Genomic Characterization of a Virulent Multidrug-Resistant Salmonella enterica Serovar Enteritidis Strain Isolated from Meat Rabbits (Oryctolagus cuniculus) in China

Genomic Characterization of a Virulent Multidrug-Resistant Salmonella enterica Serovar Enteritidis Strain Isolated from Meat Rabbits (Oryctolagus cuniculus) in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic Characterization of a Virulent Multidrug-Resistant Salmonella enterica Serovar Enteritidis Strain Isolated from Meat Rabbits (Oryctolagus cuniculus) in China Ruiming Zhang, Baoyan Wang, Yixuan Li, Jing Li, Xiangge Dong, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7540124/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted 14 You are reading this latest preprint version Abstract Background The rise of virulent, multidrug-resistant Salmonella enterica serovar Enteritidis strains threatens food safety and human health. This study reports the isolation of a Salmonella enterica serovar Enteritidis strain (SE JL228) from a meat rabbit, which was associated with a high-mortality outbreak on a rabbit farm in China in 2018. The objective of this study was to elucidate the genetic characteristics, antimicrobial resistance determinants, metal tolerance mechanisms, and virulence potential of a rabbit-derived strain, thereby providing insights into its implications for both public and animal health. Results This strain exhibited resistance to 13 antibiotics within 8 antimicrobial categories as well as to silver (Ag + ), copper (Cu 2+ ), and tellurium (Te 4+ ). Additionally, SE JL228 demonstrated high tolerance to the quaternary ammonium compound (QAC) disinfectant. In vitro assays revealed superior invasion of human brain microvascular endothelial cells and enhanced intracellular survival in activated peritoneal macrophages compared to the moderately virulent SE strain LN248. In vivo studies confirmed extensive dissemination in mice, with an LD 50 approximately 68-fold lower than that of LN248. A 4.7-megabase chromosome together with two plasmids—pSE228A (211.4 kb, IncFIB/IncHI2) and pSE228B (54.6 kb, IncN)—was identified through whole-genome sequencing. The genome encoded 17 antibiotic resistance genes (ARGs), 34 virulence factors including an intact spv operon, and heavy metal resistance operons ( copESDBAC , silPABFCRE , terEDCBAZWYX ) alongside a QAC-resistance gene ( qacE ) on pSE228A. Plasmid pSE228A shares near-identical structure with a previously-isolated IncHI2 plasmid, while pSE228B, carrying the transferable bla TEM−1 gene conferring penicillin resistance, showed limited homology to the known plasmids. Conclusions The rabbit-derived SE JL228 represents a highly virulent, multidrug-resistant, and metal-tolerant pathogen. Its ability to invade brain endothelial cells, survive within macrophages, and disseminate systemically underscores its zoonotic potential. The presence of transferable plasmids encoding both resistance and virulence factors suggests a heightened risk of spread within farm environments and beyond. These findings emphasize the urgent need for enhanced surveillance, prudent antimicrobial use, and effective biosecurity measures to mitigate the emergence and dissemination of such hybrid pathogens. Salmonella Enteritidis multidrug-resistant (MDR) high virulence metal resistance IncFIB/IncHI2 hybrid plasmid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Salmonella enterica serovar Enteritidis (SE) represents a dominant non-typhoidal Salmonella (NTS) serovar associated with foodborne gastroenteritis in people. Contaminated poultry, meat, and eggs serve as the principal vehicles of transmission, raising considerable public health issues at the global level [ 1 ]. Despite the abundant studies on SE in poultry, swine, cattle, and humans, little is known about its role in rabbit infections. Salmonella infection in rabbits tends to develop acute or peracute septicemia, resulting in high morbidity and mortality. These infections lead to major economic losses in commercial rabbit farming (known as cuniculture) and concern about zoonotic transmission since rabbits are bred for their meat, fur, and as pets [ 2 ]. Over the past few years, multidrug-resistant (MDR) SE have increasingly been reported, complicating therapeutic strategies due to resistance against multiple classes of antimicrobials [ 3 ]. Horizontal gene transfer (HGT), primarily mediated by mobile genetic elements (MGEs) such as plasmids, transposons, and integrons, underlies the widespread distribution of antimicrobial resistance (AMR) genes. As a result, MDR variants have emerged rapidly and been widely documented [ 4 , 5 ]. Among these, plasmid-driven HGT is recognized as a key mechanism enabling AMR gene exchange among Enterobacteriaceae [ 6 – 8 ]. Among MDR SE strains, plasmids belonging to incompatibility groups such as IncF, IncHI, IncI, IncA/C, IncP and IncN have been widely implicated in the spread of resistance determinants [ 9 ]. Notably, these plasmids frequently co-carry both AMR and virulence genes, enhancing bacterial adaptability and pathogenicity [ 10 ]. Besides antibiotic resistance, bacteria adapting to heavy metals like copper (Cu²⁺), silver (Ag⁺), and zinc (Zn²⁺) has become an important factor in helping MDR pathogens continue to thrive and spread. These metals are commonly used as antimicrobial agents in animal husbandry, industrial settings, and medical applications [ 11 , 12 ]. However, continuous exposure posed selective pressure and contribute to the emergence of bacterial strains carrying metal resistance genes (MRGs), which were frequently co-located with AMR genes on plasmids. This co-selection mechanism enhances bacterial survival under heavy metal stress, further driving the spread of MDR strains in livestock and food production systems [ 13 ]. Material and method Outbreak description In March 2018, a veterinarian identified an outbreak of infectious disease at a commercial rabbit farm in Nong’an County, Jilin Province, China. The farm reared New Zealand White (NZW) rabbits ( Oryctolagus cuniculus ) for meat intended for the human food chain. More than 350 kittens were affected, presenting with diarrhea, weight loss, and respiratory difficulty, leading to death in 68.5% of cases. Necropsies showed variable lesions while white multifocal lesions in livers were most common. Treatment with antibiotics tetracycline and kanamycin sulfate was ineffective. Bacterial isolation and Serotyping Liver samples from carcasses were aseptically collected and delivered to the laboratory for pathogen detection. Eight presumptive non-Typhi Salmonella spp . were recovered from ten liver samples in Xylose lysine tergitol 4 (XLT4) agars with black colonies. They were then confirmed by VITEK ® 2 Compact microbial identification system (bioMérieux, NC, USA) and designated “JL227” to “JL234”. These strains were classified as serovar Enteritidis using the White-Kauffmann-Le Minor scheme with standard agglutination methods and Salmonella antisera pools (S&A Reagents Lab, Bangkok, Thailand) [ 14 ]. Antimicrobial, disinfectant, and heavy metal susceptibility SE isolates were tested with VITEK ® 2 Compact system (bioMérieux, NC, USA) using VITEK ® 2 AST-GN65 card. Global-based Clinical and Laboratory Standards Institute (CLSI) guideline (2014 − 701) was applied for minimal inhibitory concentrations (MICs) interpretation. Susceptibilities of the strain SE JL228 to heavy metals were determined as described before [ 15 ]. These heavy metals including silver (AgNO 3 , CAS#: 7761-88-8), copper (CuCl 2 ·2H 2 O, CAS#:10125-13-0), tellurium (K 2 TeO 3 , CAS#: 7790-58-1), cobalt (CoCl 2 , CAS#: 7646-79-9), zinc (ZnSO 4 , CAS#: 7733-02-0), chromium (CrCl 3 ·6H 2 O, CAS#: 10060-12-5), cadmium (Cd, CdCl 2 , CAS#: 10108-64-2) (Macklin Biochemical, Shanghai, China). In addition, the benzyldodecyldimethylammonium bromide (DBAB, CAS#: 7281-04-1) was chosen to evaluate resistance to the quaternary ammonium compound (QAC) disinfectant of SE JL228 as well. Escherichia coli ( E. coli ) ATCC™ 25922 was chosen as a control strain. Preparation of activated peritoneal macrophages (APM) BALB/c mice (6–8 weeks, Beijing HFK Bioscience) were housed in IVC at Ludong University with ad libitum food and water. Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007). Mice received 3% thioglycollate broth intraperitoneally 72 h before cell harvest. Prior to sampling, mice were anesthetized by inhalation of 4% isoflurane, followed by intraperitoneal injection of sodium pentobarbital at a dose of 150 mg/kg to induce euthanasia. Cervical dislocation was then performed as a secondary physical method to confirm death. After disinfection of the abdominal surface, 5 mL of ice-cold sterile PBS was injected intraperitoneally, and the abdomen was gently massaged. The lavage fluid was collected and centrifuged to obtain peritoneal macrophages, which were subsequently processed under sterile conditions. Peritoneal macrophages were cultured in RPMI 1640 (Gibco, Suzhou, China) with 10% fetal bovine serum (FBS, ZETA, CA USA), washed with phosphate-buffered saline (PBS) after overnight incubation, and stimulated with 1 µg/mL LPS (Sigma-Aldrich, MO, USA) for 24 h before the Gentamicin protection invasion assay. In vitro invasion assay To evaluate bacterial invasion, gentamicin protection assays were conducted in APM and hBMEC cell models, according to established methods [ 16 , 17 ]. LN248, another SE strain with moderate invasive capability [ 18 ], and E. coli ATCC 25922™ were included as controls. Pathogenicity in mice SE JL228 and SE LN248 were grown in LB broth, subcultured at 37°C for 2.5 h with shaking. Bacteria were washed with PBS and diluted to 10 8 CFU/50 µL. The median lethal dose (LD 50 ) was determined by orally inoculating mice with serial dilutions, recording mortality at 28 days, and calculating LD50 using the Reed and Muench method [ 19 ]. For LD₅₀ and infection studies, mice were monitored at least two times daily and up to four times daily during the acute phase. Humane endpoints were predefined: severe respiratory distress, inability to eat/drink, severe neurologic signs, or moribund state. Animals meeting humane endpoint criteria were humanely euthanized immediately as described above. SE JL228 dissemination in mice was evaluated as a previously described [ 20 ]. Mice were fasted for 4 h before per os (p.o.) treatment with 20 mg streptomycin. After 20 h, they were intragastric infected with 1×10 8 CFU of SE JL228 or SE LN248. Five days post-infection, mice were anesthetized and euthanized, after which their livers, spleens, brains, and mesenteric lymph nodes were aseptically homogenized. Serial dilutions of the homogenates were plated on XLT4 agar to assess bacterial dissemination and colonization. Whole-genome sequencing (WGS) The genomic DNA of SE JL228 was extracted using an QIAamp DNA Mini Kit (Qiagen, MD, USA). Genome assembly was carried out with the Unicycler hybrid pipeline, employing the PacBio® sequencing platform (Pacific Biosciences, CA, USA) [ 21 ], and using SE strain P125109 (GenBank accession NC_011294) as the reference. This genome assembly was further proofread Illumina® Hiseq×10 (Illumina Inc., CA, USA) platform. Plasmid sequences were annotated by PLSDB database [ 9 ]. Analyses were conducted on the Majorbio Cloud Platform. Genomic sequences were deposited in GenBank under accession numbers CP094269 to CP094271. Molecular typing and prediction Chromosomal and plasmid sequences were analyzed using the Achtman multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST), and replicon sequencing typing (RST) schemes available in the PubMLST database ( http://pubmlst.org ). Predictions of antimicrobial resistance and virulence genes were conducted through ResFinder4.0 and the VFDB database [ 22 , 23 ]. Genomic comparison visualization Plasmid circular maps were created to compare a reference bacterial plasmid to all query bacterial strains using BLAST Ring Image Generator (BRIG). To search for similar metal resistance loci and genetic organization. The multi-heavy metal resistance region of plasmid pSE228A (range: 1–86200 bp) was compared against the nucleotide database using blastn program (date: April 10th, 2022). Easyfig 2.2.5 software was used for creating linear comparison figures of multiple genomic loci [ 24 ]. Plasmid Conjugal Transfer Filter and liquid mating methods were employed to assess the conjugative ability of the SE JL228. E.coli C600 as the recipient, with donor and recipient strains cultured in LB broth containing ampicillin and rifampicin. bla TEM-1 gene and penicillin resistance phenotype were chosen to assess the conjugation efficiency transconjugants selected on LB agar. Result Salmonella strains & serotyping Of 10 liver samples, 8 tested positive for non-Typhi Salmonella spp. via XLT4 agar isolation, showing black-centered colonies with yellow/pink periphery after 18 h, turning fully black by 30 h. All isolates matched Salmonella enterica serovar Enteritidis biochemical and antigenic profiles (9,12:g,m:-) based on VITEK 2 GN tests and serotyping (Additional file 1). All SE isolates are multidrug resistant (MDR) Uniform antimicrobial resistance profiles were observed across all 8 SE isolates (Table 1 ). Complete resistance was detected against Ampicillin (AMP), Piperacillin (PIP), Cephalexin (CL), Cefovecin (CEF), Ceftiofur (EFT), Amikacin (AMK), Gentamicin (GEN), Tobramycin (TOB), Trimethoprim/ Sulfamethoxazole (SXT), Chloramphenicol (CHL), Enrofloxacin (ENR), Tetracycline (TET), and Nitrofurantoin (NIT). Similarly, these isolates were all intermediately resistant to Amoxicillin/Clavulanic acid (AMC), Cefpodoxime (CPD), and Marbofloxacin (MAR), and all susceptible to Imipenem (IPM). Table 1 Antimicrobial drug susceptibility profiles of SE JL228. antimicrobial MIC (mg/L) susceptibility interpretation Penicillins Ampicillin (AMP) ≥ 32 R Amoxicillin/Clavulanic acid (AMC) 16 I Piperacillin (PIP) ≥ 128 R Cephalosporin Cephalexin (CL) ≥ 64 R Cefovecin (CEF) ≥ 8 R Ceftiofur (EFT) ≥ 8 R Cefpodoxime (CPD) 4 I Carbapenems Imipenem (IPM) ≤ 1 S Aminoglycosides Amikacin (AMK) ≥ 64 R Gentamicin (GEN) ≥ 16 R Tobramycin (TOB) ≥ 16 R Sulphonamides Trimethoprim/ Sulfamethoxazole (SXT) ≥ 320 R Fluoroquinolones Marbofloxacin (MAR) 2 I Enrofloxacin (ENR) ≥ 4 R Chloramphenicol Chloramphenicol (CHL) ≥ 64 R Tetracycline Tetracycline (TET) ≥ 16 R Nitrofurantoin Nitrofurantoin (NIT) 128 R Antimicrobial categories are shown in bold. MIC, minimum inhibitory concentration; S, susceptible; R, resistant; I, intermediate. Table 1 Antimicrobial drug susceptibility