Whole-Genome Profiling Reveals Mobile Genetic Elements Associated with Virulence and Antibiotic Resistance in Equine Lactic Acid Bacteria

Whole-Genome Profiling Reveals Mobile Genetic Elements Associated with Virulence and Antibiotic Resistance in Equine Lactic Acid Bacteria | 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 Whole-Genome Profiling Reveals Mobile Genetic Elements Associated with Virulence and Antibiotic Resistance in Equine Lactic Acid Bacteria Anran Tai, Zongmao Dai, Yabin Lu, Fangfang Yin, Ying Xiao, Haichao Guan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7684893/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background While lactic acid bacteria (LAB) are widely recognized as probiotics, their genomic safety remains understudied. Method This study integrates phenotypic characterization, whole genome sequencing (WGS), and horizontal gene transfer (HGT) analysis to evaluate the safety of two LAB strains isolated from healthy equines. Method Phenotypic assays revealed robust acid and bile salt tolerance, antimicrobial activity against pathogens ( Salmonella spp., Escherichia coli ), and α-hemolysis on horse and sheep blood agar. WGS identified multiple antibiotic resistance genes ARGs ( lmrS, optrA, vanTG ) and virulence factors (Adhesion, biofilm formation, immune evasion) in both strains. Crucially, HGT-associated mobile elements (plasmids, genomic islands) were found to carry ARGs ( vanTG, macB ) and virulence genes ( T2SS, InlJ ). For instance, F3d’s plasmid encoded 14 virulence-related genes, while F3c’s genomic island harbored TcdA (Toxin) and patB (Multidrug efflux). These findings demonstrate that mobile genetic elements (MGEs) contribute to the retention of pathogenic traits in LAB, highlighting potential safety concerns. Conclusion This study demonstrate that mobile genetic elements (MGEs) contribute to the retention of pathogenic traits in LAB, highlighting potential safety concerns and underscores the necessity of screening LAB for MGEs to ensure safety in probiotic applications. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Equine species are herbivorous mammals whose hindgut microbiota (Cecum and colon) enables efficient nutrient extraction from fibrous feed. This microbial community fulfills a substantial portion of equine daily energy requirements through fermentation of plant biomass into short-chain fatty acids (SCFAs) including acetate, propionate, and butyrate[ 1 , 2 ]. Gastrointestinal disturbances in this ecosystem may consequently induce metabolic dysregulation through altered fermentation processes[ 3 ]. Lactic acid bacteria (LAB) demonstrate host-adapted functionalities in carbohydrate metabolism and SCFA production[ 4 ]. Some studies have found that probiotic formulations promote growth and enhance resistance to diarrhea in animals[ 5 , 6 ]. Maintenance of therapeutic efficacy requires sustained high-dosage regimens due to the transient colonization patterns characteristic of lactobacilli in the animal gut[ 7 ]. LAB have a well-established history of safe consumption and are regulated by the US Food and Drug Administration (FDA) under the Generally Recognized as Safe (GRAS) designation, which contributes to their broadly unquestioned safety profile[ 8 ]. However, recent studies have identified potential safety risks associated with specific LAB strains[ 9 , 10 ] Growing evidence indicates that LAB may harbor antibiotic resistance genes (ARGs) and virulence factors capable of horizontal transfer through mobile genetic elements (MGEs), including plasmids and genomic islands[ 11 , 12 ] . Conventional probiotic screening methodologies—primarily focused on phenotypic characteristics such as acid tolerance and antimicrobial activity—frequently overlook these genomic threats[ 13 , 14 ]. Phylogenomic investigations across Lactobacillus species further demonstrate the prevalence of multidrug efflux pumps (e.g., lmrD)[ 15 ] and biofilm-associated genes, likely resulting from adaptive evolution in varied ecological environvments[ 16 ]. These insights emphasize the imperative for implementing advanced genomic screening tools in LAB safety evaluations. Horizontal gene transfer (HGT) serves as a fundamental driver of bacterial evolution by facilitating the acquisition of adaptive traits such as virulence factors and antibiotic resistance[ 17 ]. Within gut microbial communities, HGT dynamics between LAB and intestinal pathogens remain poorly understood. Despite mounting evidence of recurrent ARG transfer through MGEs — notably plasmids and genomic islands — in gut commensals, this critical knowledge deficit remains unaddressed[ 18 ]. This phenomenon is exemplified by clinical Enterococcus species that employ MGE-mediated mechanisms to disseminate resistance determinants[ 19 ]. Besides, while CRISPR-Cas systems primarily function as defensive barriers against foreign DNA, they may paradoxically preserve horizontally acquired genetic elements encoding host-adaptive features such as adhesion proteins[ 20 ]. These multifaceted genomic interactions undermine conventional assumptions regarding the intrinsic safety of LAB strains and necessitate implementation of rigorous, strain-level genomic risk evaluations[ 21 ]. In this study, we employed whole genome sequencing (WGS) and HGT-focused bioinformatics to evaluate two equine-derived LAB strains ( Weissella_confusa F3c and Lactobacillus_equi F3d). By integrating phenotypic assays, ARG/virulence factor annotation, and MGE analysis, we aimed to address a critical gap in probiotic safety research: how HGT elements contribute to the retention of pathogenic traits in ostensibly beneficial microbes. Our findings provide a framework for pre-use genomic screening of LAB, ensuring their safe application in veterinary and industrial contexts. Methods Isolation and identification of lactic acid bacteria 2.1.1 Sample Information Fresh fecal samples were collected from 10 healthy horses of different breeds, including Ili horses, Turkmenian Akhal-Teke horses, Friesian horses, Thoroughbreds, Shetland ponies, Dzungar horses and Arabian horses, at Xinjiang Wild Horse Culture Co., Ltd. Ten grams of fecal samples were collected from each horse and placed in sterilized self-sealing bags, which were numbered, sealed and stored at -20 ℃ for gut microbiota sequencing and Lactic acid bacteria isolation. 2.1.2 Gut microbiota sequencing The 10 collected fecal samples were randomly divided into two groups, which were recorded as group H1, group H2, group H3, group H4 and group H5, respectively. The total DNA of the collected samples was extracted using DNA extraction kit, and the DNA integrity was detected by 1.0% agar-gel electrophoresis and ultra-micro spectrophotometer. The qualified DNA samples were stored at -80 ℃ for later use. The purified amplified fragments were sent to Shanghai Meiji Biomedical Technology Co., Ltd. to construct a library of PE300 using Illumina MiSeq platform standard operating procedures. The sequences were manipulated into OTU clustering in UPAPSE according to 97% similarity, and the chimeras were deleted using UCHIME software. The bioinformation of OTUs at 97% similarity level was analyzed by RDP Classifier Bayesian algorithm. Each sample was operated in exactly the same steps, and the instrument was used in strict accordance with the instructions to reduce errors. (The raw data is currently being uploaded and will be promptly provided to the journal upon completion of database transfer) 2.1.3 Lactic acid bacteria isolation Take a small amount of feces and 10 mL of normal saline thoroughly mixed and diluted in equal proportion. Absorb 100µL of the mixed liquid and add it into 900 µL of normal saline and mix it to 10 − 1. Repeat this operation for 5 times, then absorb 10 − 3, 10 − 4, 10 − 5 and evenly coat on MRS Petri dish respectively. After coating, put the plate upside down into an anaerobic tank and incubate in a constant temperature incubator at 37 ℃ for 12 h. The next day, take out the petri dish, observed different colony morphology, and selected different colonies with milky white, smooth and neat edges to add to 1 mL MRS broth, make a number, and culture in 37 ℃, 180 rpm constant temperature shaking table for 12 h. The shaken bacterial solution was taken out and marked on the MRS Petri dish, and continued to be cultured in an anaerobic tank at 37 ℃. The above steps were repeated for purification culture for 3 ~ 4 times. When the single colonies on the petri dish were in the same form, the purification was completed. Each purified strain was cultured in MRS Broth and then cultured in MRS Petri dish containing calcium carbonate with lines. Strains with calcium-soluble ring were selected for Gram staining, and Gram-positive bacteria were selected for bacteria preservation. That is, the cultured bacterial solution was absorbed and 50% glycerin was mixed 1:1 and frozen in the refrigerator at -20 ℃. 2.1.4 Biochemical identification After incubation for 12 h, the strains were identified by HBI lactobacillus biochemical identification strip, and the identification indexes included: Escin, cellobiose, maltose, mannitol, salicylate, sorbitol, sucrose, raffinose, inulin, lactose. Preliminary identification was made according to Berger's Bacterial Identification Manual. The experiment was repeated three times. 2.1.5 16S rRNA sequencing Bacterial DNA extraction kit was used to extract DNA from the cultured purified strains. The extracted DNA was template DNA, and bacterial universal primers (27F: 5 '-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5 '-GGTTACCTTGTTACGACTT-3'), a 20 µL PCR system was established for amplification, and the DNA integrity was observed by gel electrophoresis with the PCR products. The PCR products with bright bands were sent to Shanghai Shengong Biological Engineering Co., LTD., for 16S rRNA sequencing. The received sequence results were compared at NCBI to identify the species. 2.1.6 Measurement of growth curve and acid production curve of lactic acid bacteria The frozen bacteria solution was removed at -20 ℃, inoculated at 1% inoculated into 1 mL broth and placed in a constant temperature shaking table at 37 ℃ for recovery for 12 h. The next day inoculated at 1% inoculated into a sterile MRS Broth culture tube, with three replicates per strain, and cultured in a constant temperature shaking table at 37 ℃. At 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, 200 µL of bacterial suspension was sampled from each culture tube for OD600 measurement, with three technical replicates. At the same time, pH value of the bacterial solution was determined by acidometer. 