Chicken eggs are considered a primary source of food and are often implicated in the transmission of Salmonella, making them a major source of foodborne illness, i.e., salmonellosis (Shaji et al., 2023). Indeed, egg-borne salmonellosis is a common worldwide public health concern that has been documented with major health and economic implications (Pal et al., 2020). Annually, the global economic burden of salmonellosis has been estimated to exceed €2.8 billion (Shekhar, 2018). Salmonellosis (Salmonella infection) affects millions of individuals globally annually, and in the United States (US) alone, it accounts for around 11% of all foodborne infections, with around 35% of those cases requiring hospitalization and about 28% developing complications that may lead to death (Billah and Rahman, 2024; Scallan et al., 2011). Recent outbreaks continue to highlight the relevance of eggs in Salmonella transmission. For instance, in 2024, a multi-state outbreak in the US linked to contaminated eggs resulted in 93 confirmed cases across 12 states, with 34 individuals hospitalized (CDC, 2025). Similarly, the European Union (EU) consistently reports eggs and egg products as among human salmonellosis (Sarno et al., 2021).

Despite the well-known public health significance of salmonellosis, recent evidence indicates that extensive national data about the prevalence, epidemiology, and transmission sources of Salmonella in Saudi Arabia are still limited, highlighting a critical knowledge gap (Alhumaidan, 2024). In Saudi Arabia, Salmonella spp. are frequently identified as significant bacterial agents linked to foodborne illnesses, resulting in a considerable number of reported cases annually (Alhumaidan, 2024). National surveillance data reveal variable incidence rates in recent years, indicating persistent exposure hazards and the possible underestimating of the actual disease burden due to underreporting and constraints in routine source attribution. In Saudi Arabia, although salmonellosis is typically documented as isolated incidents rather than persistent outbreaks, the particular food sources responsible for human infections are inadequately defined (Alhumaidan, 2024; Alhumaid et al., 2024). In the Gulf Cooperation Council (GCC) region, encompassing Saudi Arabia, salmonellosis has been linked to contaminated food products of animal origin, such as chicken, eggs, dairy, and fresh produce (Mohamed et al., 2024). In addition, local epidemiological research have revealed S. enteritidis and S. typhimurium as the most commonly detected serovars in human infections, highlighting their significance for public health surveillance (Alhumaidan, 2024). Thus, comprehending the circulating serovars, antibiotic resistance patterns, and critical virulence factors is vital for enhancing preventative tactics, directing clinical management, and shaping food safety policies (Mohamed et al., 2024; Khan et al., 2024). Salmonella species (spp.) are gram-negative, facultative anaerobic, non-spore-forming flagellated bacilli belonging to the Enterobacteriaceae family (Lamas et al., 2018). Salmonella comprises more than 2,500 serovars, and Salmonella enterica subspecies enterica is the most prevalent serovar among them, which is responsible for approximately 99% of human Salmonella infections (Lamas et al., 2018).

Salmonellosis manifests with gastrointestinal symptoms like diarrhea, abdominal pains, nausea, vomiting, and fever, often within a timeframe of 12–72 h after the ingestion of contaminated food (Giannella, 1996). While Salmonellosis often resolves on its own without treatment within a period of 3–7 days, severe illness can occur in vulnerable populations, including children under the age of 5 or elderly people of age 65 and beyond, and individuals with compromised immune systems due to specific medical illnesses, such as, diabetes, liver or kidney disease, and cancer (Lamichhane et al., 2024; CDC, 2023; Bula-Rudas et al., 2015).

Accurate identification and typing of Salmonella strains are essential for outbreak tracking and public health response. The Centers for Disease Control and Prevention CDC and other global health agencies emphasize the importance of whole-genome sequencing (WGS) technology to facilitate strain-level differentiation, detect antimicrobial resistance (AMR) genes, enhance surveillance capabilities, and better control for salmonellosis incidence and outbreaks (CDC, 2024a; Struelens and Brisse, 2013).

