Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2017.01300 Microbiology Original Research Characterization of Integrons and Resistance Genes in Salmonella Isolates from Farm Animals in Shandong Province, China Zhao Xiaonan 1 Yang Jie 1 Zhang Baozhen 2 Sun Shuhong 1 * Chang Weishan 1 * 1College of Animal Science and Technology, Shandong Agricultural University Tai'an, China 2Laboratory for Reproduction Health Institute of Biomedicine and Biotechnology Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen University Town Shenzhen, China

Edited by: Axel Cloeckaert, Institut National de la Recherche Agronomique (INRA), France

Reviewed by: Sebastian Guenther, Freie Universität Berlin, Germany; Séamus Fanning, University College Dublin, Ireland

*Correspondence: Shuhong Sun jqybfkyjs@163.com

Weishan Chang changweishan@yeah.net

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

 12 07 2017 2017 8 1300 31 03 2017 27 06 2017 Copyright © 2017 Zhao, Yang, Zhang, Sun and Chang. 2017 Zhao, Yang, Zhang, Sun and Chang

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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.

 Salmonella antimicrobial susceptibility class 1 integron antimicrobial resistance gene MLST 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 multidrug-resistant (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 antibiotic-resistant Salmonella in animal farms (Melendez et al., 2010; Graciela et al., 2016). However, the extent of antibiotic-resistant 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.

 Materials and methods Samples and <italic>Salmonella</italic> 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 pre-enrichment 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).

 <italic>Salmonella</italic> 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).

 Antimicrobial susceptibility testing

A minimal inhibition concentration (MIC) assay, as described by the Clinical and Laboratory Standard Institute (Clinical Laboratory Standards Institute, 2013), was used in this study to test the susceptibility of 12 commonly used antibiotics (Table 1), including ampicillin (AMP), amikacin (AMK), enrofloxacin (ENO), ciprofloxacin (CIP), nalidixic acid (NA), florfenicol (FFN), tetracycline (TET), ceftiofur (CEF), gentamicin (GEN), neomycin (NEO), levofloxacin (LVX), and fosfomycin (FOS). Escherichia coli (ATCC 25922) and Klebsiella pneumoniae (ATCC 700603) were used as the quality control strains in this study. Salmonella isolates resistant to more than three classes of antimicrobials were defined as MDR isolates.

Antimicrobials and the range of concentrations tested.

Antimicrobials Abbreviation Concentration range (μg/mL)
Ampicillin AMP 0.06~256
Amikacin AMK 0.5~512
Enrofloxacin ENO 0.06~512
Ciprofloxacin CIP 0.015~512
Nalidixic acid NA 0.06~512
Florfenicol FFN 0.5~512
Tetracycline TET 0.5~512
Ceftiofur CEF 0.06~512
Gentamicin GEN 0.5~512
Neomycin NEO 0.5~512
Levofloxacin LVX 0.06~512
Fosfomycin FOS 1~2.048
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 blaTEM, blaPSE−1, blaSHV, and blaCTX−M was performed as previously described (Li et al., 2013). Furthermore, PCR amplification was used to screen for plasmid-mediated 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).

 MLST

Seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were used to characterize Salmonella by MLST. MLST was performed as described online (http://mlst.warwick.ac.uk/mlst/dbs/Senterica/documents/primersEnterica_html). All polymerase chain reaction products were purified and sequenced (Invitrogen, Beijing, China), and the alleles and STs were assigned according to the MLST scheme at http://mlst.warwick.ac.uk/mlst/dbs/Senterica.

 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.

 Results Prevalence and serotypes of <italic>Salmonella</italic>

In this study, a total of 154 non-duplicate Salmonella isolates (154/1105, 13.9%) were recovered. From chickens, 78 Salmonella isolates were recovered (78/325, 24.0%) (Table 2), which was significantly higher than the Salmonella isolated from ducks and pigs (P < 0.05). Seventy-eight Salmonella isolates were divided into six serovars. The most common serovar was Salmonella enteritidis (69/78, 88.5%) (Table 3).

Prevalence of Salmonella isolates from farm animals.

Chicken Duck Pig
Locations No. of samples No. of posotive samples (%) No. of samples No. of posotive samples (%) No. of samples No. of posotive samples (%)
Qingdao 100 17 (17%) 200 22 (11.0%) 60 7 (11.7%)
Jinan 115 34 (29.6%) 200 19 (9.5%) 60 8 (13.3%)
Zibo 110 27 (24.5%) 200 15 (7.5%) 60 5 (8.3%)
Total 325 78 (24.0%) 600 56 (9.3%) 180 20 (11.1%)

Resistance phenotype, ST, incidence of class I integron, and resistance gens in Salmonella isolated from animals in farms.