profiles of S. enterica serovar Enteritidis isolate JL228. antimicrobial MIC (mg/L) susceptibility interpretation Penicillins Ampicillin (AMP) ≥ 32 R Amoxicillin/Clavulanic acid (AMC) 16 I Piperacillin (PIP) ≥ 128 R Cephalosporin Cephalexin (CL) ≥ 64 R Cefovecin (CEF) ≥ 8 R Ceftiofur (EFT) ≥ 8 R Cefpodoxime (CPD) 4 I Carbapenems Imipenem (IPM) ≤ 1 S Aminoglycosides Amikacin (AMK) ≥ 64 R Gentamicin (GEN) ≥ 16 R Tobramycin (TOB) ≥ 16 R Sulphonamides Trimethoprim/ Sulfamethoxazole (SXT) ≥ 320 R Fluoroquinolones Marbofloxacin (MAR) 2 I Enrofloxacin (ENR) ≥ 4 R Chloramphenicol Chloramphenicol (CHL) ≥ 64 R Tetracycline Tetracycline (TET) ≥ 16 R Nitrofurantoin Nitrofurantoin (NIT) 128 R Antimicrobial categories are shown in bold. MIC, minimum inhibitory concentration; S, susceptible; R, resistant; I, intermediate. The MICs for the selected heavy metal to SE JL228 as follows: Ag + , 16 mgl/L, Cu 2+ , 1024 mgl/L, TeO2 + 3,16 mgl/L, Co 2+ , 256 mgl/L, Zn 2+ , 1024 mgl/L, Cr 3+ , 512 mgl/L, Cd 2+ , 128–256 mgl/L (Table 2 ). Generally, the isolate SE JL228 is considered resistant to the Ag + , Cu 2+ and Te 4+ , with the MICs two-2-fold dilutions above the MIC 50 .The MIC value of DBAB for SE JL228 is 51.2 mg/L, suggesting the high tolerance of the isolate to DBAB, a commonly used QAC in China (Table 2 ). Table 2 Minimum inhibitory concentrations to heavy metals and QAC a of SE JL228 Strain/isolate Ag + (mg/L) Cd 2+ (mg/L) Co 2+ (mg/L) Cr 3+ (mg/L) Cu 2+ (mg/L) Te 4+ (mg/L) Zn 2+ (mg/L) DBAB b (mg/L) SE JL228 32 128–256 256 512 1024 16 1024 51.2 SE LN248 8 128 256 512 256 4 1024 25.6 E. coli ATCC 25922™ 8 128–256 64–128 512–1024 256 0.5-1 1024 25.6 MIC 50 c 10 ± 2 153 ± 46 268 ± 40 550 ± 21 256 ± 24 5 ± 0.7 1431 ± 201 nd d a QAC, quaternary ammonium compound b DBAB, benzyldodecyldimethylammonium bromide c MIC 50 shown here were determined from 113 SE isolates. d nd, not determined. Table 2 Minimum inhibitory concentrations to heavy metals and QAC of S. enterica serovar Enteritidis isolate JL228 Strain/isolate Ag + (mg/L) Cd 2+ (mg/L) Co 2+ (mg/L) Cr 3+ (mg/L) Cu 2+ (mg/L) Te 4+ (mg/L) Zn 2+ (mg/L) DBAB(mg/L) S. enterica serovar Enteritidis JL228 32 128–256 256 512 1024 16 1024 51.2 S. enterica serovar Enteritidis LN248 8 128 256 512 256 4 1024 25.6 Escherichia coli ATCC™ 25922™ 8 128–256 64–128 512–1024 256 0.5-1 1024 25.6 MIC 50 10 ± 2 153 ± 46 268 ± 40 550 ± 21 256 ± 24 5 ± 0.7 1431 ± 201 nd QAC, quaternary ammonium compound DBAB, benzyldodecyldimethylammonium bromide MIC 50 , the lowest concentration of an antimicrobial agent at which 50% of the isolates were inhibited. MIC 50 shown here were determined from 113 S. enterica serovar Enteritidis isolates. nd, not determined. SE JL228 is highly invasive in vitro Results from the gentamicin protection invasion assay indicated that SE JL228 was more invasive than SE LN248 in hBMEC (Fig. 1 ). Conversely, the two isolates showed no significant difference in their invasion of APM. Nonetheless, JL228 exhibited significantly superior intracellular survival within APM relative to LN248, while both strains presented equally poor survival rates in hBMEC. SE JL228 are high virulent in mice model The median lethal dose (LD₅₀) for SE JL228 in mice following oral inoculation was determined as 5.0 × 10⁶ CFU, which was about 68-fold lower than that observed for SE LN248 (3.4 × 10⁸ CFU) [ 18 ]. Similarly, in the streptomycin-pretreated infection model, SE JL228 was lethal in 100% of BALB/c mice. All mice succumbed to 10 8 inoculums by day 14 post-infection. Of the mice infected with LN248, however, 40% survived the infection (Fig. 2 A). Significant body mass changes were also observed in both infection groups. whilst, mice infected with SE JL228 had sharper body mass decrease in the first two days post infection than that with SE LN248 (Fig. 2 B). Interestingly, 50% SE JL228 infected mice developed overt neurological signs that resembled meningitis with balance defect and ataxia (Additional file 2). Taken together, the rabbit-originated clinical isolate SE JL228 exhibited higher virulence potential in mouse model. Dissemination capability The bacterial dissemination assay indicated that mice infected with SE JL228 harbored significantly greater bacterial loads in multiple organs compared to those infected with SE LN248 (Fig. 2 C– 2 E), implying a stronger systemic spread of JL228 following oral infection. In accordance with the findings in neurological signs, SE JL228 was more frequently recovered as well. Bacterial load was also significantly higher in SE JL228 infected mouse brains than that recovered from the SE LN248 (Fig. 2 G). However, no significant difference of cecal colonization was observed in the two groups mice (Fig. 2 F). Overview of JL228 genome The genome structure of SE JL228 consisted of a 4.7-megabase chromosome (GenBank: CP094269) along with two plasmids, 211.4 kb (pSE228A, CP094270) and 54.6 kb (pSE228B, CP094271). GC-content values were 52.17% for the chromosome, 46.28% for pSE228A, and 47.51% for pSE228B. Using NCBI’s PGAP annotation tool [ 25 ], a total of 4,896 genes were identified, with 4,612 on the chromosome, 223 on pSE228A, and 61 on pSE228B (Fig. 3 ). Molecular typing suggested chromosome of strain SE JL228 match the profiles of ST11 and cgST-7883 respectively. In silico replicon-based typing suggested plasmid pSE228A match the loci of FIB (allele 22), smr0018 (allele 1), and smr0199 (allele 2), according to the Plasmid MLST schemes [ 26 ], therefore, plasmid pSE228A is a multiple-replicon plasmid with the incompatibility group of IncFIB and IncHI2. Further subtyping by IncHI2 double locus sequence typing (DLST) scheme classified the plasmid into ST14 group. The same typing scheme suggested the plasmid pSE228B belongs to IncN group, one of the prevalent drug-resistance plasmid types in Enterobacteriaceae (Table 3 ). Table 3 Molecular characteristics of plasmids carried by the SE JL228 plasmid Size (kb) ST Inc group Replication, plasmid conjugal transfer and maintenance gene AMR gene and heavy metal resistance gene Virulence plasmid-encoded traits pSE228A 211.4 14 IncFIB-IncHI2 repB (MPK88_23535), traE, traK, trhB, trhV, traC, parA, parM, htdF, rsp, trhF, trhU, trhN, trhI, MPK88_23540 , MPK88_23545, repB (MPK88_23820), MPK88_23790 , ccdA , ccdB , trhH , trhR copper resistance: copA, copB, copC, copD, copS, copE, MPK88_23180 silver resistance: silP MPK88_23200 silA , silB silF silC silSR silE tellurite resistance: terE, terC, terD, terB, terA, terZ, terW, terY, terX antibiotic resistance: aac(6')-IIc, sul1, ere(A) a QAC resistance: qacE b spvDBAR : Salmonella plasmid virulence ibeB c : Invasion of brain endothelial cells pSE228B 54.6 23 IncN repM , MPK88_24365, MPK88_24370, MPK88_24425 , parA , parG , MPK88_24360, MPK88_24360 , ddp3 antibiotic resistance: armA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, bla TEM -1B, mph(E), sul1, sul2 QAC resistance: qacE b none a Incomplete, 85% coverage. b Incomplete, 84.7% coverage. c synonyms name, silC for this study, cusC , in E. coli . Table 3 Molecular characteristics of plasmids carried by the S. enterica serovar Enteritidis strain JL228 plasmid Size (kb) ST Inc group Replication, plasmid conjugal transfer and maintenance gene AMR gene and heavy metal resistance gene Virulence plasmid-encoded traits pSE228A 211.4 14 IncFIB-IncHI2 repB (MPK88_23535), traE, traK, trhB, trhV, traC, parA, parM, htdF, rsp, trhF, trhU, trhN, trhI, MPK88_23540 , MPK88_23545, repB (MPK88_23820), MPK88_23790 , ccdA , ccdB , trhH , trhR copper resistance: copA, copB, copC, copD, copS, copE, MPK88_23180 silver resistance: silP MPK88_23200 silA , silB silF silC silSR silE tellurite resistance: terE, terC, terD, terB, terA, terZ, terW, terY, terX antibiotic resistance: aac(6')-IIc, sul1, ere(A) a QAC b resistance: qacE c spvDBAR : Salmonella plasmid virulence ibeB d : Invasion of brain endothelial cells pSE228B 54.6 23 IncN repM , MPK88_24365, MPK88_24370, MPK88_24425 , parA , parG , MPK88_24360, MPK88_24360 , ddp3 antibiotic resistance: armA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, blaTEM-1B, mph(E), sul1, sul2 QAC resistance: qacE c none a. Incomplete, 85% coverage. b. QAC, quaternary ammonium compounds. c. Incomplete, 84.7% coverage. d. synonyms name, silC for this study, cusC , in E.coli . Structure of the plasmids carried by strain SE JL228 Plasmid pSE228A (GenBank: CP094270) contained 223 predicted genes, including two backbone repB loci located at positions 146725–147714 and 86251–87000 (complementary strand). The encoded RepB initiator proteins exhibited only 42.17% amino acid identity, although the first shared complete identity with the RepB of the FIB plasmid pS82/10 from SE [ 10 ]. Additional loci associated with IncHI-type conjugation and plasmid maintenance were also present, forming a repB-traE-traK-trhB-trhV-traC-parA-parM-htdF-rsp-trhF-trhU-trhN-trhI cluster (Fig. 4 A). Two genes, MPK88_23540 and MPK88_23545 , encoding a pili assembly chaperone and a plasmid transfer protein, respectively, were identified between repB and traE . Furthermore, a second repB locus (86251–87000), together with MPK88_23790 (DNA replication terminus site-binding protein), ccdA , ccdB , trhH , and trhR , may contribute to plasmid replication, stability, and transfer (Fig. 4 B). Plasmid pSE228B (GenBank: CP094271) was classified as an IncN replicon and contained a backbone region spanning positions (30357–43644 bp), encompassing eight genes related to replication and stability (MPK88_24360–MPK88_24425). This region encoded four replication initiation proteins, including repM and three uncharacterized replication-associated proteins (MPK88_24365, MPK88_24370, MPK88_24425). Additionally, four maintenance-related genes were identified: parA , parG , MPK88_24355 (RelE/ParE toxin), and MPK88_24360 (stabilization protein), along with ddp3, encoding a DNA distortion polypeptide. Antimicrobial and heavy metal resistance genes Using the ResFinder4.0 platform, seventeen antimicrobial resistance (AMR) genes were detected in the genome of SE strain JL228. The chromosomal gene aac(6’)-Iaa was identified, along with three genes located on plasmid pSE228A [ aac(6’)-IIc, sul1 , and ere(A) ], and ten genes carried on plasmid pSE228B [ armA , aac(6’)-Ib-cr , aac(3)-IId , aph(6)-Id , aph(3’’)-Ib , aph(3’)-Ia , blaTEM-1B , mph(E) , sul1 , and sul2 ]. (Fig. 5 A–C). AMR genes in pSE228A are clustered in the region 201393–208693, flanked by two identical IS6-like element IS26 family transposase genes and a class 1 integron integrase gene ( intl1 ), constituting a gene cluster of IS6-sul1-qacE-ere(A)-MPK88_24140-aac(3)-IIg-ereA-aac(6')-IIc-intI1-IS6 . The unassigned gene locus MPK88_24140 encodes an NAD(+)-rifampin ADP-ribosyltransferase, suggesting the rifampin resistance potential of this plasmid (Fig. 5 B). AMR genes in pSE228B scattered in four regions among the plasmid (Fig. 5 C). These AMR genes in chromosome and plasmids are predicted to confer resistance to seven class antibiotics (Additional file 3). Besides, quaternary ammonium compound-resistance gene qacE were also found in both plasmids, with the predicted phenotype of Benzylkonium Chloride, Ethidium Bromide, Chlorhexidine and Cetylpyridinium Chloride resistance (Fig. 5 B and C). The analysis of plasmid pSE228A revealed the presence of three distinct heavy metal resistance operons: the copper-associated locus copESDBAC (15559–20810), the silver resistance determinant silPABFCRE (22094–34545), and the tellurite operon terEDCBAZWYX (62141–75496). (Table 3 , Fig. 5 D). The large multi-metal resistance region of pSE228A is flanked by mobile genetic elements (MGEs) including IS6-like element IS26 family transposase (region 1652–2356 and 85494–86198), Tn3-like element Tn5403 family transposase (region 2427–4171 and 4207–4467), IS21-like element IS100 family transposase istA (region 82039–83061) and istB (region 81260–82042) (Fig. 5 D). Five plasmids carrying similar metal tolerance gene clusters were found to be highly conserved with those in pSE228A plasmid with the sequence identities over 99% (Fig. 6 ). The plasmid pR15.0430_329k, unname1 and pEC5207 carried by Salmonella Typhimurium (STm), Raoultella sp. and E.coli respectively, cover the complete collinear regions of the silver, copper and tellurite resistance loci (Fig. 6 A and B). Whilst the pKO_1 carried by Klebsiella michiganensis showed imperfect coverage of the resistance clusters with the partial copper resistance locus missing in this plasmid (Fig. 6 A). The plasmid pYUSHP2-1 carried by Enterobacter hormaechei only share the similar tellurite resistance region (Fig. 6 C). Virulence factor (VF) genes A total of 158 virulence genes were identified in virulence factor database (VFDB), of which 154 are chromosome-encoded genes. 