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Characterization of Integrons and Resistance Genes in Salmonella Isolates from Farm Animals in Shandong Province, China

A total of 154 non-duplicate Salmonella isolates were recovered from 1,105 rectal swabs collected from three large-scale chicken farms (78/325, 24.0%), three large-scale duck farms (56/600, 9.3%) and three large-scale pig farms (20/180, 11.1%) between April and July 2016. Seven serotypes were identified among the 154 isolates, with the most common serotype in chickens and ducks being Salmonella enteritidis and in pigs Salmonella typhimurium. Antimicrobial susceptibility testing revealed that high antimicrobial resistance rates were observed for tetracycline (72.0%) and ampicillin (69.4%) in all sources. Class 1 integrons were detected in 16.9% (26/154) of these isolates and contained gene cassettes aadA2, aadA1, drfA1-aadA1, drfA12-aadA2, and drfA17-aadA5. Three β-lactamase genes were detected among the 154 isolates, and most of the isolates carried blaTEM−1(55/154), followed by blaPSE−1(14/154) and blaCTX−M−55 (11/154). Three plasmid-mediated quinolone resistance genes were detected among the 154 isolates, and most of the isolates carried qnrA (113/154), followed by qnrB (99/154) and qnrS (10/154). Fifty-four isolates carried floR among the 154 isolates. Multilocus sequence typing (MLST) analysis showed that nine sequence types (STs) were identified; ST11 was the most frequent genotype in chickens and ducks, and ST19 was identified in pigs. Our findings indicated that Salmonella was widespread, and the overuse of antibiotics in animals should be reduced considerably in developing countries.


INTRODUCTION
Salmonella is an important source of foodborne diseases that cause morbidity and mortality worldwide. Among 94 million cases of non-typhoid Salmonella infections, it was presumed that approximately 85% of the cases were induced by food origin Salmonella (Chiu et al., 2010). In China, Salmonella causes an estimated 22.2% of foodborne diseases (Wang et al., 2007). Many Salmonella serovars exist. More than 2,600 serovars are classified based on the reactivity of antisera to O and H antigens (Stevens et al., 2009), and the serovars from farms have a significant overlap with those causing illnesses in humans (Alcaine et al., 2006). Animals have been recognized as an important reservoir for Salmonella, and this pathogen can be transferred to humans via the food chain, posing a serious threat to human health (Vo et al., 2006). The use of antimicrobials is important for the control and treatment of Salmonella. However, antimicrobial-and multidrug-resistant Salmonella strains have emerged, leading to treatment failure (Gong et al., 2013). The increasing prevalence of multidrug-resistance among Salmonella, not only against the front-line antimicrobials, chloramphenicol and trimethoprim/sulfamethoxazole but also against clinically important antimicrobial agents, such as β-lactams and fluoroquinolones, is also an emerging problem (Lunguya et al., 2013).
The spread of the antibiotic resistant potential in Salmonella is mainly attributed to integrons. Integrons are DNA elements, capable of capturing antimicrobial resistant genes and disseminating them using a mobile genetic element (MGE) such as a plasmid among bacteria. The class I integron is the most common integron type identified in multidrugresistant (MDR) Salmonella and plays an important role in the dissemination of resistance genes among pathogens (Wright, 2010).
In developed countries, many surveys have been conducted at the molecular level to monitor the incidence of antibioticresistant Salmonella in animal farms (Melendez et al., 2010;Graciela et al., 2016). However, the extent of antibioticresistant Salmonella in many developing countries and the molecular mechanisms underlying this resistance remain unclear. Therefore, we selected large-scale animal farms as sample sites, collected swab samples, isolated Salmonella and characterized the molecular mechanisms of antimicrobial resistance.

Samples and Salmonella Isolation
From April to July 2016, rectal swabs were collected from healthy animals on farms in Qingdao, Jinan and Zibo regions in Shandong Province, China. All of the sampling sites were visited only once. In total, 1,105 samples were collected in a random manner from chickens (n = 325), ducks (n = 600), and pigs (n = 180). The samples were independently collected from individual animals, and the sample collection conformed to the cluster random sampling principle. Farms were chosen based on their scale with the following requirements: for chickens, the breeding stock was >150,000 heads; for ducks, the breeding stock was >100,000 heads, and for pigs, the breeding stock was >1,000 heads. The owners of each farm gave permission for rectal swab samples to be collected. The animals from which samples were extracted remained alive and did not undergo any surgery. Therefore, ethical approval was not required for the study because the sampling process did not harm the animals. All of the collected samples were transported in an ice box to our laboratory within 6 h for further bacteriological analysis.
Isolation and identification of Salmonella were performed as described previously (Yan et al., 2010), with some modifications. Briefly, swabbing samples were placed into a sterile plastic bag containing 100 ml of buffered peptone water (BPW) and mixed vigorously for 3 min. The BPW mixture was then incubated for 24 h at 37 • C for pre-enrichment. Approximately 1 ml of preenrichment cultures were incubated in 10 ml of selenite cysteine (SC) broth and 10 ml of rappaport-vassiliadis (RV) broth at 42 • C for 24 h, respectively. After selective enrichment, a loop-full of SC and RV broth cultures were streaked onto xylose lysine tergitol 4 (XLT4) agar and incubated at 37 • C overnight. A minimum of two presumptive Salmonella colonies was confirmed by PCR using a previously described method (Malorny et al., 2003).

