Prevalence and antimicrobial resistance of Salmonella isolated from broilers in Shandong, China

Background Salmonella spp. are one of the most important foodborne bacterial pathogens in human beings and animals. The prevalence of Salmonella from broilers in Shandong, China and antimicrobial susceptibility of these isolates was determined. Results From May to October 2018, 600 samples collected, 67 Salmonella isolates were recovered with an isolation rate of 11.2%. The most common serovars were S. enteritidis and S. typhimurium. The highest incidence of resistance observed were for PB (100%), and AMP (68.7%), and the MDR Salmonella isolate rate was 53.7%. Four β-lactamase genes were detected among the isolates, all the isolates carried bla TEM (67/67, 100%), followed by bla OXA (19/67, 28.4%), bla CTX-M (17/67, 25.4%), and bla PSE (7/67, 10.4%); four plasmid-mediated quinolone resistance genes were detected among the isolates, the prevalent resistance genes was aac(6’)-Ib-cr (18/67, 26.9%), followed by oqxB (9/67, 13.4%), qnrB (6/67, 9.0%), and qnrD (1/67, 1.5%); the prevalent rate of mcr -1 was 6.0%(4/67). Class 1 integrons were detected in 26.9% of these isolates and contained seven groups of resistance gene cassettes. MLST analysis revealed seven sequence types, and ST11 was the most frequent sequence types. Conclusions This study indicated that reduction of Salmonella and strict control on the use of antibiotics in more than 5000 in vitally to keep public


Abstract
Background Salmonella spp. are one of the most important foodborne bacterial pathogens in human beings and animals. The prevalence of Salmonella from broilers in Shandong, China and antimicrobial susceptibility of these isolates was determined.
Results From May to October 2018, 600 samples collected, 67 Salmonella isolates were recovered with an isolation rate of 11.2%. The most common serovars were S. enteritidis and S. typhimurium.
Class 1 integrons were detected in 26.9% of these isolates and contained seven groups of resistance gene cassettes. MLST analysis revealed seven sequence types, and ST11 was the most frequent sequence types.
Conclusions This study indicated that reduction of Salmonella and strict control on the use of antibiotics in more than 5000 million broilers in Shandong are the vitally important measure to keep public health.

Background
Salmonella is a notorious human pathogen that can lead to an estimated 153 million enteric infections and 56,969 diarrheal deaths each year worldwide [1]. It has been widely reported that most of human salmonellosis is caused by infection derived from contaminated eggs, poultry meat and meatproducts [1][2][3][4].
To date, more than 2600 Salmonella serovars have been reported [5]. Poultry, especially broilers, are well known reservoirs of various Salmonella serovars, most of which are able to infect humans [6].
Therefor, it is meaning that serovar determination is extremely important for effective epidemiological surveillance and disease assessment. Antimicrobial resistance is increasingly becoming an important issue with salmonellosis infections in both animals and humans [7]. In animal husbandry, antibiotics are widely used for growth promotion, or treatment purposes which facilitated the emergence and dissemination of antibiotic resistance in Salmonella [8]. Salmonella can acquire resistance genes through mobile elements such as integrons, which contributes to the spread and distribution of antibiotic resistance genes across diverse bacterial populations [9]. The chicken can be used as a vehicle for spreading and distributing of these antimicrobial resistant strains to humans [10].
Research on the prevalence and antimicrobial resistance of Salmonella isolated from broilers is important for determining the specific distribution patterns of antimicrobial resistance for this pathogen and developing effective treatment strategies to control and prevent Salmonella infections in humans and animals. In China, broilers are widely reared and is an important sources of chicken meat [11]. Therefor, it is necessary to monitor Salmonella in broiler every year. The purpose of this study was to determine the prevalence and characteristics of Salmonella isolated from broilers in Shandong province, China.

Methods Sampling
From May to October 2018, three large-scale intensive broiler farms in Tai'an, Jinan, and Weifang areas of Shandong Province were selected as sampling points. Farms were chosen based on their scale with the following requirements that the breeding stock was > 150,000 heads. For each farm, 200 fresh fecal swabs were randomly collected from different individual animals, a total of 600 samples, which were sent to the laboratory for bacterial isolation and identification at low temperature (Table 1). Table 1 Prevalence of Salmonella isolates from broilers in Shandong province.

Locations
No

Salmonella isolation and serotype identification
Salmonella isolation was conducted using previously published protocols [12], with some modification.

Detection of class 1 integrons
All Salmonella isolates were screened for the presence of class 1 integrons based on the primers previously described [17]. The amplification fragments were purified from the agarose gel using a gel extraction kit (Tiangen, Beijing, China) and subsequently sequenced (Invitrogen, Beijing, China). Gene cassette homology searches were preformed using Basic Local Alignment Search Tool (BLAST) analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Prevalence and serovars of Salmonella
The prevalence of Salmonella in broilers form Shandong is presented in Table 1 The most common serovars were S. enteritidis (37/67, 55.2%) and S. typhimurium (18/67, 26.9 ) ( Table 2). Table 2 Resistance phenotype, ST, incidence of class 1 integron, and resistance genes in Salmonellla isolated from broilers in Shandong province.