4 plasmid-encoded VF genes distribute exclusively in plasmid pSE228A, while no VF gene found in pSE228B. Further intra-genera comparative pathogenomics using VFanalyzer tool with other 16 Salmonella genomes reveal that sefB , sefC , sefD in sef fimbrial operon are absent in SE JL228 strain, remaining only sefA gene left in chromosome. In addition, a SPI-1 T3SS gene orgB , 2 T3SS translocated effector genes sopD2 and slrP , are absent in SE JL228 genome as well. Notably, 3 members of plasmid-encoded spv locus, spvB , spvC and spvD were identified in pSE228A. Further analysis confirmed the intact bacteremia-associated locus of spvRABCD , suggesting the plasmid is a virulence plasmid (Additional file 4). Virulence determinant operons in SE JL228 genome were also compared with those in SE LN248 and other two newly characterized S. Typhimurium (ST221_31B) and Kentucky (SK222_32B). Results suggested that 3 virulence-related operons including plasmid-encoded SPV, TTSS-2 translocated effectors sseK2 and sspH2 are present in SE JL228 genome whereas absent in LN248. In addition, a Peg fimbrial adherence determinant was found exclusively in LN248. (Additional file 5). Interestingly, MPK88_23220 in pSE228A, which was assigned as silC in our submission (GenBank accession no. CP094270), was annotated to be ibeB in VFDB (Additional file 4). Its orthologous gene in Escherichia encoding a virulence factor involving in invasion of brain endothelial cells. The deduced amino acid sequence of SE JL228 shares approximate 71% identities (71.09%-71.33%) with its orthologs in pathogenic Escherichia . (Fig. 7 ). Other synonyms name of this gene in Escherichia is also known as cusC , a member in cusCFBA gene locus that encodes a Cu(+)/Ag(+) efflux system CusCFBA. Comparative Analysis of plasmids pSE228A shares near-identical backbone and loci patterns with the IncHI2 plasmid pSE_AH228 from a 2019 Salmonella isolate (Fig. 8 A). The ARG clusters are highly similar to seven plasmids, including pYUSHP2-1 ( E. hormaechei ), XY-1 ( Raoultella sp. ), pR15.0430 ( S. Typhimurium), pKA04-2 ( K. aerogenes ), pIMP26 ( E. cloacae ), p1106151-mcr ( Leclercia sp. ), and pSE_AH228. Notably, pSE228A shares the highest genetic coverage with pKA04-2, except for the latter lack of spv locus and ccdA/ccdB toxin-antitoxin genes typical of virulent Salmonella plasmids. Copper and silver resistance operons in pSE228A are homologous to those in p280_40A ( E.fergusonii ), SWHE2 (S. dysenteriae ), XY-1, pKO_1 ( K. michiganensis ), pR15.0430, pKA04-2, pEC5207 ( E. coli ), and pSE_AH228. The ter operon for tellurite resistance is conserved across all 14 plasmids, indicating its prevalence in Enterobacteriaceae plasmids. Plasmid pSE228B shows limited sequence coverage with known plasmids (Fig. 8 B). Its initial 20 kb fragment resembles pB12AN_1 from a human-derived K. pneumoniae strain in China. The 18–42 kb region, particularly 19,018–23,114 bp, harbors resistance genes ( bla TEM−1 , mph(E) , aac(6')-Ib-cr , qnrB , sul1 , sul2 ) with high similarity to Salmonella plasmids pFORC51, pSE1004837, pFORC89, p12519B, and K. pneumoniae plasmids pB12AN_1 and pB0910. The 44–54 kb region aligns with pB0910. Transferability assay Plasmid conjugation yielded eight transconjugants, three from membrane filter mating (11.11% efficiency) and five from liquid mating (15.62% efficiency). PCR confirmed the bla TEM−1 gene (850-bp amplicon) in all transconjugants, identical to SE JL228, with sequencing verifying bla TEM -1 presence (Fig. 9 ). Broth microdilution showed high-level penicillin resistance (MIC > 2048 µg/mL) in all transconjugants, consistent with SE JL228 and higher than E. coli C600 (Additional file 6). Discussion In this study, we characterize a Salmonella Enteritidis strain JL228, from a rabbit farm outbreak, revealing its high resistance to multiple antibiotics and metals mediated by plasmid and chromosomal determinants. The strain also showed increased virulence, enhanced systemic dissemination, and the ability to cross the blood-brain barrier and invade hBMECs. This highly resistant and virulent strain poses a significant threat to animal and human health, necessitating close monitoring in farms and hospitals. The SE JL228 genome consists of two plasmid pSE228A and pSE228B. pSE228A, likely a fusion of IncFIB and IncHI2, encodes multiple ARGs, MRGs, and VFs, flanked by mobile genetic elements like class 1 integrons and IS26 transposons. These features enable pSE228A to act as a reservoir for resistance and virulence genes, promoting efficient horizontal gene transfer and rapid dissemination among bacteria [ 27 ]. The presence of copper, silver, and tellurite resistance operons alongside ARGs-including sul1 , ere(A) , and aac(6')-IIc -on pSE228A suggests the co-selection of metal and antibiotic resistance. In agricultural practices, copper is widely used as additives to control infections [ 28 , 29 ]. Prolonged exposure to these metals may exert selective pressure that facilitates the persistence of MDR SE strains in agricultural environments. This observation is consistent with previous studies reporting that SE isolates from industrial farms and wastewater treatment facilities often harbor both MRGs and ARGs, enabling them to survive in metal-contaminated environments [ 30 , 31 ]. Similar patterns have been observed in other Enterobacteriaceae species, such as Vibrio cholerae and Klebsiella pneumoniae , further enhancing the contribution of heavy metal exposure to the evolution of bacterial resistance profiles [ 32 – 34 ]. In contrast, pSE228B, an IncN-type plasmid commonly found in Enterobacteriaceae , does not encode virulence factors but harbors several ARGs-including armA and bla TEM−1B , that contribute significantly to MDR phenotype of SE JL228, complicating treatment options. Comparative genomic analyses revealed high sequence identity between resistance and metal tolerance regions of pSE228A and plasmids from STm and E. coli , while pSE228B shares conserved AMR modules with plasmids from Salmonella sp. and K. pneumoniae . These findings collectively support the notion that plasmid-mediated HGT, likely occurring in polymicrobial farm environments under selective pressure, plays a central role in the dissemination of MDR traits. Notably, pSE228A encodes several key virulence genes, including ibeB , spvB , spvC , and spvD , which are known to enhance the systemic dissemination of Salmonella enterica by impairing host immune responses and promoting intracellular survival within macrophages [ 35 ]. In contrast to SE JL228, the LN248 strain lacks these genes, which may explain the markedly superior intracellular persistence and systemic infectivity of SE JL228 in APM. These genes likely contribute to observed neuroinvasive potential of SE JL228 in mice, including its ability to cross the blood-brain barrier and induce neurological manifestations [ 36 ]. Chromosomal virulence determinants play a crucial role in SE JL228 pathogenicity alongside plasmid-borne factors. Genome analysis revealed that SE JL228 possesses a full suite of SPI-1/SPI-2 effectors-including sopB , sopE , sopE2 , sseC , and sseD -that promote host cell invasion and immune subversion [ 37 , 38 ]. mgtC confers tolerance to magnesium limitation, enhancing survival in macrophages [ 39 ]. The adhesion- and persistence-associated genes pagN , pagC , and pagD contribute to bacterial attachment to host cells, invasion of immune cells, and survival within the intracellular environment [ 40 , 41 ]. Interestingly, despite the absence of orgB , sopD2 , and slrP , SE JL228 retained strong invasive and virulent phenotypes, suggesting the existence of alternative compensatory mechanisms or functional redundancies within its virulence gene repertoire. These chromosomally encoded virulence factors, in synergy with plasmid-borne elements, collectively underpin the high virulence potential of SE JL228, posing significant public health risks. The limited sequence coverage with known plasmids and the dispersed distribution of ARGs complicate accurate prediction of the origin and formation process of this plasmid. To elucidate the potential origin and dissemination pathways of the plasmids carried by SE JL228, a source-tracing investigation was conducted using BRIG-based comparative genomic analysis, which revealed that the plasmid pSE228A shared highset sequence identity with a previously reported plasmid, pSE_AH228, isolated from Salmonella spp. Although the precise geographic origin of pSE_AH228 remains undetermined, its high homology with pSE228A suggests that IncHI2-type plasmids have undergone widespread horizontal transmission across bacterial species and possibly across regional and ecological boundaries within agricultural environments. Similarly, the IncN plasmid pSE228B exhibited high sequence similarity to plasmids identified in K.pneumoniae , implying that the resistance determinants in SE JL228 likely originated from a shared plasmid pool circulating among various Enterobacteriaceae . These findings indicate that the acquisition of resistance plasmids in SE JL228 reflects not an isolated evolutionary event but an outcome of active gene flow under environmental selection pressure. While the study provides a detailed genomic and functional analysis of a single outbreak strain, further research with diverse isolates from various regions and hosts is needed to confirm broader epidemiological significance. Future studies should investigate the transmission of IncFIB-IncHI2 and IncN plasmids in environmental and clinical contexts and clarify the role of heavy metal resistance in the persistence and spread of multidrug-resistant (MDR) SE to understand environmental impacts on bacterial evolution. Declarations Ethics approval and consent to participate: Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007). Clinical trial number: not applicable. Consent for publication: Not applicable. Availability of data and materials: The complete genome sequence of Salmonella enterica serovar Enteritidis strain SEJL228A has been deposited in GenBank under accession number CP094269. The plasmid sequences are available under accession numbers CP094270 (pSE228A) and CP094271 (pSE228B). All data generated or analyzed during this study are included in this published article. Competing interests: All co-authors declare that we have no conflicts of interest. Funding: This work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2024MC148 and ZR2023MC049), the Key Research and Development Plan of Shandong Province (Grant No. 2022CXPT022, 2025CXGC010803) and the Shandong Province Poultry Industry Technology System (Grant No. SDAIT-11-10). Authors' contributions: Writing-original draft: R.Z., H.Z. Writing-review and editing: H.Z. Investigation: R.Z., B.W., Y.L., J.L., X.D., H.Z. Validation: B.W., Y.L., X.D., J.Y., J.Z. Software: R.Z., B.W. Visualization: R.Z., B.W., H.Z. Formal analysis: J.Z., Y.L., J.Y., L.J. Data curation: Y.L., L.J., H.Z. Methodology: J.L., X.Y., J.Y., H.Z. Conceptualization: H.Z. Resources: J.Z., X.Y., J.Z. Funding acquisition: X.Y., H.Z, X.Z., Y.L. Supervision: L.J., H.Z., X.Z. Project administration: X.Z. All authors read and approved the final manuscript. Acknowledgment: The conference presentation by Zhu et al. at the 20th Congress of the International Society for Animal Hygiene shares thematic similarities with this work [42]. We disclose that the language in this manuscript was polished using Grok 3, an artificial intelligence tool developed by xAI, to enhance clarity and readability without adding any data, results, ideas, or opinions by AI. References Cheng RA, Eade CR, Wiedmann M. Embracing diversity: differences in virulence mechanisms, disease severity, and host adaptations contribute to the success of nontyphoidal Salmonella as a foodborne pathogen. Front Microbiol. 2019;10. 10.3389/fmicb.2019.01368 . Bisbini P, Leoni E, Nanetti A. An outbreak of Salmonella Hadar associated with roast rabbit in a restaurant. Eur J Epidemiol. 2000;16(7):613–8. 10.1023/a:1007694605464 . Yu X, Zhu H, Bo Y, Li Y, Zhang Y, Liu Y et al. 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Supplementary Files Additionalfile.zip Additional file 1. Antigenic formula of the 8 Salmonella isolates recovered from rabbit livers by standard agglutination test. Additional file 2. Representative footage of BALB/c mice infected with SE JL228 exhibiting overt neurological signs. Additional file 3. The antimicrobial resistance (AMR) genes identified in genome of SE JL228 using ResFinder4.0 database. Additional file 4. Virulence gene profiles of SE JL228 based on VFDB and comparative pathogenomics. Additional file 5. Comparison of virulence operons in JL228, LN248, Salmonella enterica strains. Typhimurium (ST221_31B) and Kentucky (SK222_32B). Additional file 6. Penicillin Resistance Phenotype of Transconjugants. Additional file 7. The plasmid information for comparison. Additional file 8. Plasmid Information Included in the Comparative Analysis with pSE228A. Additional file 9. Plasmid Information Included in the Comparative Analysis with pSE228B. 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of Quality Safety Monitoring for Animal Products and Veterinary Drug Innovation","correspondingAuthor":false,"prefix":"","firstName":"Youzhi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-09-05 02:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7540124/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7540124/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04612-1","type":"published","date":"2026-01-06T15:58:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94481294,"identity":"7f38ef80-811d-468c-8580-2bb74084c175","added_by":"auto","created_at":"2025-10-27 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16:12:53","extension":"pdf","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1330589,"visible":true,"origin":"","legend":"","description":"","filename":"Figure8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/6f478791acb006af7d1f349d.pdf"},{"id":94481229,"identity":"10060596-c44a-4d3a-a711-2ebcd48ebf29","added_by":"auto","created_at":"2025-10-27 16:12:55","extension":"pdf","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1286253,"visible":true,"origin":"","legend":"","description":"","filename":"Figure9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/dbaaf494b9a80a7bed10fc34.pdf"},{"id":94481669,"identity":"2ab8a5bd-3be1-4010-aaef-44848c400f77","added_by":"auto","created_at":"2025-10-27 16:14:11","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169013,"visible":true,"origin":"","legend":"","description":"","filename":"9b9559e686444782823ec65c8d77d2f21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/0102916b599106e22e4bdaf3.xml"},{"id":94480950,"identity":"57f123bc-81de-46b7-945e-9703806b6ba6","added_by":"auto","created_at":"2025-10-27 16:12:17","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188279,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/b80d836e305dfd6e451b73e1.html"},{"id":94481083,"identity":"ad9e43e6-3211-446a-b714-abad2cb2612e","added_by":"auto","created_at":"2025-10-27 16:12:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvasion and intracellular survival of SE JL228 and LN248 in hBMEC and APM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfluent monolayers of hBMEC and APM were infected with SE JL228 or SE LN248 at a multiplicity of infection (MOI) of 1. The percentage of adherent and intracellular bacteria (invasion) was quantified 2 hours post-infection, expressed as a percentage of the initial inoculum (1A). while, intracellular survival of strains in hBMEC and APM were determined at 24 hours post-infection by gentamicin protection assay(1B). Data are expressed as colony-forming units (CFU) per well (mean ± SD, n = 10). Significance levels are denoted as \u003cem\u003ens\u003c/em\u003e (not significant), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/86c15dd3b4481ea5cd96c732.png"},{"id":94481239,"identity":"1e39eac5-e8c3-41c4-ac71-6a8b2ffa91c6","added_by":"auto","created_at":"2025-10-27 16:12:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial Dissemination and Colonization of SE JL228 and LN248 in Mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedian lethal dose (LD50) of SE JL228 and SE LN248 (2A). Bacteria were grown in LB broth and adjusted to 10\u003csup\u003e8\u003c/sup\u003e CFU/50 μL. Mice were orally inoculated with serial dilutions, and mortality was recorded over 28 days. LD\u003csub\u003e50 \u003c/sub\u003evalues were calculated using the Reed and Muench method [19]. Mice body weights were record and calculated as body mass changes (2B). Dissemination of SE JL228 and SE LN248 in mouse tissues (C-F). Mice were treated with 20 mg streptomycin, and intragastrically infected with 1 × 10\u003csup\u003e8\u003c/sup\u003e CFU of SE JL228 or SE LN248. Five days post-infection, tissues sample were aseptically homogenized. bacterial loads were quantified on XLT-4 plate. Data represent mean log of CFU per gram of tissue ± SD from three independent experiments (n=6). Significance levels are denoted as \u003cem\u003ens\u003c/em\u003e (not significant), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/5f3491ab065b5847db0755c6.png"},{"id":94481662,"identity":"3a75d8be-1a57-41c2-b9f1-24fe91ffd1f4","added_by":"auto","created_at":"2025-10-27 16:14:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircular map of SE JL228 chromosome and plasmids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosome SE JL228, plasmids pSE228A and pSE228B, are shown, with plasmids depicted independently of the chromosome scale. From outer to inner rings: protein-coding genes on the forward strand (colored by COG categories), forward-strand genes, reverse-strand genes, protein-coding genes on the reverse strand, G+C content, and G+C skew. The legend indicates plasmid backbone, accessory modules, virulence, metal resistance, and AMR genes with distinct colors.