Salmonellosis antibiotic therapy is recommended for severe cases and at-risk individuals, especially for cases with severe illness and a weakened immune system (Chen et al., 2013). Traditionally, β-Lactams (including penicillin derivatives, cephalosporins, and carbapenems), monobactams, and fluoroquinolones are the commonly used antibiotics to treat Salmonellosis (Lamichhane et al., 2024; Chen et al., 2013; Bhandari et al., 2023). However, the therapeutic efficacy of these drugs is increasingly threatened by the emergence of AMR. This rise in the AMR is often attributed to the extensive or unregulated use of antibiotics in animal husbandry, including poultry production (Abou-Jaoudeh et al., 2024).

AMR in poultry production represents a growing risk due to its significance for food safety, public health, and the spread of resistant bacteria across the food chain. In Saudi Arabia, government initiatives to address AMR are directed by a One Health framework that unifies the sectors of human, animal, and agrifood health. The Saudi Food and Drug Authority (SFDA) plays a central role in regulating antimicrobial usage and enhancing food safety monitoring in accordance with the Global Action Plan on Antimicrobial Resistance.

Saudi Arabia has implemented a National Action Plan on antibiotic Resistance (NAP-AMR 2022–2025), focusing on strengthening surveillance systems, improving antibiotic utilization in human and animal health, and enhancing laboratory capabilities for AMR detection (Kingdom of Saudi Arabia, 2026). The NAP-AMR emphasizes the significance of sentinel surveillance locations and laboratory-based monitoring for priority foodborne pathogens, such as Salmonella, in both human and animal sectors. Within this framework, the National Food Antimicrobial Resistance Surveillance System (NARSS), coordinated by the SFDA, has been recently established as a centralized platform to support systematic monitoring of antimicrobial resistance patterns and to facilitate evidence-based risk assessment and policy development. NARSS operates as a confidential, non-public platform, however it is essential for guiding national policy and regulatory decisions concerning food safety and antimicrobial resistance.

Despite these advancements, there is a notable lack of comprehensive genetic data on antimicrobial resistance determinants in foodborne pathogens, especially Salmonella linked to poultry eggs. The spread of AMR in Salmonella is frequently mediated by mobile genetic elements such as plasmids, integrons, and transposons, which contribute to the horizontal transfer of resistance genes across bacterial populations (Algarni et al., 2022). As a result, multidrug-resistant (MDR) Salmonella strains have become a growing concern, not only because they limit therapeutic options but also due to their association with increased morbidity, mortality, and healthcare costs (Algarni et al., 2022). For example, in the United States, an estimated 2 million people are affected annually by antibiotic-resistant bacteria, including Salmonella, leading to over 23,000 deaths (Slayton et al., 2015). This global increase underscores the urgent need for robust surveillance systems and tighter regulations on antimicrobial use in food-producing animals.

On the other hand, in Saudi Arabia, the MDR Salmonella is also a cause of concern due to the rapid growth of the egg production sector (Saudi Arabia, 2021). According to the Poultry and Products Annual Report 2021, Saudi Arabia produces approximately 5 billion eggs yearly, and exports over 5 billion eggs annually to neighboring GCC countries and recently, it reached self-sufficiency levels exceeding 100% (Saudi Arabia, 2021). However, studies on Salmonella in eggs in Saudi Arabia remain limited, regardless of their critical importance to the egg production industry. Most existing research in Saudi Arabia has focused on the prevalence of Salmonella in meat, vegetables, milk, and seafood, leaving a critical gap in understanding its presence in eggs and egg products (Moussa et al., 2010; al-Nakhli et al., 1999). Therefore, this study aims to fill that gap by assessing the prevalence, serovar distribution, antimicrobial resistance, and genomic diversity of Salmonella isolated from chicken eggs in Saudi Arabia using WGS technology.

This study is the first of its kind to focus on Salmonella from chicken eggs in Saudi Arabia, providing comprehensive genomic analysis and valuable insights into its prevalence and genetic diversity. These critical data contribute to both national surveillance and international public health efforts.

