Harnessing Native Swine Probiotics: Unveiling Their Protective Shield Against Salmonella Typhimurium – Insights from an Immune and Gut Microbiome Perspective

Harnessing Native Swine Probiotics: Unveiling Their Protective Shield Against Salmonella Typhimurium – Insights from an Immune and Gut Microbiome Perspective | 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 Harnessing Native Swine Probiotics: Unveiling Their Protective Shield Against Salmonella Typhimurium – Insights from an Immune and Gut Microbiome Perspective Kittiya Khongkool, Malai Taweechotipatr, Sunchai Payungporn, Vorthon Sawaswong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6367837/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Probiotics confer strain-specific health benefits, including protection against pathogenic infections. This study evaluated the protective efficacy of single- and multi-strain probiotics against Salmonella Typhimurium in a mouse model. Mice were administered Lactiplantibacillus plantarum TBRC-15420, Bacillus amyloliquefaciens TBRC-15434, Saccharomyces cerevisiae TBRC-19857, or their combination for 30 days prior to S. Typhimurium challenge. Protective effects were assessed through survival rates, clinical symptoms, weight changes, pathogen clearance, histopathology, secretory IgA levels, and gut microbiota shifts using 16S rRNA sequencing. No mortality was observed; however, mice exhibited varying symptoms post-infection, with some recovering by day five. Probiotics mitigated weight loss, with the multi-strain combination being most effective, while L. plantarum TBRC-15420 provided the strongest single-strain protection. Probiotics enhanced secretory IgA levels, with B. amyloliquefaciens TBRC-15434 and L. plantarum TBRC-15420 eliciting robust immune responses. All strains effectively reduced S. Typhimurium levels in the small intestine and prevented its translocation to the liver and spleen, achieving complete bacterial clearance by day five. Probiotic pretreatment preserved the structural integrity of the intestine, liver, and spleen. It also promoted beneficial bacterial phyla such as Bacteroidetes and Firmicutes while suppressing Proteobacteria , thereby maintaining gut microbiome homeostasis during infection. These findings support probiotics as antibiotic alternatives for Salmonella infection management, emphasizing their role in immune modulation and microbiota stability. Probiotics Lactiplantibacillus plantarum Bacillus amyloliquefaciens Saccharomyces cerevisiae Protective effect Salmonella Typhimurium Immune response Gut microbiome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Infections caused by Salmonella enterica pose significant challenges to the livestock industry, with S. enterica serovar Typhimurium ( S. Typhimurium ) being a leading cause of economic losses and public health concerns [ 1 ]. This serotype infects a wide range of hosts, causing gastrointestinal and systemic diseases [ 2 ], and is a common cause of severe gastroenteritis in livestock such as poultry, pigs, and cattle [ 3 ]. S. Typhimurium spreads primarily through the fecal-oral route, with contaminated feed, water, and farm environments serving as reservoirs for infection. Poor hygiene and overcrowded conditions on farms exacerbate outbreaks, leading to diarrhea, fever, and loss of appetite. Severe cases may result in septicemia, organ damage, or death, particularly in young or immunocompromised animals [ 4 – 7 ]. Antimicrobial therapy is the standard treatment; however, widespread and indiscriminate antibiotic use has resulted in the emergence of resistant Salmonella strains, posing challenges for disease control [ 8 ]. This highlights the urgent need for alternative, sustainable strategies. The gut microbiome, which regulates intestinal homeostasis and immune responses, plays a pivotal role in disease resistance. Commensal gut bacteria perform key functions such as fermenting complex carbohydrates, producing vitamins, and inhibiting pathogen growth [ 9 ]. The intestinal barrier, composed of the mucus layer, epithelial cells, and immune components, works synergistically with the microbiome to combat pathogenic organisms [ 10 , 11 ]. Gut microbiota dysbiosis has been identified as a primary contributor to post-weaning diarrhea and related infections in piglets [ 12 ]. Probiotics, live microorganisms that confer health benefits, have shown promise in restoring microbiota balance, enhancing nutrient absorption, and modulating immune defenses in swine [ 13 ]. They work by competing with pathogens for nutrients and adhesion sites, producing antimicrobial compounds, and strengthening gut integrity [ 14 – 17 ]. For instance, lincomycin-treated swine experience dysbiosis characterized by increased Clostridium and Escherichia-Shigella populations and decreased fiber-degrading microbes like Fibrobacter and Treponema , underscoring the importance of microbiota preservation [ 18 ]. This study hypothesizes that probiotics sourced from native swine feces can mitigate S. Typhimurium infections by reducing pathogen colonization, preventing systemic spread, and restoring microbiome balance. Using a mouse model, the research evaluates the protective efficacy of single-strain and multi-strain probiotics on bacterial load, immune responses, and gut microbiota composition. By investigating these aspects, this study aims to establish probiotics as a sustainable alternative to antibiotics for managing Salmonella infections, improving animal health, and enhancing food safety Methods Animals and housing Specific-pathogen-free (SPF) male Mus musculus (ICR) mice, aged 6–7 weeks, were obtained from the National Laboratory Animal Center at Mahidol University (NLAC-MU), Thailand. The mice were housed in groups of three per cage, maintained on a 12-hour light/dark cycle at a constant temperature of 24 ± 1°C and relative humidity of 60 ± 5%. The mice were fed a basal diet and given water without restriction. The bedding of mice was changed every three days, and the health status of the animals was monitored regularly. They were acclimatized for one week prior to the start of the experiments. All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of Srinakharinwirot University, under approval number COA/AE-003-2566. Probiotic strains and inoculum preparation for oral administration The probiotic strains used in this study, including Lactiplantibacillus plantarum LC5.2 (TBRC-15420), Bacillus amyloliquefaciens NL1.2 (TBRC-15434), and Saccharomyces cerevisiae YH14 (TBRC-19857), were isolated from the feces of native swine. These strains have been previously assessed and demonstrated to exhibit robust probiotic properties and safety in both in vitro and in vivo studies. Comprehensive details on the probiotic traits and safety of each strain are provided in Table S1 . To prepare the inoculum, L. plantarum TBRC-15420 was grown in De Mann, Rogosa, and Sharpe (MRS) broth and incubated anaerobically at 37°C for 24 hours. B. amyloliquefaciens TBRC-15443 was cultured in Luria-Bertani (LB) broth with shaking at 37°C and 180 rpm for 24 hours. S. cerevisiae TBRC-19857 was cultivated in Potato Dextrose Broth (PDB) at 37°C for 24 hours. After incubation, cells were harvested by centrifugation at 5,000 × g for 10 minutes at 4°C, washed twice with PBS, and resuspended to the desired concentration. The optical density (OD) at 600 nm was adjusted to 1.0 (approximately 1 × 10 11 CFU/mL) for L. plantarum , 1.3 (approximately 1 × 10 11 CFU/mL) for B. amyloliquefaciens , and 2.0 (approximately 1 × 10 10 CFU/mL) for S. cerevisiae . For the mixed culture, equal volumes (1 mL) of each strain were combined, centrifuged, and resuspended in 1 mL PBS. Pathogen and preparation of bacteria for high-dose challenge Salmonella enterica serovar Typhimurium (ESBL-producing: isolated strain, Accession no: KT12325) [ 15 ], was kindly provided by the Microbial Technology for Agriculture, Food, and Environment Research Center, Faculty of Science and Digital Innovation, Thaksin University, Thailand. The preparation of the challenge bacteria was conducted according to a previously described protocol [ 16 ]. Initially, bacteria from a frozen glycerol stock were grown on tryptic soy agar (TSA; Himedia, Mumbai, India) and incubated at 37°C for 14–18 hours. A few colonies were then transferred to tryptic soy broth (TSB; Himedia, Mumbai, India) and incubated with shaking at 200 rpm for 12–16 hours at 37°C. After centrifugation at 5,000 × g for 10 minutes, the pellet was washed twice with PBS, resuspended in PBS, and the OD at 600 nm was measured to adjust the inoculum to 1.0 (approximately 1 × 10 9 CFU/mL). Serial dilutions were plated and incubated overnight at 37°C for 24 hours, and CFUs were counted to confirm the inoculum concentration. Experimental design for probiotic administration in mouse model After 1 week of acclimatization, mice were randomly assigned to five groups (six mice per group) for 30 days of probiotic treatment. Fecal samples were collected before the challenge