Salmonella spp., as one of the most prevalent foodborne pathogen, are reportedly to cause severe and acute intestinal diseases called salmonellosis via contaminated food chain (Chu et al., 2024). The previous studies indicated that Salmonella spp. are versatile pathogens capable of infecting a wide range of host animals, including chickens, cattle, and pigs, which are closely related to anthropogenic environments and humans. The authorities in the United States estimated that Salmonella causes approximately 1.35 million cases of infections with 420 deaths reports annually (Centers for Disease Control and Prevention | CDC). It is noted that the poultry has been a major source and an important reservoir of Salmonella spp. (Andoh et al., 2017; Feasey et al., 2012; Pui et al., 2011). Contaminated poultry products such as meat and eggs, are common cues for Salmonella accumulation and subsequent salmonellosis in humans (Lamas et al., 2016; Tedersoo et al., 2022). A directly evidence is the large foodborne infection outbreak across the U.S. in 2018 had been linked to raw chicken meat, highlighting the widespread prevalence of Salmonella in the broiler industry serves as a health threat that should not be neglected (Chu et al., 2023). Additionally, Salmonella spp. demonstrated high adaptive tolerance in different harsh conditions to transmit from animals and environments to related products through fecal contamination (Smith et al., 2023). A prior study on free-ranged poultry revealed that Salmonella was able to transmit from poultry to workers within in the same ecology via daily contacts. Therefore, longitudinal epidemiological analysis of Salmonella in poultry is crucial for in-depth understanding toward prevalence and genomic insights, which helps to better control on their potential outbreak.

Currently, antibiotics like fluoroquinolones and cephalosporin are frequently used to eradicate Salmonella infections (Chiu et al., 2002). However, the misuse and overuse of such antibiotics in animals have led to a global increase of antibiotic resistance in Salmonella spp. dampening the efficacies of such agents for clinical treatment against Salmonella (Wang et al., 2021; Xu et al., 2020). Previous studies have shown that infections caused by antibiotic-resistant bacteria (ARB) greatly challenge the current treatment paradigm and are associated with higher mortality rates in clinical settings (Crump et al., 2008). Moreover, it has been confirmed that both farm animals and wildlife are able to carry the identical Salmonella serotypes, thereby leading to high level dispersion of such ARB among the different host in the same niches (Cui et al., 2019).

Innovations in genomic analysis contribute accumulative genomic data of Salmonella in the public database, which generated from cost-effective, high-throughput whole-genome sequencing (WGS) (Wang et al., 2023). WGS is progressively becoming the standard approach for in depth understanding of pathogens. As a foundational tool in current microbiology studies, WGS data also facilitate the identification of virulence factors and antimicrobial resistance (AMR) genes (Chiou et al., 2023; Chiou et al., 2022; Collineau et al., 2019; Sohail et al., 2021). Notably, Salmonella Pathogenic Islands (SPIs) that house Salmonella virulence genes play a significant role in the pathogenesis of Salmonella in divergent hosts (Hensel, 2004; Rychlik et al., 2009). The previous investigations highlighted that SPIs vary among the serotypes and may explain differences in virulence across Salmonella serotypes (Zhao et al., 2020). Therefore, the surveillance of these ARGs and virulence factors are crucial for comprehending the evolution and pathogenicity of Salmonella.

In this study, we conducted a longitudinal study on the prevalence of Salmonella spp. in chicken, duck, and goose populations across eight cities in Shandong, China from 2019 to 2022. Subsequently, we phenotypically and genotypically identified the antimicrobial resistance of collected Salmonella strains by analyzing MLST, phylogenetic relationships, antibiotic resistance genes (ARGs), and virulence factors. This study provides vital information into the molecular epidemiology and potential pathogenicity of poultry-derived Salmonella, and contributes to development of timely control before the spread of such concerning pathogens.

Salmonella spp. is considered one of the most significant foodborne pathogens and rank as the third leading cause of human mortality related to diarrheal illnesses. Animals serve as the primary reservoir for Salmonella, with animal-derived foods being the essential route for the transmission to humans via food chain (Ferrari et al., 2019). This study aimed to assess the prevalence and distribution of Salmonella isolates from poultry species in the Shandong province, which is one of the major bases of agricultural and livestock industry in China.