No. Location Farms Serovar ST Resistance phenotype Integrons/resistance genes
1 Qingdao Chicken S. Enteritidis 11 AMP, CEF, ENO, TET blaTEM−1,qnrA
2 Qingdao Chicken S. Enteritidis 11 AMP, TET qnrA
3 Qingdao Chicken S. Enteritidis 11 AMP, CEF, NA, NEO, TET Class I (drfA1-aadA1), blaTEM−1,qnrA, qnrS
4 Qingdao Chicken S. Enteritidis 11 AMP, ENO, NA, TET blaTEM−1, qnrA, qnrB
5 Qingdao Chicken S. Enteritidis 11 AMP, CEF, CIP, ENO, GEN, NA, NEO Class I (aadA2), blaPSE−1, qnrB, qnrS
6 Qingdao Chicken S. Enteritidis 11 AMP, CIP, ENO, FFN, GEN, NA, NEO blaTEM−1, blaCTX−M−55, qnrB, floR
7 Qingdao Chicken S. Enteritidis 11 AMP, TET blaTEM−1
8 Qingdao Chicken S. Enteritidis 11 AMP, ENO, NA, TET blaCTX−M−55, qnrB
9 Qingdao Chicken S. Enteritidis 11 AMP, CEF, CIP, ENO, NA, TET qnrA, qnrB
10 Qingdao Chicken S. Enteritidis 11 AMP, CIP, TET blaTEM−1,qnrA, qnrB
11 Qingdao Chicken S. Indiana 17 AMP, CEF, CIP, ENO, FFN, NA, TET Class I (aadA2), blaTEM−1, qnrA, qnrB, qnrS, floR
12 Qingdao Chicken S. Enteritidis 11 AMP, CEF, ENO, GEN, NA, TET blaPSE−1, qnrA, qnrS
13 Qingdao Chicken S. Enteritidis 11
14 Qingdao Chicken S. Enteritidis 11 AMP, CIP, TET qnrA
15 Qingdao Chicken S. Enteritidis 11 AMP, NA qnrA
16 Qingdao Chicken S. Enteritidis 11 AMP, ENO, NA, TET blaTEM−1, qnrA, qnrB
17 Qingdao Chicken S. Enteritidis 11 NA
18 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET qnrA
19 Jinan Chicken S. Thompson 26 AMP, CEF, GEN, NA, TET qnrA, qnrB
20 Jinan Chicken S. Enteritidis 11 AMP, CIP, ENO, FFN, GEN, NA, NEO Class I (aadA2), blaTEM−1, qnrA, qnrB, floR
21 Jinan Chicken S. Enteritidis 11 AMP, CEF, CIP, NA, TET blaTEM−1, qnrA, qnrB
22 Jinan Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrA
23 Jinan Chicken S. Enteritidis 11 AMP, TET
24 Jinan Chicken S. Enteritidis 11 AMP, CIP, ENO, FFN, GEN, NA, NEO Class I (aadA2), blaTEM−1, qnrA, qnrB, floR
25 Jinan Chicken S. Enteritidis 11 AMP, CEF, FFN, ENO, NA, TET Class I (aadA2), qnrA, qnrB, floR
26 Jinan Chicken S. Thompson 26 AMP, ENO, NA, NEO, TET blaTEM−1, qnrA, qnrB
27 Jinan Chicken S. Enteritidis 11 AMP, CEF, FFN, NEO, NA, TET Class I (drfA1-aadA1), blaPSE−1, qnrA, floR
28 Jinan Chicken S. Enteritidis 11 AMP, CEF, NA, NEO, TET qnrA, qnrB
29 Jinan Chicken S. Enteritidis 11
30 Jinan Chicken S. Enteritidis 11 AMP, CEF, FFN, NEO, NA, TET Class I (aadA1), blaTEM−1, qnrA, floR
31 Jinan Chicken S. Enteritidis 11 AMP, CEF, CIP, ENO, NA, TET qnrA, qnrB
32 Jinan Chicken S. Enteritidis 11
33 Jinan Chicken S. Enteritidis 11 AMP, GEM, NA, TET qnrA
34 Jinan Chicken S. Typhimurium 19 AMP, CEF, CIP, FFN, NA, TET qnrB, floR
35 Jinan Chicken S. Enteritidis 11 AMP, CEF, ENO, TET qnrA
36 Jinan Chicken S. Enteritidis 11 AMP, CEF, CIP, FFN, NA, TET Class I (drfA17-aadA5), blaPSE−1, qnrA, qnrB, floR
37 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET qnrA, qnrB