2.1.7 Acid resistance of two strains of Lactic acid bacteria was determined The pH value in the horse's stomach is between 2.5 and 5.6, so MRS Broth with pH 2.5, 3.6, 4.6 and 5.6 was prepared with concentrated hydrochloric acid as spare. Transfer 100 µL of thawed bacterial solution into 900 µL of broth, and incubate the culture with shaking until it reaches the logarithmic growth phase. Centrifuge the suspension at 6000 rpm for 3 minutes, discard the supernatant, and resuspend the pellet in 1 mL of PBS (pH 6.5). The bacteria were inoculated into the liquid medium with adjusted pH at 5% inoculation rate, and the normal liquid medium was used as the control. OD600 was measured after culture for 3 h. 2.1.8 Determination of bile salt tolerance of two strains of lactic acid bacteria Based on the measured bile salt concentration of 0.3% in equine small intestine, MRS broth containing 0.3% and 0.5% (w/v) bovine bile salts was prepared alongside PBS controls. The test lactic acid bacterial cultures were initially grown overnight, harvested through centrifugation (6000 rpm, 10 min), and subsequently transferred to fresh media with respective bile salt concentrations (0%, 0.3%, 0.5%) for an additional overnight incubation. After secondary centrifugation, pellets were resuspended in phosphate-buffered saline containing matched bile salt levels. Bacterial growth was monitored by optical density measurements (OD600) at 1, 2, and 4 hours post-resuspension. The experiment was repeated three times. 2.1.9 Determination of antibacterial activity of two strains of lactic acid bacteria Three pathogenic bacterial strains ( Escherichia coli, Staphylococcus aureus, and Salmonella) were cultured overnight in LB broth. Bacterial suspensions were uniformly spread on LB agar plates using sterile cotton swabs. Sterile Oxford cups (prepared by high-pressure sterilization) were aseptically placed on the inoculated agar surfaces using tweezers. Test groups received activated lactic acid bacterial cultures, while control groups contained sterile MRS broth. The plates were incubated at 37°C for 18–24 hours, after which inhibition zone diameters were measured using vernier calipers. The experiment was repeated three times. 2.1.10 Evaluation of drug resistance of lactic acid bacteria The agar diffusion method was employed for antimicrobial evaluation. Activated test strains were uniformly inoculated onto MRS agar plates using sterile cotton swabs. After complete drying of bacterial lawns, antibiotic susceptibility discs were aseptically positioned on the agar surface with specified spatial parameters: 10–15 mm from plate periphery, 24 mm inter-disc spacing, and 24 mm distance from disc center to plate edge. Discs were secured using sterilized forceps through gentle central pressure. Inverted plates were incubated at 37°C for 16–18 hours prior to zone of inhibition measurement with vernier calipers. Antimicrobial susceptibility was determined through comparison with standardized reference tables. The experiment was repeated three times. 2.1.11 Hemolytic evaluation of lactic acid bacteria Sterilized MRS broth was cooled to 50–60°C before aseptic addition of 6% defibrinated horse blood and 6% defibrinated sheep blood,, followed by gentle mixing and subsequent pouring of horse blood agar plates and sheep blood agar plates, respectively. Test strains were streaked onto both prepared horse blood agar and sheeep blood agar using inoculating loops. Following 24-hour incubation at 37°C under anaerobic conditions, hemolytic responses were systematically evaluated: α-hemolytic activity was characterized by partial erythrocyte lysis exhibiting greenish peripheries (1–2 mm width), β-hemolysis demonstrated complete erythrocyte clearance with > 2 mm transparent zones, while γ-hemolytic strains showed colony morphologies identical to those on aerobically incubated MRS control plates. The experiment was repeated three times. 2 Whole genome sequencing and analysis 2.2.1 Extract genomic DNA from lactic acid bacteria (F3c and F3d) The F3c and F3d strains preserved at -40℃ were revived by streaking onto MRS agar plates followed by incubation at 37℃ for 24 h in a temperature-controlled chamber. Well-isolated colonies demonstrating robust growth were subsequently transferred to MRS broth and cultured at 37℃ until reaching mid-logarithmic phase. Bacterial cells were harvested through centrifugation at 8,000 rpm for 10 min using a refrigerated centrifuge maintained at 4℃. Genomic DNA was then isolated from the cell pellets using the Wizard® Genomic DNA Purification Kit according to the manufacturer's instructions. Purified DNA samples were delivered to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for subsequent whole genome sequencing. 2.2.2 Whole genome sequencing and assembly Genome sequencing was performed using Illumina HiSeq and PacBio Sequel platforms, with achieved coverages of 2812.84× (F3C) and 1891.57× (F3d), both passing stringent contamination screening protocols. Bacterial genome assembly involved optimization through multiple k-mer parameter analysis using SOAPdenovo2 for short-read assembly, generating optimal contigs. Sequencing reads were aligned to contigs, with subsequent scaffold construction achieved through local reassembly guided by paired-end relationships and read overlaps. The Unicycler assembly software was employed for hybrid assembly incorporating third-generation sequencing data. Assembly refinement was conducted using Pilon software for sequence correction. When terminal sequence overlaps exceeding specified lengths were detected in final assemblies, circularization was achieved by trimming redundant overlapping regions. This pipeline ultimately yielded complete chromosomal and plasmid sequences. 2.2.3 Prediction of whole genome features Genome annotation was performed through a multi-software pipeline. Protein-coding genes (CDS) were predicted using Glimmer ( http://ccb.jhu.edu/software/glimmer/index.shtml ), GeneMarkS, and Prodigal in parallel. Specifically, Glimmer was employed for initial CDS prediction in draft genome assemblies, chromosomal CDS annotation in complete assemblies, while GeneMarkS was designated for plasmid genome prediction. tRNA identification was conducted using tRNAscan-SE v2.0 ( http://trna.ucsc.edu/software/ ), providing nucleotide sequences, anticodon information, and secondary structure predictions. rRNA operons were mapped with Barrnap ( https://github.com/tseemann/barrnap ), generating complete inventories of rRNA types with their genomic coordinates and sequences. Final genome visualization was achieved using CGView for circular genome map construction. 2.2.4 Gene function analysis The predicted protein sequences were functionally annotated against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups (COG) databases, with priority given to the highest-scoring alignment ratios from comparative analyses. Virulence-associated genes and antimicrobial resistance determinants were identified through the Virulence Factor Database (VFDB) and Comprehensive Antibiotic Resistance Database (CARD), respectively. MGEs were systematically characterized using the VRprofile 2 online platform. Thresholds for gene annotation: identity ≥ 40%, coverage ≥ 70%, E-value ≤ 1×10⁻⁵. Results 3.1 Isolation and identification of lactic acid bacteria Sequencing analysis revealed significant enrichment of lactic acid bacteria in fecal samples (Fig. 1 A, B), from which two strains (F3c and F3d) were isolated. On MRS agar, strain F3c formed white colonies with smooth margins and appeared as purple-staining short rods under microscopy, while strain F3d developed convex colonies and displayed slightly curved bacilli (Fig. 1 C). Phylogenetic analysis based on 16S rRNA sequences revealed that F3d showed the closest phylogenetic relationship to Lactobacillus equi , whereas F3c was most closely related to Weissella confusa (Fig. 1 D, E). Metabolic characterization demonstrated strain-specific profiles: F3d tested positive for esculin hydrolysis and fermentation of maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose, but negative for cellobiose utilization. In contrast, F3c utilized only esculin, cellobiose, and salicin (Table 1 ). These biochemical and molecular features conform to the taxonomic standards established in Bergey's Manual of Determinative Bacteriology (8th edition), validating both isolates as LAB. Table 1 Bacterial Biochemical Identification Type ID Esculin Cellobiose Maltose Mannitol Salicin Sorbitol Sucrose Raffinose Inulin Lactose F3c + + - - + - - - - - F3d + - + + + + + + + + 3.2 Biological characteristics of F3c and F3d Both isolates demonstrated divergent growth kinetics and acidogenesis patterns (Fig. 2 A, B). Strain F3c initiated logarithmic growth between 2–6 h with stationary phase attainment at 8 h, exhibiting concurrent rapid acidification (pH 4.4–4.6) within the initial 2 h. In contrast, F3d displayed accelerated acid production (pH 4.0-4.5 by 6 h) with transient pH rebound during 6–8 h. (pH 2.5–5.6 OD₆₀₀: F3c 0.125–0.556, F3d 0.153–0.750) and bile salt (0.3–0.5% OD₆₀₀: F3c 0.383–0.663, F3d 0.554–0.824) (Fig. 2 D, E). Hemolytic profiling identified α-hemolysis on both horse and sheep blood agar plates, with measured hemolysis zones ≤ 2 mm in diameter (Fig. 2 F). Antimicrobial efficacy testing showed F3c's superior inhibitory action against Salmonella spp . and both strains effectively suppressed Staphylococcus aureus and Escherichia coli (Table 2 ). Identical antibiotic susceptibility patterns were recorded: susceptibility to ampicillin, erythromycin, tetracycline, chloramphenicol, clindamycin, cephalexin, and cephradine; Resistance to kanamycin, streptomycin, norfloxacin, and vancomycin (Table 3 ). Table 2 Bacteriostasis results of three strains of bacteria Pathogenic bacteria F3c/mm F3d/mm Staphylococcus aureus 9.755 9.99 Escherichia coli 10.535 9.46 Salmonella 11.95 9.71 Table 3 Antibiotic susceptibility of strains Antibiotics F3c F3d Ampicillin S S Kanamycin R R Streptomycin R R Gentamicin I I Erythromycin S S Tetracycline S S Vancomycin R R Penicillin I I Cephalothin S S Cephalexin S S Ciprofloxacin R R Cefradine S I Amoxicillin S S Oxacillin S S 3.3 Genome-wide profiles of F3c and F3d WGS uncovered distinct genomic organizations between the strains. F3c harbored a 2,247,119 bp genome configuration consisting of a singular circular chromosome (2,198,644 bp; 45.11% GC content) accompanied by three plasmids measuring 32,174 bp (43.36% GC), 11,751 bp (40.43% GC), and 4,550 bp (36.79% GC), collectively encoding 2,186 predicted genes. Conversely, F3d displayed a more compact 2,114,783 bp genomic structure featuring a reduced circular chromosome (1,641,382 bp; 40.04% GC) paired with four plasmids of 415,366 bp (38.96% GC), 48,923 bp (37.98% GC), 4,687 bp (41.03% GC), and 4,425 bp (35.66% GC), containing 1,943 annotated coding sequences (Tables 4 and 5 ). Table 4 Detail of F3c and F3d gene assembly results Characteristics F3c F3d Chromosome No. 1 1 Plasmid No. 3 4 Genome Size (bp) 2 247 119 2 114 783 G + C(%) 45.04 39.77 Depth 2 812.84 1 891.57 Table 5 Basic characteristics of the F3c and F3d gene assembly results Genomic characterstics F3c F3d Number of CDS 2 186 1 943 Number of tRNA 87 106 Number of rRNA 9 10 Number of sRNA 25 34 3.4 F3c&F3d function annotation COG analysis revealed 23 functional categories for both strains (Fig. 3 A, B). F3c and F3d exhibited strong protein synthesis capacity, with 193 and 190 genes respectively annotated in translation, ribosome strcture biogenesis. Metabolic versatility was evidenced by enrichment in carbohydrate transport and metabolism (F3c:163 genes, F3d:123 genes), amino acid transport and metabolism (F3c:118 genes, F3d: 80 genes). Both isolates exhibited adaptive genomic features, including replication, recombination and repair (F3c:107 genes; F3d:108 genes) as well as biofilm formation-associated genes (F3c:116 genes; F3d:136 genes). Notably, the mobilome was significantly represented, with F3c harboring type II secretion system (T2SS)-associated virulence genes and F3d encoding vancomycin resistance-related vanTG loci. GO term categorization resolved 49 functional subcategories, with F3c (1,421 genes) and F3d (2,000 genes) displaying conserved molecular architecture (Fig. 3 C, D). Membrane components dominated cellular components (F3c:421 genes, F3d:372 genes), while molecular function analysis demonstrated significant enrichment in ATP-binding (F3c:201; F3d:234) and DNA-binding (F3c:153; F3d:179) functional classes. KEGG pathways highlighted metabolic specialization (Fig. 3 E, F). F3c prioritized carbohydrate (116 genes) and nucleotide metabolism (70 genes), whereas F3d emphasized carbohydrate (123 genes) and amino acid metabolism (80 genes). Both strains showed strong environmental adaptation through membrane transport genes (F3c:115 genes, F3d:85 genes). 3.5 The annotation of CARD & VFDB CARD database analysis identified 118 genes (F3c) and 131 genes (F3d) multidrug resistance genes distributed across chromosomes and plasmids (Fig. 4 A, B), including LmrS, lmrP , and optrA conferring resistance to macrolides, tetracyclines, fluoroquinolones, and β-lactams through target alteration, efflux pumps, and enzymatic inactivation. VFDB annotation revealed 180 virulence factors in F3d, dominated by immune modulation (62 genes), nutrient metabolism (35 genes), and exotoxins (10 genes). Comparatively, F3c contained 164 virulence-associated genes showing dominance in iron scavenging systems (37 genes), magnesium acquisition (21 genes), and phagocytosis evasion mechanisms (19 genes) (Fig. 4 C, D). GCView-generated high-resolution maps delineated chromosomal architecture, including COG-functional zonation, prophage integration sites, CRISPR-cas arrays, noncoding RNAs, and GC-skew patterns for both genomes (Fig. 4 E, F). 3.6 Plasmid-Borne Mobile Elements: Functional Annotation, CARD, and VFDB Bacterial evolution is characterized by the ability to acquire genetic material from the environment through HGT, with common mobile genetic elements involved in this process including plasmids, prophages, genomic islands, CRISPR-Cas, transposons, and insertion sequences .This study focuses on analyzing plasmids, prophages, CRISPR-Cas and genomic islands. Functional annotation of the F3c plasmid identified (Fig. 5 A) only two virulence genes (pB_gene0006 and pB_gene0007), with the latter additionally carrying the ARGs macB. Lysin-related functions were associated with pB_gene0046 and pB_gene0053, while pB_gene0029 was annotated as Cag24 pathogenicity island protein and pC_gene0002 as alkaline shock protein (Asp23 family). No further pathogenicity-related features were detected. In contrast, the F3d plasmid (Fig. 5 B) exhibited broader annotations, with 25 genes linked to CARD, 14 to VFDB, and 14 overlapping between both databases. 3.7 GI -Borne Mobile Elements: Functional Annotation, CARD, and VFDB Genomic island (GI) analysis of F3c (Fig. 6 A) revealed four AMR genes (patB, rpoB2, vanYM, LRA-8) and seven VFDB-associated virulence factors, including InlJ (VF0445), HitABC (VF0268), and TcdA (VF0376). Functional annotations highlighted gene1381 (lysozyme), gene1399 (Cag24 pathogenicity island protein), and gene1662-1664 (trimeric autotransporter adhesin), alongside diverse enzymatic roles such as gene1706 (rhodanese-like domain-containing protein), gene1713 (penicillin-binding protein), and gene1937 (CtsR family transcriptional regulator). For F3d (Fig. 6 B), GI annotation identified three ARGs ( PmrF, macB, eFrA ) and 16 VFDB entries, including LPS, Capsule, and LOS-related genes. Functional features included gene0226 (flippase), gene0254 (thioredoxin), and gene0837 (L,D-transpeptidase). 3.8 Prophage -Borne Mobile Elements: Functional Annotation, CARD, and VFDB Prophage annotation of both plasmids showed no virulence or AMR features in VFDB or CARD. However, F3d (Fig. 7 B) harbored pA_gene0120 (cell wall-associated hydrolase), while F3c (Fig. 7 A) carried pB_gene0047 (lysozyme M1) and pB_gene0053 (lysin-related function). No additional pathogenicity-associated annotations were observed. 3.9 CRISPR-Cas -Borne Mobile Elements: Functional Annotation, CARD, and VFDB CRISPR-Cas system analysis (Fig. 8 A) identified a single feature in F3c (gene0312: KxYKxGKxW signal peptide domain-containing protein). F3d (Fig. 8 B-F) exhibited six annotated components: gene0836 (Mucin-4), gene0910 (cell wall-associated hydrolase), pB_gene0009 (cysteine-rich acidic membrane protein), pA_gene0320 (zonadhesin), and pA_gene0322 (Sec-dependent serine-rich adhesin). Discussion This study finds potential safety concerns regarding two LAB strains (F3c: Weissella confusa ; F3d: Lactobacillus equi ) isolated from healthy equine hosts. Despite their probiotic potential, both strains, however, harbored horizontally acquired antibiotic resistance genes (ARGs) and virulence factors, posing potential safety risks. Significantly, these risk elements were mechanistically linked to MGEs—specifically plasmids, genomic islands, and prophages—that act as reservoirs for pathogenic traits. For instance, F3d’s plasmid carried vanTG (conferring vancomycin resistance) alongside 14 virulence-related genes involved in processes such as lipopolysaccharide biosynthesis. Similarly, F3c’s genomic island encoded TcdA (a toxin gene) and patB (a multidrug efflux determinant). These findings underscore how MGEs facilitate the persistence of hazardous genomic elements in ostensibly beneficial microbes. The identified ARGs— lmrS , optrA , and vanTG —correlate with documented intrinsic or acquired resistance mechanisms in LAB[ 22 , 23 ]. Genus-level analyses of Lactobacillus spp . demonstrate the ubiquity of multidrug efflux pumps such as lmrS , likely attributable to environmental selection pressures across diverse niches[ 24 ]. Of particular concern is the vanTG detected in strain F3d, as vancomycin resistance remains uncommon in LAB and poses potential complications for clinical therapies[ 25 ]. The plasmid localization of these ARGs reinforces HGT risks[ 26 ], emphasizing the imperative for MGE screening in LAB prior to industrial deployment. This is illustrated by two key findings: F3c’s plasmid encodes Cag pathogenicity island protein 24—a virulence determinant typically linked to Helicobacter pylori[ 27 ]—while F3d’s genomic island harbors PmrF, associated with polymyxin resistance[ 28 ]. These examples collectively demonstrate LAB’s genomic adaptability via HGT mechanisms[ 18 ]. Virulence factors such as adhesion proteins, biofilm-related genes, and immune evasion markers (e.g., T2SS in F3c) were identified through VFDB annotation. While LAB are generally recognized as safe[ 29 ], these findings echo recent warnings that certain strains may retain pathogenic traits. Phenotypic analyses validated genomic risk indicators. Both strains exhibited tolerance to acidic conditions (pH 2.5–5.6) and elevated bile salt concentrations (0.3–0.5%), traits advantageous for probiotic survival but potentially concerning when coexisting with virulence factors. While their antimicrobial activity against Salmonella spp. supports probiotic applications, the concurrent observation of α-hemolysis on horse blood agar warrants caution, as hemolytic capacity is a recognized pathogenic hallmark. This α-hemolytic activity correlates with hemolysin-associated genes annotated in F3c’s GI, directly connecting genomic predictions to measurable phenotypic risks. The identical antibiotic susceptibility profiles (Table 3 ) further highlight shared resistance mechanisms. Observed resistance to kanamycin, streptomycin, and vancomycin—contrasting with susceptibility to ampicillin and erythromycin—mirrors LAB resistance patterns documented by Goldstein et al[ 14 ]. Collectively, these results support growing evidence that LAB reservoirs of ARGs may act as conduits for resistance dissemination. Notably, the distinct genomic localization of resistance determinants— lmrS (multidrug resistance) on chromosomes versus optrA (oxazolidinone resistance) on plasmids—underscores the complementary roles of vertical inheritance and HGT in shaping LAB resistance profiles[ 30 ] . In general, while F3c and F3d display probiotic characteristics, their genomic repertoire of ARGs and virulence factors calls into question their unrestricted application. HGT-centric analyses demonstrate that MGEs serve as vectors for transferring pathogenic traits—even among ostensibly beneficial strains[ 31 ]. While these studies provide foundational insights into HGT mechanisms, the phenotypic consequences of transferred genes remain uncharacterized. Future work must bridge this knowledge gap by correlating genetic acquisitions with functional outcomes, such as pathogenicity or metabolic adaptation. These findings emphasize the need for strain-specific safety evaluations, particularly for equine probiotics, to minimize inadvertent hazards. Critical next steps include functional characterization of MGE-linked genes (e.g. vanTG efflux activity), real-time tracking of HGT dynamics in equine gut ecosystems to quantify resistance transmission risk, and systematic investigation of HGT-derived phenotypic expressions. Given that environmental ARG reservoirs may facilitate cross-boundary resistance proliferation [ 32 ], rigorous scrutiny of LAB strains such as F3c/F3d is imperative for both veterinary practice and public health agendas. Conclusion While strains F3c and F3d