to confirm the absence of Salmonella . Following the treatment, mice were challenged with S. Typhimurium SC2451-3 via oral gavage. The experimental design for evaluating probiotic protection against S. Typhimurium infection is shown in Fig. 1 . Each group received specific treatments for 30 days before the challenge. The control group received sterile PBS, the TBRC-15420 group received L. plantarum TBRC-15420 (1 × 10 11 CFU/mL), the TBRC-15434 group received B. amyloliquefaciens TBRC-15434 (1 × 10 11 CFU/mL), and the TBRC-19857 group received S. cerevisiae TBRC-19857 (1 × 10 10 CFU/mL). The multi-strain group received a combination of all three strains, and all groups were challenged with S. Typhimurium (1 × 10 9 CFU/mL). Monitoring clinical symptoms, body weight, and survival rate Body weight was measured and recorded both before and after the challenge. After infection, mice were monitored daily for signs of distress or illness, and mortality was tracked for each group. Euthanasia was performed early if mice exhibited signs of imminent death, such as rapid weight loss (> 20%), lack of food or water intake, failure to groom, or unresponsiveness to stimuli. Sample collection The samples were collected following a procedure described in a previous study [ 17 ], with minor modifications. After the probiotic treatment period, three mice from each group were randomly selected for pre-challenge assessment of gut microbiome, and the data obtained were used to compare results before and after the challenge. Subsequently, after the challenge, one mouse from each group was randomly selected, anesthetized with isoflurane, and humanely euthanized on days 1, 3, and 5 of the post-challenge periods. For intestinal fluid collection, the small intestine was carefully extracted from the gastro-duodenal to the ileocecal junction. The intestinal contents were flushed with 2 mL of sterile PBS. The fluid was then divided into two tubes (1 mL each), with one tube designated for bacterial load quantification and the other for total secretory IgA antibody determination. The organs (small intestine, colon, liver, and spleen) were carefully isolated and placed in separate Petri dishes with sterile PBS to remove any blood and contaminants. These organs for bacterial translocation and histological analysis. Determination of bacterial load The bacterial load in the small intestine and its translocation to liver and spleen were assessed following the method described in a previous study [ 18 ] with minor modifications. To evaluate the bacterial load in the small intestine, 1 mL of intestinal fluid was serially diluted in PBS followed by serial 10-fold dilutions. For determination of the bacterial translocation, the liver and spleen were aseptically cut into 1 g pieces, finely minced using sterile scissors, and homogenized in 1 mL of sterile PBS. This mixture was thoroughly mixed and subjected to serial 10-fold dilutions. Suitable dilutions from each sample were plated in triplicate on Salmonella-Shigella (SS) agar and incubated at 37°C for 24 hours. Colony-forming units (CFU) of S. Typhimurium were then counted. Histological analysis Mice organs were collected for histopathological analysis according to standard protocols [ 19 ]. Prior to necropsy, mice were fasted overnight, anesthetized with isoflurane, humanely euthanized, and placed on a sterile dissection bench. A midline incision was made from the navel to the mouth, with additional lateral cuts. The skin, muscle layers, and abdominal membrane were removed to expose the internal organs. The small intestine, colon, liver, and spleen were carefully separated, rinsed in sterile phosphate-buffered saline to remove blood and contaminants, and prepared for histological analysis. Organs were fixed in 4% paraformaldehyde phosphate buffer for 24 hours, dehydrated in graded ethanol solutions (70–100%), and cleared with xylene using the Leica TP1020 Tissue Processor. The tissues were embedded in paraffin with the MEDITE TES Valida system and solidified. Thin sections (4–6 µm) were cut using the HistoCore MULTICUT microtome, flattened in a warm water bath, mounted on glass slides, deparaffinized with xylene, rehydrated, and stained with hematoxylin and eosin. The stained sections were then dehydrated, cleared, and mounted with coverslips for examination under a light microscope (Olympus UC50, Japan). Determination of total secretory immunoglobulin A in the intestinal fluids The intestinal fluid from each mouse was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was filtered through sterile 0.22 µm syringe filters and stored at -80°C until analysis. Secretory IgA levels were measured using an ELISA kit, following the manufacturer’s protocol (IgA Mouse Uncoated ELISA Kit, Invitrogen, Thermo Fisher Scientific). An anti-mouse IgA monoclonal antibody was coated onto a microplate and incubated overnight at 4°C. After washing, the plate was blocked with blocking buffer and incubated at room temperature for 2 hours, followed by two washes with wash buffer (1x PBS, 0.05% Tween 20). Each sample or standard were added to the appropriate wells and incubated at room temperature for 2 hours. The plate was washed four times, then HRP-conjugated anti-mouse IgA polyclonal antibody was added to each well and incubated at room temperature for 1 hour, followed by another four washes. Tetramethylbenzidine (TMB) substrate solution was added to each well and incubated at room temperature for 15 minutes. The reaction was stopped by adding 2 N H₂SO₄ to each well, and absorbance was measured at 450 nm for data analysis. Gut microbiome analysis Three mice from each group were randomly selected for analysis before and after the S. Typhimurium challenge. Fecal samples were collected from the colon, and total DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) according to the manufacturer’s instructions. Bacterial composition of the gut microbiome was analyzed through next-generation sequencing, following established protocols [ 20 ]. Full-length 16S rDNA was amplified via PCR using universal 16S rRNA primers (27F/1492R) with nanopore adaptor tails. Multiplexing barcodes were then incorporated, followed by purification and quantification. The pooled DNA samples were further purified, subjected to adaptor ligation, and sequenced using a MinION Mk1C device. Bioinformatic analysis included base calling, quality assessment, demultiplexing, adaptor trimming, clustering, polishing, and taxonomic identification. Raw FAST5 sequencing data were basecalled using the Guppy basecaller in super-accuracy (SUP) mode. FASTQ sequences were quality-checked with MinIONQC, demultiplexed, and trimmed using Porechop. The processed reads were analyzed with NanoCLUST for clustering, polishing, and taxonomic classification. The resulting taxonomic profiles and abundance data were further examined using MicrobiomeAnalyst [ 21 ]. Statistical analysis All values were expressed as means ± standard deviation. Statistical differences between the various groups were evaluated by Student’s t-test. P values < 0.05 were considered statistically significant. Results Survival and clinical symptoms after Salmonella Typhimurium infection No mortality occurred in any group during the five-day experiment. Probiotic administration mitigated symptoms of infection, promoting recovery and reducing disease severity. On day 1 post-infection, all mice appeared normal. By day 2, infected mice in all groups showed decreased activity, lethargy, reduced food intake, and ruffled fur, with some control mice also exhibiting mild diarrhea. By day 5, some mice had recovered, while others still displayed symptoms, though less severe than on day 2. Effect of probiotics on weight loss in mice exposed to Salmonella Typhimurium Probiotics mitigated weight loss in mice exposed to S. Typhimurium, as shown in Fig. 2 . The control group experienced the greatest weight loss, averaging 4.03 ± 0.50 g (10.67 ± 0.81%). Conversely, mice treated with probiotics showed significantly less weight loss. The multi-strain group exhibited the strongest protective effect, with only 0.86 ± 0.94 g (2.33 ± 2.43%, P = 0.003) of weight loss. Among the single-strain treatments, L. plantarum TBRC-15420 provided the most protection, with a weight loss of 1.37 ± 0.60 g (3.56 ± 1.53%, P = 0.002), followed by S. cerevisiae TBRC-19857 at 1.77 ± 0.99 g (4.65 ± 2.55%, P = 0.012) and B. amyloliquefaciens TBRC-15434 at 2.13 ± 0.47 g (6.25 ± 0.96%, P = 0.004). No significant differences were found among the probiotic-treated groups. Bacterial load in the gut, liver, and spleen Probiotic treatment significantly reduced S. Typhimurium SC2451-3 levels in the gut and limited its spread to the liver and spleen (Table 1 ). On day 1 post-infection, all groups had high bacterial loads in the gut. The control group showed notable bacterial presence in the liver and spleen, while probiotic-treated groups showed bacteria only in the liver of the S. cerevisiae TBRC-19857 group. The L. plantarum TBRC-15420 and