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/a8eb6b8bd0de24e6d6bade44.png"},{"id":94481332,"identity":"b4c21812-873f-4c2c-b3c5-beb17b47ddd1","added_by":"auto","created_at":"2025-10-27 16:13:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic organization of plasmids pSE228A and pSE228B in strain SE JL228.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Genetic structure of the \u003cem\u003erepB\u003c/em\u003e-\u003cem\u003etraE\u003c/em\u003e-\u003cem\u003etraK\u003c/em\u003e-\u003cem\u003etrhB\u003c/em\u003e-\u003cem\u003etrhV\u003c/em\u003e-\u003cem\u003etraC\u003c/em\u003e-\u003cem\u003eparA\u003c/em\u003e-\u003cem\u003eparM\u003c/em\u003e-\u003cem\u003ehtdF\u003c/em\u003e-\u003cem\u003ersp\u003c/em\u003e-\u003cem\u003etrhF\u003c/em\u003e-\u003cem\u003etrhU\u003c/em\u003e-\u003cem\u003etrhN\u003c/em\u003e-\u003cem\u003etrhI\u003c/em\u003eloci in pSE228A, which are involved in plasmid replication, maintenance, and conjugation. In addition, two related genes encoding pili assembly chaperone and plasmid transfer protein, corresponding to gene MPK88_23540 and MPK88_23545 were also found between \u003cem\u003erepB\u003c/em\u003e and \u003cem\u003etraE\u003c/em\u003e. (B) Additional replication and stability-related genes in pSE228A. This section includes a second \u003cem\u003erepB\u003c/em\u003egene, along MPK88_23790, \u003cem\u003eccdA\u003c/em\u003e, \u003cem\u003eccdB\u003c/em\u003e, \u003cem\u003etrhH\u003c/em\u003e, \u003cem\u003etrhR\u003c/em\u003e. (C) The IncN-type plasmid pSE228B carries key replication (\u003cem\u003erepM\u003c/em\u003e, MPK88_24365, MPK88_24370, MPK88_24425) and maintenance (\u003cem\u003eparA\u003c/em\u003e, \u003cem\u003eparG\u003c/em\u003e, MPK88_24355 (RelE/ParE toxin), MPK88_24360 (plasmid stabilization protein)) genes. Additionally, \u003cem\u003eddp3\u003c/em\u003e, encoding a DNA distortion polypeptide, is present.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/504f046a5211fdbac8e0e6e2.png"},{"id":94481726,"identity":"7eeaa421-d2df-4f11-add8-4fff7dc53d2b","added_by":"auto","created_at":"2025-10-27 16:14:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of antimicrobial resistance (AMR) genes in the chromosome and plasmids of JL228.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chromosomal AMR gene. The \u003cem\u003eaac(6‘)-Iaa\u003c/em\u003e gene, conferring resistance to aminoglycosides, is located within the chromosome. Flanking genes (gene2476 and \u003cem\u003erspA\u003c/em\u003e) are shown in gray, while the AMR gene is highlighted in cyan. (B) AMR gene cluster in plasmid pSE228A. A cluster of AMR genes, including \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003eere(A)\u003c/em\u003e and \u003cem\u003eaac(6')-IIc\u003c/em\u003e, is present within the pSE228A. This cluster is flanked by IS6-like transposase genes and contains a class 1 integron integrase gene (\u003cem\u003eintI1\u003c/em\u003e), facilitating gene mobility. The presence of MPK88_24140, encoding an NAD (+)-rifampin ADP-ribosyltransferase, suggests potential rifampin resistance. (C) AMR genes in plasmid pSE228B. The 54.6-kb IncN-type plasmid pSE228B carries eleven AMR genes (\u003cem\u003earmA\u003c/em\u003e, \u003cem\u003eaac(6')-Ib-cr\u003c/em\u003e, \u003cem\u003eaac(3)-IId\u003c/em\u003e, \u003cem\u003eaph(6)-Id\u003c/em\u003e, \u003cem\u003eaph(3'')-Ib\u003c/em\u003e, \u003cem\u003eaph(3')-Ia\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1B\u003c/em\u003e, \u003cem\u003emph(E)\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003emsr(E)\u003c/em\u003e), scattered across four regions. The \u003cem\u003eqacE\u003c/em\u003e gene, conferring resistance to quaternary ammonium compounds, is also present. (D) Plasmid pSE228A harbors three heavy metal resistance operons: (Ⅰ) Copper resistance locus (\u003cem\u003ecopESDBAC\u003c/em\u003e, region 15,559–20,810)–Genes involved in copper resistance are shown in green. (Ⅱ) Silver resistance locus (\u003cem\u003esilPABFCRE\u003c/em\u003e, region 22,094–34,545)–Genes responsible for silver resistance are depicted in white. (Ⅲ) Tellurite resistance locus (\u003cem\u003eterEDCBAZWYX\u003c/em\u003e, region 62,141–75,496)–Tellurite resistance genes are marked in purple.\u003c/p\u003e","description":"","filename":"Figure15.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/f413c5d7750dd2d16e349aaf.png"},{"id":94481226,"identity":"3b7dc1ee-3338-424e-aae4-834b5f5d270e","added_by":"auto","created_at":"2025-10-27 16:12:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":85513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasmid Comparison Reveals Conserved Multi-Metal Resistance Locus in pSE228A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The gene collinearity alignment of pSE228A with pKO_1 and XY-1 plasmid unnamed1. (B) The gene collinearity alignment of pSE228A with pEC5207 and pR15.0430. (C) The gene collinearity alignment of pSE228A with pYUSHP2-1. The multi-heavy metal resistance region of pSE228A (Red label, 1-86200) was compared with the resistance region in other plasmids from different bacterial sources. (Green: copper resistance gene; Purple: tellurite resistance gene; Gray: silver resistance gene; Red: other). The plasmid information involved in the comparison is detailed in Additional file 7.\u003c/p\u003e","description":"","filename":"Figure16.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/cbce88ecaa47a4cf8ab09474.png"},{"id":94481678,"identity":"a42cf87c-5a20-4147-b232-28c1bd360e23","added_by":"auto","created_at":"2025-10-27 16:14:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":401810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved Amino Acid Alignment of JL228 and Orthologs from Pathogenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences include \u003cem\u003eECP_0603_UPEC\u003c/em\u003e, \u003cem\u003eEC55989_056_EAEC\u003c/em\u003e, \u003cem\u003eO3K_18755_StxEAEC\u003c/em\u003e, \u003cem\u003eAPECO1_1476_APEC\u003c/em\u003e, \u003cem\u003eZ0711_EHEC\u003c/em\u003e, and \u003cem\u003eUMNK88_601_ETEC\u003c/em\u003e. Identical residues are highlighted, showing a high degree of sequence conservation. The alignment reveals approximately 71% sequence identity (70.72%–71.15%) between JL228 and its orthologs. Gaps indicate sequence variations among different strains.\u003c/p\u003e","description":"","filename":"Figure17.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/7488f045177d99cc8dc6c08a.png"},{"id":94481098,"identity":"46aaac52-5b3a-4f89-9142-fffd4a8caeba","added_by":"auto","created_at":"2025-10-27 16:12:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1095170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative plasmid maps for SE JL228 and related plasmids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Comparison of plasmid pSE228A. From outer to inner circle: CP091471.2, CP073772, CP067067, CP055064, CP031284, AP028562, CP139029, CP115835, CP113164, MH399264, KT347600, MN423361, ON960346, OW849257, GC skew−, GC skew+, GC content. (B) Comparison of plasmid pSE228B. From outer to inner circle: CP041175, CP031234, CP029683, CP026570, CP026156, CP017233, CP096599, CP063509, GC skew−, GC skew+, GC content. Detailed plasmid information are provided in Additional file 8 and 9.\u003c/p\u003e","description":"","filename":"Figure18.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/973047f5d06f9ad34b32dfad.png"},{"id":94481220,"identity":"26133c72-c1cf-4930-8f0e-f998137e6511","added_by":"auto","created_at":"2025-10-27 16:12:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":446780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfirmation of plasmid conjugation and resistance gene transfer in transconjugants. \u003c/strong\u003e(A) PCR amplification of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1\u003c/em\u003e gene in all eight transconjugants. Lane M: DNA\u003cstrong\u003e \u003c/strong\u003emarker; Lane 1: negative control; Lane 2: \u003cem\u003eE. coli\u003c/em\u003e C600; Lane 3: SE JL228 (positive control);\u003cstrong\u003e \u003c/strong\u003eLanes 4–11: transconjugants-1–8. All transconjugants produced an 850 bp amplicon\u003cstrong\u003e \u003c/strong\u003eidentical to the donor strain SE JL228. (B) Nucleotide sequence alignment of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1\u003c/em\u003e amplicon from transconjugants with the reference \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1\u003c/em\u003e gene sequence. The\u003cstrong\u003e \u003c/strong\u003ealignment confirmed 100% sequence identity between the transconjugants and the donor\u003c/p\u003e\n\u003cp\u003estrain JL228, validating the successful horizontal transfer\u003c/p\u003e","description":"","filename":"Figure19.png","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/355ebea2d6d75573886cb636.png"},{"id":100070502,"identity":"dfe3090b-84b0-4412-aeaf-fc931a08342d","added_by":"auto","created_at":"2026-01-12 16:17:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4209118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/8e1f471c-daae-40f9-8a1a-d202593e696b.pdf"},{"id":94481722,"identity":"4aae6847-b922-4d42-8323-6d26409d7577","added_by":"auto","created_at":"2025-10-27 16:14:46","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6976811,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1. Antigenic formula of the 8 Salmonella isolates recovered from rabbit livers by standard agglutination test.\u003c/p\u003e\n\u003cp\u003eAdditional file 2. Representative footage of BALB/c mice infected with SE JL228 exhibiting overt neurological signs.\u003c/p\u003e\n\u003cp\u003eAdditional file 3. The antimicrobial resistance (AMR) genes identified in genome of SE JL228 using ResFinder4.0 database.\u003c/p\u003e\n\u003cp\u003eAdditional file 4. Virulence gene profiles of SE JL228 based on VFDB and comparative pathogenomics.\u003c/p\u003e\n\u003cp\u003eAdditional file 5. Comparison of virulence operons in JL228, LN248, \u003cem\u003eSalmonella enterica\u003c/em\u003estrains. Typhimurium (ST221_31B) and Kentucky (SK222_32B).\u003c/p\u003e\n\u003cp\u003eAdditional file 6. Penicillin Resistance Phenotype of Transconjugants.\u003c/p\u003e\n\u003cp\u003eAdditional file 7. The plasmid information for comparison.\u003c/p\u003e\n\u003cp\u003eAdditional file 8. Plasmid Information Included in the Comparative Analysis with pSE228A.\u003c/p\u003e\n\u003cp\u003eAdditional file 9. Plasmid Information Included in the Comparative Analysis with pSE228B.\u003c/p\u003e","description":"","filename":"Additionalfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-7540124/v1/c00e1c47ff3b38af09771c4e.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic Characterization of a Virulent Multidrug-Resistant Salmonella enterica Serovar Enteritidis Strain Isolated from Meat Rabbits (Oryctolagus cuniculus) in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis (SE) represents a dominant non-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e (NTS) serovar associated with foodborne gastroenteritis in people. Contaminated poultry, meat, and eggs serve as the principal vehicles of transmission, raising considerable public health issues at the global level [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the abundant studies on SE in poultry, swine, cattle, and humans, little is known about its role in rabbit infections. \u003cem\u003eSalmonella\u003c/em\u003e infection in rabbits tends to develop acute or peracute septicemia, resulting in high morbidity and mortality. These infections lead to major economic losses in commercial rabbit farming (known as cuniculture) and concern about zoonotic transmission since rabbits are bred for their meat, fur, and as pets [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOver the past few years, multidrug-resistant (MDR) SE have increasingly been reported, complicating therapeutic strategies due to resistance against multiple classes of antimicrobials [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Horizontal gene transfer (HGT), primarily mediated by mobile genetic elements (MGEs) such as plasmids, transposons, and integrons, underlies the widespread distribution of antimicrobial resistance (AMR) genes. As a result, MDR variants have emerged rapidly and been widely documented [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, plasmid-driven HGT is recognized as a key mechanism enabling AMR gene exchange among \u003cem\u003eEnterobacteriaceae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among MDR SE strains, plasmids belonging to incompatibility groups such as IncF, IncHI, IncI, IncA/C, IncP and IncN have been widely implicated in the spread of resistance determinants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, these plasmids frequently co-carry both AMR and virulence genes, enhancing bacterial adaptability and pathogenicity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBesides antibiotic resistance, bacteria adapting to heavy metals like copper (Cu\u0026sup2;⁺), silver (Ag⁺), and zinc (Zn\u0026sup2;⁺) has become an important factor in helping MDR pathogens continue to thrive and spread. These metals are commonly used as antimicrobial agents in animal husbandry, industrial settings, and medical applications [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, continuous exposure posed selective pressure and contribute to the emergence of bacterial strains carrying metal resistance genes (MRGs), which were frequently co-located with AMR genes on plasmids. This co-selection mechanism enhances bacterial survival under heavy metal stress, further driving the spread of MDR strains in livestock and food production systems [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e"},{"header":"Material and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eOutbreak description\u003c/h2\u003e\u003cp\u003eIn March 2018, a veterinarian identified an outbreak of infectious disease at a commercial rabbit farm in Nong\u0026rsquo;an County, Jilin Province, China. The farm reared New Zealand White (NZW) rabbits (\u003cem\u003eOryctolagus cuniculus\u003c/em\u003e) for meat intended for the human food chain. More than 350 kittens were affected, presenting with diarrhea, weight loss, and respiratory difficulty, leading to death in 68.5% of cases. Necropsies showed variable lesions while white multifocal lesions in livers were most common. Treatment with antibiotics tetracycline and kanamycin sulfate was ineffective.