38 Jinan Chicken S. Enteritidis 11 AMP, CEF, CIP, ENO, FFN, GEN, NA Class I (drfA17-aadA5), blaTEM−1, blaPSE−1, qnrA, qnrB, floR
39 Jinan Chicken S. Enteritidis 11 TET
40 Jinan Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrA, qnrB
41 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET blaTEM−1, qnrA, qnrB, floR
42 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET qnrA, qnrB
43 Jinan Chicken S. Enteritidis 11 AMP, CEF, FFN, NA, TET qnrA, qnrB, floR
44 Jinan Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrB
45 Jinan Chicken S. Enteritidis 11 NA qnrB
46 Jinan Chicken S. Enteritidis 11 AMP, ENO, NA, TET blaPSE−1, qnrA, qnrB
47 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET qnrA
48 Jinan Chicken S. Enteritidis 11
49 Jinan Chicken S. Enteritidis 11 AMP, CIP, TET qnrA, qnrB
50 Jinan Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrA, qnrB
51 Jinan Chicken S. Indiana 17 AMP, CEF, CIP, ENO, FFN, NA, TET Class I (drfA17-aadA5), qnrA, qnrB, floR
52 Zibo Chicken S. Enteritidis 11 AMP, CEF, ENO, GEN, NA, TET Class I (aadA1), qnrA, qnrB, qnrS
53 Zibo Chicken S. Agona 28 AMP, CEF, ENO, NA, TET blaTEM−1, qnrA, qnrB
54 Zibo Chicken S. Enteritidis 11 AMP, CEF, ENO, FFN, GEN, NA, TET Class I (drfA1-aadA1), blaTEM−1, blaCTX−M−55, qnrA, qnrS, floR
55 Zibo Chicken S. Senftenberg 14 AMP, ENO, NA, TET qnrA, qnrB
56 Zibo Chicken S. Enteritidis 11 AMP, CEF, FFN, NEO, NA, TET Class I (drfA1-aadA1), qnrB, floR
57 Zibo Chicken S. Enteritidis 11 AMP, CIP, TET blaCTX−M−55, qnrA
58 Zibo Chicken S. Enteritidis 11 AMP, CEF, ENO, NEO, NA, TET blaTEM−1, qnrA, floR
59 Zibo Chicken S. Enteritidis 11 AMP, CEF, FFN, GEN, NA, TET blaTEM−1, blaPSE−1, qnrA, floR
60 Zibo Chicken S. Enteritidis 11 AMP, CIP, TET qnrA, qnrB
61 Zibo Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrB
62 Zibo Chicken S. Enteritidis 11
63 Zibo Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrA
64 Zibo Chicken S. Enteritidis 11 AMP, TET
65 Zibo Chicken S. Enteritidis 11 AMP, ENO, NA, TET blaTEM−1
66 Zibo Chicken S. Enteritidis 11 AMP, ENO, NA, TET qnrA
67 Zibo Chicken S. Enteritidis 11 AMP, CEF,GEN, NA, TET blaTEM−1, qnrB, floR
68 Zibo Chicken S. Enteritidis 11 AMP, CIP, TET qnrB
69 Zibo Chicken S. Enteritidis 11 AMP, CEF, NA, NEO, TET qnrA, qnrB, floR
70 Zibo Chicken S. Enteritidis 11
71 Zibo Chicken S. Enteritidis 11 AMP, CIP, TET
72 Zibo Chicken S. Enteritidis 11 AMP, NA
73 Zibo Chicken S. Indiana 17 AMP, CEF, CIP, FFN, GEN, NA, TET Class I (drfA1-aadA1), blaTEM−1, qnrA, floR
74 Zibo Chicken S. Enteritidis 11 AMP, CEF, CIP, ENO, GEN, NA, NEO blaTEM−1, qnrA, qnrB
75 Zibo Chicken S. Indiana 17 AMP, CEF, CIP, ENO, FFN, GEN, NA Class I (drfA1-aadA1), blaTEM−1, qnrA, qnrS, floR
76 Zibo Chicken S. Enteritidis 11 AMP, CEF, CIP, FFN, GEN, TET qnrA, qnrB, floR
77 Zibo Chicken S. Enteritidis 11 AMP, CEF, CIP, FFN, GEN, TET Class I (drfA1-aadA1), blaTEM−1, qnrB, floR
78 Zibo Chicken S. Enteritidis 11 AMP, GEN, NA, TET qnrA, qnrB
