Salmonella, a foodborne pathogen, causes gastroenteritis with severity ranging from diarrheal symptoms to death (Bae et al., 2013). Salmonella is one of the most common pathogens causing foodborne poisoning and is listed as a significant cause of illness and death by the World Health Organization (Chang et al., 2022). To date, approximately 2,600 serotypes have been identified and the majority of them can cause cross-infection between animals and humans (Raufu et al., 2021). Among these, Salmonella Enteritidis (S. Enteritidis) and Salmonella Typhimurium (S. Typhimurium) are the frequently isolated as the most common serovars worldwide (Monte et al., 2021; Raufu et al., 2021). Some studies indicated that Salmonella can be transmitted to humans along the farm-to-fork continuum, commonly through contaminated foods of animal origin, namely poultry, swine, pigeon, cattle, fish etc. (Jajere, 2019; Stevens and Kingsley, 2021). Over the last few years, periodic outbreaks of Salmonella have been reported worldwide to result in tremendous economic losses.

Antibiotic therapy acts as the first-line approach to treat and control Salmonella infection, and antibiotic-resistance in Salmonella has received global attention (Vinueza-Burgos et al., 2019). Cephalosporins and fluoroquinolones are the conventionally used for the treatment against Salmonella infection. Unfortunately, the emergence of extended-spectrum β-lactamase (ESBLs) producers and fluoroquinolone resistant strains among Salmonella posed a challenge for clinical treatment of Salmonella infection by dampening the antibiotic efficacy (Mąka and Popowska, 2016). The major ESBLs families are found as the bla_TEM, bla_SHV, and bla_CTX-M clinically, of which, the bla_CTX-M group presented as most commonly identified ESBL type in Salmonella spp. (Bai et al., 2016). The plasmid-mediated quinolone resistance (PMQR) involves acquisition of (i) qnr genes (qnrA, qnrB, qnrS, qnrC, qnrD), (ii) the aac(6^，)-lb-cr gene, and (iii) oqxAB and qepA genes (Robicsek et al., 2006; Correia et al., 2017). They predominantly contributed spreading of fluoroquinolone resistance Salmonella spp. at human–animal interface (Cuypers et al., 2018). And it is even worse after irrational usage of antibiotics in agriculture for decades, leading to further development of resistances to diverse antibiotics in Salmonella (Cuypers et al., 2018). These multidrug resistant (MDR) Salmonella now become a life-threatening concern to public health with increased morbidity and mortality (Economou and Gousia, 2015; Bai et al., 2016). With growing awareness to tackle the further development of antibiotic resistance in Salmonella, longitudinal surveillance programs have been launched by governments or researchers, providing valuable epidemiological data for risk assessment and medication guidance.

As mentioned above, colonized Salmonella exert harmful impact on host health. The pathogenicity is generally dependent upon their virulence which responsible for bacterial adhesion, invasion and replication within the host, thereafter damage infected tissues. The virulence can be encoded by genes presented either on the bacterial chromosome or plasmids, to work symmetrically to infect the hosts (Raufu et al., 2021). To date, 24 SPIs have been identified and characterized, which are involved in different stages of Salmonella infection. Of which, SPIs-1-5 were common to all serotypes of Salmonella. SPI-1 and SPI-2 contain a large number of virulence genes associated with the intracellular pathogenesis and co-encode T3SS, a molecular syringe (Wang et al., 2020; Sedrakyan et al., 2022). Other SPIs are present to varying degrees in S. Enteritidis subspecies, some encoding other secretion systems such as T1SS and T6SS, other effector molecules, and fimbriae (Sedrakyan et al., 2022). Among the virulence associated genes, different virulence factors played different role in pathogenesis. Fimbriae virulence genes (sefA, lpfA, lpfC, csgA, and pefA) promote bacterial binding to intestinal epithelium (Webber et al., 2019). Fimbriae are essential for the synthesis of extracellular polymeric substances (EPS), which are involved in organism formation and environmental persistence. SpvB associated with the Salmonella virulence plasmid, responsible for intracellular maintenance and bacterial survival (Kong-Ngoen et al., 2022). The invA, orgA, sipB, prgH, and spaN genes are related to the structure of TTSS (Type Three Secretion System) (Khajanchi and Foley, 2022). SifA, avrA, sopE, sopB and sivH genes associated to Salmonellosis outbreaks (Webber et al., 2019). CsgA is associated with pathogenic mechanisms and autoagglutination, promoting inflammation and increasing invasion.

