence gene profiles (VPs) were identified, of which VP1 (avrA-ssaQ-mgtC-siiD-sopB-sodC1-sopE-bcfC, 82.7%) accounted for the majority, followed by VP2 (avrA-ssaQ-mgtC-siiD-sopB-sopE-bcfC, 5.26%) and VP3 (avrA-ssaQ-mgtC-siiD-sopB-gipA-sopE-bcfC, 3.76%). The rest of the VPs (VP4, VP5, VP6, VP7, VP8, VP9, VP10, and VP11) contained only one or two strains (Table 2).

Table 2.

Virulence Profiles of Salmonella Derby Isolated from Human Patients and Foods of Animal Origin

	Virulence gene
Virulence profiles	avrA	ssaQ	mgtC	siiD	sopB	gipA	sodC1	sopE	spvC	bcfC	No. of isolates
VP1	■	■	■	■	■	□	■	■	□	■	Human (61), food (49)
VP2	■	■	■	■	■	□	□	■	□	■	Human (2), food (5)
VP3	■	■	■	■	■	■	□	■	□	■	Human (4), food (1)
VP4	■	■	□	■	■	□	■	■	□	■	Human (2)
VP5	■	■	■	■	□	□	■	■	□	■	Human (1), food (1)
VP6	■	■	■	■	■	□	■	□	□	■	Human (1), food (1)
VP7	■	■	□	■	■	■	■	■	□	■	Human (1)
VP8	■	■	■	■	■	■	■	□	■	■	Food (1)
VP9	■	■	■	□	■	□	■	■	□	■	Human (1)
VP10	■	■	■	□	□	□	■	■	□	■	Human (1)
VP11	■	□	■	■	■	□	■	□	□	■	Food (1)
Prevalence (%)	100	99.25	97.74	98.5	97.74	5.26	94.74	96.99	0.75	100

“■” indicates the designated virulence gene was present in every strain of the Salmonella Derby tested.

“□” indicates the designated virulence gene was not present in any strain of the Salmonella Derby tested.

Typing by PFGE

The genetic relatedness of the 133 Salmonella Derby isolates in this study was evaluated on the basis of the PFGE pattern analysis (Fig. 1). Most of the strains (127, 95.49%) were typeable by PFGE with XbaI macrorestriction endonuclease, but six isolates could not be subtyped. PFGE typing of the 127 isolates resulted in 68 distinct banding patterns with a similarity index ranging from 53.8% to 100%. The PFGE pattern of isolates from foods of animal origin and human clinical samples showed considerable overlap. At 85% similarity threshold, the dendrogram revealed nine clusters (A to I) with at least two strains. Cluster E was the largest and comprised 33 (24.81%) strains: 20 isolated from human patients and 13 isolated from foods of animal origin (10 pork, 1 mutton, 1 beef, and 1 duck). Cluster B comprised 30 (22.56%) strains isolated from humans and pork. Cluster A consisted of human, pork, and egg isolates, with an 87.65% similarity. Interestingly, isolates from different sources possessed the same fingerprints in each cluster, which strongly suggests the possibility that foods of animal origin serve as a source for human infection.

FIG. 1.

XbaI pulsed-field gel electrophoresis patterns, virulence gene profiles, plasmid replicon types, and antimicrobial resistance profiles of Salmonella Derby isolates from human patients and foods of animal origin. For the virulence and plasmid replicon results, a black box indicates that the isolate was positive for a particular plasmid replicon type or virulence gene. For the susceptibility results, a black box indicates resistance to a particular antimicrobial agent and a gray box indicates intermediate resistance to a particular antimicrobial agent. At 85% similarity threshold, the dendrogram revealed nine clusters (A to I) with at least two strains.

Combination of profiles

Significant associations were observed between PFGE and ARPs (p < 0.05) when isolates from major clusters were compared (Fig. 1). For instance, the main ARP in cluster B was TET-SUL-STR, while that in cluster E was TET-AMP-SXT-CHL-NAL-TMP-GEN-SUL-STR. Furthermore, segregation of plasmid replicon types was noted among clusters. Cluster A had a significantly higher content (p < 0.05) of replicon type FIC (84.21%) compared with cluster B (3.33%), cluster E (21.21%), and cluster F (23.08%). Cluster B had a significantly higher content (p < 0.05) of replicon type P (83.33%) than cluster E (30.30%) and cluster F (23.08%), which in turn had significantly higher content (p < 0.05) than cluster A (0%). Furthermore, the main virulence profile VP1 (avrA-ssaQ-mgtC-siiD-sopB-sodC1-sopE-bcfC, 82.7%) appeared in every cluster, and other profiles were not distributed in the same cluster. However, interestingly, only three isolates did not carry mgtC, and two of them had a similarity index of 96%. SH14SF287 was the only strain that carried spvC and it did not belong to any cluster.

Discussion

Salmonella Derby infection has been a long-standing major problem in humans and is associated with animal products, particularly pork, in many parts of the world (Hauser et al., 2011). However, the epidemic characteristic and biological features are far from being completely understood, especially in China. Therefore, our study will provide the most detail to date of the phenotypic characteristics and genetic diversity of clinical and food strains of Salmonella Derby isolated in Shanghai, China.