79 Qingdao Duck S. Typhimurium 34 CIP, ENO, GEN, NA, NEO, TET qnrA, qnrB
80 Qingdao Duck S. Typhimurium 34 CIP, NA, NEO, TET qnrA
81 Qingdao Duck S. Typhimurium 19 AMP blaTEM−1
82 Qingdao Duck S. Enteritidis 11 AMP, CEF, CIP, ENO, FFN, NEO, GEN, NA, TET Class I (drfA1-aadA1), blaTEM−1, qnrB, floR
83 Qingdao Duck S. Enteritidis 11 AMP blaTEM−1
84 Qingdao Duck S. Typhimurium 19 AMP, NA blaTEM−1, qnrA, qnrB
85 Qingdao Duck S. Enteritidis 11 AMP, CEF, ENO, GEN, NA, NEO, TET Class I (drfA1-aadA1), blaTEM−1, blaCTX−M−55, qnrB
86 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET Class I (aadA2), qnrA, qnrB
87 Qingdao Duck S. Enteritidis 11 AMP, TET blaTEM−1
88 Qingdao Duck S. Enteritidis 11 blaTEM−1
89 Qingdao Duck S. Typhimurium 34 CIP, ENO, GEN, NA, NEO, TET qnrA, qnrB
90 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, floR
91 Qingdao Duck S. Enteritidis 11 AMP, CEF, CIP, ENO, FFN, NEO, GEN, NA, TET qnrA, qnrB, floR
92 Qingdao Duck S. Typhimurium 34 CEF, CIP, ENO, GEN, NA, NEO, TET Class I (aadA2), qnrB
93 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, NEO, TET qnrA
94 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB
95 Qingdao Duck S. Enteritidis 11 AMP, CEF, CIP, GEN, NA, NEO, TET blaTEM−1, qnrA
96 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, NA, TET qnrA, qnrB
97 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, NA, NEO, TET qnrA
98 Qingdao Duck S. Enteritidis 11 CIP, ENO, GEN, NA, TET qnrA, qnrB
99 Qingdao Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, NEO, TET blaTEM−1, qnrA, floR
100 Qingdao Duck S. Enteritidis 11 CEF, CIP, NA, TET qnrB
101 Jinan Duck S. Typhimurium 19 CIP, ENO, FFN, GEN, NA, NEO, TET qnrA, floR
102 Jinan Duck S. Enteritidis 11 AMP, CIP, ENO, FFN, NA, NEO, TET blaTEM−1, qnrB
103 Jinan Duck S. Typhimurium 19 AMP, CEF, CIP, NA, NEO, TET qnrA
104 Jinan Duck S. Typhimurium 19 CIP, NA, NEO, TET qnrA, qnrB
105 Jinan Duck S. Typhimurium 19 AMP, CEF, ENO, GEN, NA, NEO, TET Class I (aadA2), blaTEM−1, qnrB
106 Jinan Duck S. Typhimurium 19 CIP, ENO, GEN, NA, TET qnrA, qnrB
107 Jinan Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, NEO, TET qnrA, qnrB
108 Jinan Duck S. Enteritidis 11 AMP, CEF, CIP, ENO, FFN, GEN, NA, TET blaTEM−1, qnrA, floR
109 Jinan Duck S. Enteritidis 11 CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB
110 Jinan Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB, floR
111 Jinan Duck S. Enteritidis 11 AMP, CEF, CIP, ENO, NA, TET blaTEM−1, qnrB
112 Jinan Duck S. Enteritidis 11 AMP, CEF, CIP, GEN, NA, NEO, TET blaTEM−1, blaPSE−1, qnrA
113 Jinan Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB, floR
114 Jinan Duck S. Enteritidis 11 AMP, CEF, NA, NEO, TET blaTEM−1, qnrA, qnrB
115 Jinan Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, NEO, TET qnrA, qnrB, floR
116 Jinan Duck S. Enteritidis 11 CIP, ENO, GEN, NA, TET qnrA, qnrB