B. amyloliquefaciens TBRC-15434 groups had lower bacterial counts in the spleen compared to the multi-strain group. By day 3, probiotic groups, especially L. plantarum TBRC-15420 and B. amyloliquefaciens TBRC-15434, showed significant reductions in gut bacteria, with no bacteria in the liver or spleen. The multi-strain and S. cerevisiae TBRC-19857 groups also showed reduced bacterial presence in the liver and spleen. On day 5, the control group still harbored bacteria in the liver and spleen, while all probiotic-treated groups completely eliminated the pathogen from the gut, liver, and spleen, highlighting the probiotics' role in pathogen clearance. Table 1 Bacterial load of Salmonella Typhimurium in mice at different days post-infection for various probiotic treatments DPI* Organ Bacterial load (CFU/ml or CFU/g tissue) Control TBRC-15420 TBRC-15434 TBRC-19857 Multi-strains 1 Intestinal fluid > 3.00E + 04 1.95E + 04 > 3.00E + 04 > 3.00E + 04 > 3.00E + 04 Liver > 3.00E + 04 ND ND 2.00E + 02 ND Spleen > 3.00E + 04 2.00E + 02 7.00E + 02 ND 1.13E + 03 3 Intestinal fluid > 3.00E + 04 9.20E + 03 4.70E + 03 3.00E + 04 ND Liver 1.60E + 03 ND ND ND ND Spleen 4.90E + 03 ND ND ND ND 5 Intestinal fluid ND ND ND ND ND Liver 8.00E + 02 ND ND ND ND Spleen 2.80E + 03 ND ND ND ND *DPI refers to days post-infection and ND indicates no detection. Histopathological examination of mice infected with Salmonella Typhimurium The small intestine of all groups remained unaffected by the S. Typhimurium challenge. The control group exhibited normal intestinal structure, with intact villi, crypts, and muscle layers (Fig. 3 A- 3 B). Similarly, all probiotic-treated groups maintained well-preserved intestinal architecture without signs of inflammation or tissue damage (Fig. 3 C- 2 F). In contrast, the liver of control mice showed severe pathological changes, including focal necrosis, neutrophil clusters, inflammatory cell infiltration, perivascular leukocyte accumulation, vascular dilation, congestion, hepatocyte swelling, and vacuolization, indicating significant inflammation (Fig. 4 A– 3 B). Probiotic-treated mice, however, exhibited normal liver histology with smooth tissue structure, well-organized hepatocytes, and intact blood vessels, free of necrosis or swelling (Fig. 4 C– 3 F). Similarly, the spleen of control mice displayed marked lymphoid hyperplasia, with dense lymphocyte accumulation, disrupted splenic architecture, indistinct red and white pulp boundaries, and increased vacuolization, reflecting an intense immune response (Fig. 5 A– 4 B). In contrast, probiotic-treated groups maintained normal splenic morphology, with well-defined red and white pulp regions separated by a clear marginal zone. Macrophages were prominently observed in the red pulp and marginal zone across all probiotic-treated groups (Fig. 5 C– 4 F). Secretory IgA levels Secretory IgA (sIgA) levels in intestinal fluid samples were measured on days 1, 3, and 5 post-infection (Fig. 6 ). On day 1 post-infection, the control group had the lowest sIgA level (15.95 ng/mL). Among probiotic-treated groups, L. plantarum TBRC-15420 exhibited the highest sIgA level (21.04 ng/mL), followed by B. amyloliquefaciens TBRC-15434 (17.88 ng/mL), the multi-strain group (16.85 ng/mL), and S. cerevisiae TBRC-19857 (16.67 ng/mL). On day 3, sIgA levels in the control group increased slightly to 16.13 ng/mL. In comparison, B. amyloliquefaciens TBRC-15434 peaked at 21.43 ng/mL, while L. plantarum TBRC-15420 maintained a high level (18.93 ng/mL). The multi-strain and S. cerevisiae TBRC-19857 groups showed moderate increases at 16.92 and 16.87 ng/mL, respectively. On day 5, sIgA levels declined in all groups, but probiotic-treated mice maintained higher levels than the control (15.76 ng/mL). B. amyloliquefaciens TBRC-15434 retained the highest level (21.42 ng/mL), followed by L. plantarum TBRC-15420 (18.56 ng/mL), the multi-strain group (17.08 ng/mL), and S. cerevisiae TBRC-19857 (16.53 ng/mL). Gut microbiome analysis This study assessed the impact of probiotics on gut microbiome composition before and after S. Typhimurium infection, with fecal samples analyzed by 16S rRNA gene sequencing. Rarefaction curves showed sufficient sampling, with all samples demonstrating 100% Good’s coverage (Fig. S1 ). Relative abundance of bacteria Bacterial relative abundance was analyzed at phylum levels, as show in Fig. 7 . Prior to infection, Bacteroidetes was the most abundant phylum in all groups, followed by Firmicutes , Proteobacteria , Deferribacteres , and Verrucomicrobia . After infection, shifts in microbial composition were observed. In the control group, Bacteroidetes decreased from 46.31–41.02%, while Firmicutes rose from 44.59–46.44%. Proteobacteria increased from 6.78–10.43%, and Deferribacteres grew from 1.38–2.11%. Verrucomicrobia was absent post-infection (from 0.95%) In the L. plantarum TBRC-15420 group, Bacteroidetes increased significantly from 47.29–57.13%. In contrast, Firmicutes decreased from 43.62–36.82%, Proteobacteria dropped from 7.10–4.31%, and Deferribacteres showed a slight reduction from 1.98–1.18%. Verrucomicrobia , which was undetected before infection, emerged at 0.57% post-infection. In the B. amyloliquefaciens TBRC-15434 group, Bacteroidetes declined from 59.64–53.66%, Firmicutes increased from 31.97–41.00%, and Proteobacteria decreased from 8.09–4.71%. Verrucomicrobia remained stable at 0.31%, and Deferribacteres emerged at 0.32%. For the S. cerevisiae TBRC-19857 group, Bacteroidetes decreased from 54.41–42.24%, Firmicutes rose from 34.80–48.23%, and Proteobacteria slightly decreased from 8.67–7.50%. Verrucomicrobia dropped from 1.44–0.70%, and Deferribacteres doubled from 0.68–1.34%. In the multi-strain group, Bacteroidetes increased from 50.34–51.97%, Firmicutes decreased from 44.59–40.65%, and Proteobacteria showed a slight rise from 4.40–5.73%. Deferribacteres grew from 0.67–1.31%, and Verrucomicrobia appeared at 0.34%. Post-infection, Firmicutes became the most abundant phylum in the control and S. cerevisiae TBRC-19857 groups, while Bacteroidetes remained dominant in the other groups. Single-strain probiotics caused a decrease in Proteobacteria , while the control group saw an increase in this phylum. Verrucomicrobia and Deferribacteres remained relatively stable across all groups. Alpha diversity Alpha diversity, which assesses the richness and evenness of bacterial species within the gut ecosystem, was evaluated using the Shannon, Simpson, and Chao1 indices at the phylum and genus levels (Fig. 8 ). No significant changes in bacterial diversity were observed before and after the S. Typhimurium challenge in all groups. Beta diversity Beta diversity, which measures differences in microbial composition between samples, was analyzed using Bray-Curtis and Jaccard indices at the phylum and genus levels, with results visualized through PCoA plots (Fig. 9 ). Statistical analysis using PERMANOVA ( P < 0.05) revealed no significant differences in microbial composition between groups. Discussion The increasing use of antimicrobial drugs in livestock contributes to the development of drug-resistant Salmonella strains, which can pose significant risks for zoonotic transmission. Probiotics have emerged as a promising alternative to antibiotics, with potential to promote gut health and reduce reliance on antimicrobial treatments. This study demonstrates that probiotic supplementation, whether as a single strain or a multi-strain combination, offers protective effects against S. Typhimurium infection and supports gut homeostasis in a mouse model. In this study, mice were pre-treated with Lactiplantibacillus plantarum TBRC-15420, Bacillus amyloliquefaciens TBRC-15434, Saccharomyces cerevisiae TBRC-19857, or a multi-strain combination for 30 days before being challenged with S. Typhimurium SC2451-3. None of the infected mice succumbed to the infection. However, variations in clinical signs were noted, particularly in the control group, which exhibited mild diarrhea. Despite controlling for genetic background, environmental factors, and infection dose, the mice showed diverse physiological responses. This suggests that individual variations in immune function or microbial resilience could influence the response to infection. Previous research has shown that S. Typhimurium infection is typically associated with significant weight loss in livestock [ 22 – 25 ]. In this study, probiotic supplementation, especially the multi-strain treatment, notably reduced weight loss in infected mice, indicating a possible synergistic interaction between the bacterial and yeast strains that enhances immune modulation and inhibits pathogen growth. Among the single-strain treatments, L. plantarum TBRC-15420 consistently showed the greatest protective effect, likely due to its specific probiotic properties. These findings align with previous studies, which reported that probiotics reduce weight loss during bacterial infections. For example, pre-treatment with Lentilactobacillus buchneri or