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacterial isolation and Serotyping\u003c/h3\u003e\n\u003cp\u003eLiver samples from carcasses were aseptically collected and delivered to the laboratory for pathogen detection. Eight presumptive non-Typhi \u003cem\u003eSalmonella spp\u003c/em\u003e. were recovered from ten liver samples in Xylose lysine tergitol 4 (XLT4) agars with black colonies. They were then confirmed by VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e2 Compact microbial identification system (bioM\u0026eacute;rieux, NC, USA) and designated \u0026ldquo;JL227\u0026rdquo; to \u0026ldquo;JL234\u0026rdquo;. These strains were classified as serovar Enteritidis using the White-Kauffmann-Le Minor scheme with standard agglutination methods and \u003cem\u003eSalmonella\u003c/em\u003e antisera pools (S\u0026amp;A Reagents Lab, Bangkok, Thailand) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAntimicrobial, disinfectant, and heavy metal susceptibility\u003c/h3\u003e\n\u003cp\u003eSE isolates were tested with VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e2 Compact system (bioM\u0026eacute;rieux, NC, USA) using VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e2 AST-GN65 card. Global-based Clinical and Laboratory Standards Institute (CLSI) guideline (2014\u0026thinsp;\u0026minus;\u0026thinsp;701) was applied for minimal inhibitory concentrations (MICs) interpretation. Susceptibilities of the strain SE JL228 to heavy metals were determined as described before [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These heavy metals including silver (AgNO\u003csub\u003e3\u003c/sub\u003e, CAS#: 7761-88-8), copper (CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, CAS#:10125-13-0), tellurium (K\u003csub\u003e2\u003c/sub\u003eTeO\u003csub\u003e3\u003c/sub\u003e, CAS#: 7790-58-1), cobalt (CoCl\u003csub\u003e2\u003c/sub\u003e, CAS#: 7646-79-9), zinc (ZnSO\u003csub\u003e4\u003c/sub\u003e, CAS#: 7733-02-0), chromium (CrCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, CAS#: 10060-12-5), cadmium (Cd, CdCl\u003csub\u003e2\u003c/sub\u003e, CAS#: 10108-64-2) (Macklin Biochemical, Shanghai, China). In addition, the benzyldodecyldimethylammonium bromide (DBAB, CAS#: 7281-04-1) was chosen to evaluate resistance to the quaternary ammonium compound (QAC) disinfectant of SE JL228 as well. \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) ATCC\u0026trade; 25922 was chosen as a control strain.\u003c/p\u003e\n\u003ch3\u003ePreparation of activated peritoneal macrophages (APM)\u003c/h3\u003e\n\u003cp\u003eBALB/c mice (6\u0026ndash;8 weeks, Beijing HFK Bioscience) were housed in IVC at Ludong University with \u003cem\u003ead libitum\u003c/em\u003e food and water. Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007). Mice received 3% thioglycollate broth intraperitoneally 72 h before cell harvest. Prior to sampling, mice were anesthetized by inhalation of 4% isoflurane, followed by intraperitoneal injection of sodium pentobarbital at a dose of 150 mg/kg to induce euthanasia. Cervical dislocation was then performed as a secondary physical method to confirm death. After disinfection of the abdominal surface, 5 mL of ice-cold sterile PBS was injected intraperitoneally, and the abdomen was gently massaged. The lavage fluid was collected and centrifuged to obtain peritoneal macrophages, which were subsequently processed under sterile conditions. Peritoneal macrophages were cultured in RPMI 1640 (Gibco, Suzhou, China) with 10% fetal bovine serum (FBS, ZETA, CA USA), washed with phosphate-buffered saline (PBS) after overnight incubation, and stimulated with 1 \u0026micro;g/mL LPS (Sigma-Aldrich, MO, USA) for 24 h before the Gentamicin protection invasion assay.\u003c/p\u003e\n\u003ch3\u003eIn vitro invasion assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate bacterial invasion, gentamicin protection assays were conducted in APM and hBMEC cell models, according to established methods [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. LN248, another SE strain with moderate invasive capability [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u0026trade; were included as controls.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePathogenicity in mice\u003c/h2\u003e\u003cp\u003eSE JL228 and SE LN248 were grown in LB broth, subcultured at 37\u0026deg;C for 2.5 h with shaking. Bacteria were washed with PBS and diluted to 10\u003csup\u003e8\u003c/sup\u003e CFU/50 \u0026micro;L. The median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) was determined by orally inoculating mice with serial dilutions, recording mortality at 28 days, and calculating LD50 using the Reed and Muench method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For LD₅₀ and infection studies, mice were monitored at least two times daily and up to four times daily during the acute phase. Humane endpoints were predefined: severe respiratory distress, inability to eat/drink, severe neurologic signs, or moribund state. Animals meeting humane endpoint criteria were humanely euthanized immediately as described above. SE JL228 dissemination in mice was evaluated as a previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Mice were fasted for 4 h before \u003cem\u003eper os\u003c/em\u003e (p.o.) treatment with 20 mg streptomycin. After 20 h, they were intragastric infected with 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU of SE JL228 or SE LN248. Five days post-infection, mice were anesthetized and euthanized, after which their livers, spleens, brains, and mesenteric lymph nodes were aseptically homogenized. Serial dilutions of the homogenates were plated on XLT4 agar to assess bacterial dissemination and colonization.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWhole-genome sequencing (WGS)\u003c/h3\u003e\n\u003cp\u003eThe genomic DNA of SE JL228 was extracted using an QIAamp DNA Mini Kit (Qiagen, MD, USA). Genome assembly was carried out with the Unicycler hybrid pipeline, employing the PacBio\u0026reg; sequencing platform (Pacific Biosciences, CA, USA) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and using SE strain P125109 (GenBank accession NC_011294) as the reference. This genome assembly was further proofread Illumina\u0026reg; Hiseq\u0026times;10 (Illumina Inc., CA, USA) platform. Plasmid sequences were annotated by PLSDB database [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Analyses were conducted on the Majorbio Cloud Platform. Genomic sequences were deposited in GenBank under accession numbers CP094269 to CP094271.\u003c/p\u003e\n\u003ch3\u003eMolecular typing and prediction\u003c/h3\u003e\n\u003cp\u003eChromosomal and plasmid sequences were analyzed using the Achtman multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST), and replicon sequencing typing (RST) schemes available in the PubMLST database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pubmlst.org\u003c/span\u003e\u003cspan address=\"http://pubmlst.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Predictions of antimicrobial resistance and virulence genes were conducted through ResFinder4.0 and the VFDB database [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eGenomic comparison visualization\u003c/h2\u003e\u003cp\u003ePlasmid circular maps were created to compare a reference bacterial plasmid to all query bacterial strains using BLAST Ring Image Generator (BRIG). To search for similar metal resistance loci and genetic organization. The multi-heavy metal resistance region of plasmid pSE228A (range: 1\u0026ndash;86200 bp) was compared against the nucleotide database using \u003cem\u003eblastn\u003c/em\u003e program (date: April 10th, 2022). Easyfig 2.2.5 software was used for creating linear comparison figures of multiple genomic loci [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePlasmid Conjugal Transfer\u003c/h2\u003e\u003cp\u003eFilter and liquid mating methods were employed to assess the conjugative ability of the SE JL228. \u003cem\u003eE.coli\u003c/em\u003e C600 as the recipient, with donor and recipient strains cultured in LB broth containing ampicillin and rifampicin. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM-1\u003c/em\u003e\u003c/sub\u003e gene and penicillin resistance phenotype were chosen to assess the conjugation efficiency transconjugants selected on LB agar.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e\u003cem\u003eSalmonella strains \u0026amp; serotyping\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eOf 10 liver samples, 8 tested positive for non-Typhi \u003cem\u003eSalmonella\u003c/em\u003e spp. via XLT4 agar isolation, showing black-centered colonies with yellow/pink periphery after 18 h, turning fully black by 30 h. All isolates matched \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis biochemical and antigenic profiles (9,12:g,m:-) based on VITEK 2 GN tests and serotyping (Additional file 1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAll\u003c/b\u003e \u003cb\u003eSE\u003c/b\u003e \u003cb\u003eisolates are multidrug resistant (MDR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUniform antimicrobial resistance profiles were observed across all 8 SE isolates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Complete resistance was detected against Ampicillin (AMP), Piperacillin (PIP), Cephalexin (CL), Cefovecin (CEF), Ceftiofur (EFT), Amikacin (AMK), Gentamicin (GEN), Tobramycin (TOB), Trimethoprim/ Sulfamethoxazole (SXT), Chloramphenicol (CHL), Enrofloxacin (ENR), Tetracycline (TET), and Nitrofurantoin (NIT). Similarly, these isolates were all intermediately resistant to Amoxicillin/Clavulanic acid (AMC), Cefpodoxime (CPD), and Marbofloxacin (MAR), and all susceptible to Imipenem (IPM).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial drug susceptibility profiles of SE JL228.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eantimicrobial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003esusceptibility interpretation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePenicillins\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmpicillin (AMP)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmoxicillin/Clavulanic acid (AMC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePiperacillin (PIP)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCephalosporin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCephalexin (CL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCefovecin (CEF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCeftiofur (EFT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCefpodoxime (CPD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCarbapenems\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eImipenem (IPM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAminoglycosides\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmikacin (AMK)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGentamicin (GEN)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTobramycin (TOB)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSulphonamides\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrimethoprim/ Sulfamethoxazole (SXT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFluoroquinolones\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMarbofloxacin (MAR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnrofloxacin (ENR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eChloramphenicol\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChloramphenicol (CHL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTetracycline\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTetracycline (TET)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNitrofurantoin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNitrofurantoin (NIT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eAntimicrobial categories are shown in bold.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eMIC, minimum inhibitory concentration; S, susceptible; R, resistant; I, intermediate.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial drug susceptibility profiles of \u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis isolate JL228.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eantimicrobial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003esusceptibility interpretation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePenicillins\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmpicillin (AMP)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmoxicillin/Clavulanic acid (AMC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePiperacillin (PIP)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCephalosporin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCephalexin (CL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCefovecin (CEF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCeftiofur (EFT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCefpodoxime (CPD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCarbapenems\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eImipenem (IPM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAminoglycosides\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmikacin (AMK)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGentamicin (GEN)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTobramycin (TOB)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSulphonamides\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrimethoprim/ Sulfamethoxazole (SXT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFluoroquinolones\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMarbofloxacin (MAR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnrofloxacin (ENR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eChloramphenicol\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChloramphenicol (CHL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTetracycline\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTetracycline (TET)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNitrofurantoin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNitrofurantoin (NIT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eAntimicrobial categories are shown in bold.