117 Jinan Duck S. Enteritidis 11 CIP, GEN, NA, TET qnrA, qnrB
118 Jinan Duck S. Enteritidis 11 AMP, CEF, CIP, NA, NEO, TET Class I (aadA2), blaTEM−1, qnrA, qnrB
119 Jinan Duck S. Enteritidis 11 CIP, ENO, GEN, NA, TET qnrA, qnrB
120 Zibo Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB, floR
121 Zibo Duck S. Enteritidis 11 AMP, CEF, CIP, ENO, FFN, NEO, GEN, NA, TET Class I (drfA1-aadA1), blaTEM−1, qnrA, qnrB, floR
122 Zibo Duck S. Enteritidis 11 AMP, CEF, FFN, GEN, NA, NEO, TET qnrA, qnrB
123 Zibo Duck S. Typhimurium 19 AMP, CEF, CIP, FFN, NA, NEO, TET qnrA, qnrB
124 Zibo Duck S. Typhimurium 19 CEF, CIP, ENO, GEN, NA, TET qnrA, qnrB
125 Zibo Duck S. Typhimurium 34 CIP, ENO, GEN, NA, NEO, TET qnrA, qnrB
126 Zibo Duck S. Enteritidis 11 AMP, CEF, ENO, GEN, NA, NEO, TET blaTEM−1, blaPSE−1, qnrA, qnrB
127 Zibo Duck S. Enteritidis 11 AMP, CEF, CIP, GEN, NA, NEO, TET qnrA, qnrB
128 Zibo Duck S. Enteritidis 11 AMP, CEF, ENO, FFN, GEN, NA, NEO, TET Class I (drfA12-aadA2), blaTEM−1, qnrB, qnrS
129 Zibo Duck S. Typhimurium 34 CEF, CIP, ENO, GEN, NA, TET qnrA
130 Zibo Duck S. Typhimurium 34 AMP, CEF, CIP, NA, TET qnrA, qnrB
131 Zibo Duck S. Enteritidis 11 AMP, CIP, ENO, FFN, GEN, NA, NEO, TET blaTEM−1, qnrA, qnrS, floR
132 Zibo Duck S. Typhimurium 19 CIP, ENO, GEN, NA, NEO, TET qnrA, qnrB
133 Zibo Duck S. Typhimurium 19 AMP, CEF, CIP, NA, TET qnrA, qnrB
134 Zibo Duck S. Enteritidis 11 CEF, CIP, ENO, FFN, GEN, NA, TET qnrA, qnrB, floR
135 Qingdao Pig S. Typhimurium 19 AMP, NA qnrA, qnrB, floR
136 Qingdao Pig S. Typhimurium 19 AMP, TET blaTEM−1, qnrA, qnrB, floR
137 Qingdao Pig S. Typhimurium 19 qnrB, floR
138 Qingdao Pig S. Typhimurium 34 AMP, CIP, ENO, FFN, LVX, NA, NEO, TET blaTEM−1, blaCTX−M−55,qnrA, qnrB, floR
139 Qingdao Pig S. Derby 40 TET floR
140 Qingdao Pig S. Derby 40 AMP blaTEM−1,qnrA, floR
141 Qingdao Pig S. Typhimurium 19 AMP, CIP, FFN blaCTX−M−55, blaPSE−1, qnrA, qnrB, floR
142 Jinan Pig S. Typhimurium 19 FFN, FOS, TET qnrA,qnrB, floR
143 Jinan Pig S. Derby 40 AMP qnrA, floR
144 Jinan Pig S. Derby 40
145 Jinan Pig S. Typhimurium 19 AMP, CIP, FFN, LVX, NA, NEO, TET Class I (aadA2), blaTEM−1,blaPSE−1, qnrA, qnrB, floR
146 Jinan Pig S. Typhimurium 34 AMP, CIP, FFN blaTEM−1, blaPSE−1, qnrA, qnrB, floR
147 Jinan Pig S. Enteritidis 11 AMP, CEF, CIP, FFN, FOS, NA blaTEM−1, blaCTX−M−55, qnrA, qnrB, floR
148 Jinan Pig S. Typhimurium 34 AMP, TET blaCTX−M−55, qnrA, qnrB, floR
149 Jinan Pig S. Typhimurium 34
150 Zibo Pig S. Enteritidis 11 AMP, CIP, FFN, NA blaTEM−1, blaPSE−1, qnrA, qnrB, floR
151 Zibo Pig S. Typhimurium 34 AMP, CIP, ENO, FFN blaTEM−1, blaCTX−M−55, qnrA, qnrB, floR
152 Zibo Pig S. Typhimurium 19 AMP, CIP, TET blaTEM−1, blaPSE−1, qnrA, qnrB, floR
153 Zibo Pig S. Typhimurium 19 AMP, TET blaTEM−1, qnrA, qnrB, qnrS, floR
154 Zibo Pig S. Enteritidis 3,007 AMP, TET blaCTX−M−55, qnrA, qnrB, floR