Saccharomyces boulardii alleviated weight loss and improved survival rates in S. Typhimurium-infected mice [ 26 , 27 ]. Further investigation into the impact of probiotics on infection and translocation of S. Typhimurium revealed that probiotic supplementation significantly reduced bacterial loads in intestinal fluid, liver, and spleen across different time points post-infection. On day 1 post-infection, bacterial levels were high in all groups, including the liver and spleen of the control group. However, probiotic-treated groups exhibited reduced bacterial loads, with L. plantarum TBRC-15420 and B. amyloliquefaciens TBRC-15434 showing early systemic protection against S. Typhimurium. By day 3, these probiotic groups demonstrated significant reduction in bacterial loads in the gut, with no detectable bacteria in the liver or spleen, indicating effective pathogen clearance. By day 5, all probiotic-treated groups had cleared S. Typhimurium from the intestinal fluid, liver, and spleen, whereas the control group continued to show bacterial presence. The immune response in the liver, particularly through Kupffer cells, may have contributed to the reduced pathogen levels observed in probiotic-treated groups [ 28 ]. The spleen, which plays a key role in pathogen storage and filtration, had higher bacterial loads than the liver, further emphasizing the importance of the immune system in controlling pathogen spread [ 29 ]. These results align with previous studies that have shown probiotics' ability to reduce bacterial loads and prevent systemic spread of pathogens. For example, Lactobacillus rhamnosus HN001 has been shown to reduce S. Typhimurium loads by 100-fold in the spleen and liver [ 30 ], Similarly, Lactobacillus casei and Bifidobacterium lactis have been demonstrated to prevent bacterial translocation and significantly reduce S. Typhimurium infections [ 31 ]. In addition to bacterial load reduction, we examined the intestinal histology for signs of enteritis, typically characterized by neutrophil infiltration, goblet cell depletion, and epithelial damage [ 2 ]. Interestingly, despite a high bacterial load in the intestine, no evidence of enteritis was observed, and the small intestine remained structurally intact. This aligns with previous studies showing that while S. Typhimurium-infected mice often develop systemic infections, gastrointestinal symptoms such as enteritis may not always occur [ 32 ]. To confirm the effect of probiotics in preventing pathogen translocation, histological analysis of the liver and spleen was performed. The liver, which plays a crucial role in pathogen elimination [ 33 , 34 ], showed normal histology in probiotic-treated mice, suggesting that probiotics help prevent liver inflammation and damage. The spleen complements liver function by filtering blood and coordinating adaptive immunity [ 35 ]. It consists of the red pulp, which houses macrophages that remove pathogens, and the white pulp, which facilitates immune responses [ 29 ]. After S. Typhimurium infection, control mice displayed severe splenic tissue damage, whereas probiotic-treated mice maintained normal morphology, with active macrophages combating infection. These findings suggest that probiotics help preserve splenic function and reduce inflammation. Consistent with previous studies, our results demonstrate that probiotics reduce bacterial translocation to systemic sites. Lactobacillus casei CRL431 and Lactobacillus paracasei CNCMI-1518 significantly lowered S. Typhimurium loads in the spleen and liver [ 36 ], while Bifidobacterium lactis INL1 prevented bacterial dissemination [ 37 ]. These findings underscore the potential of probiotics in mitigating systemic complications from enteric infections. Probiotic supplementation also increased the production of secretory IgA (sIgA), which plays a crucial role in mucosal immunity by binding and preventing the adhesion of enteric pathogens, and facilitating their clearance via mucus [ 38 , 39 ]. Among the probiotic-treated groups, B. amyloliquefaciens TBRC-15434 and L. plantarum TBRC-15420 were most effective in enhancing sIgA production. The increased sIgA levels observed in probiotic-treated groups likely contributed to bacterial clearance by preventing adhesion of S. Typhimurium to the intestinal epithelium, thereby reducing its ability to colonize and translocate to systemic sites. These findings highlight the potential of probiotics to boost mucosal immune defenses during infection, as supported by previous studies demonstrating that probiotic supplementation elevates sIgA levels and modulates immune responses. For instance, L. casei and Bifidobacterium animalis supplementation led to significant increases in IgA + cells in the small intestine and higher sIgA levels in the intestines following S. Typhimurium challenge [ 40 ]. Moreover, total sIgA levels increased in the intestinal fluid of mice fed with L. casei CRL 431 following a challenge with S. Typhimurium [ 41 ]. In addition to immune modulation, probiotic pre-treatment in this study led to shifts in gut microbiota composition. Notably, probiotics reduced the abundance of Proteobacteria , which includes pathogens like Salmonella [ 42 ], while maintaining Bacteroidetes , which are beneficial for gut health. Bacteroidetes play a critical role in immune modulation and short-chain fatty acid (SCFA) production, supporting gut homeostasis [ 43 ]. The reduction in Proteobacteria and maintenance of Bacteroidetes suggest that probiotics contribute to gut stability during infection. However, alpha and beta diversity analyses showed no significant differences between pre- and post-infection groups, indicating that the gut microbiome composition remained stable despite S. Typhimurium challenge. This stability may reflect microbiome resilience or the protective effects of probiotics in maintaining gut homeostasis. Although overall microbial diversity was not significantly altered, the observed reduction in Proteobacteria and the maintenance of Bacteroidetes, particularly in single-strain probiotic groups, suggest that probiotics may support gut health by modulating specific bacterial taxa. Further research should investigate how probiotic strain selection, dosage, and host factors influence microbial diversity and gut health outcomes. Our findings provide further support for probiotics as a viable strategy to mitigate S. Typhimurium infection without relying on antibiotics. This is particularly relevant in livestock production, where reducing antibiotic use is a global priority to combat antimicrobial resistance. Further studies should assess the long-term stability of probiotic colonization and its implications for pathogen resistance over extended periods. Conclusions This study demonstrates that probiotics from native swine feces, including Lactiplantibacillus plantarum TBRC-15420, Bacillus amyloliquefaciens TBRC-15434, Saccharomyces cerevisiae TBRC-19857, and their combination, provide significant protection against Salmonella Typhimurium infection in mice. Both single-strain and multi-strain treatments effectively reduced weight loss, with the most notable effects in the multi-strain combination and TBRC-15420. These probiotics reduced pathogen burden, prevented systemic dissemination, and maintained the integrity of intestinal and systemic organs, including the liver and spleen. They also enhanced secretory IgA levels, particularly in TBRC-15434 and TBRC-15420. Additionally, they promoted gut microbiome stability by increasing beneficial bacteria and reducing harmful ones. These findings highlight the potential of probiotics as an effective, antibiotic-free approach to improving livestock health and resilience to infections. Future studies should explore the long-term effects of these probiotics in different animal models and their potential for broader applications. Declarations Ethics approval and consent to participate All animal experiments received approval from the Ethics and Research Standardization Section, Srinakharinwirot University (Approval number: COA/AE-003-2566). Consent for publication Not applicable. Availability of data and material The probiotic strains have been deposited at the Thailand Bioresources Research Center (TBRC) under the following accession numbers: Lactiplantibacillus plantarum LC5.2 (TBRC-15420), Bacillus amyloliquefaciens NL1.2 (TBRC-15434), and Saccharomyces cerevisiae YH14 (TBRC-19857). All the sequencing data presented in this study is available at the Sequence Read Archive (SRA), under Bioproject accession number ID PRJNA1190112. Competing interests The authors declare that they have no competing interests. Funding This research is supported by the National Research Council of Thailand (NRCT): NRCT5-RGJ63005-085. Authors’ contributions KK and ML contributed to the conceptualization and methodology of the study. KK, ML, and MT conducted the mouse model experiments and interpreted the results. KK, VS, and SP performed the gut microbiome analysis and interpreted the data. KK was responsible for the original draft preparation, while ML, MT, VS, and SP contributed to the review and editing of the manuscript by critically evaluating and revising it. All authors read and approved the final manuscript. Acknowledgements The authors sincerely thank the Central Equipment Center and the Laboratory Animal Center of the Research and International Affairs, Faculty of Medicine, Srinakharinwirot University, for providing research facilities, equipment, and laboratory support for animal experiments. We also extend our special thanks to Dr. Akkanee Pewhom, Department of Biological Science, Faculty of Science and Digital Innovation, Thaksin University, for his expertise in histology. References Kuang X, Hao H, Dai M, et al. Serotypes and antimicrobial susceptibility of Salmonella spp. isolated from farm animals in China. Front Microbiol. 