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eMIC, minimum inhibitory concentration; S, susceptible; R, resistant; I, intermediate.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe MICs for the selected heavy metal to SE JL228 as follows: Ag\u003csup\u003e+\u003c/sup\u003e, 16 mgl/L, Cu\u003csup\u003e2+\u003c/sup\u003e, 1024 mgl/L, TeO2\u0026thinsp;+\u0026thinsp;3,16 mgl/L, Co\u003csup\u003e2+\u003c/sup\u003e, 256 mgl/L, Zn\u003csup\u003e2+\u003c/sup\u003e, 1024 mgl/L, Cr\u003csup\u003e3+\u003c/sup\u003e, 512 mgl/L, Cd\u003csup\u003e2+\u003c/sup\u003e, 128\u0026ndash;256 mgl/L (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Generally, the isolate SE JL228 is considered resistant to the Ag\u003csup\u003e+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Te\u003csup\u003e4+\u003c/sup\u003e, with the MICs two-2-fold dilutions above the MIC\u003csub\u003e50\u003c/sub\u003e.The MIC value of DBAB for SE JL228 is 51.2 mg/L, suggesting the high tolerance of the isolate to DBAB, a commonly used QAC in China (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMinimum inhibitory concentrations to heavy metals and QAC\u003csup\u003ea\u003c/sup\u003e of SE JL228\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain/isolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAg\u003csup\u003e+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCd\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCo\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCr\u003csup\u003e3+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTe\u003csup\u003e4+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eDBAB\u003csup\u003eb\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSE JL228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u0026ndash;256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e51.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSE LN248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u0026trade;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u0026ndash;256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64\u0026ndash;128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u0026ndash;1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e153\u0026thinsp;\u0026plusmn;\u0026thinsp;46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e268\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e550\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1431\u0026thinsp;\u0026plusmn;\u0026thinsp;201\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003end\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003eQAC, quaternary ammonium compound\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eb\u003c/sup\u003eDBAB, benzyldodecyldimethylammonium bromide\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ec\u003c/sup\u003eMIC\u003csub\u003e50\u003c/sub\u003e shown here were determined from 113 SE isolates.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ed\u003c/sup\u003end, not determined.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMinimum inhibitory concentrations to heavy metals and QAC of \u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis isolate JL228\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain/isolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAg\u003csup\u003e+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCd\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCo\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCr\u003csup\u003e3+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTe\u003csup\u003e4+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eDBAB(mg/L)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis JL228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u0026ndash;256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e51.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis LN248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e ATCC\u0026trade; 25922\u0026trade;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128\u0026ndash;256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64\u0026ndash;128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e512\u0026ndash;1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e153\u0026thinsp;\u0026plusmn;\u0026thinsp;46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e268\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e550\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1431\u0026thinsp;\u0026plusmn;\u0026thinsp;201\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eQAC, quaternary ammonium compound\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eDBAB, benzyldodecyldimethylammonium bromide\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eMIC\u003csub\u003e50\u003c/sub\u003e, the lowest concentration of an antimicrobial agent at which 50% of the isolates were inhibited. MIC\u003csub\u003e50\u003c/sub\u003e shown here were determined from 113 \u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis isolates.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003end, not determined.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSE JL228 is highly invasive in vitro\u003c/h2\u003e\u003cp\u003eResults from the gentamicin protection invasion assay indicated that SE JL228 was more invasive than SE LN248 in hBMEC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Conversely, the two isolates showed no significant difference in their invasion of APM. Nonetheless, JL228 exhibited significantly superior intracellular survival within APM relative to LN248, while both strains presented equally poor survival rates in hBMEC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSE JL228 are high virulent in mice model\u003c/h2\u003e\u003cp\u003eThe median lethal dose (LD₅₀) for SE JL228 in mice following oral inoculation was determined as 5.0 \u0026times; 10⁶ CFU, which was about 68-fold lower than that observed for SE LN248 (3.4 \u0026times; 10⁸ CFU) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, in the streptomycin-pretreated infection model, SE JL228 was lethal in 100% of BALB/c mice. All mice succumbed to 10\u003csup\u003e8\u003c/sup\u003e inoculums by day 14 post-infection. Of the mice infected with LN248, however, 40% survived the infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Significant body mass changes were also observed in both infection groups. whilst, mice infected with SE JL228 had sharper body mass decrease in the first two days post infection than that with SE LN248 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Interestingly, 50% SE JL228 infected mice developed overt neurological signs that resembled meningitis with balance defect and ataxia (Additional file 2). Taken together, the rabbit-originated clinical isolate SE JL228 exhibited higher virulence potential in mouse model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDissemination capability\u003c/h2\u003e\u003cp\u003eThe bacterial dissemination assay indicated that mice infected with SE JL228 harbored significantly greater bacterial loads in multiple organs compared to those infected with SE LN248 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), implying a stronger systemic spread of JL228 following oral infection. In accordance with the findings in neurological signs, SE JL228 was more frequently recovered as well. Bacterial load was also significantly higher in SE JL228 infected mouse brains than that recovered from the SE LN248 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). However, no significant difference of cecal colonization was observed in the two groups mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eOverview of JL228 genome\u003c/h2\u003e\u003cp\u003eThe genome structure of SE JL228 consisted of a 4.7-megabase chromosome (GenBank: CP094269) along with two plasmids, 211.4 kb (pSE228A, CP094270) and 54.6 kb (pSE228B, CP094271). GC-content values were 52.17% for the chromosome, 46.28% for pSE228A, and 47.51% for pSE228B. Using NCBI\u0026rsquo;s PGAP annotation tool [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], a total of 4,896 genes were identified, with 4,612 on the chromosome, 223 on pSE228A, and 61 on pSE228B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMolecular typing suggested chromosome of strain SE JL228 match the profiles of ST11 and cgST-7883 respectively. \u003cem\u003eIn silico\u003c/em\u003e replicon-based typing suggested plasmid pSE228A match the loci of \u003cem\u003eFIB\u003c/em\u003e (allele 22), \u003cem\u003esmr0018\u003c/em\u003e (allele 1), and \u003cem\u003esmr0199\u003c/em\u003e (allele 2), according to the Plasmid MLST schemes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], therefore, plasmid pSE228A is a multiple-replicon plasmid with the incompatibility group of IncFIB and IncHI2. Further subtyping by IncHI2 double locus sequence typing (DLST) scheme classified the plasmid into ST14 group. The same typing scheme suggested the plasmid pSE228B belongs to IncN group, one of the prevalent drug-resistance plasmid types in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular characteristics of plasmids carried by the SE JL228\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eplasmid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSize (kb)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInc group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReplication, plasmid conjugal transfer and maintenance gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMR gene and heavy metal resistance gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eVirulence plasmid-encoded traits\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epSE228A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e211.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIncFIB-IncHI2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003erepB (MPK88_23535), traE, traK, trhB, trhV, traC, parA, parM, htdF, rsp, trhF, trhU, trhN, trhI, MPK88_23540\u003c/em\u003e, \u003cem\u003eMPK88_23545, repB\u003c/em\u003e (MPK88_23820), \u003cem\u003eMPK88_23790\u003c/em\u003e, \u003cem\u003eccdA\u003c/em\u003e, \u003cem\u003eccdB\u003c/em\u003e, \u003cem\u003etrhH\u003c/em\u003e, \u003cem\u003etrhR\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecopper resistance: \u003cem\u003ecopA, copB, copC, copD, copS, copE, MPK88_23180\u003c/em\u003e\u003c/p\u003e\u003cp\u003esilver resistance: \u003cem\u003esilP MPK88_23200 silA\u003c/em\u003e, \u003cem\u003esilB\u003c/em\u003e silF \u003cem\u003esilC silSR silE\u003c/em\u003e\u003c/p\u003e\u003cp\u003etellurite resistance: \u003cem\u003eterE, terC, terD, terB, terA, terZ, terW, terY, terX\u003c/em\u003e\u003c/p\u003e\u003cp\u003eantibiotic resistance: \u003cem\u003eaac(6')-IIc, sul1, ere(A)\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eQAC resistance: \u003cem\u003eqacE\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003espvDBAR\u003c/em\u003e: Salmonella plasmid virulence\u003c/p\u003e\u003cp\u003eibeB\u003csup\u003ec\u003c/sup\u003e: Invasion of brain endothelial cells\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epSE228B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIncN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003erepM\u003c/em\u003e, \u003cem\u003eMPK88_24365, MPK88_24370, MPK88_24425\u003c/em\u003e, \u003cem\u003eparA\u003c/em\u003e, \u003cem\u003eparG\u003c/em\u003e, \u003cem\u003eMPK88_24360, MPK88_24360\u003c/em\u003e, \u003cem\u003eddp3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eantibiotic resistance: \u003cem\u003earmA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, bla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1B, mph(E), sul1, sul2\u003c/em\u003e\u003c/p\u003e\u003cp\u003eQAC resistance: \u003cem\u003eqacE\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003enone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003eIncomplete, 85% coverage.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003eIncomplete, 84.7% coverage.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ec\u003c/sup\u003esynonyms name, \u003cem\u003esilC\u003c/em\u003e for this study, \u003cem\u003ecusC\u003c/em\u003e, in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular characteristics of plasmids carried by the \u003cem\u003eS. enterica\u003c/em\u003e serovar Enteritidis strain JL228\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eplasmid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSize (kb)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInc group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReplication, plasmid conjugal transfer and maintenance gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMR gene and heavy metal resistance gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eVirulence plasmid-encoded traits\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epSE228A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e211.