Prevalence and Characteristics of Salmonella from a Laying hen Production Chain in Jiangsu Province, China

1. Introduction

Salmonella belongs to the Enterobacteriaceae family and comprised two species: Salmonella bongori and Salmonella enterica. The species Salmonella enterica include 6 subspecies, with a total of approximately 2,600 serotypes [1]. In poultry, infections caused by S. pullorum are referred to as pulorosis, those caused by S. gallinarum are called typhus, and infections by numerous other non-host-adapted serovars are termed paratyphoid [2]. Pulorosis typically leads to the death of chicks aged 1 to 3 weeks. In adult chickens, S. pullorum infection usually display mild or asymptomatic symptoms but can result in decreased production performance and increased mortality [3]. Typhus is generally considered as a disease primarily affecting adult chickens, but it can also lead to significant mortality in chicks [4]. Paratyphoid infections often present as subclinical infections that go unnoticed, thereby posing a significant threat to human health due to the contamination of food, particularly eggs and egg products [5].

Egg, as a high-protein, low-fat, multi-nutrient food, has become an important part of a healthy dietary pattern [6] and the consumption of eggs is increasing year by year in the world [7]. According to statistics, over a billion cases of gastroenteritis caused by Salmonella are reported worldwide annually [8]. Each year in America, there are one million cases of foodborne illness related to non-typhoid Salmonella, specifically the S. Enteritidis, which leads to human salmonellosis mainly through contaminated eggs or egg products [9]. Between 2015 and 2018, a total of 1,209 large-scale infections caused by S. Enteritidis-contaminated eggs were reported in 16 european countries [10]. In the poultry production chain, Salmonella can be present at various stages of production, ultimately contaminating the entire chain through both vertical and horizontal transmission [11]. Lei et al. [12] demonstrated that 148 strains of Salmonella were isolated from 2100 fecal swab samples and the isolation rate of Salmonella was 5.3% at breeder house, 4.2% at hatchery, and 10.4% at commercial chicken farm. Liu et al. [13] conducted continuous monitoring of Salmonella on a large-scale intensive laying hen farm from 2020 to 2023, isolating 105 strains from 1,537 samples, and the commercial areas were identified as the most contaminated with Salmonella. Therefore, comprehensive prevention and control measures should be implemented across the entire farming process to mitigate the risk of contamination.

Recent years have witnessed substantial advancements in the molecular typing analysis of foodborne pathogens with whole-genome sequencing (WGS). WGS has become an effective tool for investigating serotyping, antimicrobial resistance genes, pathogenicity characteristics and the e source of Salmonella. Some researchers [14] employed WGS to study phylogenetic relationships of Salmonella at different stages of chicken slaughter in East China and the slaughter environment was recognized as the source of cross-contamination during processing. Liu et al. [13] conducted a source-trace analysis of S. Kentucky in a laying hen farm using WGS in conjunction with epidemiological investigations, revealing that S. Kentucky entered the farm via an introduction link.

In this study, we conducted a surveillance study to assess the prevalence of Salmonella within a laying hen production chain. Samples were collected from a breeder house and hatchery in Yangzhou, Jiangsu Province, as well as from a commercial house and market in Taizhou, Jiangsu Province. Subsequently, the drug resistance of Salmonella isolates was investigated to provide a scientific basis for optimizing production management models. WGS was used to investigate the genetic relationships among the isolates, thereby offering a theoretical foundation for the prevention and control of Salmonella.

2. Materials and Methods

2.1. Sample Collection

In this study, 979 samples were collected from the laying hen industry chain, which includes one breeder house and one hatchery in Yangzhou, Jiangsu Province, as well as one commercial house (a family farm) and one market in Taizhou, Jiangsu Province, for Salmonella isolation. Parent breeder chickens are raised in the breeder house for egg production, and the eggs are subsequently hatched. Once the chicks reach a certain age, they are transferred to commercial houses for egg laying. Eggs was then transported to the retail market for sale.

A total of 242 samples were collected from breeder house, which included samples from sick or dead chicken, environment, stool, feed and water. A total of 170 samples were collected from hatchery, including dead embryos samples and environment samples. A total of 484 samples were collected from commercial house, which included samples from sick or dead chicken, environment, stool, egg, feed and water. A total of 83 samples were collected from market, including egg samples and environmental samples. All samples were stored on ice and transported to the laboratory within 24 hours. Upon arrival at the laboratory, the samples were processed immediately.