The findings of this study reveal the prevalence, serovar diversity, and genomic profiles of Salmonella in all sites of commercial chicken egg samples, including eggshell and egg content, sold in Riyadh, Saudi Arabia. Salmonella was detected in 9% of the 260 examined egg samples, more frequently observed on eggshells (5%) than within the egg contents (3%). This variation between the eggshells and egg contents is expected due to the direct exposure of the eggshells to fecal matter, bedding, and environmental dust within poultry houses, while the internal contamination of the egg contents occurred due to the vertical transmission or trans-shell penetration (Arnold et al., 2014). From a public health standpoint, eggshell contamination poses a considerable danger for indirect transmission via cross-contamination during food preparation, especially when raw eggs are managed in household or food service environments (Alsufyani et al., 2025). In contrast, the detection of Salmonella within egg contents, though less frequent, raises significant concern as it signifies internal contamination that cannot be remedied by surface washing or shell hygiene. Therefore, in this study, the isolates obtained from egg contents were primarily linked to clinically significant serovars and exhibited an elevated presence of antibiotic resistance determinants. This finding indicates a potentially increased risk of consumer exposure to more virulent and antimicrobial-resistant Salmonella strains.

The results of Salmonella prevalence underscore the necessity for dual risk mitigation strategies, encompassing stringent hygienic practices during egg handling to minimize shell-to-food cross-contamination, and enhanced control measures at the production level to avert internal egg contamination and restrict the spread of resistant strains (Alsufyani et al., 2025).

The global distribution of Salmonella serovars varies significantly between countries and across regions within the same country (Jamal et al., 2021; European Food Safety Authority and European Centre for Disease Prevention and Control, 2024; Wang et al., 2024). This variation is influenced by multiple factors such as host-pathogen transmission dynamics, agricultural practices, and food production and processing (Ferrari et al., 2019). For instance, S. enteritidis stands out as a major serotype linked to poultry and eggs, contributing to numerous foodborne outbreaks across Europe, including France, Spain, the Netherlands, and the United Kingdom during the years 2021 and 2022 (European Centre for Disease Prevention and Control, European Food Safety Authority, 2022). Nonetheless, the prevalence of other serovars differs geographically. For example, Australia and New Zealand reported elevated contamination levels of S. typhimurium and S. infantis in eggs and poultry products (Chousalkar et al., 2018).

Our investigation reflects this global diversity in which revealed that the predominant serotypes identified were S. enteritidis (17%) and S. typhimurium (13%), in line with previous reports from Riyadh city and global literature (Moussa et al., 2010). Both serovars were commonly linked to egg-associated outbreaks and harbor virulence genes that have been associated with various public health threats, including meningitis (Bahramianfard et al., 2021; ÓhAiseadha et al., 2010). Notably, S. enteritidis was detected in four samples from eggshells only. This observation is consistent with its established association with surface contamination in eggs and poultry products. However, according to the literature, it has been shown that S. enteritidis has the ability to inhabit and colonize the reproductive tissues of chicken, specifically in the ovaries and oviducts, enabling vertical transmission and persistence inside the egg (Foley et al., 2013). This underscores its relevance as a foodborne pathogen and the importance of monitoring both external and internal contamination routes when assessing egg safety.

Other serovars, including S. Heidelberg, S. Bareilly, and S. Minnesota, were also detected, which adds to the significance of this study. Both S. Heidelberg and S. Bareilly were rank among the leading cause of salmonellosis in the US and were linked to outbreaks reported in eggs and poultry (European Centre for Disease Prevention and Control, European Food Safety Authority, 2022; Bahramianfard et al., 2021; ÓhAiseadha et al., 2010; Foley et al., 2013; Chittick et al., 2006). The identification of less commonly reported serovars like S. Braenderup, S. Concord, and S. Minnesota indicates a wider range of Salmonella strains in poultry environments in Saudi Arabia which is remarkable (Bayu et al., 2013; Garcia et al., 2022). Of note, S. Minnesota is gaining attention for its multidrug resistance and potential to cause invasive illness (83–85.93). The observed variations in Salmonella strains could be a result of discrepancies in management practices, environmental conditions, or additional factors that support the persistence of various Salmonella serovars within egg production facilities (Park et al., 2022).