Considering the consumption of animal-derived food accumulatively increase for decades, the prevalence and characterization of Salmonella in contaminated food of animal source is of interest. However, the previous works majorly focused on certain provinces or territories, rarely reaching to the in-depth understating towards evolutionary trajectories and transmission dynamics of Salmonella nationwide (Li et al., 2013; Zhu et al., 2019; Chen et al., 2020). Therefore, we investigated the distribution characteristics, phenotypic and genotypic antimicrobial resistance and virulence profiling of Salmonella isolates from 4 major food animals in China.

In this study, a total of 617 Salmonella isolates were obtained from 4 major food animals in 23 provinces, China. As shown in the section above, animal products in Guangdong and Shandong demonstrated more Salmonella contamination In a previous study, we found that food animals, especially the chickens, pigs, ducks, and geese, in Shandong and Guangdong have been massively contaminated by antibiotics-resistant Enterobacteriaceae (Wang et al., 2021). Therefore, we initiated this study to further investigate the Salmonella colonization on these food animals. Similar to previous reports, Salmonella isolates have been frequently recovered from foods like chicken, eggs, pork, duck, beef, peanut, and vegetables (Viana et al., 2019; von Hertwig et al., 2019; Chen et al., 2020; Hai et al., 2020; Yang et al., 2020b). As indicated in this study, prevalence of Salmonella has accumulated significantly since 2015 by 5.51% to 2020 by 27.23%. This interestingly increasing trend in Salmonella prevalence is similar to human non-typhoidal Salmonella (NTS) infections from 2004 to 2016 by 17% (Bharat et al., 2021).

There were 41 serotypes identified among the 617 Salmonella isolates, from which S. Derby, was the most common serovar followed by S. Enteritidis and S. Typhimurium. S. Derby were also previously found to be the predominant serovar in retail meat, particularly in pork meat (Xu et al., 2020). In several provinces of China, this serovar was also considered as the most prevalent Salmonella species from pig borne food (Jiang et al., 2021). Nonetheless, S. Enteritidis, and S. Typhimurium were the most common non-typhoidal Salmonella serovars in surveillance for invasive Salmonella disease from 2002 to 2018 (Still et al., 2020). In another study, 35 serovars were identified among 667 Salmonella isolates from retail poultry meat, and predominant serovars were S. Enteritidis, S. Indiana and S. Typhimurium (Yang et al., 2020a). Also, there were 19 isolates whose serotypes were not clearly determined, indicating that there were possibly new serotypes in development and the causes of the contamination is complicated (Xu et al., 2020).

Widespread and improper usage of antimicrobials accelerate the development and spread of AMR (Raufu et al., 2021). Although the presence of infection caused by antimicrobial resistant Salmonella reported worldwide, the cases in developing countries is remarkably increasing at an alarming rate (Jiang et al., 2021). Antibiotics selected for this study were all first-line drugs currently in clinical use in veterinary medicine. Our results suggested that antimicrobial resistance was present in the majority of Salmonella isolates, 80% isolates were resistant to ampicillin, neomycin, nalidixic acid, tetracycline, doxycycline, and sulfadiazine/trimethoprim. The outcome was consistent with those reports (Fang et al., 2019; Asefa Kebede and Duga, 2022; Borah et al., 2022; Hassena et al., 2022). Noteworthy, meropenem resistance appeared in fewer isolates (0.5%) in our study. These outcome was in disagreement with other reports that indicated no resistance for colistin, nalidixic acid, meropenem, gentamicin, florfenicol and chloramphenicol in Australian (Abraham et al., 2022), no resistance for meropenem in Eastern China (Tang et al., 2022), fewer resistance for ampicillin and sulfonamide-trimethoprim in Ethiopia (Asefa Kebede and Duga, 2022), These disagreement may result from geographical and biological differences exist among various strains. In addition, colistin is considered one of the last resort for the treatment of multidrug-resistant Enterobacteriaceae (Elbediwi et al., 2019), 4.7% of Salmonella isolates showed resistance to colistin in our study, which was consistent with other reports (Li et al., 2022; Tang et al., 2022) from poultry and pigs. In present study, more antimicrobial resistance was identified in S. Derby and S. Typhimurium than those in S. Enteritidis. It is different from a previous study which characterized the S. Indiana with most enriched antimicrobial resistance genes among all serovars (Yang et al., 2020a). In this study, 99.8% of the Salmonella isolates were MDR, which was consistent with Tang’s report (Tang et al., 2022). The majority of Salmonella isolates were resistant to 8 ~ 12 antibiotics, highlighting the worsening situation of multidrug resistance development in Salmonella in China. Therefore, the continuous surveillance of antimicrobial resistance in Salmonella and political implements are conducive to safeguard consumer health (Chen et al., 2020).