From the samples collected in eight major cities in Shandong during 2019 to 2022, a total of 53 Salmonella isolates were identified. This isolation rate was higher than that previously reported in Xinjiang (11.0%, 8/73) and Guangdong Province (3.58%, 301/8405), yet much lower than that in Tibet (30.4%, 70/230) and Henan (48.7%, 131/269) (Perry et al., 2024). However, the exact prevalence rates of Salmonella were observed heterogeneous in the different sampling sites of the eight cities, ranging from 8.3 to 25% accordingly. A previous study reported Salmonella detection rates in 12 provinces were ranging from 3.6 to 12.9% (Gong et al., 2014), indicating the Salmonella prevalence might be under certain geographic influences. The study also showed a notable presence of Salmonella contamination in poultry in Shandong Province, particularly in chickens which was with an alarming rate of 18.2% (41/225) (Liyuan et al., 2023). These results implied a wide presence of Salmonella contaminations in varied poultry breeds. Moreover, the isolation rate of Salmonella was also reported to be varied across the different environments, as some studies evidencing that the detection of Salmonella increased from poultry farms (9.1%) to slaughterhouses (9.5%) and finally enriched in the retail markets (41.8%) (Dargatz et al., 2016; Papadopoulos et al., 2016; Shang et al., 2019). To conclude, these differences were under impact of many factors such as geographical factors, environmental conditions and poultry species/breeds (Cao et al., 2017).

The prior study highlighted the increasing prevalence of Salmonella were potentially influenced by climate changing and dietary supplementations. For example, investigation by Sonora showed that Salmonella isolation rates were found to elevate in concert with the increment of temperature (Davies and Breslin, 2002). This suggests that global warming and climate change may promote Salmonella dispersion in nature especially in the anthropogenic environments. Factors such as antibiotic use, poultry density, and environmental pollution are as well believed to contribute to the global rise of Salmonella prevalence (Davies and Breslin, 2002; Sohail et al., 2021). It underscores the urgent necessity to monitor the Salmonella spp. and the preventive measures by relevant authorities.

This study identified S. Enteritidis as the predominant serotype among the collected isolates, followed by S. Pullorum and S. Typhimurium. In recent years, the contamination of S. Enteritidis has been a critical health concern in many provinces of China (Gong et al., 2014; Yang et al., 2019). This indicates that the widespread of S. Enteritidis is plausibly linked to the extensive contamination of poultry and poultry products. Cases of salmonellosis due to the consumption of contaminated poultry products, including meat, eggs and fresh products, have been reported as early in 2011 (Collineau et al., 2020; Middleton et al., 2014; Varga et al., 2012). S. Typhimurium, one of the most common serotypes in humans, have been frequently associated with severe infections on pigs, cattle, and other livestock in the European Union and the United States (Kuus et al., 2021). However, currently, this species has spread extensively worldwide, affecting not only fresh pork and beef but also poultry meat and poultry-related products. The consumption of contaminated livestock and poultry products has been proposed as a significant driver for bacterial infections in human (Perry et al., 2019; Ploton et al., 2017; Shen et al., 2022).