This diversity of Salmonella serovars highlights the necessity for risk-based surveillance strategies that prioritize serovars according to prevalence, their correlation with human disease, potential for outbreaks, virulence traits, and antimicrobial resistance profiles. Serovars like S. enteritidis and S. typhimurium require specific focus due to their persistent link to egg-related outbreaks, capacity to induce serious human infections, and established significance in both global and local epidemiological data (Moussa et al., 2010; Foley et al., 2013). The identification of serovars such as S. infantis, S. Heidelberg, and S. Minnesota raises further concerns, as these serovars have been increasingly associated with antibiotic resistance and the global spread of high-risk clones via poultry production systems (European Centre for Disease Prevention and Control, European Food Safety Authority, 2022; Bahramianfard et al., 2021; ÓhAiseadha et al., 2010; Foley et al., 2013; Chittick et al., 2006).

Virulence factors play a crucial role in the ability of Salmonella to survive in the host and cause disease. In this study, 137 virulence-related genes were identified across all isolates, with 80 of them consistently present in every serovar. pefA, fyuA, and rck were among the most notable ones that contribute to adhesion, iron acquisition, and immune evasion, which are key traits that enhance persistence and pathogenicity of Salmonella (Figure 8). The pefA gene, found in S. typhimurium, Typhimurium/Lagos, S. enteritidis, and S. Concord, encodes plasmid-encoded fimbriae and involved in intestinal adhesion and biofilm formation, potentially increasing the strains’ ability to colonize host tissues and survive in the environment (Weng et al., 2022). The fyuA gene, which enables bacteria to acquire iron in low-iron environments through the yersiniabactin system, was detected in S. Minnesota, S. infantis, S. Heidelberg, and S. Braenderup. The presence of this gene may contribute to increased virulence and a greater risk of invasive disease (Weng et al., 2022). The rck gene, associated with resistance to complement-mediated cell lysis, was found in S. typhimurium, S. enteritidis, and S. Concord, supporting these strains’ ability to evade host immune responses and potentially cause systemic infections (Silva et al., 2017).

Another concerning finding was the presence of the cdtB gene in S. Minnesota and S. Montevideo, which is a part of the cytolethal distending toxin (CDT-B). This gene interferes with host immune function and promotes deeper tissue invasion, increasing the likelihood of severe illness (Ho and Slauch, 2001). Its detection in S. Minnesota, a serovar already known for its multidrug resistance, raises significant public health concerns, as it suggests the strain may be both harder to treat and more capable of causing invasive disease.

Certain genes were identified in S. typhimurium, including the Salmonella virulence plasmid genes (e.g., spvC and spvR), fimbrial adhesion factors (e.g., pefA–D), and intestinal persistence gene (e.g., ratB), suggest a strong adaptation for systemic infection and intestinal persistence (Ho and Slauch, 2001; Castilla et al., 2006; Aljindan and Alkharsah, 2020; Cherubin et al., 1986). These virulence genes, particularly those associated with Salmonella virulence plasmids (e.g., spvR, spvB, spvC), were supported by the consistent detection of IncFIB(S)_1 and IncFII(S)_1 plasmids in all S. typhimurium and S. enteritidis isolates that are well known to carry the spv operon (Ho and Slauch, 2001; Castilla et al., 2006; Aljindan and Alkharsah, 2020; Cherubin et al., 1986). Moreover, smaller Col-type plasmids were also identified, notably Col440I and ColRNAI in S. typhimurium and S. enteritidis isolates, respectively. While these Col plasmids typically encode bacteriocins rather than direct resistance or virulence genes, their presence may enhance environmental survival and bacterial competitiveness (Silva et al., 2017). Together, the presence of virulence genes associated with IncF plasmids (i.e., spv) with Col plasmids underscores the genetic complexity, adaptability, and pathogenic potential of these Salmonella strains.

In contrast, S. Heidelberg carried specific genes, such as grvA (a stress response regulator) and sspH1/sspH2 (ubiquitin ligase effectors), which may contribute to improved survival mechanisms in both host environments and food production settings, rendering these strains resilient and adaptive (Ho and Slauch, 2001). Meanwhile, S. Cerro and S. Bareilly demonstrated reduced virulence profiles, with a limited presence of SPI-1 and adhesion-related genes such as pefA, and sopA, indicating that these serovars may have the tendency toward environmental persistence rather than high pathogenicity (Srednik et al., 2023; Alvarez et al., 2023).

Overall, all the virulence factors emphasize the capacity of specific serovars to induce more severe or invasive infections and stress the necessity of integrating virulence profiling into genetic surveillance and public health risk evaluation.