2.2. Isolation and Identification of Salmonella

The buffered peptone water was added to each sample at a volume ratio of 1:9 relative to the sample volume, followed by incubation at 37℃ for 16-20 h for preliminary enrichment. Then, 1 mL of preliminary enrichment culture was added to 10 mL Rappaport-Vassiliadis R10 (RvR10) broth and incubated at 42 °C for 24 h. Upon completion of incubation, selective enrichment culture was streaked onto xylose lysine tergitol 4 (XLT4) agar plates and incubated at 37 °C for 24 h. The polymerase chain reaction (PCR) was employed to determine whether the isolates were Salmonella spp. using specific primers for stn amplification [13]. The serotype of isolates was confirmed by agglutination test using Salmonella antisera (Tianrun, Ningbo, China) according to the Kauffmann–White scheme.

2.3. Antimicrobial Susceptibility Testing

According to the Clinical and Laboratory Standards Institute (CLSI 2021), the antimicrobial susceptibility of 30 Salmonella isolates were tested with disk diffusion method. In brief, a single colony was injected into 4 mL of MH liquid medium and incubated at 37 °C for 16-18 hours. The bacterial suspension was calibrated to 0.5 McFarland units with sterile PBS solution and uniformly distributed onto MH solid agar plates. Antibiotic discs were placed on the infected MH solid agar surface with a disc dispenser. Plates were incubated at 37 °C for 24 hours, after which the diameters of the zones were measured and compared to standard criteria. A total of 16 antimicrobial agents were used: ampicillin (AMP), amoxicillin (AMC), cefazolin (CFZ), cefotaxime(CTX), aztreonam (ATM), meropenem (MEM), gentamicin (GEN), amikacin (AK), streptomycin (STR), enrofloxacin (ENR), nalidixic acid (NAL), ciprofloxacin(CIP), tetracycline (TET), trimethoprim-sulfamethoxazole (SXT), chloramphenicol (CHL), nitrofurantoin (F). Escherichia coli ATCC 25922 served as a control strain.

2.4. WGS

A total of 35 S. Enteritidis isolates from different production stages were selected for WGS. The genomic DNA of the isolated strains was extracted using the TIAN amp Bacteria DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. All genomes were fragmented to an insertion size of 500 bp for library construction. WGS was conducted using the Illumina NovaSeq 6000 platform, producing 150 bp paired-end reads. Genomes were de novo assembled using SPAdes 3.15.5. Multilocus Sequence Typing (MLST), antimicrobial resistance genes, and core genome SNP analysis were performed according to the methods described by Kang et al. [15]. WGS data of all Salmonella isolates were submitted to the NCBI database with the accession number PRJNA1479310.

2.5. Statistical Analysis

Data of Salmonella isolation prevalence was analyzed using SPSS 22.0 software. The chi-square test was employed to assess the significance of differences, with p< 0.05 indicating significant differences.

3. Results

3.1. Prevalence of Salmonella in the Laying Hen Production Chain

In this study, samples were collected from different stages of a laying hen production chain in Jiangsu Province, including breeder house, hatchery, commercial house, and market, followed by isolation and identification of Salmonella. Among the 979 samples collected, 84 Salmonella isolates were recovered, yielding an overall prevalence rate of 8.58% (Table 1). The highest isolation rate was observed in commercial house (11.2%), followed by market (10.8%). In contrast, the prevalence in breeder house and hatchery was 4.6% and 5.9%, respectively. The isolation rate in commercial house were significantly higher than those in breeder house and hatchery (p < 0.05). Environmental samples from commercial house and markets exhibited relatively high isolation rates. The isolation rate of environmental samples reached 21.4% in markets and 17.3% in commercial house. Among egg samples, the highest prevalence was detected in markets (5.5%). Salmonella was not detected in any feed or water samples.

3.2. Serotyping of Salmonella Isolates

Serotyping of the 84 Salmonella isolates identified six distinct serovars. S. Enteritidis was the predominant serovar, accounting for 41.7% of the isolates, followed by S. Typhimurium (22.6%) and S. Infantis (21.4%) (Figure 1). In addition, seven isolates were identified as S. Kentucky, three as S. Brenderup, and two as S. Agona. S. Enteritidis was the dominant serovar throughout the entire laying hen production chain and was detected in breeder house, hatchery, commercial house, and market.