demonstrate probiotic potential, their HGT-associated genomic risks necessitate cautious evaluation. These findings underscore that even purportedly "beneficial" microorganisms require rigorous safety assessments to mitigate unintended ecological and clinical consequences in probiotic utilization. Declarations Ethics approval and consent to participate All animal experimental protocols were approved by the Research Ethics Committee of Xinjiang Agricultural University with an ID of 2024012. Consent for Publication All authors read and approved the final manuscript. We confirm that this work is original, has not been published elsewhere, and is not under consideration by another journal. Competing interests All other authors declare they have no competing interests. Funding This research was funded by National Natural Science Foundation of China, grant number 32360868. Author Contribution TA: Data curation, Investigation, Methodology, Validation, Writing–original draft. DZ: Data curation, Investigation, Methodology, Validation, Writing–original draft. LY: Conceptualization, Data curation, Investigation, Methodology. XY& YF: Conceptualization, Data curation, Investigation, Methodology. LR: Formal Analysis, Methodology, Writing-review and editing. HY: Formal Analysis, Methodology, Writing-review and editing. HS: Formal Analysis, Methodology, Writing-review and editing. HZ: Formal Analysis, Methodology, Writing-review and editing. WJ: Data curation, Investigation, Methodology, Supervision, Validation, Writing-review and editing. All authors contributed to the article and approved the submitted version. Availability of data and materials All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.The sequencing data generated in this study have been submitted to the NCBI SRA under submission IDs SUB15706789 and SUB15707400. The assembled genome sequences are being processed and the accession numbers will be updated upon completion of the deposition process. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 05 Nov, 2025 Reviews received at journal 26 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor assigned by journal 15 Oct, 2025 Editor invited by journal 15 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 14 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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(F)F3c\u0026amp;F3d\u003cem\u003e \u003c/em\u003e– Hemolysis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/ca518697309db4c9faec5e70.png"},{"id":94731080,"identity":"2066a75b-c120-465c-8091-922c7f74e6fa","added_by":"auto","created_at":"2025-10-30 07:07:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":378655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eF3c\u0026amp;F3d function annotation.\u003c/strong\u003e (A\u0026amp;B) COG function classification. (C\u0026amp;D) GOfunction classification. (E\u0026amp;F) KEGG function classification.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/64a927b6a06f4cef4a16315c.png"},{"id":94731113,"identity":"28edcd25-4e12-4f4e-a2be-0b234ac912a0","added_by":"auto","created_at":"2025-10-30 07:07:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":495049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe annotation of CARD \u0026amp; VFDB. \u003c/strong\u003e(A\u0026amp;B) ARDB function classification. (C\u0026amp;D) VFDB function classification. (E\u0026amp;F) GCView.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/348d60ecaca72e3b378acd5d.png"},{"id":94731100,"identity":"a5bb3272-9749-497a-8cc4-edd73bae4685","added_by":"auto","created_at":"2025-10-30 07:07:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":268671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasmid-Borne Mobile Elements: Functional Annotation, CARD, and VFDB. \u003c/strong\u003e(A)F3c.(B)F3d.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/624e82336f3e21c8c7df25dc.png"},{"id":94731108,"identity":"e1293ab5-a72f-4dd8-9c16-00f96dba6497","added_by":"auto","created_at":"2025-10-30 07:07:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":314433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGI-Borne Mobile Elements: Functional Annotation, CARD, and VFDB. \u003c/strong\u003e(A)F3c.(B)F3d.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/6701a7ef37b0d6054c96f4e5.png"},{"id":94731144,"identity":"e3301391-53ea-4bf5-9605-8bb12fe93938","added_by":"auto","created_at":"2025-10-30 07:07:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":241509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProphage-Borne Mobile Elements: Functional Annotation, CARD, and VFDB. \u003c/strong\u003e(A)F3c.(B)F3d.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/942ae44eb4dbea8b84e748e8.png"},{"id":94731147,"identity":"5eb8ed96-c596-44bc-a23c-d1d385dfd26a","added_by":"auto","created_at":"2025-10-30 07:07:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":170553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR-Cas-Borne Mobile Elements: Functional Annotation, CARD, and VFDB. \u003c/strong\u003e(A) F3c-CRISPR1-gene0312. (B) F3d-CRISPR1-gene0836. (C) F3d-CRISPR2-gene0910. (D) F3d-CRISPR3-pAgene0320. (E) F3d-CRISPR4\u0026amp; CRISPR5-pAgene0322. (F) F3d-CRISPR6-gene0009.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/e9a5051b5851aebb24766263.png"},{"id":94827157,"identity":"48256bce-92a8-4a46-a742-ea7b9f8fe9bd","added_by":"auto","created_at":"2025-10-31 06:55:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3591226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7684893/v1/72fe5769-6a99-4a8b-b3b7-8dddcf64038c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-Genome Profiling Reveals Mobile Genetic Elements Associated with Virulence and Antibiotic Resistance in Equine Lactic Acid Bacteria","fulltext":[{"header":"Background","content":"\u003cp\u003eEquine species are herbivorous mammals whose hindgut microbiota (Cecum and colon) enables efficient nutrient extraction from fibrous feed. This microbial community fulfills a substantial portion of equine daily energy requirements through fermentation of plant biomass into short-chain fatty acids (SCFAs) including acetate, propionate, and butyrate[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Gastrointestinal disturbances in this ecosystem may consequently induce metabolic dysregulation through altered fermentation processes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eLactic acid bacteria\u003c/em\u003e (LAB) demonstrate host-adapted functionalities in carbohydrate metabolism and SCFA production[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Some studies have found that probiotic formulations promote growth and enhance resistance to diarrhea in animals[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Maintenance of therapeutic efficacy requires sustained high-dosage regimens due to the transient colonization patterns characteristic of \u003cem\u003elactobacilli\u003c/em\u003e in the animal gut[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLAB have a well-established history of safe consumption and are regulated by the US Food and Drug Administration (FDA) under the Generally Recognized as Safe (GRAS) designation, which contributes to their broadly unquestioned safety profile[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, recent studies have identified potential safety risks associated with specific LAB strains[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Growing evidence indicates that LAB may harbor antibiotic resistance genes (ARGs) and virulence factors capable of horizontal transfer through mobile genetic elements (MGEs), including plasmids and genomic islands[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eConventional probiotic screening methodologies\u0026mdash;primarily focused on phenotypic characteristics such as acid tolerance and antimicrobial activity\u0026mdash;frequently overlook these genomic threats[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Phylogenomic investigations across \u003cem\u003eLactobacillus\u003c/em\u003e species further demonstrate the prevalence of multidrug efflux pumps (e.g., lmrD)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and biofilm-associated genes, likely resulting from adaptive evolution in varied ecological environvments[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These insights emphasize the imperative for implementing advanced genomic screening tools in LAB safety evaluations.\u003c/p\u003e\u003cp\u003eHorizontal gene transfer (HGT) serves as a fundamental driver of bacterial evolution by facilitating the acquisition of adaptive traits such as virulence factors and antibiotic resistance[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Within gut microbial communities, HGT dynamics between LAB and intestinal pathogens remain poorly understood. Despite mounting evidence of recurrent ARG transfer through MGEs \u0026mdash; notably plasmids and genomic islands \u0026mdash; in gut commensals, this critical knowledge deficit remains unaddressed[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This phenomenon is exemplified by clinical Enterococcus species that employ MGE-mediated mechanisms to disseminate resistance determinants[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Besides, while CRISPR-Cas systems primarily function as defensive barriers against foreign DNA, they may paradoxically preserve horizontally acquired genetic elements encoding host-adaptive features such as adhesion proteins[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These multifaceted genomic interactions undermine conventional assumptions regarding the intrinsic safety of LAB strains and necessitate implementation of rigorous, strain-level genomic risk evaluations[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we employed whole genome sequencing (WGS) and HGT-focused bioinformatics to evaluate two equine-derived LAB strains (\u003cem\u003eWeissella_confusa F3c\u003c/em\u003e and \u003cem\u003eLactobacillus_equi\u003c/em\u003e F3d). By integrating phenotypic assays, ARG/virulence factor annotation, and MGE analysis, we aimed to address a critical gap in probiotic safety research: how HGT elements contribute to the retention of pathogenic traits in ostensibly beneficial microbes. Our findings provide a framework for pre-use genomic screening of LAB, ensuring their safe application in veterinary and industrial contexts.