The present study observed an increased antimicrobial resistance (MDR = 69.92%), compared with reports from America (MDR = 47.27%) (Valdezate et al., 2005), Europe (MDR = 46.15%) (Bonardi et al., 2016), and Asia (MDR = 31.00%) (Li et al., 2013), especially to current first-line antibiotics that are used for the treatment of bacterial diarrhea, such as cefepime, cefotaxime, and ofloxacin. In particular, the emergence of fluoroquinolone-resistant and third- or fourth-generation cephalosporin-resistant Salmonella Derby has become a critical issue for public health. Resistance to these antimicrobials was already reported for Salmonella Kentucky, Salmonella Newport, and Salmonella Enteritidis isolates (Wasyl et al., 2015). Fluoroquinolones and cephalosporins have been widely prescribed for a diverse range of infections, including bacterial enteritis and typhoid fever. However, the widespread use and misuse of these agents, such as the treatment of infections caused by bacteria that were only marginally susceptible, has been predictably followed by the emergence of resistance (Fang, 2015).

Mobilizable plasmids have a tremendous impact in horizontal gene transfer in nature, including the spread of antibiotic resistance and virulence (Carattoli, 2013). Plasmid replicon typing has proved helpful for distinction and characterization of Salmonella serotypes (Sanad et al., 2016). Previous studies have reported that the IncFIA replicon type of plasmid, which carries an extended-spectrum beta-lactamase (ESBL) gene or bla _NDM-1gene, was found in Klebsiella pneumoniae and E. coli (Wang et al., 2013; Chen et al., 2014). F family and P types, which were predominant in this study, are reportedly harbored by plasmids of E. coli, K. pneumoniae, and some S. enterica species worldwide. IncP-type plasmids have been associated with the spread of the ESBL resistance genes, bla _CTX-M and bla _SHV (Pouget et al., 2013; Zhang et al., 2016). The IncF group plasmids (FIA, FIB, FIC, Frep, and FIIs) are frequently encountered in clinical enterobacterial strains associated with the spread of relevant antimicrobial resistance and virulence genes such as bla _KPC, bla _SHV, bla _CTX-M, quinolone resistance genes, plasmid-mediated aminoglycoside genes, and Salmonella plasmid virulence; other Inc types were also noted as vehicles for the spread of resistance or virulence genes (Villa et al., 2010).

PFGE is currently a valuable tool for assessing relatedness among Salmonella isolates from different sources and has been used for fingerprinting of S. enterica for both national and international outbreak investigations because of its remarkable discriminatory power and high reproducibility (Lienemann et al., 2015). However, in our study, six isolates could not be subtyped. In a previous study, only 80 of 127 Salmonella Derby isolates could be typed by XbaI restriction. This was presumably because of the production of a DNase that degraded the chromosomal DNA before digestion with restriction endonuclease was complete (Ling et al., 2001). Among the 127 Salmonella Derby isolates tested, 68 distinct banding patterns were identified, and nine main individual clusters (A to I) were grouped at an 85% pattern similarity threshold. Interestingly, there were many isolates from humans and foods that possessed the same fingerprints in each cluster. Some of the strains (100% similarity with PFGE) could be differentiated by combining the PFGE data with plasmid replicon types, ARP, and VP methods, which suggests that the resolution ratio of composite analysis was higher than that of one single analysis in the current study. However, there were still some strains from foods of animal origin and humans that could not be differentiated by the composite analysis among the top three biggest clusters (A, B, and E). For example, there were 16 strains with same PFGE profile in the first profile of cluster B, which could be divided into three groups under the composite analysis. In addition, there were two strains, three strains, and seven strains in the three groups, respectively, and these strains showed the same PFGE pattern, plasmid replicon type, ARP, and VP in each group. These findings allowed us to infer the possibility that foods of animal origin, especially pork, serve as a source for human Salmonella Derby infection.

Molecular subtyping of Salmonella isolates is essential when monitoring the transmission of bacterial strains across various sources. By comparing PFGE, plasmid replicon types, ARP, and VP methods using the same strains, we found that PFGE subtyping was more discriminative than the other methods; nevertheless, we still found some correlation between the factors analyzed. For example, composite analysis of PFGE and ARP showed that most of the Salmonella isolates representing similar ARPs clustered together, irrespective of their source of isolation. Similar results were reported by several previous studies (Lynne et al., 2009; Dionisi et al., 2011). Furthermore, we found significant associations between PFGE and plasmid replicons, especially in cluster A and cluster B, and these clusters carried a higher percentage of IncFIC (84.21%) and IncP (83.33%), respectively, when isolates from major clusters were compared. Similar findings were previously reported in Salmonella Kentucky (Ladely et al., 2016). Moreover, VP has been suggested to be a good discriminatory tool for typing S. enterica, and a significant association between certain PFGE clusters and VP has been reported (Kuang et al., 2015). However, our results do not support this finding, as most isolates belonged to a single VP (VP1, 82.7%), and comparison of the PFGE clusters showed no direct correlation. Our results indicate that virulotyping may be more appropriate for different serotypes of Salmonella isolates rather than a single serotype.

Conclusions

This study revealed a high frequency of resistance and MDR among Salmonella Derby strains isolated from different sources, especially the emergence of fluoroquinolone resistance and third- or fourth-generation cephalosporin resistance. The similarity in PFGE and other profiles between isolates from humans and foods of animal origin suggests the possibility that foods of animal origin, especially pork, serve as an important source for human infection in China. Moreover, PFGE subtyping was more discriminative than the other methods analyzed. However, composite analysis provided better discrimination among Salmonella isolates than individual typing methods. Our results indicate that surveillance on this serotype should be strengthened to prevent the pathogenicity and potential dissemination of Salmonella Derby that carries antimicrobial and virulence genes and harbors plasmids from foods to the environment and humans.

Footnotes

Acknowledgments

This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (grant No. 201403054) and the National Natural Science Foundation of China (grant No. 31402193).

Disclosure Statement

No competing financial interests exist.

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