2015;6:602. https://doi.org/10.3389/fmicb.2015.00602 . Fàbrega A. Vila. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6367837","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472058345,"identity":"52f75c7d-3b43-4380-acd1-5294a4249b4b","order_by":0,"name":"Kittiya Khongkool","email":"","orcid":"","institution":"Thaksin University","correspondingAuthor":false,"prefix":"","firstName":"Kittiya","middleName":"","lastName":"Khongkool","suffix":""},{"id":472058346,"identity":"5959561b-6de5-4470-8a7f-7932c65c4bba","order_by":1,"name":"Malai Taweechotipatr","email":"","orcid":"","institution":"Srinakharinwirot University","correspondingAuthor":false,"prefix":"","firstName":"Malai","middleName":"","lastName":"Taweechotipatr","suffix":""},{"id":472058347,"identity":"aea9b3fb-3228-4f2c-a826-68f057cb8ba7","order_by":2,"name":"Sunchai Payungporn","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Sunchai","middleName":"","lastName":"Payungporn","suffix":""},{"id":472058348,"identity":"210476cf-6a23-44d8-b5d4-c9ba6858c94c","order_by":3,"name":"Vorthon Sawaswong","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Vorthon","middleName":"","lastName":"Sawaswong","suffix":""},{"id":472058349,"identity":"f68b7a0a-94e5-4e2d-8844-f56a055c5dd7","order_by":4,"name":"Monthon Lertworapreecha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYFADCQbGB0CKh48ULcwGIC1spGhhkwDRBLUYHO99+Llwj40c/+zmZ5Vfc+xk2BiYHz66gU/LmePG0jOepRlL3Dlmdlt2WzLQYWzGxjl4tJjdSGOQ5jlwOHGDRILZbcltzEAtPGzSBLQw/+Y58L9+g0T6t2LJbfVEaWED2nIgwUAix4zx47bDhLXYnznGZj3jQLLhjBs5xdKM247zsDET8Itkexvz7YIDdvL8M9I3fvy5rdqen7354WN8WkCAGc7gQeESo4XxBxGqR8EoGAWjYOQBACI2Q+SrCHhMAAAAAElFTkSuQmCC","orcid":"","institution":"Thaksin University","correspondingAuthor":true,"prefix":"","firstName":"Monthon","middleName":"","lastName":"Lertworapreecha","suffix":""}],"badges":[],"createdAt":"2025-04-03 09:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6367837/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6367837/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84892867,"identity":"29b742d2-b692-4848-954d-90a78122f000","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98907,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for evaluating probiotic protection against \u003cem\u003eSalmonella\u003c/em\u003e typhimurium infection\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/841bd658af1eb3c048ca20a8.jpeg"},{"id":84892866,"identity":"2b97ae96-9e05-4e3c-bfc0-cf5f2bfd92d1","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31047,"visible":true,"origin":"","legend":"\u003cp\u003eChange in body weight of mice after exposure to \u003cem\u003eSalmonella\u003c/em\u003eTyphimurium SC2451-3. The plot compares the weight loss among the control group and groups treated with various probiotic strains.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/384bf2e87375a508e11fe2e7.png"},{"id":84892876,"identity":"36d5ed75-9d3f-46b4-9a2f-e53cc610d938","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1772695,"visible":true,"origin":"","legend":"\u003cp\u003eHistological examination of the small intestine in control and probiotic-treated mice. \u003cstrong\u003e(A, B)\u003c/strong\u003e Control group, \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e TBRC-15420, \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e TBRC-15434, \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, and \u003cstrong\u003e(F)\u003c/strong\u003eMulti-strains group. All images were captured at 200× magnification.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/a69945ce0f2d537b72529caa.png"},{"id":84892874,"identity":"8b66403a-4302-4cb9-ba2b-f3a0115f5346","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2620409,"visible":true,"origin":"","legend":"\u003cp\u003eHistological examination of the liver in control and probiotic-treated mice. \u003cstrong\u003e(A, B)\u003c/strong\u003e The control group shows perivascular leukocyte accumulation (yellow dashed line), vascular dilation, and congestion of leukocytes and erythrocytes within blood vessels (yellow arrows). The probiotic-treated groups—\u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eLactiplantibacillusplantarum\u003c/em\u003e TBRC-15420, \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003eTBRC-15434, \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, and \u003cstrong\u003e(F)\u003c/strong\u003ethe multi-strains group—exhibit normal histological structures. All images were captured at 100× magnification.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/0ca57bb802166ed3a724e6ae.png"},{"id":84893062,"identity":"2c8a1a65-871c-41d5-916d-e5f9c5b95770","added_by":"auto","created_at":"2025-06-18 13:19:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2972437,"visible":true,"origin":"","legend":"\u003cp\u003eHistological examination of the spleen in control and probiotic-treated mice. \u003cstrong\u003e(A, B)\u003c/strong\u003e The control group showed a dense accumulation of lymphocytes, numerous small vacuoles (red arrows), and significant disorganization of splenic architecture. The probiotic-treated groups—\u003cstrong\u003e(C) \u003c/strong\u003e\u003cem\u003eLactiplantibacillusplantarum\u003c/em\u003e TBRC-15420, \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003eTBRC-15434, \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, and \u003cstrong\u003e(F)\u003c/strong\u003ethe multi-strains group—exhibited a normal histological structure with well-defined white pulp (WP) (outlined by the yellow dashed line) and red pulp (RP) regions, separated by a marginal zone with active macrophages (yellow arrows). All images were captured at 100× magnification.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/b75e1049205b691ed0068d15.png"},{"id":84892881,"identity":"114156eb-c16e-48b8-9f98-1edddb6831d0","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":135171,"visible":true,"origin":"","legend":"\u003cp\u003eIntestinal Secretory IgA Levels in Mice After \u003cem\u003eSalmonella\u003c/em\u003eTyphimurium Infection\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/47086c2fe743cf9ecb37d4b1.png"},{"id":84892878,"identity":"e9ad96c8-b005-4009-9f55-d68e29a9d2b9","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":177186,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of bacterial phyla in mice feces before and after \u003cem\u003eS. \u003c/em\u003eTyphimurium challenge\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/65c3e4cb3d5cbd6876e500dc.png"},{"id":84892884,"identity":"ae28dbfc-61c4-4791-89c0-4a1ca4742115","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":510067,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity of fecal microbiota before and after \u003cem\u003eSalmonella\u003c/em\u003eTyphimurium infection in mice. Box plots show the Shannon, Simpson, and Chao1 alpha diversity indices at the phylum and genus levels for fecal microbiota in mice treated with different probiotics and a control group, before and after \u003cem\u003eS.\u003c/em\u003eTyphimurium SC2451-3 challenge. The median is represented by the line within each box, with whiskers indicating the range of values. Outliers and individual samples are marked by dots.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/14a782244fc0f9986954ad6f.png"},{"id":84893061,"identity":"7100cacb-943d-473e-b9f8-e6dac054c046","added_by":"auto","created_at":"2025-06-18 13:19:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":454373,"visible":true,"origin":"","legend":"\u003cp\u003eBeta diversity of fecal microbiota before and after \u003cem\u003eSalmonella\u003c/em\u003eTyphimurium infection in mice. Beta diversity of the microbiome in mice treated with probiotics or a control group, shown through PCoA plots based on Bray–Curtis distance and Jaccard index at the phylum and genus levels. Red dots represent pre-challenge samples, while blue dots indicate post-challenge samples with \u003cem\u003eS.\u003c/em\u003e Typhimurium SC2451-3.