The tremendous use of antibiotics in animal production to promote growth and combat infection contributes to the rapid development of antibiotic resistance among pathogens (Liyuan et al., 2023). Our study revealed high levels of resistance in Salmonella isolates to clindamycin, lincomycin, ampicillin, enrofloxacin, azithromycin, and doxycycline, possibly owing to large-scale antibiotic usage for poultry farming. The S9 Salmonella strain exhibits resistance to 15 different antibiotics, making it a typical example of multidrug-resistant (MDR). Salmonella strains in the late 1980s challenged treatment choices amoxicillin. The MDR strains of Salmonella exhibit various resistance pattern, including ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline) and AKSSuT (ampicillin, kanamycin, streptomycin, sulfamethoxazole, tetracycline) resistance pattern has been commonly reported. And this often includes first-line antibiotics (β-lactamases, macrolides), and the rise of MDR strains has compelled clinicians to resort to second-line agents (quinolones, tetracyclines and third-generation cephalosporins) (Rahman et al., 2023; Ramatla et al., 2021; Rowe et al., 1997; Samia et al., 2021). It is of particular concern that β-lactams and fluoroquinolones, which are typically the primary options for treating salmonellosis, were found to be lack of potency to the strains collected in our study due to carriage of corresponding ARGs. As observed with the emergence of Salmonella Infantis (ESI) clone in 2014, which was first detected in retail meats in Tennessee, by 2019, it had spread across the United States. The pESI plasmid underwent significant recombination, carrying not only the extended-spectrum β-lactamase gene bla_CTX-M-65, but also the gyrA mutation, which confers resistance to fluoroquinolones, further limiting treatment options (Perry et al., 2024). Additionally, Salmonella strains are often transmitted through contaminated food, wastewater, animals, and other sources (Cosby et al., 2019; Gebreyes et al., 2008). This suggests that MDR strains have the potential for spread, which undoubtedly poses a significant threat to public health, exacerbates the burden on clinical medicine, and imposes a tremendous economic strain on society. However, the resistances to cephalosporins and fluoroquinolones have been observed to increase in many countries, possibly due to incorrect antibiotic usage. Additionally, Shea (2004) highlighted that prolonged antibiotic therapy contributed to development of antimicrobial resistance. Ciprofloxacin and enrofloxacin-resistant Salmonella isolates have been widely found in aquaculture, poultry, poultry meat, and pig manure (Ubeyratne et al., 2023; Wang et al., 2022), which threaten both animal wellbeing and human health under One Health concept.

MLST analysis revealed that all S. Enteritidis, S. Typhimurium, S. Kentucky, and S. indiana isolates belonging to ST11, ST19, ST198, and ST17, respectively. ST11 emerged as a predominant serotype, particularly prevalent chicken samples in our cases. Notably, 95% of S. Enteritidis isolates in England and Wales from April 2014 to March 2015 were classified as ST11 (Aung et al., 2022). The European Food Safety Authority (EFSA) indicated that the outbreak of infection of Salmonella ST11 in 2022 in Europe was associated with eggs and egg products, underscoring the urgent need for controlling the Salmonella contamination in poultry and its supply chain. The ST19 is the predominant genotype of Salmonella isolate with broad geographic distribution (Achtman et al., 2012; Gómez-Baltazar et al., 2023). The ST19 Salmonella is commonly found in patients with diarrhea and is always detected in various origins including animals, food, and humans (Sun et al., 2014; Wong et al., 2013). S. Kentucky, an emerging pathogen, has been increasingly responsible for human non-typhoidal Salmonella infections since 2005 (Mahindroo et al., 2019). While S. Kentucky isolates are frequently found in poultry and livestock yet rarely associated with human infection. Furthermore, the ST198 has been observed to spread independently among poultry and other livestock, occasionally leading to human infections (Samper-Cativiela et al., 2022). In this study, S. Pullorum was found with ST92 and ST3717.

Phylogenetic analyses are crucial for understanding microbial evolution and infectious disease transmission. Bacterial phylogenies are often inferred from SNP alignments, with SNPs as the method in the genetic relationship between isolates, allowing for comparison of genetic information at the genome level for each isolate (Dallman et al., 2018; Mona et al., 2025). In this study, the core-genome sequences of S. Kentucky isolate from Yantai (S9) demonstrated high similarity with an isolate from Liaocheng (S51) by showing only 20 single nucleotide polymorphisms (SNPs) in the genome. The genetic distance between these isolates is small, suggesting that they may originate from the same clonal group, which is consistent with previous Salmonella studies. Specifically, isolates with a small SNP distance are highly likely to come from the same clonal lineage (Wang et al., 2018). Additionally, SNP analysis revealed significant genetic diversity among these isolates. For example, isolate S50 showed over 30,000 SNP differences compared to several other isolates from the same sampling site (Liaocheng), including S45, S46, S47, S5, S52, S53, S54, S83, S84, and S86. This finding suggests that the genetic variation may be related to the source and time of isolation, with the primary factor being the origin of the Salmonella isolates (Levent et al., 2021). With this estimate of genetic similarity, bioinformaticians can better identify isolates that likely have a recent common source, and provides essential data for public health management.