Discussion
The prevalence and distribution of Salmonella constitute a threat to human health and present a major financial burden [18]. In this study, The Salmonella isolation rate of 11.2%from broilers showed a lower prevalence than that from poultry slaughterhouses in Shandong province (23.5 ) [19] and in Ireland (26.4 ) from the poultry carcasses [20], and was similar to the previous reports from poultry slaughterhouses that in Sichuan province (10.7 ) [14], and in Germany (13.2 ) [21]. Although different sampling procedures, sample sizes, collection seasons, region difference and bacteria isolation and identification methods could affect the isolation rates of Salmonella, this level of contamination indicates a potential breakdown of hygiene at broiler farms. In many countries all over the world, a wide range of different Salmonella serotypes have been found to contaminate the broilers [22]. Additionally, S. enteritidis was the most common serotype identified in broilers, which was similar to the previous reports from Sichuan province and Henan province [23,24], and has been isolated in chicken eggs and chicken meat (11), but contrasts with our previous report from chickens that the most common serovar was S. indiana [25]. This difference may be associated with geographic variation and the chicken breeds. Of note, high isolation rates of S. typhimurium was also noticed. S. typhimurium remains one of the main serotype that can lead to sever human and animal diseases [26].
Antibiotic resistant in Salmonella has been a major problem in animal farms especially in broilers.
Though relevant departments have been emphasizing the limited use of antibiotics in animal feeding, the effect was small. In this study, all the Salmonella isolates were resistance to PB, which was much higher than that previously reported in Salmonella from food-producing animals (5.2%) at slaughter in Europe [27]. This high resistant rate of PB in broilers may be attributed to the widely use of this antibiotic in animals during breeding, and disease control and it suggesting that farm managers should reduce antibiotic usage. Resistance to AMP (68.7 ) frequently observed in Salmonella isolates, which was higher than that previously reported in Jiangsu province (19.3 ) [28] and in the South Africa and Brazil (47.0 ) [29]. In this study, the MDR Salmonella isolate rate was 53.7 which was lower than that our previously reported from Shandong province (80.8 ) in 2016 [25] and similar to the report from pigs in Southern Brazil [30]. In addition, S. enteritidis showed a high multidrug resistance rate, contrasts with the report that most of S. Indiana showed multidrug resistance [24].
Production of β-lactamases has been identified as the main plasmid-mediated mechanisms of resistance to third-generation cephalosporins, and is currently considered a major concern both in human and veterinary medicine [31]. In this study, all the Salmonella isolates carried bla TEM which was higher than the report in Egypt (41.5%) [32]. Of note, all the Salmonella isolates carried bla TEM , but 68.7 showed phenotypical resistance to AMP, indicating that there existed another resistance mechanism. It has been reported that bla OXA was considered to be the most commonly identified βlactamase gene in Salmonella isolates from China, and in portugal, bla CTX−M was commonly detected in Salmonella isolates from poultry, swine and food products of animal origin [33], which was different from our result.
Colistin are often used to treat food-producing animals and has been considered the last resort antibiotics for the rapidly increasing MDR gram-negative pathogens [37]. However, colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella [38]. In this study, the prevalent rate of mcr−1 was 6.0% which was higher than that in Europe (0.1 ).
In this study, all the Salmonella resistance to PB, however, only 6.0 of the isolates carried mcr−1 which was different from the report that there was a close positive correlation between the resistance phenotypes and genotypes of the isolates [39].
The presence of genetic element such as integrons is often associated with multi-resistant phenotypes among Salmonella isolates and plays an important role in spread of antimicrobial resistance genes among gram-negarive bacteria [40]. In this study, class 1 integron detected in 26.9% of Salmonella isolates, which was higher than that previously study from raw chicken and beef meat in Egypt (13.3%) [36] and from poultry in Korea (9.1 ) [41], but lower when compared with a report from meat and dairy products in Egypt (39.1 ) [32]. The predominant gene cassette was dfrA17-aadA5 which conferring resistance to trimethoprim (dfrA) and spectinomycin (aadA) and has been reported worldwide in isolates from different origins, might be associated with the extensive use of trimethoprim and spectinomycin in broiler breeding. In addition, 61.1% class 1 integron was detected in MDR Salmonella isolates which was different the reports that all the class 1 integron exhibited resistance to at least three classes of antimicrobials [42,43].
MLST results showed that seven STs were generated from all Salmonella isolates belonging to six serotypes. ST11 was the most frequent genotype that was recovered in this study and this ST corresponded to S. enteritidis, which was coincide with our previously report from chickens in Shandong [25]. ST19 and ST 34 corresponded to S. typhimurium have contiually been reported to cause human salnellosis in resent years [44]. Another case that should be considered is ST92,a rarely reported ST which appeared in Shandong province that corresponds to S. pullorumm which often reported to cause pullorum disease of chickens in China [42].

Conclusion
In summary, we examined the epidemiology of Salmonella from broilers in Shandong, China. The results showed that the prevalence of Salmonella was found to be high in the broilers. S. enteritidis and S. typhimurium were the most common serotypes that reported to cause human salmonellosis, and the high rate of MDR Salmonella were recovered in this study. This finding highlights the fact that broilers remain to be an potential reservoir of antimicrobial-resistant Salmonella. Therefore, prudent use of antibiotics and management in broilers breeding should be enacted by the authorities to ensure food safety. Academy of Agricultural Sciences "Young Talent" Program.

Availability of data and materials
The data used and analyzed during the present study are accessible from the corresponding author on request.

Ethics approval and consent to participate
Verbal consent for all the sampling procedures was obtained from the owners of the animals. All procedures were approved by the Animal Care and Use of Shandong Academy of Agricultural Sciences