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eIsolation and identification of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.1 Sample Information\u003c/div\u003e\u003cp\u003eFresh fecal samples were collected from 10 healthy horses of different breeds, including Ili horses, Turkmenian Akhal-Teke horses, Friesian horses, Thoroughbreds, Shetland ponies, Dzungar horses and Arabian horses, at Xinjiang Wild Horse Culture Co., Ltd. Ten grams of fecal samples were collected from each horse and placed in sterilized self-sealing bags, which were numbered, sealed and stored at -20 ℃ for gut microbiota sequencing and Lactic acid bacteria isolation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.2 Gut microbiota sequencing\u003c/div\u003e\u003cp\u003eThe 10 collected fecal samples were randomly divided into two groups, which were recorded as group H1, group H2, group H3, group H4 and group H5, respectively. The total DNA of the collected samples was extracted using DNA extraction kit, and the DNA integrity was detected by 1.0% agar-gel electrophoresis and ultra-micro spectrophotometer. The qualified DNA samples were stored at -80 ℃ for later use. The purified amplified fragments were sent to Shanghai Meiji Biomedical Technology Co., Ltd. to construct a library of PE300 using Illumina MiSeq platform standard operating procedures. The sequences were manipulated into OTU clustering in UPAPSE according to 97% similarity, and the chimeras were deleted using UCHIME software. The bioinformation of OTUs at 97% similarity level was analyzed by RDP Classifier Bayesian algorithm. Each sample was operated in exactly the same steps, and the instrument was used in strict accordance with the instructions to reduce errors. (The raw data is currently being uploaded and will be promptly provided to the journal upon completion of database transfer)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.3 \u003cem\u003eLactic acid bacteria\u003c/em\u003e isolation\u003c/div\u003e\u003cp\u003eTake a small amount of feces and 10 mL of normal saline thoroughly mixed and diluted in equal proportion. Absorb 100\u0026micro;L of the mixed liquid and add it into 900 \u0026micro;L of normal saline and mix it to 10\u0026thinsp;\u0026minus;\u0026thinsp;1. Repeat this operation for 5 times, then absorb 10\u0026thinsp;\u0026minus;\u0026thinsp;3, 10\u0026thinsp;\u0026minus;\u0026thinsp;4, 10\u0026thinsp;\u0026minus;\u0026thinsp;5 and evenly coat on MRS Petri dish respectively. After coating, put the plate upside down into an anaerobic tank and incubate in a constant temperature incubator at 37 ℃ for 12 h. The next day, take out the petri dish, observed different colony morphology, and selected different colonies with milky white, smooth and neat edges to add to 1 mL MRS broth, make a number, and culture in 37 ℃, 180 rpm constant temperature shaking table for 12 h. The shaken bacterial solution was taken out and marked on the MRS Petri dish, and continued to be cultured in an anaerobic tank at 37 ℃. The above steps were repeated for purification culture for 3\u0026thinsp;~\u0026thinsp;4 times. When the single colonies on the petri dish were in the same form, the purification was completed. Each purified strain was cultured in MRS Broth and then cultured in MRS Petri dish containing calcium carbonate with lines. Strains with calcium-soluble ring were selected for Gram staining, and Gram-positive bacteria were selected for bacteria preservation. That is, the cultured bacterial solution was absorbed and 50% glycerin was mixed 1:1 and frozen in the refrigerator at -20 ℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.4 Biochemical identification\u003c/div\u003e\u003cp\u003eAfter incubation for 12 h, the strains were identified by HBI lactobacillus biochemical identification strip, and the identification indexes included: Escin, cellobiose, maltose, mannitol, salicylate, sorbitol, sucrose, raffinose, inulin, lactose. Preliminary identification was made according to Berger's Bacterial Identification Manual. The experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.5 16S rRNA sequencing\u003c/div\u003e\u003cp\u003eBacterial DNA extraction kit was used to extract DNA from the cultured purified strains. The extracted DNA was template DNA, and bacterial universal primers (27F: 5 '-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5 '-GGTTACCTTGTTACGACTT-3'), a 20 \u0026micro;L PCR system was established for amplification, and the DNA integrity was observed by gel electrophoresis with the PCR products. The PCR products with bright bands were sent to Shanghai Shengong Biological Engineering Co., LTD., for 16S rRNA sequencing. The received sequence results were compared at NCBI to identify the species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.6 Measurement of growth curve and acid production curve of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/div\u003e\u003cp\u003eThe frozen bacteria solution was removed at -20 ℃, inoculated at 1% inoculated into 1 mL broth and placed in a constant temperature shaking table at 37 ℃ for recovery for 12 h. The next day inoculated at 1% inoculated into a sterile MRS Broth culture tube, with three replicates per strain, and cultured in a constant temperature shaking table at 37 ℃. At 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, 200 \u0026micro;L of bacterial suspension was sampled from each culture tube for OD600 measurement, with three technical replicates. At the same time, pH value of the bacterial solution was determined by acidometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.7 Acid resistance of two strains of \u003cem\u003eLactic acid bacteria\u003c/em\u003e was determined\u003c/div\u003e\u003cp\u003eThe pH value in the horse's stomach is between 2.5 and 5.6, so MRS Broth with pH 2.5, 3.6, 4.6 and 5.6 was prepared with concentrated hydrochloric acid as spare. Transfer 100 \u0026micro;L of thawed bacterial solution into 900 \u0026micro;L of broth, and incubate the culture with shaking until it reaches the logarithmic growth phase. Centrifuge the suspension at 6000 rpm for 3 minutes, discard the supernatant, and resuspend the pellet in 1 mL of PBS (pH 6.5). The bacteria were inoculated into the liquid medium with adjusted pH at 5% inoculation rate, and the normal liquid medium was used as the control. OD600 was measured after culture for 3 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.8 Determination of bile salt tolerance of two strains of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/div\u003e\u003cp\u003eBased on the measured bile salt concentration of 0.3% in equine small intestine, MRS broth containing 0.3% and 0.5% (w/v) bovine bile salts was prepared alongside PBS controls. The test lactic acid bacterial cultures were initially grown overnight, harvested through centrifugation (6000 rpm, 10 min), and subsequently transferred to fresh media with respective bile salt concentrations (0%, 0.3%, 0.5%) for an additional overnight incubation. After secondary centrifugation, pellets were resuspended in phosphate-buffered saline containing matched bile salt levels. Bacterial growth was monitored by optical density measurements (OD600) at 1, 2, and 4 hours post-resuspension. The experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.9 Determination of antibacterial activity of two strains of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/div\u003e\u003cp\u003eThree pathogenic bacterial strains (\u003cem\u003eEscherichia coli, Staphylococcus aureus, and Salmonella)\u003c/em\u003e were cultured overnight in LB broth. Bacterial suspensions were uniformly spread on LB agar plates using sterile cotton swabs. Sterile Oxford cups (prepared by high-pressure sterilization) were aseptically placed on the inoculated agar surfaces using tweezers. Test groups received activated lactic acid bacterial cultures, while control groups contained sterile MRS broth. The plates were incubated at 37\u0026deg;C for 18\u0026ndash;24 hours, after which inhibition zone diameters were measured using vernier calipers. The experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.10 Evaluation of drug resistance of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/div\u003e\u003cp\u003eThe agar diffusion method was employed for antimicrobial evaluation. Activated test strains were uniformly inoculated onto MRS agar plates using sterile cotton swabs. After complete drying of bacterial lawns, antibiotic susceptibility discs were aseptically positioned on the agar surface with specified spatial parameters: 10\u0026ndash;15 mm from plate periphery, 24 mm inter-disc spacing, and 24 mm distance from disc center to plate edge. Discs were secured using sterilized forceps through gentle central pressure. Inverted plates were incubated at 37\u0026deg;C for 16\u0026ndash;18 hours prior to zone of inhibition measurement with vernier calipers. Antimicrobial susceptibility was determined through comparison with standardized reference tables. The experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.1.11 Hemolytic evaluation of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/div\u003e\u003cp\u003eSterilized MRS broth was cooled to 50\u0026ndash;60\u0026deg;C before aseptic addition of 6% defibrinated horse blood and 6% defibrinated sheep blood,, followed by gentle mixing and subsequent pouring of horse blood agar plates and sheep blood agar plates, respectively. Test strains were streaked onto both prepared horse blood agar and sheeep blood agar using inoculating loops. Following 24-hour incubation at 37\u0026deg;C under anaerobic conditions, hemolytic responses were systematically evaluated: α-hemolytic activity was characterized by partial erythrocyte lysis exhibiting greenish peripheries (1\u0026ndash;2 mm width), β-hemolysis demonstrated complete erythrocyte clearance with \u0026gt;\u0026thinsp;2 mm transparent zones, while γ-hemolytic strains showed colony morphologies identical to those on aerobically incubated MRS control plates. The experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2 Whole genome sequencing and analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.2.1 Extract genomic DNA from \u003cem\u003elactic acid bacteria\u003c/em\u003e (F3c and F3d)\u003c/div\u003e\u003cp\u003eThe F3c and F3d strains preserved at -40℃ were revived by streaking onto MRS agar plates followed by incubation at 37℃ for 24 h in a temperature-controlled chamber. Well-isolated colonies demonstrating robust growth were subsequently transferred to MRS broth and cultured at 37℃ until reaching mid-logarithmic phase. Bacterial cells were harvested through centrifugation at 8,000 rpm for 10 min using a refrigerated centrifuge maintained at 4℃. Genomic DNA was then isolated from the cell pellets using the Wizard\u0026reg; Genomic DNA Purification Kit according to the manufacturer's instructions. Purified DNA samples were delivered to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for subsequent whole genome sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.2.2 Whole genome sequencing and assembly\u003c/div\u003e\u003cp\u003eGenome sequencing was performed using Illumina HiSeq and PacBio Sequel platforms, with achieved coverages of 2812.84\u0026times; (F3C) and 1891.57\u0026times; (F3d), both passing stringent contamination screening protocols. Bacterial genome assembly involved optimization through multiple k-mer parameter analysis using SOAPdenovo2 for short-read assembly, generating optimal contigs. Sequencing reads were aligned to contigs, with subsequent scaffold construction achieved through local reassembly guided by paired-end relationships and read overlaps. The Unicycler assembly software was employed for hybrid assembly incorporating third-generation sequencing data. Assembly refinement was conducted using Pilon software for sequence correction. When terminal sequence overlaps exceeding specified lengths were detected in final assemblies, circularization was achieved by trimming redundant overlapping regions. This pipeline ultimately yielded complete chromosomal and plasmid sequences.