In this study, Salmonella susceptibility results revealed notable resistance in the β-lactamase inhibitor class, particularly in two important combination antibiotics: ampicillin/sulbactam and amoxicillin/clavulanic acid, across six isolates. The same isolates that were resistant to ampicillin/sulbactam were also resistant to ampicillin alone. A previous study by Aljindan and Alkharsah (2020) reported antimicrobial susceptibility patterns in clinical Salmonella isolates from the Eastern Province of Saudi Arabia, showing that penicillin and penicillin combined with β-lactamase inhibitors, including ampicillin and ampicillin/sulbactam, still exhibited good activity against Salmonella over 8 years (2014–2018) (Aljindan and Alkharsah, 2020). The susceptibility pattern of our isolates to penicillin antibiotics is consistent with those findings.

Cephalosporins, also known as cephems, are widely used antibiotics for treating severe salmonellosis in humans (Cherubin et al., 1986). In this study, we tested representatives from the second generation (cefoxitin), third generation (cefotaxime, ceftazidime, ceftriaxone), and fourth generation (cefepime). All Salmonella isolates were susceptible to cefepime, the first cephalosporin of the fourth generation.

A high incidence of resistance was observed against third-generation cephalosporins, which raising substantial medical concern. Persistent resistance to cephalosporins over time suggests the presence of extended-spectrum β-lactamases (ESBLs), which frequently confer resistance to this antibiotic class (Shaikh et al., 2014). Fortunately, all Salmonella isolates in this study were screened for ESBL production, and only one isolate (S. infantis) tested positive. This isolate carried the IncFIB(K)_1_Kpn3 plasmid, which is strongly associated with the dissemination of bla_CTX-M-65 in S. infantis. This genetic profile aligns with findings by Alzahrani et.al, who reported S. infantis isolates from chicken meat in Saudi Arabia harboring the same resistance genes and plasmid types, suggesting a potential link between contaminated poultry and the spread of MDR S. infantis (Alzahrani et al., 2023).

The repeated detection of the IncFIB(K)_1_Kpn3 plasmid in multiple studies underscores its role as a mobile genetic element that facilitates persistence and dissemination of resistance genes in Salmonella, particularly in S. infantis (Srednik et al., 2023). Globally, S. infantis has recently emerged as a major public health concern, particularly in Europe, England, and Wales, where strains carrying the megaplasmid pESI (plasmid of emerging S. enterica Infantis) have been reported. These plasmids enhance both multidrug resistance and virulence traits (Alvarez et al., 2023; Lee et al., 2021).

Maintaining the effectiveness of widely used third-generation cephalosporins, while ensuring the continued efficacy of fourth-generation agents such as cefepime, remains crucial in safeguarding treatment options against Salmonella (Wilson, 1998).

Quinolones and fluoroquinolones are considered as the first line treatment for adult worldwide. However, Salmonella isolates have been increasingly developing resistance to fluoroquinolones (CDC, 2024c). A study by Aljindan et al. (2018) reported a resistance prevalence of 19%, a high rise compared to 3% reported in 2013 (Aljindan and Alkharsah, 2020; Elhadi et al., 2013). Our findings, consistent with these and other studies, confirm the presence of fluoroquinolone-resistant Salmonella in Saudi Arabia (Aljindan and Alkharsah, 2020; Elhadi et al., 2013; Aljindan et al., 2018). In particular, we observed that the same isolates resistant to nalidixic acid also displayed intermediate to resistant levels against ciprofloxacin and levofloxacin. This resistance is a significant public health concern, especially if such strains cause salmonellosis in children, adults with invasive infections, or immunocompromised individuals (CDC, 2025).

The CDC has long recognized Salmonella resistance to conventional antimicrobial drugs such as ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. None of these agents should be considered first-line treatments, which is consistent with our findings, as we also observed resistance to these antibiotics in our isolates (CDC, 2024c). Another notable MDR serovar identified in this study was S. enteritidis. As previously noted, S. enteritidis is the most common serovar associated with human infections in Riyadh, Saudi Arabia (Alghoribi et al., 2019).