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIncFIB-IncHI2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003erepB (MPK88_23535), traE, traK, trhB, trhV, traC, parA, parM, htdF, rsp, trhF, trhU, trhN, trhI, MPK88_23540\u003c/em\u003e, \u003cem\u003eMPK88_23545, repB\u003c/em\u003e (MPK88_23820), \u003cem\u003eMPK88_23790\u003c/em\u003e, \u003cem\u003eccdA\u003c/em\u003e, \u003cem\u003eccdB\u003c/em\u003e, \u003cem\u003etrhH\u003c/em\u003e, \u003cem\u003etrhR\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecopper resistance: \u003cem\u003ecopA, copB, copC, copD, copS, copE, MPK88_23180\u003c/em\u003e\u003c/p\u003e\u003cp\u003esilver resistance: \u003cem\u003esilP MPK88_23200 silA\u003c/em\u003e, \u003cem\u003esilB\u003c/em\u003e silF \u003cem\u003esilC silSR silE\u003c/em\u003e\u003c/p\u003e\u003cp\u003etellurite resistance: \u003cem\u003eterE, terC, terD, terB, terA, terZ, terW, terY, terX\u003c/em\u003e\u003c/p\u003e\u003cp\u003eantibiotic resistance: \u003cem\u003eaac(6')-IIc, sul1, ere(A)\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eQAC\u003csup\u003eb\u003c/sup\u003e resistance: \u003cem\u003eqacE\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003espvDBAR\u003c/em\u003e: Salmonella plasmid virulence\u003c/p\u003e\u003cp\u003eibeB\u003csup\u003ed\u003c/sup\u003e: Invasion of brain endothelial cells\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epSE228B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIncN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003erepM\u003c/em\u003e, \u003cem\u003eMPK88_24365, MPK88_24370, MPK88_24425\u003c/em\u003e, \u003cem\u003eparA\u003c/em\u003e, \u003cem\u003eparG\u003c/em\u003e, \u003cem\u003eMPK88_24360, MPK88_24360\u003c/em\u003e, \u003cem\u003eddp3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eantibiotic resistance: \u003cem\u003earmA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, blaTEM-1B, mph(E), sul1, sul2\u003c/em\u003e\u003c/p\u003e\u003cp\u003eQAC resistance: \u003cem\u003eqacE\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003enone\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003ea. Incomplete, 85% coverage.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eb. QAC, quaternary ammonium compounds.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003ec. Incomplete, 84.7% coverage.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003ed. synonyms name, \u003cem\u003esilC\u003c/em\u003e for this study, \u003cem\u003ecusC\u003c/em\u003e, in \u003cem\u003eE.coli\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eStructure of the plasmids carried by strain SE JL228\u003c/b\u003e\u003c/h2\u003e\u003cp\u003ePlasmid pSE228A (GenBank: CP094270) contained 223 predicted genes, including two backbone \u003cem\u003erepB\u003c/em\u003e loci located at positions 146725\u0026ndash;147714 and 86251\u0026ndash;87000 (complementary strand). The encoded RepB initiator proteins exhibited only 42.17% amino acid identity, although the first shared complete identity with the \u003cem\u003eRepB\u003c/em\u003e of the FIB plasmid pS82/10 from SE [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additional loci associated with IncHI-type conjugation and plasmid maintenance were also present, forming a \u003cem\u003erepB-traE-traK-trhB-trhV-traC-parA-parM-htdF-rsp-trhF-trhU-trhN-trhI\u003c/em\u003e cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Two genes, \u003cem\u003eMPK88_23540\u003c/em\u003e and \u003cem\u003eMPK88_23545\u003c/em\u003e, encoding a pili assembly chaperone and a plasmid transfer protein, respectively, were identified between \u003cem\u003erepB\u003c/em\u003e and \u003cem\u003etraE\u003c/em\u003e. Furthermore, a second \u003cem\u003erepB\u003c/em\u003e locus (86251\u0026ndash;87000), together with \u003cem\u003eMPK88_23790\u003c/em\u003e (DNA replication terminus site-binding protein), \u003cem\u003eccdA\u003c/em\u003e, \u003cem\u003eccdB\u003c/em\u003e, \u003cem\u003etrhH\u003c/em\u003e, and \u003cem\u003etrhR\u003c/em\u003e, may contribute to plasmid replication, stability, and transfer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlasmid pSE228B (GenBank: CP094271) was classified as an IncN replicon and contained a backbone region spanning positions (30357\u0026ndash;43644 bp), encompassing eight genes related to replication and stability (MPK88_24360\u0026ndash;MPK88_24425). This region encoded four replication initiation proteins, including \u003cem\u003erepM\u003c/em\u003e and three uncharacterized replication-associated proteins (MPK88_24365, MPK88_24370, MPK88_24425). Additionally, four maintenance-related genes were identified: \u003cem\u003eparA\u003c/em\u003e, \u003cem\u003eparG\u003c/em\u003e, \u003cem\u003eMPK88_24355\u003c/em\u003e (RelE/ParE toxin), and \u003cem\u003eMPK88_24360\u003c/em\u003e (stabilization protein), along with ddp3, encoding a DNA distortion polypeptide.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial and heavy metal resistance genes\u003c/h2\u003e\u003cp\u003eUsing the ResFinder4.0 platform, seventeen antimicrobial resistance (AMR) genes were detected in the genome of SE strain JL228. The chromosomal gene \u003cem\u003eaac(6\u0026rsquo;)-Iaa\u003c/em\u003e was identified, along with three genes located on plasmid pSE228A [\u003cem\u003eaac(6\u0026rsquo;)-IIc, sul1\u003c/em\u003e, and \u003cem\u003eere(A)\u003c/em\u003e], and ten genes carried on plasmid pSE228B [\u003cem\u003earmA\u003c/em\u003e, \u003cem\u003eaac(6\u0026rsquo;)-Ib-cr\u003c/em\u003e, \u003cem\u003eaac(3)-IId\u003c/em\u003e, \u003cem\u003eaph(6)-Id\u003c/em\u003e, \u003cem\u003eaph(3\u0026rsquo;\u0026rsquo;)-Ib\u003c/em\u003e, \u003cem\u003eaph(3\u0026rsquo;)-Ia\u003c/em\u003e, \u003cem\u003eblaTEM-1B\u003c/em\u003e, \u003cem\u003emph(E)\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, and \u003cem\u003esul2\u003c/em\u003e]. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). AMR genes in pSE228A are clustered in the region 201393\u0026ndash;208693, flanked by two identical IS6-like element IS26 family transposase genes and a class 1 integron integrase gene (\u003cem\u003eintl1\u003c/em\u003e), constituting a gene cluster of \u003cem\u003eIS6-sul1-qacE-ere(A)-MPK88_24140-aac(3)-IIg-ereA-aac(6')-IIc-intI1-IS6\u003c/em\u003e. The unassigned gene locus \u003cem\u003eMPK88_24140\u003c/em\u003e encodes an NAD(+)-rifampin ADP-ribosyltransferase, suggesting the rifampin resistance potential of this plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). AMR genes in pSE228B scattered in four regions among the plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These AMR genes in chromosome and plasmids are predicted to confer resistance to seven class antibiotics (Additional file 3). Besides, quaternary ammonium compound-resistance gene \u003cem\u003eqacE\u003c/em\u003e were also found in both plasmids, with the predicted phenotype of Benzylkonium Chloride, Ethidium Bromide, Chlorhexidine and Cetylpyridinium Chloride resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe analysis of plasmid pSE228A revealed the presence of three distinct heavy metal resistance operons: the copper-associated locus \u003cem\u003ecopESDBAC\u003c/em\u003e (15559\u0026ndash;20810), the silver resistance determinant \u003cem\u003esilPABFCRE\u003c/em\u003e (22094\u0026ndash;34545), and the tellurite operon \u003cem\u003eterEDCBAZWYX\u003c/em\u003e (62141\u0026ndash;75496). (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The large multi-metal resistance region of pSE228A is flanked by mobile genetic elements (MGEs) including IS6-like element IS26 family transposase (region 1652\u0026ndash;2356 and 85494\u0026ndash;86198), Tn3-like element Tn5403 family transposase (region 2427\u0026ndash;4171 and 4207\u0026ndash;4467), IS21-like element IS100 family transposase \u003cem\u003eistA\u003c/em\u003e (region 82039\u0026ndash;83061) and \u003cem\u003eistB\u003c/em\u003e (region 81260\u0026ndash;82042) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eFive plasmids carrying similar metal tolerance gene clusters were found to be highly conserved with those in pSE228A plasmid with the sequence identities over 99% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The plasmid pR15.0430_329k, unname1 and pEC5207 carried by \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium (STm), \u003cem\u003eRaoultella sp.\u003c/em\u003e and \u003cem\u003eE.coli\u003c/em\u003e respectively, cover the complete collinear regions of the silver, copper and tellurite resistance loci (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Whilst the pKO_1 carried by \u003cem\u003eKlebsiella michiganensis\u003c/em\u003e showed imperfect coverage of the resistance clusters with the partial copper resistance locus missing in this plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The plasmid pYUSHP2-1 carried by \u003cem\u003eEnterobacter hormaechei\u003c/em\u003e only share the similar tellurite resistance region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eVirulence factor (VF) genes\u003c/h2\u003e\u003cp\u003eA total of 158 virulence genes were identified in virulence factor database (VFDB), of which 154 are chromosome-encoded genes. 4 plasmid-encoded VF genes distribute exclusively in plasmid pSE228A, while no VF gene found in pSE228B. Further intra-genera comparative pathogenomics using VFanalyzer tool with other 16 \u003cem\u003eSalmonella\u003c/em\u003e genomes reveal that \u003cem\u003esefB\u003c/em\u003e, \u003cem\u003esefC\u003c/em\u003e, \u003cem\u003esefD\u003c/em\u003e in \u003cem\u003esef\u003c/em\u003e fimbrial operon are absent in SE JL228 strain, remaining only \u003cem\u003esefA\u003c/em\u003e gene left in chromosome. In addition, a SPI-1 T3SS gene \u003cem\u003eorgB\u003c/em\u003e, 2 T3SS translocated effector genes \u003cem\u003esopD2\u003c/em\u003e and \u003cem\u003eslrP\u003c/em\u003e, are absent in SE JL228 genome as well. Notably, 3 members of plasmid-encoded \u003cem\u003espv\u003c/em\u003e locus, \u003cem\u003espvB\u003c/em\u003e, \u003cem\u003espvC\u003c/em\u003e and \u003cem\u003espvD\u003c/em\u003e were identified in pSE228A. Further analysis confirmed the intact bacteremia-associated locus of \u003cem\u003espvRABCD\u003c/em\u003e, suggesting the plasmid is a virulence plasmid (Additional file 4). Virulence determinant operons in SE JL228 genome were also compared with those in SE LN248 and other two newly characterized \u003cem\u003eS.\u003c/em\u003e Typhimurium (ST221_31B) and Kentucky (SK222_32B). Results suggested that 3 virulence-related operons including plasmid-encoded SPV, TTSS-2 translocated effectors sseK2 and sspH2 are present in SE JL228 genome whereas absent in LN248. In addition, a Peg fimbrial adherence determinant was found exclusively in LN248. (Additional file 5). Interestingly, MPK88_23220 in pSE228A, which was assigned as \u003cem\u003esilC\u003c/em\u003e in our submission (GenBank accession no. CP094270), was annotated to be \u003cem\u003eibeB\u003c/em\u003e in VFDB (Additional file 4). Its orthologous gene in \u003cem\u003eEscherichia\u003c/em\u003e encoding a virulence factor involving in invasion of brain endothelial cells. The deduced amino acid sequence of SE JL228 shares approximate 71% identities (71.09%-71.33%) with its orthologs in pathogenic \u003cem\u003eEscherichia\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Other synonyms name of this gene in \u003cem\u003eEscherichia\u003c/em\u003e is also known as \u003cem\u003ecusC\u003c/em\u003e, a member in \u003cem\u003ecusCFBA\u003c/em\u003e gene locus that encodes a Cu(+)/Ag(+) efflux system CusCFBA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eComparative Analysis of plasmids\u003c/h2\u003e\u003cp\u003epSE228A shares near-identical backbone and loci patterns with the IncHI2 plasmid pSE_AH228 from a 2019 \u003cem\u003eSalmonella\u003c/em\u003e isolate (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The ARG clusters are highly similar to seven plasmids, including pYUSHP2-1 (\u003cem\u003eE. hormaechei\u003c/em\u003e), XY-1 (\u003cem\u003eRaoultella sp.\u003c/em\u003e), pR15.0430 (\u003cem\u003eS.\u003c/em\u003e Typhimurium), pKA04-2 (\u003cem\u003eK. aerogenes\u003c/em\u003e), pIMP26 (\u003cem\u003eE. cloacae\u003c/em\u003e), p1106151-mcr (\u003cem\u003eLeclercia sp.\u003c/em\u003e), and pSE_AH228. Notably, pSE228A shares the highest genetic coverage with pKA04-2, except for the latter lack of \u003cem\u003espv\u003c/em\u003e locus and \u003cem\u003eccdA/ccdB\u003c/em\u003e toxin-antitoxin genes typical of virulent \u003cem\u003eSalmonella\u003c/em\u003e plasmids. Copper and silver resistance operons in pSE228A are homologous to those in p280_40A (\u003cem\u003eE.fergusonii\u003c/em\u003e), SWHE2 \u003cem\u003e(S. dysenteriae\u003c/em\u003e), XY-1, pKO_1 (\u003cem\u003eK. michiganensis\u003c/em\u003e), pR15.0430, pKA04-2, pEC5207 (\u003cem\u003eE. coli\u003c/em\u003e), and pSE_AH228. The \u003cem\u003eter\u003c/em\u003e operon for tellurite resistance is conserved across all 14 plasmids, indicating its prevalence in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e plasmids.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlasmid pSE228B shows limited sequence coverage with known plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Its initial 20 kb fragment resembles pB12AN_1 from a human-derived \u003cem\u003eK. pneumoniae\u003c/em\u003e strain in China. The 18\u0026ndash;42 kb region, particularly 19,018\u0026ndash;23,114 bp, harbors resistance genes (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u0026minus;1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003emph(E)\u003c/em\u003e, \u003cem\u003eaac(6')-Ib-cr\u003c/em\u003e, \u003cem\u003eqnrB\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e) with high similarity to \u003cem\u003eSalmonella\u003c/em\u003e plasmids pFORC51, pSE1004837, pFORC89, p12519B, and \u003cem\u003eK. pneumoniae\u003c/em\u003e plasmids pB12AN_1 and pB0910. The 44\u0026ndash;54 kb region aligns with pB0910.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eTransferability assay\u003c/h2\u003e\u003cp\u003ePlasmid conjugation yielded eight transconjugants, three from membrane filter mating (11.11% efficiency) and five from liquid mating (15.62% efficiency). PCR confirmed the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u0026minus;1\u003c/em\u003e\u003c/sub\u003e gene (850-bp amplicon) in all transconjugants, identical to SE JL228, with sequencing verifying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-1\u003c/em\u003e presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Broth microdilution showed high-level penicillin resistance (MIC\u0026thinsp;\u0026gt;\u0026thinsp;2048 \u0026micro;g/mL) in all transconjugants, consistent with SE JL228 and higher than \u003cem\u003eE. coli\u003c/em\u003e C600 (Additional file 6).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we characterize a \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis strain JL228, from a rabbit farm outbreak, revealing its high resistance to multiple antibiotics and metals mediated by plasmid and chromosomal determinants. The strain also showed increased virulence, enhanced systemic dissemination, and the ability to cross the blood-brain barrier and invade hBMECs. This highly resistant and virulent strain poses a significant threat to animal and human health, necessitating close monitoring in farms and hospitals.