Biofilm formation is a crucial strategy for Salmonella survival under unfavorable environmental conditions (Merino et al., 2019). Studies have found a range of bacterial cell surface components such as cellulose, flagella and fimbriae contributing to the attachment of Salmonella to different surfaces (Kroupitski et al., 2009). In this study, we found that 88.49% of Salmonella isolates were able to produce biofilms at different degree. A total 236 Salmonella isolates (38.3%) were strong biofilm producer. Some studies show that all of the Salmonella isolates from the chain of beef production and retail were able to form biofilm (Yin et al., 2018; Manafi et al., 2020). Also, we found that the detection rates of strong biofilm producer were higher in S. Enteritidis (48.2%) and S. Typhimurium (55.7%) isolates. This finding is consistent with a previous study that S. Enteritidis was the strongest biofilm producer (Manafi et al., 2020). The enhanced biofilm producing ability demonstrated high co-occurrence with the resistance to azithromycin, colistin, cefotaxime levofloxacin, gatifloxacin. These may be due to co-localization of genes encoding biofilm and antibiotics resistance in Salmonella isolates (Shi et al., 2018). Voss-Rech et al. reported that 65% of the isolates showed the ability to produce biofilm in Brazil (Voss-Rech et al., 2022), and Manafi et al. reported that all isolates were able to form biofilm (75.86% moderate and 24.14% strong) (Manafi et al., 2020). These previous results are consistent with our findings, showed that antimicrobial resistance may correlate with biofilm formation. Biofilm formation will enhance resistance to antibiotics and disinfectants, enhance the ability of Salmonella to survive in animals and the environment, leading to big harm to animals (Dai et al., 2021). This study underlined the ability of Salmonella to contaminate food possibly attributed by their biofilm producing capability. Therefore, it is important to study the mechanism of biofilm formation to prevent the spread and infection of Salmonella.

Salmonella harbor diverse antibiotic resistance genes along with mobile genetic elements, which accelerate the dissemination of resistance to other serotypes or even bacteria of different genera (Sharma et al., 2019). Five β-lactam resistance genes were detected among these Salmonella isolates, and bla_TEM was the most predominant, which is consistent with prior studies that bla_TEM was the most frequent genotype to confer the β-lactam resistance to Salmonella from retail chicken meat, poultry, pig and humans (Djeffal et al., 2017; Trongjit et al., 2017; Sharma et al., 2019). Seven types of quinolone resistance genes were detected including oqxAB, qnrBCDS, and qeqA. The co-existence of quinolone resistance genes with other clinically important ARGs including ESBL genes was observed in current and previous study (Fang et al., 2019). In this study, tetB and sul2 were the most frequent in tetracycline and sulfonamide resistance genes, respectively. Contrary to previous publications, Sharma and coworkers reported that tetA and sul1 were the most frequent resistance genes to protect Salmonella from bactericidal effect of tetracycline and sulfonamide antibiotics (Sharma et al., 2019). The studies on the number of ARGs carried by strains, results showed that all strains carried more than three ARGs, and one strain carried 18 ARGs, which warranted in-depth study. These results are consistent with the reported results (Akinyemi et al., 2011; Ammar et al., 2016a; Abraham et al., 2022; Hassena et al., 2022; Kong-Ngoen et al., 2022; Tang et al., 2022), which all concluded that Salmonella carries multiple ARGs. The results of our analysis for the three main prevalent serotypes carried ARGs, showed that S. Derby mainly carried 11 ~ 14 genes, S. Typhimurium mainly carried 4 ~ 7 genes, and S. Enteritidis mainly carried 3 ~ 4 ARGs. This result revealed that there is no correlation between serotype and ARGs. The occurrence of this may be related to the resistance genes mainly associated with antibiotic resistance (Ammar et al., 2016a; Abraham et al., 2022).