From ducks, 56 Salmonella isolates were recovered (56/600, 9.3%) (Table 2), and they were divided into two serovars. The most common serovar was Salmonella enteritidis (38/56, 67.9%) (Table 3).

From pigs, 20 Salmonella isolates were recovered (20/180, 11.1%) (Table 2), and they were divided into three serovars. The most common serovar was Salmonella typhimurium (13/20, 65.0%) (Table 3).

 Antimicrobial susceptibility testing

Among 78 isolates from chickens, they were susceptible to amikacin, levofloxacin and fosfomycin. Most isolates were resistant to ampicillin (69/78, 88.5%) and tetracycline (61/78, 78.2%). In addition, 63 isolates (63/78, 80.8%) exhibited MDR (Table 3).

Among 56 isolates from ducks, they were susceptible to amikacin, levofloxacin and fosfomycin. Most isolates were resistant to tetracycline (52/56, 92.9%) and ciprofloxacin (45/56, 80.4%). In addition, 50 isolates (50/56, 89.3%) exhibited MDR (Table 3), which was significantly higher than the Salmonella isolated from chickens and pigs (P < 0.05).

Among 20 isolates from pigs, they were susceptible to amikacin and levofloxacin. Most isolates were resistant to ampicillin (15/20, 75.0%) and tetracycline (9/20, 45.0%). In addition, 9 isolates (9/20, 45.0%) exhibited MDR (Table 3).

 Characteristics of class I integrons and antimicrobial resistance genes

Among the 78 isolates recovered from chickens, 17 isolates (17/78, 21.8%) contained four groups of resistance gene cassettes, consisting of drfA1-aadA1 (1.7 kb, n = 7), aadA2 (1.2 kb, n = 5), drfA17-aadA5 (2 kb, n = 3), and aadA1 (1.2 kb, n = 2). Three β-lactamase genes were detected among the isolates, and blaTEM−1 (n = 25) was the most commonly isolated β-lactamase gene, followed by blaPSE−1 (n = 7) and blaCTX−M−55 (n = 4). Three plasmid-mediated quinolone resistance genes were detected among the isolates. qnrA (n = 53) was the most commonly isolated plasmid-mediated quinolone resistance gene, followed by qnrB (n = 44) and qnrS (n = 7). In addition, 23 isolates carried floR (Table 3).

Among the 56 isolates recovered from ducks, eight isolates (8/56, 14.3%) contained three groups of resistance gene cassettes, consisting of aadA2 (1.2 kb, n = 4), drfA1-aadA1 (1.7 kb, n = 3), and drfA12-aadA2 (2 kb, n = 1). Three β-lactamase genes were detected among the isolates. blaTEM−1 was the most commonly isolated β-lactamase gene (n = 20), followed by blaPSE−1(n = 2) and blaCTX−M−55 (n = 1). Three plasmid-mediated quinolone resistance genes were detected among the isolates. qnrA was the most commonly isolated plasmid-mediated quinolone resistance gene (n = 44), followed by qnrB (n = 40) and qnrS (n = 2). In addition, 13 isolates carried floR (Table 3).

Among the 20 isolates recovered from pigs, one isolate (1/20, 5.0%) contained one group of a resistance gene cassette, consisting of aadA2 (1.2 kb, n = 1). Three β-lactamase genes were detected among the isolates. blaTEM−1 was the most commonly isolated β-lactamase gene (n = 10), followed by blaCTX−M−55 (n = 6) and blaPSE−1 (n = 5). Three plasmid-mediated quinolone resistance genes were detected among the isolates. qnrA was the most commonly isolated plasmid-mediated quinolone resistance gene (n = 16), followed by qnrB (n = 15) and qnrS (n = 1). In addition, 18 isolates carried floR (Table 3).

 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 blaTEM and blaCTX−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: blaTEM−1, blaPSE−1, and blaCTX−M−55. Most of the isolates carried blaTEM−1, which was similar to the report in South Africa that blaTEM−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 blaTEM−1 and blaCTX−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.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This work was supported by the National key R&D project (2016YFD0501608) and (2016 YFD0500510). Taishan Scholars Program (201511023); Funds of Shandong “Double Tops” program.

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