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/9fb3c738b5a2ed0b433733d0.png"},{"id":100371885,"identity":"70a10b5e-f0cf-44a9-a40a-5e701d336bf1","added_by":"auto","created_at":"2026-01-16 08:11:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8703537,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/6f657e37-67a1-4832-8833-4b7c054f1906.pdf"},{"id":84892868,"identity":"a6f2c120-2fbb-4430-b925-3715d6a24103","added_by":"auto","created_at":"2025-06-18 13:11:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":257787,"visible":true,"origin":"","legend":"","description":"","filename":"3SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6367837/v1/656618bd74ff80470be0dd46.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Harnessing Native Swine Probiotics: Unveiling Their Protective Shield Against Salmonella Typhimurium – Insights from an Immune and Gut Microbiome Perspective","fulltext":[{"header":"Background","content":"\u003cp\u003eInfections caused by \u003cem\u003eSalmonella enterica\u003c/em\u003e pose significant challenges to the livestock industry, with \u003cem\u003eS. enterica\u003c/em\u003e serovar Typhimurium (\u003cem\u003eS. Typhimurium\u003c/em\u003e) being a leading cause of economic losses and public health concerns [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This serotype infects a wide range of hosts, causing gastrointestinal and systemic diseases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and is a common cause of severe gastroenteritis in livestock such as poultry, pigs, and cattle [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. Typhimurium\u003c/em\u003e spreads primarily through the fecal-oral route, with contaminated feed, water, and farm environments serving as reservoirs for infection. Poor hygiene and overcrowded conditions on farms exacerbate outbreaks, leading to diarrhea, fever, and loss of appetite. Severe cases may result in septicemia, organ damage, or death, particularly in young or immunocompromised animals [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimicrobial therapy is the standard treatment; however, widespread and indiscriminate antibiotic use has resulted in the emergence of resistant \u003cem\u003eSalmonella\u003c/em\u003e strains, posing challenges for disease control [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This highlights the urgent need for alternative, sustainable strategies. The gut microbiome, which regulates intestinal homeostasis and immune responses, plays a pivotal role in disease resistance. Commensal gut bacteria perform key functions such as fermenting complex carbohydrates, producing vitamins, and inhibiting pathogen growth [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The intestinal barrier, composed of the mucus layer, epithelial cells, and immune components, works synergistically with the microbiome to combat pathogenic organisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGut microbiota dysbiosis has been identified as a primary contributor to post-weaning diarrhea and related infections in piglets [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Probiotics, live microorganisms that confer health benefits, have shown promise in restoring microbiota balance, enhancing nutrient absorption, and modulating immune defenses in swine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. They work by competing with pathogens for nutrients and adhesion sites, producing antimicrobial compounds, and strengthening gut integrity [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For instance, lincomycin-treated swine experience dysbiosis characterized by increased \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e populations and decreased fiber-degrading microbes like \u003cem\u003eFibrobacter\u003c/em\u003e and \u003cem\u003eTreponema\u003c/em\u003e, underscoring the importance of microbiota preservation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study hypothesizes that probiotics sourced from native swine feces can mitigate\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. Typhimurium\u003c/em\u003e infections by reducing pathogen colonization, preventing systemic spread, and restoring microbiome balance. Using a mouse model, the research evaluates the protective efficacy of single-strain and multi-strain probiotics on bacterial load, immune responses, and gut microbiota composition. By investigating these aspects, this study aims to establish probiotics as a sustainable alternative to antibiotics for managing \u003cem\u003eSalmonella\u003c/em\u003e infections, improving animal health, and enhancing food safety\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and housing\u003c/h2\u003e \u003cp\u003eSpecific-pathogen-free (SPF) male Mus musculus (ICR) mice, aged 6\u0026ndash;7 weeks, were obtained from the National Laboratory Animal Center at Mahidol University (NLAC-MU), Thailand. The mice were housed in groups of three per cage, maintained on a 12-hour light/dark cycle at a constant temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and relative humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. The mice were fed a basal diet and given water without restriction. The bedding of mice was changed every three days, and the health status of the animals was monitored regularly. They were acclimatized for one week prior to the start of the experiments. All animal procedures were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of Srinakharinwirot University, under approval number COA/AE-003-2566.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProbiotic strains and inoculum preparation for oral administration\u003c/h3\u003e\n\u003cp\u003eThe probiotic strains used in this study, including \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e LC5.2 (TBRC-15420), \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e NL1.2 (TBRC-15434), and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e YH14 (TBRC-19857), were isolated from the feces of native swine. These strains have been previously assessed and demonstrated to exhibit robust probiotic properties and safety in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. Comprehensive details on the probiotic traits and safety of each strain are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. To prepare the inoculum, \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 was grown in De Mann, Rogosa, and Sharpe (MRS) broth and incubated anaerobically at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15443 was cultured in Luria-Bertani (LB) broth with shaking at 37\u0026deg;C and 180 rpm for 24 hours. \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 was cultivated in Potato Dextrose Broth (PDB) at 37\u0026deg;C for 24 hours. After incubation, cells were harvested by centrifugation at 5,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C, washed twice with PBS, and resuspended to the desired concentration. The optical density (OD) at 600 nm was adjusted to 1.0 (approximately 1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e CFU/mL) for \u003cem\u003eL. plantarum\u003c/em\u003e, 1.3 (approximately 1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e CFU/mL) for \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e, and 2.0 (approximately 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e CFU/mL) for \u003cem\u003eS. cerevisiae\u003c/em\u003e. For the mixed culture, equal volumes (1 mL) of each strain were combined, centrifuged, and resuspended in 1 mL PBS.\u003c/p\u003e\n\u003ch3\u003ePathogen and preparation of bacteria for high-dose challenge\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhimurium (ESBL-producing: isolated strain, Accession no: KT12325) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], was kindly provided by the Microbial Technology for Agriculture, Food, and Environment Research Center, Faculty of Science and Digital Innovation, Thaksin University, Thailand. The preparation of the challenge bacteria was conducted according to a previously described protocol [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Initially, bacteria from a frozen glycerol stock were grown on tryptic soy agar (TSA; Himedia, Mumbai, India) and incubated at 37\u0026deg;C for 14\u0026ndash;18 hours. A few colonies were then transferred to tryptic soy broth (TSB; Himedia, Mumbai, India) and incubated with shaking at 200 rpm for 12\u0026ndash;16 hours at 37\u0026deg;C. After centrifugation at 5,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes, the pellet was washed twice with PBS, resuspended in PBS, and the OD at 600 nm was measured to adjust the inoculum to 1.0 (approximately 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/mL). Serial dilutions were plated and incubated overnight at 37\u0026deg;C for 24 hours, and CFUs were counted to confirm the inoculum concentration.\u003c/p\u003e\n\u003ch3\u003eExperimental design for probiotic administration in mouse model\u003c/h3\u003e\n\u003cp\u003eAfter 1 week of acclimatization, mice were randomly assigned to five groups (six mice per group) for 30 days of probiotic treatment. Fecal samples were collected before the challenge to confirm the absence of \u003cem\u003eSalmonella\u003c/em\u003e. Following the treatment, mice were challenged with \u003cem\u003eS.