The currently known virulence factors of Salmonella include toxin production, capsule, flagellum, fimbriae, secretory system and other factors which are involved in various stages of infection (Sedrakyan et al., 2022). Salmonella virulence factors encode products to assist invading procedures in the host (Raufu et al., 2021). PipA was an effector protein, which redundantly target components of the NF signaling pathway to cause inflammation (Sun et al., 2016). Salmonella invasion protein B (SipB) initiates the invasion process which belongs to Salmonella type 3 secretion system (Chen et al., 2018). sodC1 and sodC2 were two genes encoding periplasmic superoxide dismutase, locating on lambdoid prophage and chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide radicals generated by host’s phagocytes (Ammendola et al., 2005). SpvB presented on the plasmid and facilitated Salmonella survival and replication within macrophages via perturbing cellular iron metabolism (Deng et al., 2021). The role of some of these virulence-associated plasmids in the dissemination of increased virulence in food-animal environments and humans (Ammar et al., 2016b; Khajanchi and Foley, 2022). While the main differences between isolates were attributed to the serotype-specific diversity of virulence genes, SPIs, virulence plasmids, and phages (Hassena et al., 2022). The presence of some virulence genes was serotype specific (Sedrakyan et al., 2022). In this study, 17 virulence genes were identified among these Salmonella isolates. The results suggested that the isolates regularly harboring most of virulence genes selected in this study with a detection rate of over 80%. This is in agreement with the findings in previous studies (Hai et al., 2020; Borah et al., 2022; Siddiky et al., 2022). By contrast, 6 virulence genes (pipA, sipB, sodC, spvB and spvC) were detected at lower presence. The diversity of virulence genes may provide important characterization clues for the further study of Salmonella pathogenicity (Sedrakyan et al., 2022). In this study, the high detection rate of these virulence genes may explain why Salmonella infection is prone to cause significant morbidity or mortality in animals. The action mechanism of virulence genes in Salmonella needs to be further investigated. Continuous surveillance of the prevalence, resistance and virulence of Salmonella in food animals will greatly enhance the control surveillance and future outbreak investigation of the infection.

In conclusion, this study investigated the prevalence and distribution of Salmonella isolates from food animals in China. A total of 41 serotypes were identified, and S. Derby, S. Enteritidis, and S. Typhimurium were the most prevalent serotypes. The high rates of antimicrobial resistance were observed among the majority of the Salmonella isolates, and biofilm formation ability can enhance the resistance of Salmonella to antibiotics. In addition, these isolates carried abundant antibiotics resistance genes and virulence genes. This study provided useful information regarding the epidemiological characteristics of Salmonella in the food animals in China and may help the policy-making to better control the fast development Salmonella contaminations China.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

YZ and XL designed and coordinated this research and drafted the manuscript. LG conducted experiments. YL, JS, and HR modified the manuscript. TX, LW and WL carried out the data analysis. KL and WJ conceived of this study. YL and XD revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was jointly supported by grants from PhD Fund of Qingdao Agricultural University (663-1119017), Shandong Provincial the Development of new raw materials and preparations for Animal Respiratory Disease Control under Grant (662-2321018), Taishan Industrial Experts Program (TS20220701), Qingdao Science and Technology for the People Demonstration Special-Creation of monitoring and early warning platform and demonstration of purification and prevention and control for important animal diseases (2023), Innovative Research Groups of the National Natural Science Foundation of China(32121004), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02N054), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT_17R39) and Innovation Team Project of Guang dong University (2019KCXTD001).

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.

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