Salmonella Serotyping
According to the manufacturer's instructions, the serogroup and serovars of Salmonella isolates were determined according to the Kauffmann-White scheme by slide agglutination with O and H antigens (Tianrun Bio-Pharmaceutical, Ningbo, China).

Detection of Class I Integrons and Antimicrobial Resistance Genes
Bacterial DNA was extracted using a TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Conserved primers were used for the detection and identification of class I integrons using previously described primers and procedures (Kerrnet et al., 2002). PCR screening for β-lactamase-encoding genes bla TEM , bla PSE−1 , bla SHV , and bla CTX−M was performed as previously described (Li et al., 2013). Furthermore, PCR amplification was used to screen for plasmidmediated quinolone resistance genes, qnrA, qnrB, qnrC, qnrD, and qnrS, which were the most frequently observed in China, using previously described primers (Ahmed et al., 2013). Finally, the florfenicol resistance gene, floR, was detected using previously described primers (Ahmed et al., 2013). The PCR products were purified and subsequently sequenced (Invitrogen, Beijing, China). The obtained DNA sequences were compared with those in GenBank using Basic Local Alignment Search Tool (BLAST).

Data Analysis
The statistical package SPSS (version 15.0, SPSS, Chicago, IL, USA) was used to compare the prevalence and MDR resistance rate of Salmonella isolated from chickens, ducks and pigs, and a P-value less than 0.05 was considered significant.

MLST
A total of nine STs among the 154 isolates were found. ST11 was the most common ST in both chickens and ducks, and it was represented by 69 and 38 Salmonella isolates, respectively. ST19 was the most common ST in pigs, and it was represented by eight Salmonella isolates ( Table 3). The STs in this study were correlated with specific serovars, such as ST11 with Salmonella enteritidis, ST19 and ST34 with Salmonella typhimurium, and ST40 with Salmonella derby.