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.2.3 Prediction of whole genome features\u003c/div\u003e\u003cp\u003eGenome annotation was performed through a multi-software pipeline. Protein-coding genes (CDS) were predicted using Glimmer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccb.jhu.edu/software/glimmer/index.shtml\u003c/span\u003e\u003cspan address=\"http://ccb.jhu.edu/software/glimmer/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GeneMarkS, and Prodigal in parallel. Specifically, Glimmer was employed for initial CDS prediction in draft genome assemblies, chromosomal CDS annotation in complete assemblies, while GeneMarkS was designated for plasmid genome prediction. tRNA identification was conducted using tRNAscan-SE v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trna.ucsc.edu/software/\u003c/span\u003e\u003cspan address=\"http://trna.ucsc.edu/software/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), providing nucleotide sequences, anticodon information, and secondary structure predictions. rRNA operons were mapped with Barrnap (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/barrnap\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/barrnap\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), generating complete inventories of rRNA types with their genomic coordinates and sequences. Final genome visualization was achieved using CGView for circular genome map construction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003cdiv class=\"Heading\"\u003e2.2.4 Gene function analysis\u003c/div\u003e\u003cp\u003eThe predicted protein sequences were functionally annotated against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups (COG) databases, with priority given to the highest-scoring alignment ratios from comparative analyses. Virulence-associated genes and antimicrobial resistance determinants were identified through the Virulence Factor Database (VFDB) and Comprehensive Antibiotic Resistance Database (CARD), respectively. MGEs were systematically characterized using the VRprofile 2 online platform. Thresholds for gene annotation: identity\u0026thinsp;\u0026ge;\u0026thinsp;40%, coverage\u0026thinsp;\u0026ge;\u0026thinsp;70%, E-value\u0026thinsp;\u0026le;\u0026thinsp;1\u0026times;10⁻⁵.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Isolation and identification of \u003cem\u003elactic acid bacteria\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eSequencing analysis revealed significant enrichment of lactic acid bacteria in fecal samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B), from which two strains (F3c and F3d) were isolated. On MRS agar, strain F3c formed white colonies with smooth margins and appeared as purple-staining short rods under microscopy, while strain F3d developed convex colonies and displayed slightly curved bacilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Phylogenetic analysis based on 16S rRNA sequences revealed that F3d showed the closest phylogenetic relationship to \u003cem\u003eLactobacillus equi\u003c/em\u003e, whereas F3c was most closely related to \u003cem\u003eWeissella confusa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Metabolic characterization demonstrated strain-specific profiles: F3d tested positive for esculin hydrolysis and fermentation of maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose, but negative for cellobiose utilization. In contrast, F3c utilized only esculin, cellobiose, and salicin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These biochemical and molecular features conform to the taxonomic standards established in Bergey's Manual of Determinative Bacteriology (8th edition), validating both isolates as LAB.\u003c/p\u003e\u003cp\u003e\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\u003eBacterial Biochemical Identification\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType\u003c/p\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEsculin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCellobiose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMaltose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMannitol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSalicin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSorbitol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSucrose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRaffinose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eInulin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eLactose\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Biological characteristics of F3c and F3d\u003c/h2\u003e\u003cp\u003eBoth isolates demonstrated divergent growth kinetics and acidogenesis patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Strain F3c initiated logarithmic growth between 2\u0026ndash;6 h with stationary phase attainment at 8 h, exhibiting concurrent rapid acidification (pH 4.4\u0026ndash;4.6) within the initial 2 h. In contrast, F3d displayed accelerated acid production (pH 4.0-4.5 by 6 h) with transient pH rebound during 6\u0026ndash;8 h. (pH 2.5\u0026ndash;5.6 OD₆₀₀: F3c 0.125\u0026ndash;0.556, F3d 0.153\u0026ndash;0.750) and bile salt (0.3\u0026ndash;0.5% OD₆₀₀: F3c 0.383\u0026ndash;0.663, F3d 0.554\u0026ndash;0.824) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Hemolytic profiling identified α-hemolysis on both horse and sheep blood agar plates, with measured hemolysis zones\u0026thinsp;\u0026le;\u0026thinsp;2 mm in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Antimicrobial efficacy testing showed F3c's superior inhibitory action against \u003cem\u003eSalmonella spp\u003c/em\u003e. and both strains effectively suppressed \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Identical antibiotic susceptibility patterns were recorded: susceptibility to ampicillin, erythromycin, tetracycline, chloramphenicol, clindamycin, cephalexin, and cephradine; Resistance to kanamycin, streptomycin, norfloxacin, and vancomycin (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\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 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBacteriostasis results of three strains of bacteria\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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePathogenic bacteria\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF3c/mm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF3d/mm\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\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.755\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.99\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\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.535\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\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 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibiotic susceptibility of strains\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\u003eAntibiotics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF3c\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF3d\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmpicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eKanamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\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\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\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\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\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\u003eErythromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eTetracycline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eVancomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\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\u003ePenicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\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\u003eCephalothin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eCephalexin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eCiprofloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\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\u003eCefradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eAmoxicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\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\u003eOxacillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Genome-wide profiles of F3c and F3d\u003c/h2\u003e\u003cp\u003eWGS uncovered distinct genomic organizations between the strains. F3c harbored a 2,247,119 bp genome configuration consisting of a singular circular chromosome (2,198,644 bp; 45.11% GC content) accompanied by three plasmids measuring 32,174 bp (43.36% GC), 11,751 bp (40.43% GC), and 4,550 bp (36.79% GC), collectively encoding 2,186 predicted genes. Conversely, F3d displayed a more compact 2,114,783 bp genomic structure featuring a reduced circular chromosome (1,641,382 bp; 40.04% GC) paired with four plasmids of 415,366 bp (38.96% GC), 48,923 bp (37.98% GC), 4,687 bp (41.03% GC), and 4,425 bp (35.66% GC), containing 1,943 annotated coding sequences (Tables\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\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 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetail of F3c and F3d gene assembly results\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\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF3c\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF3d\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChromosome No.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlasmid No.