3.3. Antimicrobial Resistance Phenotypes and Genotypes

Thirty Salmonella isolates were selected for antimicrobial susceptibility testing against 16 antibiotics (Table 2). The highest resistance rates were observed for NAL and CIP (both 100%), followed by AMP, AMC, and STR (56.7%). Resistance to TET was 53.3%, and 33.3% of the isolates were resistant to ENR. Resistance to the remaining antibiotics was primarily detected in S. Kentucky isolates recovered from commercial layer farms. When analyzed by different stages of the production chain, S. Enteritidis isolates from breeder house and hatchery exhibited 100% resistance to NAL and CIP and 30% resistance to ENR, while remaining susceptible to the other 13 tested antibiotics. In contrast, S. Enteritidis isolates from commercial layer farms and retail markets showed 100% resistance to NAL and CIP, and 76.9% resistance to AMP, AMC, and STR. Additionally, these isolates demonstrated resistance to CFZ (7.7%), TET (69.2%), and F (10.5%). S. Kentucky isolates from commercial house exhibited resistance to at least 13 antibiotics, indicating a multidrug-resistant (MDR) phenotype.

WGS was subsequently performed to detect 16 antimicrobial resistance genes among the 30 isolates. All isolates harbored the aminoglycoside resistance gene aac(6’)-Iaa. The detection rates of aph(6)-Id (aminoglycoside resistance) and sul2 (sulfonamide resistance) were both 33.33%, while tet(A) (tetracycline resistance) and bla_TEM-1B (β-lactam resistance) were detected in 53.33% and 56.67% of isolates, respectively (Table 3). The resistance genes aadA7，rmtB，sul1，bla_CTX-M-55，qnrS1，dfrA14，lnu(F)，ARR-2，floR，mph(A) and fosA3 were exclusively identified in the seven S. Kentucky isolates. Resistance to quinolones is commonly associated with mutations in the quinolone resistance-determining regions (QRDRs). WGS analysis revealed that 33.3% (10/30) of the isolates carried a substitution at codon 87 of gyrA, resulting in an Asp87Tyr (D87Y) mutation, while 43.3% (13/30) exhibited an Asp87Asn (D87N) mutation. Furthermore, 23.3% (7/30) of the isolates harbored dual mutations in gyrA, including Asp87Asn (D87N) and Ser83Phe (S83F).

3.4. Traceability Analysis of S. Enteritidis

Whole-genome sequencing was performed on 35 S. Enteritidis isolates. Multilocus sequence typing (MLST) analysis revealed that all isolates belonged to sequence type 11 (ST11) (Figure 2). An evolutionary tree was constructed based on core genome single nucleotide polymorphism (cgSNP), and the isolates were divided into two distinct clades (A1 and A2). Clade A1 included strains recovered from breeder farms, hatcheries, commercial layer farms and retail markets. These isolates shared high genetic relatedness (the pairwise SNP distance values<10 ) and belonged to the same clonal cluster, suggesting that this clone may disseminate along the egg production chain.

4. Discussion

Salmonellosis remains one of the most prevalent foodborne zoonotic diseases worldwide, with poultry and poultry products serving as the primary reservoirs and transmission vehicles for human infections [16,17,18]. This study provides a comprehensive epidemiological analysis of Salmonella contamination across the laying hen production chain in Jiangsu Province, China, revealing an overall isolation rate of 8.58% from samples collected at breeder house, hatchery, commercial house and market. The higher prevalence in commercial house and market compared to breeder house and hatchery aligns with findings from other studies, where commercial and downstream stages often show elevated contamination due to increased environmental exposure, multi-age management, and potential cross-contamination [13,19]. Moreover, environmental samples from commercial house and market in the present study exhibited relatively high isolation rates, suggesting that environmental contamination may contribute substantially to the maintenance and transmission of Salmonella within the production chain.