\u003c/em\u003e Typhimurium SC2451-3 via oral gavage. The experimental design for evaluating probiotic protection against \u003cem\u003eS.\u003c/em\u003e Typhimurium infection is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Each group received specific treatments for 30 days before the challenge. The control group received sterile PBS, the TBRC-15420 group received \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 (1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e CFU/mL), the TBRC-15434 group received \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 (1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e CFU/mL), and the TBRC-19857 group received \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 (1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e CFU/mL). The multi-strain group received a combination of all three strains, and all groups were challenged with \u003cem\u003eS.\u003c/em\u003e Typhimurium (1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/mL).\u003c/p\u003e \n\u003ch3\u003eMonitoring clinical symptoms, body weight, and survival rate\u003c/h3\u003e\n\u003cp\u003eBody weight was measured and recorded both before and after the challenge. After infection, mice were monitored daily for signs of distress or illness, and mortality was tracked for each group. Euthanasia was performed early if mice exhibited signs of imminent death, such as rapid weight loss (\u0026gt;\u0026thinsp;20%), lack of food or water intake, failure to groom, or unresponsiveness to stimuli.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThe samples were collected following a procedure described in a previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], with minor modifications. After the probiotic treatment period, three mice from each group were randomly selected for pre-challenge assessment of gut microbiome, and the data obtained were used to compare results before and after the challenge. Subsequently, after the challenge, one mouse from each group was randomly selected, anesthetized with isoflurane, and humanely euthanized on days 1, 3, and 5 of the post-challenge periods. For intestinal fluid collection, the small intestine was carefully extracted from the gastro-duodenal to the ileocecal junction. The intestinal contents were flushed with 2 mL of sterile PBS. The fluid was then divided into two tubes (1 mL each), with one tube designated for bacterial load quantification and the other for total secretory IgA antibody determination. The organs (small intestine, colon, liver, and spleen) were carefully isolated and placed in separate Petri dishes with sterile PBS to remove any blood and contaminants. These organs for bacterial translocation and histological analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of bacterial load\u003c/h3\u003e\n\u003cp\u003eThe bacterial load in the small intestine and its translocation to liver and spleen were assessed following the method described in a previous study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] with minor modifications. To evaluate the bacterial load in the small intestine, 1 mL of intestinal fluid was serially diluted in PBS followed by serial 10-fold dilutions. For determination of the bacterial translocation, the liver and spleen were aseptically cut into 1 g pieces, finely minced using sterile scissors, and homogenized in 1 mL of sterile PBS. This mixture was thoroughly mixed and subjected to serial 10-fold dilutions. Suitable dilutions from each sample were plated in triplicate on Salmonella-Shigella (SS) agar and incubated at 37\u0026deg;C for 24 hours. Colony-forming units (CFU) of \u003cem\u003eS.\u003c/em\u003e Typhimurium were then counted.\u003c/p\u003e\n\u003ch3\u003eHistological analysis\u003c/h3\u003e\n\u003cp\u003eMice organs were collected for histopathological analysis according to standard protocols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Prior to necropsy, mice were fasted overnight, anesthetized with isoflurane, humanely euthanized, and placed on a sterile dissection bench. A midline incision was made from the navel to the mouth, with additional lateral cuts. The skin, muscle layers, and abdominal membrane were removed to expose the internal organs. The small intestine, colon, liver, and spleen were carefully separated, rinsed in sterile phosphate-buffered saline to remove blood and contaminants, and prepared for histological analysis. Organs were fixed in 4% paraformaldehyde phosphate buffer for 24 hours, dehydrated in graded ethanol solutions (70\u0026ndash;100%), and cleared with xylene using the Leica TP1020 Tissue Processor. The tissues were embedded in paraffin with the MEDITE TES Valida system and solidified. Thin sections (4\u0026ndash;6 \u0026micro;m) were cut using the HistoCore MULTICUT microtome, flattened in a warm water bath, mounted on glass slides, deparaffinized with xylene, rehydrated, and stained with hematoxylin and eosin. The stained sections were then dehydrated, cleared, and mounted with coverslips for examination under a light microscope (Olympus UC50, Japan).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of total secretory immunoglobulin A in the intestinal fluids\u003c/h2\u003e \u003cp\u003eThe intestinal fluid from each mouse was centrifuged at 12,000 rpm for 10 minutes at 4\u0026deg;C. The supernatant was filtered through sterile 0.22 \u0026micro;m syringe filters and stored at -80\u0026deg;C until analysis. Secretory IgA levels were measured using an ELISA kit, following the manufacturer\u0026rsquo;s protocol (IgA Mouse Uncoated ELISA Kit, Invitrogen, Thermo Fisher Scientific). An anti-mouse IgA monoclonal antibody was coated onto a microplate and incubated overnight at 4\u0026deg;C. After washing, the plate was blocked with blocking buffer and incubated at room temperature for 2 hours, followed by two washes with wash buffer (1x PBS, 0.05% Tween 20). Each sample or standard were added to the appropriate wells and incubated at room temperature for 2 hours. The plate was washed four times, then HRP-conjugated anti-mouse IgA polyclonal antibody was added to each well and incubated at room temperature for 1 hour, followed by another four washes. Tetramethylbenzidine (TMB) substrate solution was added to each well and incubated at room temperature for 15 minutes. The reaction was stopped by adding 2 N H₂SO₄ to each well, and absorbance was measured at 450 nm for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiome analysis\u003c/h2\u003e \u003cp\u003eThree mice from each group were randomly selected for analysis before and after the \u003cem\u003eS.\u003c/em\u003e Typhimurium challenge. Fecal samples were collected from the colon, and total DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) according to the manufacturer\u0026rsquo;s instructions. Bacterial composition of the gut microbiome was analyzed through next-generation sequencing, following established protocols [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Full-length 16S rDNA was amplified via PCR using universal 16S rRNA primers (27F/1492R) with nanopore adaptor tails. Multiplexing barcodes were then incorporated, followed by purification and quantification. The pooled DNA samples were further purified, subjected to adaptor ligation, and sequenced using a MinION Mk1C device. Bioinformatic analysis included base calling, quality assessment, demultiplexing, adaptor trimming, clustering, polishing, and taxonomic identification. Raw FAST5 sequencing data were basecalled using the Guppy basecaller in super-accuracy (SUP) mode. FASTQ sequences were quality-checked with MinIONQC, demultiplexed, and trimmed using Porechop. The processed reads were analyzed with NanoCLUST for clustering, polishing, and taxonomic classification. The resulting taxonomic profiles and abundance data were further examined using MicrobiomeAnalyst [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll values were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical differences between the various groups were evaluated by Student\u0026rsquo;s t-test. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSurvival and clinical symptoms after\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003eTyphimurium infection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNo mortality occurred in any group during the five-day experiment. Probiotic administration mitigated symptoms of infection, promoting recovery and reducing disease severity. On day 1 post-infection, all mice appeared normal. By day 2, infected mice in all groups showed decreased activity, lethargy, reduced food intake, and ruffled fur, with some control mice also exhibiting mild diarrhea. By day 5, some mice had recovered, while others still displayed symptoms, though less severe than on day 2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of probiotics on weight loss in mice exposed to\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003eTyphimurium\u003c/b\u003e\u003c/p\u003e \u003cp\u003eProbiotics mitigated weight loss in mice exposed to \u003cem\u003eS.