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenome Size (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 247 119\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 114 783\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG\u0026thinsp;+\u0026thinsp;C(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDepth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 812.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 891.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\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 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic characteristics of the F3c and F3d gene assembly results\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\u003eGenomic characterstics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF3c\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF3d\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of CDS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 943\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of tRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of rRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of sRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4 F3c\u0026amp;F3d function annotation\u003c/h2\u003e\u003cp\u003eCOG analysis revealed 23 functional categories for both strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). F3c and F3d exhibited strong protein synthesis capacity, with 193 and 190 genes respectively annotated in translation, ribosome strcture biogenesis. Metabolic versatility was evidenced by enrichment in carbohydrate transport and metabolism (F3c:163 genes, F3d:123 genes), amino acid transport and metabolism (F3c:118 genes, F3d: 80 genes). Both isolates exhibited adaptive genomic features, including replication, recombination and repair (F3c:107 genes; F3d:108 genes) as well as biofilm formation-associated genes (F3c:116 genes; F3d:136 genes). Notably, the mobilome was significantly represented, with F3c harboring type II secretion system (T2SS)-associated virulence genes and F3d encoding vancomycin resistance-related \u003cem\u003evanTG\u003c/em\u003e loci.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGO term categorization resolved 49 functional subcategories, with F3c (1,421 genes) and F3d (2,000 genes) displaying conserved molecular architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Membrane components dominated cellular components (F3c:421 genes, F3d:372 genes), while molecular function analysis demonstrated significant enrichment in ATP-binding (F3c:201; F3d:234) and DNA-binding (F3c:153; F3d:179) functional classes.\u003c/p\u003e\u003cp\u003eKEGG pathways highlighted metabolic specialization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). F3c prioritized carbohydrate (116 genes) and nucleotide metabolism (70 genes), whereas F3d emphasized carbohydrate (123 genes) and amino acid metabolism (80 genes). Both strains showed strong environmental adaptation through membrane transport genes (F3c:115 genes, F3d:85 genes).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.5 The annotation of CARD \u0026amp; VFDB\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eCARD database analysis identified 118 genes (F3c) and 131 genes (F3d) multidrug resistance genes distributed across chromosomes and plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), including \u003cem\u003eLmrS, lmrP\u003c/em\u003e, and \u003cem\u003eoptrA\u003c/em\u003e conferring resistance to macrolides, tetracyclines, fluoroquinolones, and β-lactams through target alteration, efflux pumps, and enzymatic inactivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eVFDB annotation revealed 180 virulence factors in F3d, dominated by immune modulation (62 genes), nutrient metabolism (35 genes), and exotoxins (10 genes). Comparatively, F3c contained 164 virulence-associated genes showing dominance in iron scavenging systems (37 genes), magnesium acquisition (21 genes), and phagocytosis evasion mechanisms (19 genes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003eGCView-generated high-resolution maps delineated chromosomal architecture, including COG-functional zonation, prophage integration sites, CRISPR-cas arrays, noncoding RNAs, and GC-skew patterns for both genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.6 Plasmid-Borne Mobile Elements: Functional Annotation, CARD, and VFDB\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eBacterial evolution is characterized by the ability to acquire genetic material from the environment through HGT, with common mobile genetic elements involved in this process including plasmids, prophages, genomic islands, CRISPR-Cas, transposons, and insertion sequences .This study focuses on analyzing plasmids, prophages, CRISPR-Cas and genomic islands.\u003c/p\u003e\u003cp\u003eFunctional annotation of the F3c plasmid identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) only two virulence genes (pB_gene0006 and pB_gene0007), with the latter additionally carrying the ARGs macB. Lysin-related functions were associated with pB_gene0046 and pB_gene0053, while pB_gene0029 was annotated as Cag24 pathogenicity island protein and pC_gene0002 as alkaline shock protein (Asp23 family). No further pathogenicity-related features were detected. In contrast, the F3d plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) exhibited broader annotations, with 25 genes linked to CARD, 14 to VFDB, and 14 overlapping between both databases.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.7 GI -Borne Mobile Elements: Functional Annotation, CARD, and VFDB\u003c/h2\u003e\u003cp\u003eGenomic island (GI) analysis of F3c (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) revealed four AMR genes (patB, rpoB2, vanYM, LRA-8) and seven VFDB-associated virulence factors, including InlJ (VF0445), HitABC (VF0268), and TcdA (VF0376). Functional annotations highlighted gene1381 (lysozyme), gene1399 (Cag24 pathogenicity island protein), and gene1662-1664 (trimeric autotransporter adhesin), alongside diverse enzymatic roles such as gene1706 (rhodanese-like domain-containing protein), gene1713 (penicillin-binding protein), and gene1937 (CtsR family transcriptional regulator). For F3d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), GI annotation identified three ARGs (\u003cem\u003ePmrF, macB, eFrA\u003c/em\u003e) and 16 VFDB entries, including LPS, Capsule, and LOS-related genes. Functional features included gene0226 (flippase), gene0254 (thioredoxin), and gene0837 (L,D-transpeptidase).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.8 Prophage -Borne Mobile Elements: Functional Annotation, CARD, and VFDB\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eProphage annotation of both plasmids showed no virulence or AMR features in VFDB or CARD. However, F3d (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) harbored pA_gene0120 (cell wall-associated hydrolase), while F3c (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) carried pB_gene0047 (lysozyme M1) and pB_gene0053 (lysin-related function). No additional pathogenicity-associated annotations were observed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.9 CRISPR-Cas -Borne Mobile Elements: Functional Annotation, CARD, and VFDB\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eCRISPR-Cas system analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) identified a single feature in F3c (gene0312: KxYKxGKxW signal peptide domain-containing protein). F3d (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-F) exhibited six annotated components: gene0836 (Mucin-4), gene0910 (cell wall-associated hydrolase), pB_gene0009 (cysteine-rich acidic membrane protein), pA_gene0320 (zonadhesin), and pA_gene0322 (Sec-dependent serine-rich adhesin).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study finds potential safety concerns regarding two LAB strains (F3c: \u003cem\u003eWeissella confusa\u003c/em\u003e; F3d: \u003cem\u003eLactobacillus equi\u003c/em\u003e) isolated from healthy equine hosts. Despite their probiotic potential, both strains, however, harbored horizontally acquired antibiotic resistance genes (ARGs) and virulence factors, posing potential safety risks. Significantly, these risk elements were mechanistically linked to MGEs\u0026mdash;specifically plasmids, genomic islands, and prophages\u0026mdash;that act as reservoirs for pathogenic traits. For instance, F3d\u0026rsquo;s plasmid carried \u003cem\u003evanTG\u003c/em\u003e (conferring vancomycin resistance) alongside 14 virulence-related genes involved in processes such as lipopolysaccharide biosynthesis. Similarly, F3c\u0026rsquo;s genomic island encoded \u003cem\u003eTcdA\u003c/em\u003e (a toxin gene) and \u003cem\u003epatB\u003c/em\u003e (a multidrug efflux determinant). These findings underscore how MGEs facilitate the persistence of hazardous genomic elements in ostensibly beneficial microbes.\u003c/p\u003e\u003cp\u003eThe identified ARGs\u0026mdash;\u003cem\u003elmrS\u003c/em\u003e, \u003cem\u003eoptrA\u003c/em\u003e, and \u003cem\u003evanTG\u003c/em\u003e\u0026mdash;correlate with documented intrinsic or acquired resistance mechanisms in LAB[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Genus-level analyses of \u003cem\u003eLactobacillus spp\u003c/em\u003e. demonstrate the ubiquity of multidrug efflux pumps such as \u003cem\u003elmrS\u003c/em\u003e, likely attributable to environmental selection pressures across diverse niches[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Of particular concern is the \u003cem\u003evanTG\u003c/em\u003e detected in strain F3d, as vancomycin resistance remains uncommon in LAB and poses potential complications for clinical therapies[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The plasmid localization of these ARGs reinforces HGT risks[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], emphasizing the imperative for MGE screening in LAB prior to industrial deployment. This is illustrated by two key findings: F3c\u0026rsquo;s plasmid encodes Cag pathogenicity island protein 24\u0026mdash;a virulence determinant typically linked to Helicobacter pylori[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u0026mdash;while F3d\u0026rsquo;s genomic island harbors PmrF, associated with polymyxin resistance[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These examples collectively demonstrate LAB\u0026rsquo;s genomic adaptability via HGT mechanisms[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVirulence factors such as adhesion proteins, biofilm-related genes, and immune evasion markers (e.g., T2SS in F3c) were identified through VFDB annotation. While LAB are generally recognized as safe[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], these findings echo recent warnings that certain strains may retain pathogenic traits.