The phenotypic resistances are closely associated with ARGs. Genotypic analysis revealed that the most prevalent ARG gene was aac, followed by aph, sul2, and tet (A/B). These genes conferred the resistances to aminoglycosides, sulfonamides, and tetracyclines (Ubeyratne et al., 2023). Further analyses also uncovered the presence of resistance genes against β-lactamase, including bla_TEM, bla_OXA, bla_CTX-M-65, bla_CTX-M-55, and bla_CMY. Among them, bla_TEM was the most prevalent (Olesen et al., 2004). These β-lactamases are active to hydrolyze the cephalosporins, dampening the efficacies of such antibiotics. Notably, genes like aadA rendered the isolates resistant to streptomycin, gentamicin, and tobramycin (Chu et al., 2023; Samia et al., 2021). Previous studies have also identified these genes and their impact on antimicrobial resistance in Salmonella spp.

Plasmids have been reported as a key player in the dissemination of ARGs, virulence genes, and other traits that provide a fitness advantage to host bacteria. Analysis on plasmid in the current study revealed the presence of 9 different plasmid replicons existing in the Salmonella isolates, with the IncFII as the dominant replicons. These plasmids were found in various host bacteria, indicating their ability to spread the harbored genes with their transmissions. IncFII and IncFIB are the most common variants within the IncF plasmid family. They are important vectors for the carriage and transmission of antibiotic resistance and virulence genes (Kline, 1985; Ravi et al., 2017; Villa et al., 2010). They are important vectors for the transmission of antibiotic resistance and virulence genes. In particular, IncFII plasmids are also the main vector of mcr-1 in E. coli isolates (Sugawara et al., 2019; Xavier et al., 2016). The IncF plasmids in Enterobacteriaceae are of particular interest since they contribute to the carriage and spread of ARGs and virulence genes (Carattoli, 2011). IncF plasmids have been isolated that contain ARGs genes. IncF plasmids in Salmonella isolated in China carried fluoroquinolone resistance genes (Chen et al., 2018a). In Salmonella isolated in the United States, IncF plasmids have been associated with strAB, tetA, tetC, tetD, aphA (aminoglycosides), and sul2 (Han et al., 2012; McMillan et al., 2019; McMillan et al., 2020), and they were most frequently described carrying ESBL genes, encoding carbapenemases genes, and plasmid-mediated quinolone resistance (PMQR) genes (Rozwandowicz et al., 2018). In addition, they also carry specific virulence traits such as cytotoxins and adhesion factors as accessory genes (Johnson and Nolan, 2009). In this study, IncF family plasmids were isolated from multiple serotypes of Salmonella, primarily S. Enteritidis and S. Typhimurium, across different regions. This suggests that IncF family plasmids may be associated with specific regions and serotypes. These findings are in accordance with previous studies, where the IncF family was detected in multiple serotypes of Salmonella across different regions and farms (NCF3, −5, and − 6) (Pornsukarom and Thakur, 2017; Wang et al., 2013). The association between plasmid types and specific serotypes or geographical regions may lead to the spread of antibiotic resistance and treatment limitations, exacerbating the antibiotic resistance crisis and posing a significant threat to global public health. Furthermore, IncHI2, as part of the backbone element of the pSE380T plasmid, in conjunction with the IncFIA plasmid, produces a rare fusion product encoding both virulence and resistance genes with high transmissibility (Villa et al., 2010; Wong et al., 2017). Therefore, monitoring the prevalence of ARGs together with plasmids profiling is crucial for the prevention and control of antibiotic-resistant Salmonella.