Salmonella Minnesota was detected once in this study, and it was MDR. In 2021, Saudi Arabia reported that a strain of S. Minnesota isolated from chicken carried the mcr-9 gene, which confers colistin resistance (Alzahrani et al., 2021). In that report, the strain was phenotypically sensitive to colistin but resistant to amoxicillin-clavulanic acid, ampicillin, sulfisoxazole, and tetracycline. Our results for the two S. Minnesota isolates were consistent with those findings, except that colistin resistance was interpreted as intermediate, following the Clinical and Laboratory Standards Institute (CLSI) 2024 M100, 33th edition, which updated the MIC breakpoints for colistin from “sensitive” to “intermediate” (Alzahrani et al., 2021; Clinical and Laboratory Standards Institute, 2024). Additionally, S. Minnesota isolates from Saudi poultry have been shown to carry the cdtB toxin gene, which induces DNA damage and cell cycle arrest, potentially leading to typhoid-like disease in humans, in agreement with findings that one of the two isolates S. Minnesota was harboring the same gene (Alzahrani et al., 2023).

Recent genomic surveillance in Riyadh detected two S. Minnesota ST548 isolates from retail chicken meat that showed phenotypic colistin resistance and harbored the plasmid-borne mcr-1.1 gene, alongside multiple resistance genes (sul2, tetA, blaTEM-1B, qnrB19, aac(6′)-Iaa, floR) and various plasmid replicons (Mukhtar et al., 2022). These data collectively indicate that Saudi S. Minnesota strains possess a broad resistome and plasmids that support horizontal gene transfer. In our analysis, S. Minnesota isolate demonstrated multidrug resistance profiles and possessed the cdtB virulence gene, confirming existing literature and highlighting concerns that this serovar may combine heightened virulence with the ability to acquire and transmit clinically significant resistance genes. Collectively, our observations emphasize S. Minnesota as a serovar requiring prioritized focus in national AMR surveillance and poultry food safety initiatives. In 2023, S. Minnesota and S. Heidelberg were investigated in Brazil, where isolates from poultry and other sources were compared with international strains (Rodrigues et al., 2020). The study confirmed that both serovars are highly infectious and MDR, posing significant public health concerns worldwide (Rodrigues et al., 2020).

In our study, S. Montevideo was also identified and found to harbor the cdtB toxin gene. This serovar can infect chickens, often without causing noticeable symptoms (Lalsiamthara and Lee, 2017). Infected birds may carry S. Montevideo asymptomatically, creating a transmission risk through direct contact or consumption of contaminated poultry products, including eggs (Lalsiamthara and Lee, 2017). Notably, the S. Montevideo isolate in our findings was detected in the egg content, suggesting systemic infection in the hen.

The antimicrobial resistance gene profiles determined in this study align with widely documented patterns in poultry-associated Salmonella. The prevalent identification of aminoglycoside resistance genes, especially aac(6′)-Iaa among various serovars, indicates a frequently documented intrinsic resistance characteristic in Salmonella isolates globally (Abdelhamid and Yousef, 2023). Comparable distributions of aminoglycoside-modifying enzymes, including aph and ant gene families, have been consistently reported in poultry-derived Salmonella from Europe, Asia, and North America (Zhang et al., 2023).

The detection of plasmid-mediated β-lactamase genes, specifically bla_CMY-2 and bla_CTX-M-65, in serovars such as S. infantis, S. Minnesota, S. Heidelberg, and S. Concord corresponds with global accounts of the widespread occurrence of AmpC and extended-spectrum β-lactamases in foodborne Salmonella. These resistance determinants are frequently linked to multidrug-resistant clones prevalent in chicken production systems (Aravena et al., 2024).

The identification of plasmid-mediated fluoroquinolone resistance genes (qnrB19 and qnrS1), alongside sulfonamide (sul1, sul2), tetracycline [tet(A)], and trimethoprim (dfrA14) resistance genes, reflects resistance patterns often documented in poultry-associated Salmonella worldwide (Aravena et al., 2024). The significant genetic resistance load identified in S. infantis in this investigation aligns with its designation as a globally distributed high-risk serovar linked to antibiotic resistance and intensive chicken farming (Alvarez et al., 2023).