\u003c/p\u003e\u003cp\u003ePhenotypic analyses validated genomic risk indicators. Both strains exhibited tolerance to acidic conditions (pH 2.5\u0026ndash;5.6) and elevated bile salt concentrations (0.3\u0026ndash;0.5%), traits advantageous for probiotic survival but potentially concerning when coexisting with virulence factors. While their antimicrobial activity against Salmonella spp. supports probiotic applications, the concurrent observation of α-hemolysis on horse blood agar warrants caution, as hemolytic capacity is a recognized pathogenic hallmark. This α-hemolytic activity correlates with hemolysin-associated genes annotated in F3c\u0026rsquo;s GI, directly connecting genomic predictions to measurable phenotypic risks.\u003c/p\u003e\u003cp\u003eThe identical antibiotic susceptibility profiles (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) further highlight shared resistance mechanisms. Observed resistance to kanamycin, streptomycin, and vancomycin\u0026mdash;contrasting with susceptibility to ampicillin and erythromycin\u0026mdash;mirrors LAB resistance patterns documented by Goldstein et al[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Collectively, these results support growing evidence that LAB reservoirs of ARGs may act as conduits for resistance dissemination. Notably, the distinct genomic localization of resistance determinants\u0026mdash;\u003cem\u003elmrS\u003c/em\u003e (multidrug resistance) on chromosomes versus \u003cem\u003eoptrA\u003c/em\u003e (oxazolidinone resistance) on plasmids\u0026mdash;underscores the complementary roles of vertical inheritance and HGT in shaping LAB resistance profiles[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eIn general, while F3c and F3d display probiotic characteristics, their genomic repertoire of ARGs and virulence factors calls into question their unrestricted application. HGT-centric analyses demonstrate that MGEs serve as vectors for transferring pathogenic traits\u0026mdash;even among ostensibly beneficial strains[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While these studies provide foundational insights into HGT mechanisms, the phenotypic consequences of transferred genes remain uncharacterized. Future work must bridge this knowledge gap by correlating genetic acquisitions with functional outcomes, such as pathogenicity or metabolic adaptation. These findings emphasize the need for strain-specific safety evaluations, particularly for equine probiotics, to minimize inadvertent hazards. Critical next steps include functional characterization of MGE-linked genes (e.g.\u003cem\u003evanTG\u003c/em\u003e efflux activity), real-time tracking of HGT dynamics in equine gut ecosystems to quantify resistance transmission risk, and systematic investigation of HGT-derived phenotypic expressions. Given that environmental ARG reservoirs may facilitate cross-boundary resistance proliferation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], rigorous scrutiny of LAB strains such as F3c/F3d is imperative for both veterinary practice and public health agendas.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWhile strains F3c and F3d demonstrate probiotic potential, their HGT-associated genomic risks necessitate cautious evaluation. These findings underscore that even purportedly \"beneficial\" microorganisms require rigorous safety assessments to mitigate unintended ecological and clinical consequences in probiotic utilization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eAll animal experimental protocols were approved by the Research Ethics Committee of Xinjiang Agricultural University with an ID of 2024012.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript. We confirm that this work is original, has not been published elsewhere, and is not under consideration by another journal.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAll other authors declare they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was funded by National Natural Science Foundation of China, grant number 32360868.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eTA: Data curation, Investigation, Methodology, Validation, Writing\u0026ndash;original draft. DZ: Data curation, Investigation, Methodology, Validation, Writing\u0026ndash;original draft. LY: Conceptualization, Data curation, Investigation, Methodology. XY\u0026amp; YF: Conceptualization, Data curation, Investigation, Methodology. LR: Formal Analysis, Methodology, Writing-review and editing. HY: Formal Analysis, Methodology, Writing-review and editing. HS: Formal Analysis, Methodology, Writing-review and editing. HZ: Formal Analysis, Methodology, Writing-review and editing. WJ: Data curation, Investigation, Methodology, Supervision, Validation, Writing-review and editing. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.The sequencing data generated in this study have been submitted to the NCBI SRA under submission IDs SUB15706789 and SUB15707400. The assembled genome sequences are being processed and the accession numbers will be updated upon completion of the deposition process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWunderlich G, Bull M, Ross T, Rose M, Chapman B. 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O'Toole PW: Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp. Appl Environ Microbiol 2019, 85(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRozman V, Mohar Lorbeg P, Treven P, Accetto T, Janezic S, Rupnik M, Bogovic Matijasic B. Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria. Life Sci Alliance 2023, 6(4).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGundolf T, Kalb R, Rossmanith P, Mester P. Bacterial Resistance Toward Antimicrobial Ionic Liquids Mediated by Multidrug Efflux Pumps. Front Microbiol. 2022;13:883931.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAfshari A, Taheri S, Hashemi M, Norouzy A, Nematy M, Mohamadi S. Methicillin- and Vancomycin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci Isolated from Hospital Foods: Prevalence and Antimicrobial Resistance Patterns. 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Microbiol Spectr. 2023;11(1):e0273622.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMunoz-Atienza E, Gomez-Sala B, Araujo C, Campanero C, del Campo R, Hernandez PE, Herranz C, Cintas LM. Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture. BMC Microbiol. 2013;13:15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen W, Wang Q, Wu H, Xia P, Tian R, Li R, Xia L. Molecular epidemiology, phenotypic and genomic characterization of antibiotic-resistant enterococcal isolates from diverse farm animals in Xinjiang, China. Sci Total Environ. 2024;912:168683.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLang AS, Buchan A, Burrus V. Interactions and evolutionary relationships among bacterial mobile genetic elements. Nat Rev Microbiol. 2025;23(7):423\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaddamsetti R, Yao Y, Wang T, Gao J, Huang VT, Hamrick GS, Son HI, You L. Duplicated antibiotic resistance genes reveal ongoing selection and horizontal gene transfer in bacteria. Nat Commun. 2024;15(1):1449.\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":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-7684893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7684893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eWhile \u003cem\u003elactic acid bacteria\u003c/em\u003e (LAB) are widely recognized as probiotics, their genomic safety remains understudied.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e\u003cp\u003eThis study integrates phenotypic characterization, whole genome sequencing (WGS), and horizontal gene transfer (HGT) analysis to evaluate the safety of two LAB strains isolated from healthy equines.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e\u003cp\u003ePhenotypic assays revealed robust acid and bile salt tolerance, antimicrobial activity against pathogens (\u003cem\u003eSalmonella spp., Escherichia coli\u003c/em\u003e), and \u003cem\u003eα-hemolysis\u003c/em\u003e on horse and sheep blood agar. WGS identified multiple antibiotic resistance genes ARGs (\u003cem\u003elmrS, optrA, vanTG\u003c/em\u003e) and virulence factors (Adhesion, biofilm formation, immune evasion) in both strains. Crucially, HGT-associated mobile elements (plasmids, genomic islands) were found to carry ARGs (\u003cem\u003evanTG, macB\u003c/em\u003e) and virulence genes (\u003cem\u003eT2SS, InlJ\u003c/em\u003e). For instance, F3d\u0026rsquo;s plasmid encoded 14 virulence-related genes, while F3c\u0026rsquo;s genomic island harbored \u003cem\u003eTcdA\u003c/em\u003e (Toxin) and \u003cem\u003epatB\u003c/em\u003e (Multidrug efflux). These findings demonstrate that mobile genetic elements (MGEs) contribute to the retention of pathogenic traits in LAB, highlighting potential safety concerns.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study demonstrate that mobile genetic elements (MGEs) contribute to the retention of pathogenic traits in LAB, highlighting potential safety concerns and underscores the necessity of screening LAB for MGEs to ensure safety in probiotic applications.\u003c/p\u003e","manuscriptTitle":"Whole-Genome Profiling Reveals Mobile Genetic Elements Associated with Virulence and Antibiotic Resistance in Equine Lactic Acid Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 22:20:21","doi":"10.21203/rs.3.rs-7684893/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T18:15:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T15:03:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T15:40:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18952212808075655482590002798181587223","date":"2025-10-18T13:53:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297412299097421752457791438589497311021","date":"2025-10-16T06:50:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T17:51:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T17:40:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-15T15:38:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T11:14:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-10-14T10:56:33+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":"ccbc11b5-bd6c-4594-83a9-03638db6d20c","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T09:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 22:20:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7684893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7684893","identity":"rs-7684893","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}