Analysis of WGS data revealed that the Salmonella isolates were found with multiple virulence factors including invABCEFGHIJ, prgHIJK, hilACD, sicAP, sipABCD, sopABD/D2E2, spaOPQRS, sptP, and mgtBC. Within these, virulence genes like invA, mgtC, and sopB are clustered in the Salmonella Pathogenicity Island (SPI) on the chromosomes, help Salmonella to establish systematic infection by mediating cell invasion, intracellular survival, and inflammatory responses (Marcus et al., 2000). For instance, hilACD are able to activate transcription of key virulence genes on SPI-1, facilitating the release of essential effector proteins for invading the host intestine (Bosire et al., 2020). Furthermore, the spv genes located on the Salmonella virulence plasmid (IncF), which is involved in survival and systemic infection in host cells, were detected in S. Enteritidis and S. Typhimurium. The spvABCD genes are located upstream of the genes pefA (plasmid-encoded fimbriae) and rck (resistance to complement killing) in a virulence plasmid by the upstream spvR gene (Monte et al., 2020; Silva et al., 2017). However, spvR, which is needed for the regulation of the spv locus (Guiney and Fierer, 2011), was absent in our strains. Previous studies have indicated that the spv operon, which plays a role Salmonella survival within the host cell during systemic infection, affects the formation ofautophagosomes, as well as highlight its association in killing of macrophages and neutrophils (Wu et al., 2016), being crucially required for virulence in vivo (Passaris et al., 2018), including aggravated damage in zebrafish infection model (Cao et al., 2023; Hsu et al., 2019; Passaris et al., 2018). Moreover, other virulence factors like pefABCD, lpfABCDE, cdtB, pltAB and rck were detected. The lpfABCDE and pefABCD gene clusters on pSV are reported to regulate the formation of long polar fimbriae fimbriae. The cdtB is a common virulence gene found in certain Gram-negative bacteria, and the expression of cdtB promotes toxin production in non-typhoidal Salmonella (Wójcicki et al., 2021). In addition, cdtB was characterized in S. typhi as one component of the cytolethal distending toxin (CDT) (Haghjoo and Galán, 2004). Several Gram-negative bacterial pathogens, including S. typhi, produce CDT, which arrests growth, induces apoptosis of infected host cells, and enhances the persistence of pathogenic bacteria in the host. The cdtB genes in S. typhi is located on a pathogenicity island upstream of the pltA and pltB genes, which encode pertussis-like toxins A and B (PltA and PltB). These toxins form a complex with cdtB, with the pltB gene having no apparent effect on cellular distension. However, when cdtB is combined with pltA, it induces noticeable distension in both the cytoplasm and the nucleus (Mezal et al., 2014). In S. Javiana, cdtB may require PltA for the typical signs of cytolethal distension, such as cytoplasmic and nuclear swelling. The cdtB gene, in conjunction with pltA (pertussis-like toxin A) and pltB (pertussis-like toxin B), is necessary to induce intoxication signs in eukaryotic cells, such as cellular distension and cell cycle arrest (Mezal et al., 2014; Spanò et al., 2008). In the other hand, cdtB, with the assistance of cdtAC, is translocated into the nucleus of target cells, where it induces double-strand breaks in DNA, resulting in cell apoptosis (Ohara, 2004; Thakur et al., 2022).

This study has identified a total of 53 Salmonella isolates from 300 samples collected during 2019 to 2022 in Shandong, China. Among the 53 Salmonella isolates, seven serotypes were identified wherein the S. Enteritidis was found to be most prevalent, followed by S. Pullorum and S. Typhimurium. WGS analysis revealed that ST11, ST92, and ST19 were the dominant sequence types in Salmonella in this study. The findings underscore the importance of epidemiological surveillance in the major agricultural area like Shandong Province. Furthermore, the majority of Salmonella isolates exhibited features of multi-drug resistance, highlighting the need for precision controlling strategies against such resistant Salmonella.

The majority of Salmonella isolates in this study exhibited multidrug resistance, highlighting the need for precise control strategies to address these resistant strains. This underscores the critical role of the One Health concept, which recognizes the interconnectedness of human, animal, and environmental health. The emergence and spread of multidrug-resistant Salmonella are influenced by various factors, including human, animal, and environmental elements. Therefore, an integrated approach should not only monitor human health but also coordinate animal health management and environmental surveillance. Such a strategy is essential for curbing the spread of resistant bacteria and mitigating the public health risks posed by multidrug-resistant Salmonella.

However, several limitations exist in this study. Firstly, it was region-specific, focusing solely on Shandong Province, which limits the generalizability of the findings. Additionally, the sample size was relatively small, and the study did not include Salmonella strains from other animals, such as cattle, sheep, and pigs, which may provide insights into cross-species transmission. Moreover, the impact of the rearing environment on Salmonella was not explored. To address these gaps, future research should expand the study to broader regions, investigate cross-species transmission, and examine the influence of environmental factors and farming practices. Incorporating the One Health approach into such studies will be crucial for developing more effective strategies to explore and combat antimicrobial resistance.