DISCUSSION
In this study, Salmonella spp. were recovered from chickens, ducks and pigs in Qingdao, Jinan and Zibo regions. For the chickens, the prevalence (24.0%) was significantly higher than that reported in Shanghai, China (4.5%) (Liu et al., 2010) but was lower than that reported from chicken farms in Egypt (41.0%) (Hanem et al., 2017). The prevalence (9.3%) in ducks was similar to that obtained from duck farms in Sichuan province (12.0%) (Li et al., 2013) but was lower than those reported in Penang, Malaysia (39.0%) (Adzitey et al., 2012), and in South Korea (65.2%) (Cha et al., 2013). For pigs, the occurrence ratio (11.1%) was similar to those reported in previous studies of Salmonella spp. in food products of animal origin in China (Jiang et al., 2006;Li et al., 2013) but was higher than that reported from conventional farms (3.5%) in Korea (Migma et al., 2015). Data on the prevalence of Salmonella in different studies were difficult to compare based on differences in regions, collection seasons, sample types, isolation methodologies, culture methods, culture media, and environmental conditions. For serotyping, a total of seven serovars were found among the 154 isolates, including six from chickens, two from ducks, and three from pigs. The most common serotype in chickens and ducks was Salmonella enteritidis. This result was consistent with those from Shanxi province (Yang et al., 2010), but it was different from other reports that the dominant serotype in chicken farms was Salmonella Colindale in Chad (Tabo et al., 2013). The most common serotype in duck farms was Salmonella typhimurium (Martelli et al., 2016). The dominant serotype in pigs was Salmonella typhimurium, which was the most common serovar isolated from humans and it can lead to severe human and animal diseases (Deng et al., 2012), but it was different from other studies, where the dominant serotype in pig farms was Salmonella IIIb in Henan province (Kuang et al., 2015), and Salmonella derby in England and Wales (Miller et al., 2011). The difference in dominant serotype among animals may be due to differences in the pathogenicity of two serovars, geographical regions and diversities (Volf et al., 2010;European Centre for Disease Prevention Control, 2013).
Antimicrobial resistance in Salmonella is a threat to human public health. As shown in Table 3, the high rates of antimicrobial resistance were against tetracycline (72.0%) and ampicillin (69.4%) in all sources, which was similar to reports of Salmonella isolates from Africa, in which chickens exhibited resistance to tetracycline (93.0%) and ampicillin (47.0%) (Zishiri et al., 2016). These high resistance rates are due to its wide use in animal feed and were consistent with other reports (Piras et al., 2011;Shao, 2011;Bai et al., 2015). In addition, resistance to ciprofloxacin in 35.9% of chickens, 80.4% of ducks, and 30.0% of pigs deserves our attention because resistance to this antimicrobial agent may lead to the delay or failure of fluoroquinolone therapies (Van et al., 2007). In this study, all of the isolates were susceptible to amikacin, which may be because this antimicrobial is not used for therapeutic purposes in veterinary medicine or as a growth promoter in conventional animal fattening, and the result was consistent with other reports (Eva et al., 2015). In this study, MDR Salmonella isolates were frequently observed among chickens, ducks and pigs. In addition, MDR Salmonella is serotype-dependent (Clemente et al., 2014): the data provided evidence that Salmonella indiana, Salmonella typhimurium and Salmonella enteritidis were strongly associated with MDR phenotypes. Of particular concern, MDR strains could transfer to humans via animal or animal-derived products and pose a great risk to public health (Rosangela et al., 2016).
In this study, our results related to the incidence of class I integrons (26/154, 16.9%) were similar to the report in Sichuan (Li et al., 2013) but were higher than those reported in the USA, as class I integrons were identified in only 2.8% of the Salmonella isolates from bulk milk and milk filters ( Van et al., 2013). In the present study, the incidence of class I integrons was significantly higher in Salmonella from chickens (21.8%) than Salmonella from pigs (5.0%). In addition, in this study, the Salmonella isolates carrying class I integrons included Salmonella enteritidis, typhimurium and indiana.
Production of β-lactamases is considered to be the main mechanism of resistance in Gram-negative bacteria to overcome penicillin-derived antibiotics, and the bla TEM and bla CTX−M ESBLs can hydrolyse third and fourth generation cephalosporins. In this study, a total of three β-lactamase genes were detected among the Salmonella isolates recovered from chickens, ducks and pigs: bla TEM−1 , bla PSE−1 , and bla CTX−M−55 . Most of the isolates carried bla TEM−1, which was similar to the report in South Africa that bla TEM−1 was the most commonly identified β-lactamase gene in Salmonella isolates from food-producing animals (Igbinosa, 2015). In addition, in this study, most isolates carried bla TEM−1 and bla CTX−M−55 , which confer resistance to ampicillin.
Quinolones are the first choice for the treatment of invasive and systemic salmonellosis that occurs in humans and animals (Dimitrov et al., 2007). A total of three quinolone resistance genes were detected among the Salmonella isolates recovered from chickens, ducks and pigs: qnrA, qnrB and qnrS. qnrA was the most commonly isolated plasmid-mediated quinolone resistance gene consistent with a report in Henan, where qnrA, qnrB and qnrS were identified in Salmonella strains isolated from retail food with an incidence of 46.6, 12.7, and 19.5%, respectively (Yang et al., 2013). It is well known that qnr genes confer only low-level resistance to fluoroquinolones, and accumulation of quinolone resistance-determining region (QRDR) mutations is necessary for S. enterica to be resistant to fluoroquinolone, especially ciprofloxacin (Eaves et al., 2004). In this study, most Salmonella isolates containing a plasmid-mediated quinolone resistance gene were resistant to ciprofloxacin, nalidixic acid and gentamicin.
Florfenicol, a new chemosynthesis broad spectrum antibiotic of chloramphenicol analogs, is a fluorinated derivative of thiamphenicol. It is not approved for human use. In this study, floR was identified in 35.1% of Salmonella strains isolated from chickens, ducks and pigs, which was significantly higher than that reported in Egypt (1.0%) (Ahmed and Shimamoto, 2012). In addition, floR was identified in 90.0% of Salmonella strains isolated from pigs in this study. In this study, most Salmonella isolates containing the floR gene were resistant to florfenicol.
MLST results reveal that a total of nine STs were identified in this study. ST11 was the most frequent genotype that was recovered in chickens and ducks, and ST19 was the most frequent genotype that was recovered in pigs. ST11 belongs to Salmonella enteritidis, and ST19 belongs to Salmonella typhimurium; they all have continually been reported to cause human salmonellosis in recent years (Cai et al., 2016;Kang et al., 2017). In addition, our results revealed that the MLST patterns were generally associated with serotypes and provided a reliable prediction of the Salmonella serovars, which was consistent with previous research (Achtman et al., 2012).

CONCLUSION
The prevalence of Salmonella was higher in the animal farms. Moreover, many serovars reported in humans and MDR Salmonella were recovered in this study. The high rates of MDR Salmonella, class I integrons and antibiotic resistance gene positive isolates detected suggest that measures must be taken to facilitate the reasonable use of antimicrobials in animal husbandry. Therefore, continuous surveillance of Salmonella and associated antimicrobial resistance in Salmonella of animals is essential to detect emerging Salmonella serovars and associated resistance genes.

AUTHOR CONTRIBUTIONS
SS, XZ, contributed to the conception of the study; WC, XZ; contributed significantly to analysis and manuscript preparation; XZ, performed the data analyses and wrote the manuscript; XZ, JY, BZ: helped perform the analysis with constructive discussions.