\u003c/p\u003e\u003cp\u003eThe SE JL228 genome consists of two plasmid pSE228A and pSE228B. pSE228A, likely a fusion of IncFIB and IncHI2, encodes multiple ARGs, MRGs, and VFs, flanked by mobile genetic elements like class 1 integrons and IS26 transposons. These features enable pSE228A to act as a reservoir for resistance and virulence genes, promoting efficient horizontal gene transfer and rapid dissemination among bacteria [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The presence of copper, silver, and tellurite resistance operons alongside ARGs-including \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003eere(A)\u003c/em\u003e, and \u003cem\u003eaac(6')-IIc\u003c/em\u003e-on pSE228A suggests the co-selection of metal and antibiotic resistance. In agricultural practices, copper is widely used as additives to control infections [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Prolonged exposure to these metals may exert selective pressure that facilitates the persistence of MDR SE strains in agricultural environments. This observation is consistent with previous studies reporting that SE isolates from industrial farms and wastewater treatment facilities often harbor both MRGs and ARGs, enabling them to survive in metal-contaminated environments [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Similar patterns have been observed in other \u003cem\u003eEnterobacteriaceae\u003c/em\u003e species, such as \u003cem\u003eVibrio cholerae\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, further enhancing the contribution of heavy metal exposure to the evolution of bacterial resistance profiles [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In contrast, pSE228B, an IncN-type plasmid commonly found in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, does not encode virulence factors but harbors several ARGs-including \u003cem\u003earmA\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u0026minus;1B\u003c/em\u003e\u003c/sub\u003e, that contribute significantly to MDR phenotype of SE JL228, complicating treatment options.\u003c/p\u003e\u003cp\u003eComparative genomic analyses revealed high sequence identity between resistance and metal tolerance regions of pSE228A and plasmids from \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSTm\u003c/span\u003e and \u003cem\u003eE. coli\u003c/em\u003e, while pSE228B shares conserved AMR modules with plasmids from \u003cem\u003eSalmonella sp.\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e. These findings collectively support the notion that plasmid-mediated HGT, likely occurring in polymicrobial farm environments under selective pressure, plays a central role in the dissemination of MDR traits.\u003c/p\u003e\u003cp\u003eNotably, pSE228A encodes several key virulence genes, including \u003cem\u003eibeB\u003c/em\u003e, \u003cem\u003espvB\u003c/em\u003e, \u003cem\u003espvC\u003c/em\u003e, and \u003cem\u003espvD\u003c/em\u003e, which are known to enhance the systemic dissemination of \u003cem\u003eSalmonella enterica\u003c/em\u003e by impairing host immune responses and promoting intracellular survival within macrophages [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast to SE JL228, the LN248 strain lacks these genes, which may explain the markedly superior intracellular persistence and systemic infectivity of SE JL228 in APM. These genes likely contribute to observed neuroinvasive potential of SE JL228 in mice, including its ability to cross the blood-brain barrier and induce neurological manifestations [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Chromosomal virulence determinants play a crucial role in SE JL228 pathogenicity alongside plasmid-borne factors. Genome analysis revealed that SE JL228 possesses a full suite of SPI-1/SPI-2 effectors-including \u003cem\u003esopB\u003c/em\u003e, \u003cem\u003esopE\u003c/em\u003e, \u003cem\u003esopE2\u003c/em\u003e, \u003cem\u003esseC\u003c/em\u003e, and \u003cem\u003esseD\u003c/em\u003e-that promote host cell invasion and immune subversion [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cem\u003emgtC\u003c/em\u003e confers tolerance to magnesium limitation, enhancing survival in macrophages [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The adhesion- and persistence-associated genes \u003cem\u003epagN\u003c/em\u003e, \u003cem\u003epagC\u003c/em\u003e, and \u003cem\u003epagD\u003c/em\u003e contribute to bacterial attachment to host cells, invasion of immune cells, and survival within the intracellular environment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Interestingly, despite the absence of \u003cem\u003eorgB\u003c/em\u003e, \u003cem\u003esopD2\u003c/em\u003e, and \u003cem\u003eslrP\u003c/em\u003e, SE JL228 retained strong invasive and virulent phenotypes, suggesting the existence of alternative compensatory mechanisms or functional redundancies within its virulence gene repertoire. These chromosomally encoded virulence factors, in synergy with plasmid-borne elements, collectively underpin the high virulence potential of SE JL228, posing significant public health risks.\u003c/p\u003e\u003cp\u003eThe limited sequence coverage with known plasmids and the dispersed distribution of ARGs complicate accurate prediction of the origin and formation process of this plasmid. To elucidate the potential origin and dissemination pathways of the plasmids carried by SE JL228, a source-tracing investigation was conducted using BRIG-based comparative genomic analysis, which revealed that the plasmid pSE228A shared highset sequence identity with a previously reported plasmid, pSE_AH228, isolated from \u003cem\u003eSalmonella\u003c/em\u003e spp. Although the precise geographic origin of pSE_AH228 remains undetermined, its high homology with pSE228A suggests that IncHI2-type plasmids have undergone widespread horizontal transmission across bacterial species and possibly across regional and ecological boundaries within agricultural environments. Similarly, the IncN plasmid pSE228B exhibited high sequence similarity to plasmids identified in \u003cem\u003eK.pneumoniae\u003c/em\u003e, implying that the resistance determinants in SE JL228 likely originated from a shared plasmid pool circulating among various \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. These findings indicate that the acquisition of resistance plasmids in SE JL228 reflects not an isolated evolutionary event but an outcome of active gene flow under environmental selection pressure.\u003c/p\u003e\u003cp\u003eWhile the study provides a detailed genomic and functional analysis of a single outbreak strain, further research with diverse isolates from various regions and hosts is needed to confirm broader epidemiological significance. Future studies should investigate the transmission of IncFIB-IncHI2 and IncN plasmids in environmental and clinical contexts and clarify the role of heavy metal resistance in the persistence and spread of multidrug-resistant (MDR) SE to understand environmental impacts on bacterial evolution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe complete genome sequence of Salmonella enterica serovar Enteritidis strain SEJL228A has been deposited in GenBank under accession number CP094269. The plasmid sequences are available under accession numbers CP094270 (pSE228A) and CP094271 (pSE228B). All data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e All co-authors declare that we have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2024MC148 and ZR2023MC049), the Key Research and Development Plan of Shandong Province (Grant No. 2022CXPT022, 2025CXGC010803) and the Shandong Province Poultry Industry Technology System (Grant No. SDAIT-11-10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e Writing-original draft: R.Z., H.Z. Writing-review and editing: H.Z. Investigation: R.Z., B.W., Y.L., J.L., X.D., H.Z. Validation: B.W., Y.L., X.D., J.Y., J.Z. Software: R.Z., B.W. Visualization: R.Z., B.W., H.Z. Formal analysis: J.Z., Y.L., J.Y., L.J. Data curation: Y.L., L.J., H.Z. Methodology: J.L., X.Y., J.Y., H.Z. Conceptualization: H.Z. Resources: J.Z., X.Y., J.Z. Funding acquisition: X.Y., H.Z, X.Z., Y.L. Supervision: L.J., H.Z., X.Z. Project administration: X.Z. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e The conference presentation by Zhu et al. at the 20th Congress of the International Society for Animal Hygiene shares thematic similarities with this work [42]. We disclose that the language in this manuscript was polished using Grok 3, an artificial intelligence tool developed by xAI, to enhance clarity and readability without adding any data, results, ideas, or opinions by AI.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheng RA, Eade CR, Wiedmann M. 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Genetic insight into a \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis strain circulating in a rabbit farm reveals IncFIB\u0026ndash;IncHI2 and IncN plasmids mediated hybrid virulence resistance mechanism. In: 20th Congress of the International Society for Animal Hygiene (ISAH). 2022: 135\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Salmonella Enteritidis, multidrug-resistant (MDR), high virulence, metal resistance, IncFIB/IncHI2 hybrid plasmid","lastPublishedDoi":"10.21203/rs.3.rs-7540124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7540124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe rise of virulent, multidrug-resistant \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis strains threatens food safety and human health. This study reports the isolation of a \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis strain (SE JL228) from a meat rabbit, which was associated with a high-mortality outbreak on a rabbit farm in China in 2018. The objective of this study was to elucidate the genetic characteristics, antimicrobial resistance determinants, metal tolerance mechanisms, and virulence potential of a rabbit-derived strain, thereby providing insights into its implications for both public and animal health.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThis strain exhibited resistance to 13 antibiotics within 8 antimicrobial categories as well as to silver (Ag\u003csup\u003e+\u003c/sup\u003e), copper (Cu\u003csup\u003e2+\u003c/sup\u003e), and tellurium (Te\u003csup\u003e4+\u003c/sup\u003e). Additionally, SE JL228 demonstrated high tolerance to the quaternary ammonium compound (QAC) disinfectant. \u003cem\u003eIn vitro\u003c/em\u003e assays revealed superior invasion of human brain microvascular endothelial cells and enhanced intracellular survival in activated peritoneal macrophages compared to the moderately virulent SE strain LN248. \u003cem\u003eIn vivo\u003c/em\u003e studies confirmed extensive dissemination in mice, with an LD\u003csub\u003e50\u003c/sub\u003e approximately 68-fold lower than that of LN248. A 4.7-megabase chromosome together with two plasmids\u0026mdash;pSE228A (211.4 kb, IncFIB/IncHI2) and pSE228B (54.6 kb, IncN)\u0026mdash;was identified through whole-genome sequencing. The genome encoded 17 antibiotic resistance genes (ARGs), 34 virulence factors including an intact \u003cem\u003espv\u003c/em\u003e operon, and heavy metal resistance operons (\u003cem\u003ecopESDBAC\u003c/em\u003e, \u003cem\u003esilPABFCRE\u003c/em\u003e, \u003cem\u003eterEDCBAZWYX\u003c/em\u003e) alongside a QAC-resistance gene (\u003cem\u003eqacE\u003c/em\u003e) on pSE228A. Plasmid pSE228A shares near-identical structure with a previously-isolated IncHI2 plasmid, while pSE228B, carrying the transferable \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eTEM\u0026minus;1\u003c/em\u003e\u003c/sub\u003e gene conferring penicillin resistance, showed limited homology to the known plasmids.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe rabbit-derived SE JL228 represents a highly virulent, multidrug-resistant, and metal-tolerant pathogen. Its ability to invade brain endothelial cells, survive within macrophages, and disseminate systemically underscores its zoonotic potential. The presence of transferable plasmids encoding both resistance and virulence factors suggests a heightened risk of spread within farm environments and beyond. These findings emphasize the urgent need for enhanced surveillance, prudent antimicrobial use, and effective biosecurity measures to mitigate the emergence and dissemination of such hybrid pathogens.\u003c/p\u003e","manuscriptTitle":"Genomic Characterization of a Virulent Multidrug-Resistant Salmonella enterica Serovar Enteritidis Strain Isolated from Meat Rabbits (Oryctolagus cuniculus) in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 15:25:46","doi":"10.21203/rs.3.rs-7540124/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-21T12:36:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T18:56:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T00:03:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T22:51:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60334841373000513720617180722708536805","date":"2025-10-15T15:54:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172989255819283282199022715611852832978","date":"2025-10-15T13:39:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106427080619303892801304210434363545775","date":"2025-10-14T21:21:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258628504223243678568894151720893448296","date":"2025-10-14T13:16:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280085060851407410610207714084765301647","date":"2025-10-13T15:04:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-13T13:08:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T07:28:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-25T07:17:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-25T05:26:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-09-25T04:46:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1bb2eec4-bf27-4758-b63f-036313972a3d","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:13:04+00:00","versionOfRecord":{"articleIdentity":"rs-7540124","link":"https://doi.org/10.1186/s12866-025-04612-1","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-06 15:58:55","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-10-27 15:25:46","video":"","vorDoi":"10.1186/s12866-025-04612-1","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04612-1","workflowStages":[]},"version":"v1","identity":"rs-7540124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7540124","identity":"rs-7540124","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}