Serotyping analysis identified six distinct Salmonella serovars among the 84 isolates, with S. Enteritidis being the predominant serovar (41.7%), followed by S. Typhimurium (22.6%) and S. Infantis (21.4%). This serotype distribution is consistent with global and regional trends, where S. Enteritidis has long been recognized as the leading cause of egg-associated salmonellosis worldwide [20,21]. The U.S. Centers for Disease Control and Prevention (CDC) has repeatedly documented S. Enteritidis as a primary serovar in egg-linked outbreaks in the United States [22,23]. It is estimated that approximately 3.25 million eggs in the United States are contaminated with S. enteritidis each year [24]. Similarly, the European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC) have reported numerous multi-country outbreaks of S. Enteritidis linked to eggs and egg products, including prolonged outbreaks affecting up to 18 countries between 2015 and 2020 [10,25,26]. Importantly, our study found that S. Infantis accounted for 21.4% of the isolates, indicating a notable presence that is consistent with its reported emergence in Chinese poultry production. Recent surveillance in China has shown S. Infantis rising in prevalence within poultry and egg-related chains, often ranking among the top serovars alongside S. Enteritidis [19,27]. The rapid spread of S. Infantis is of particular concern because this serovar has been frequently associated with multidrug resistance and enhanced virulence potential in both poultry and humans [28]. Furthermore, S. Enteritidis was detected in all production stages, confirming its ability to disseminate throughout the entire laying hen production chain. In contrast, commercial house harbored the greatest serovar diversity. This pattern supports the hypothesis that open, small-scale production systems are more susceptible to the introduction of Salmonella from external sources such as wildlife, rodents, personnel, or environmental contamination [29,30].

Antimicrobial resistance (AMR) is a major global public health threat, and the emergence of multidrug-resistant Salmonella in poultry production has significantly complicated the treatment of salmonellosis [31]. In this study, all 30 tested Salmonella isolates exhibited 100% resistance to NAL and CIP. NAL and CIP have not been recently applied to control bacterial contamination. Hence, the emergence of drug resistance can be largely explained by the historical use of quinolone antibiotics. The increasing prevalence of NAL resistance raises major concerns, as it may impair the efficacy of fluoroquinolone treatments and ultimately result in delayed or failed therapy [32]. WGS revealed that quinolone resistance was primarily mediated by mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene, with 33.3% of isolates carrying the Asp87Tyr (D87Y) mutation, 43.3% carrying the Asp87Asn (D87N) mutation, and 23.3% harboring dual mutations (Asp87Asn and Ser83Phe). Single mutations at codons 83 or 87 of gyrA typically confer high-level resistance to nalidixic acid, while dual mutations significantly elevate resistance to ciprofloxacin and other fluoroquinolones [33,34]. Notably, Salmonella isolates from commercial house and market displayed broader resistance spectra than those from breeder house and hatchery, which may reflect long-term antimicrobial selection pressure during commercial production and environmental exposure. In particular, S. Kentucky isolated from commercial house exhibited resistance to 15 distinct antibiotics, representing a typical extensively drug-resistant (XDR) phenotype. Meanwhile, several resistance genes such as bla_CTX-M-55, qnrS1, fosA3, and rmtB were detected in these isolates. Multidrug-resistant S. Kentucky strains have been regarded as a serious global public health concern in recent years [35,36], with spread in poultry and humans. The detection of multidrug-resistant S. Kentucky in the present study highlights the potential risk of antimicrobial resistance transmission through the egg production chain and underscores the necessity for prudent antimicrobial usage and continuous surveillance in poultry production systems.

WGS-based phylogenetic analysis demonstrated that all S. Enteritidis isolates belonged to ST11, which is recognized as one of the dominant epidemic sequence types associated with poultry and human infections worldwide [37,38]. Core genome SNP analysis further showed that several strains isolated from breeder house, hatchery, commercial house and market clustered into the same clonal lineage with pairwise SNP distances of fewer than 10 SNPs. These results strongly suggest the existence of clonal transmission along the laying hen production chain. Similar findings have been reported in previous studies, which demonstrated that highly related Salmonella isolates can disseminate from upstream breeding systems to downstream retail products through vertical transmission, environmental contamination, personnel movement, and transportation processes. Therefore, this study offered meaningful data regarding the presence and outbreaks of Salmonella in poultry farms and further elucidated the underlying causes for its persistence throughout the subsequent production chain.

5. Conclusions

This study demonstrated that Salmonella, especially S. Enteritidis, is prevalent throughout the laying hen production chain in Jiangsu Province, China and can disseminate among different production stages. The high prevalence of quinolone resistance and the emergence of multidrug-resistant S. Kentucky further underline the potential public health risks associated with antimicrobial-resistant Salmonella in poultry production. These findings provide important epidemiological evidence supporting the implementation of comprehensive surveillance, strict biosecurity measures, and rational antimicrobial use across the entire egg production chain.