These findings situate the identified resistance gene patterns within a global framework and emphasize the role of poultry-associated Salmonella in Saudi Arabia in the global reservoir of antimicrobial resistance determinants, highlighting the necessity of incorporating whole-genome sequencing into national and international surveillance systems.

In this study, we also investigated the association between ARGs and plasmids among the Salmonella isolates (Figure 7). It was found that certain ARGs were primarily linked to specific plasmid types, suggesting possible horizontal gene transfer (Aviv et al., 2016). Interestingly, we found no clear correlation between the IncFIB(K)_1_Kpn3 plasmid and ARGs, despite the S. infantis isolate carrying about nine resistance genes. This discrepancy could be explained by mutations or partial deletions in the plasmid that disrupted its expected association with ARGs (Aviv et al., 2016). Alternatively, the resistance genes may have been integrated into the bacterial chromosome, making them independent of plasmid carriage (García-Soto et al., 2020).

Moreover, the IncFIB(K)_1_Kpn3 plasmid was associated with virulence factors. Although ARGs were not directly linked to this plasmid, it carried virulence determinants, consistent with reports that such plasmids can facilitate the horizontal transfer of virulence factors (Hull et al., 2022). The presence of virulence genes, even in the absence of resistance determinants, suggests that IncFIB(K)_1_Kpn3 enhances pathogen fitness by promoting adhesion, invasion, or immune evasion (García-Soto et al., 2020). These factors may indirectly support antimicrobial resistance by improving bacterial survival and persistence in hosts (García-Soto et al., 2020). Further investigations, including transcriptome analysis, plasmid profiling, and functional analysis of bacterial pathogenicity, are necessary to understand the functional significance of this plasmid. Especially, growing evidence indicates that large plasmids in S. infantis can acquire extra resistance genes and expand into megaplasmids, thereby increasing bacterial survival and accelerating the dissemination of AMR (García-Soto et al., 2020).

In this study, we also observed that the only isolate resistant to colistin and polymyxin B lacked the recognized genes typically associated with resistance (such as mcr genes, pmrA, pmrB, phoPQ, and mgrB) (Moghadam et al., 2022; Leite et al., 2020). In addition, the absence of the plasmids IncI2, IncX4, and IncHI2, which are the most frequently reported carriers of mcr genes, suggests a potential alternative resistance mechanism (Moghadam et al., 2022). Interestingly, the presence of IncFII(S)_1 plasmid indicates that plasmid-associated regulatory elements may contribute to this resistance, as it is reported that its backbones are involved in the dissemination of colistin resistance, as they are known to commonly carry mcr genes and other resistance determinants (Zelendova et al., 2021). Although no mcr genes were detected, the isolate’s resistance to colistin, along with the carriage of this plasmid and the aac(6′)-Iaa gene (which encodes resistance to aminoglycoside antibiotics), raises major concern. This genetic configuration could facilitate the horizontal transmission of antimicrobial resistance along the food chain.

The serovar S. Heidelberg was isolated three times, and according to the phylogenetic tree (Figure 2), the isolates clustered within the same branch. Among them, two closely related isolates harbored the bla_CMY gene. The presence of bla_CMY on IncX1 plasmids is particularly important, as these plasmids are recognized as key vectors for the transmission of AmpC-type β-lactamase resistance genes, conferring resistance to multiple β-lactam antibiotics, including third-generation cephalosporins (Dobiasova and Dolejska, 2016). The identification of these resistance determinants on IncX1 plasmids underscores their ability to mediate rapid horizontal gene transfer among bacterial populations. This highlights their role in the dissemination of multidrug-resistant organisms from food sources to humans, representing a significant public health concern.

Pan-genome analysis enhances outbreak investigations and molecular epidemiological studies by facilitating high-resolution comparisons of gene content among isolates (Barh et al., 2020). In addition, it offers a structure for understanding the genetic diversity and epidemiological patterns of Salmonella, beyond single-reference genome methodologies (Barh et al., 2020; Muigano and Musila, 2025). The pangenome structure of all Salmonella in this study demonstrated a majority of core genes (45.3%), conserved across all serovars and critical for Salmonella survival. The conserved genes confirm their significance for species identification and molecular diagnostic applications, ensuring precise and consistent detection across several serovars (Barh et al., 2020). The significant percentage of auxiliary genes, comprising cloud genes (31.8%) and shell genes (20.2%), underscores notable genomic heterogeneity among isolates. This variable gene pool is significant because it often contains factors linked to antimicrobial resistance, virulence, and environmental adaptation, elucidating the strain-specific variations in pathogenic potential and resistance profiles identified in this study. These findings collectively illustrate that incorporating pan-genome analysis into Salmonella surveillance enhances diagnosis, improves predictions of resistance and virulence, and offers a transferable and scalable framework applicable to food safety and public health monitoring programs in Saudi Arabia and other regions.

The findings of this study have significant implications within a One Health framework, highlighting the interconnected roles of environmental, agricultural, and human health sectors in the epidemiology of Salmonella and antibiotic resistance. The identification of many Salmonella serovars and resistance factors in chicken eggs underscores the complex nature of contamination routes throughout the food production chain and emphasizes the necessity for comprehensive surveillance strategies.

Furthermore, the findings underscore the importance of risk-based monitoring and management strategies in egg production and distribution from an agricultural and food safety standpoint. The genomic characterization of Salmonella serovars and their antibiotic resistance profiles yields data that can facilitate targeted surveillance techniques and improve the early detection of serovars pertinent to public health.

The existence of clinically significant and antimicrobial-resistant Salmonella serovars in eggs emphasizes the importance of integrated surveillance between food and clinical sectors in accordance with human health. The utilization of whole-genome sequencing for foodborne pathogens provides a significant resource for enhancing risk assessment, promoting antimicrobial stewardship, and improving collaboration within environmental, agricultural, and public health domains. Therefore, integrating genetic data from foodborne isolates with human health surveillance enhances source attribution and fortifies epidemic investigation capabilities. In Saudi Arabia, these results correspond with government initiatives to improve food safety regulations and monitor antibiotic resistance through a One Health framework.

While this study provides the first in-depth genomic characterization of Salmonella in chicken eggs in Saudi Arabia, several limitations should be acknowledged, the sampling was limited to chicken eggs from selected regions, which may not fully represent the national distribution of Salmonella; thus, expanding the sampling across other regions and times would enhance future surveillance efforts. In addition, the cross-sectional approach offers essentially a snapshot of contamination and antimicrobial resistance patterns, highlighting the necessity for long-term monitoring to observe developing clones and resistance trends.

Lastly, it would be valuable to expand future research under a One Health framework, including animal, food, environmental, and human Salmonella isolates. This would offer a more thorough comprehension of the transmission dynamics of Salmonella and associated antimicrobial resistance factors throughout the food chain and public health domains.

This study provides the first of its kind a detailed genomic analysis of Salmonella contamination in chicken eggs in Saudi Arabia by combining whole-genome sequencing with phenotypic antimicrobial susceptibility testing. The paper provides a comprehensive analysis of serovar diversity, antimicrobial resistance, and virulence profiles linked to egg production. The identification of epidemiologically significant serovars, including S. enteritidis and S. typhimurium, as well as emerging serovars such as S. Minnesota, S. Heidelberg, and S. Montevideo, which possess clinically pertinent resistance determinants, emphasizes the potential public health hazards linked to poultry eggs and accentuates the increasing apprehension regarding antimicrobial resistance in poultry settings.

These findings underscore the necessity for enhanced, risk-based surveillance of Salmonella in egg production systems, focusing specifically on serovars of public health significance and those exhibiting multidrug resistance and virulence factors. Incorporating genetic surveillance into standard monitoring methods can facilitate the early identification of high-risk clones, refine source attribution, and guide targeted actions throughout production, processing, and distribution phases. Moreover, strengthening antimicrobial stewardship procedures in chicken production is crucial to curtail the selection and spread of resistant strains across the food chain.

Considering the evolving nature of antimicrobial resistance, future study needs to concentrate on continuous and multi-regional investigations to observe dynamic trends in serovar distribution, resistance patterns, and virulence profiles. These approaches will provide essential insights into the development and existence of antimicrobial-resistant Salmonella in Saudi poultry systems, thereby enhancing national risk assessment, One Health surveillance, and evidence-based policymaking to mitigate foodborne salmonellosis and antimicrobial resistance.