\u003c/em\u003e Typhimurium, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The control group experienced the greatest weight loss, averaging 4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 g (10.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81%). Conversely, mice treated with probiotics showed significantly less weight loss. The multi-strain group exhibited the strongest protective effect, with only 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 g (2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) of weight loss. Among the single-strain treatments, \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 provided the most protection, with a weight loss of 1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 g (3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), followed by \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 at 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 g (4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 at 2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 g (6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). No significant differences were found among the probiotic-treated groups.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacterial load in the gut, liver, and spleen\u003c/h2\u003e \u003cp\u003eProbiotic treatment significantly reduced \u003cem\u003eS.\u003c/em\u003e Typhimurium SC2451-3 levels in the gut and limited its spread to the liver and spleen (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On day 1 post-infection, all groups had high bacterial loads in the gut. The control group showed notable bacterial presence in the liver and spleen, while probiotic-treated groups showed bacteria only in the liver of the \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 group. The \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 groups had lower bacterial counts in the spleen compared to the multi-strain group. By day 3, probiotic groups, especially \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434, showed significant reductions in gut bacteria, with no bacteria in the liver or spleen. The multi-strain and \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 groups also showed reduced bacterial presence in the liver and spleen. On day 5, the control group still harbored bacteria in the liver and spleen, while all probiotic-treated groups completely eliminated the pathogen from the gut, liver, and spleen, highlighting the probiotics' role in pathogen clearance.\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 load of \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium in mice at different days post-infection for various probiotic treatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDPI*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eBacterial load (CFU/ml or CFU/g tissue)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eTBRC-15420\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eTBRC-15434\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eTBRC-19857\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eMulti-strains\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntestinal fluid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.95E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.00E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.00E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.13E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntestinal fluid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.20E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.70E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.00E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.60E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.90E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntestinal fluid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.00E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.80E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\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*DPI refers to days post-infection and ND indicates no detection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopathological examination of mice infected with\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003eTyphimurium\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe small intestine of all groups remained unaffected by the \u003cem\u003eS.\u003c/em\u003e Typhimurium challenge. The control group exhibited normal intestinal structure, with intact villi, crypts, and muscle layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, all probiotic-treated groups maintained well-preserved intestinal architecture without signs of inflammation or tissue damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In contrast, the liver of control mice showed severe pathological changes, including focal necrosis, neutrophil clusters, inflammatory cell infiltration, perivascular leukocyte accumulation, vascular dilation, congestion, hepatocyte swelling, and vacuolization, indicating significant inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Probiotic-treated mice, however, exhibited normal liver histology with smooth tissue structure, well-organized hepatocytes, and intact blood vessels, free of necrosis or swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Similarly, the spleen of control mice displayed marked lymphoid hyperplasia, with dense lymphocyte accumulation, disrupted splenic architecture, indistinct red and white pulp boundaries, and increased vacuolization, reflecting an intense immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, probiotic-treated groups maintained normal splenic morphology, with well-defined red and white pulp regions separated by a clear marginal zone. Macrophages were prominently observed in the red pulp and marginal zone across all probiotic-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSecretory IgA levels\u003c/h2\u003e \u003cp\u003eSecretory IgA (sIgA) levels in intestinal fluid samples were measured on days 1, 3, and 5 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). On day 1 post-infection, the control group had the lowest sIgA level (15.95 ng/mL). Among probiotic-treated groups, \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 exhibited the highest sIgA level (21.04 ng/mL), followed by \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 (17.88 ng/mL), the multi-strain group (16.85 ng/mL), and \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 (16.67 ng/mL). On day 3, sIgA levels in the control group increased slightly to 16.13 ng/mL. In comparison, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 peaked at 21.43 ng/mL, while \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 maintained a high level (18.93 ng/mL). The multi-strain and \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 groups showed moderate increases at 16.92 and 16.87 ng/mL, respectively. On day 5, sIgA levels declined in all groups, but probiotic-treated mice maintained higher levels than the control (15.76 ng/mL). \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 retained the highest level (21.42 ng/mL), followed by \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 (18.56 ng/mL), the multi-strain group (17.08 ng/mL), and \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 (16.53 ng/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiome analysis\u003c/h2\u003e \u003cp\u003eThis study assessed the impact of probiotics on gut microbiome composition before and after \u003cem\u003eS.\u003c/em\u003e Typhimurium infection, with fecal samples analyzed by \u003cem\u003e16S rRNA\u003c/em\u003e gene sequencing. Rarefaction curves showed sufficient sampling, with all samples demonstrating 100% Good\u0026rsquo;s coverage (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRelative abundance of bacteria\u003c/h2\u003e \u003cp\u003eBacterial relative abundance was analyzed at phylum levels, as show in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Prior to infection, \u003cem\u003eBacteroidetes\u003c/em\u003e was the most abundant phylum in all groups, followed by \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eDeferribacteres\u003c/em\u003e, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e. After infection, shifts in microbial composition were observed. In the control group, \u003cem\u003eBacteroidetes\u003c/em\u003e decreased from 46.31\u0026ndash;41.02%, while \u003cem\u003eFirmicutes\u003c/em\u003e rose from 44.59\u0026ndash;46.44%. \u003cem\u003eProteobacteria\u003c/em\u003e increased from 6.78\u0026ndash;10.43%, and \u003cem\u003eDeferribacteres\u003c/em\u003e grew from 1.38\u0026ndash;2.11%. \u003cem\u003eVerrucomicrobia\u003c/em\u003e was absent post-infection (from 0.95%) In the \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 group, \u003cem\u003eBacteroidetes\u003c/em\u003e increased significantly from 47.29\u0026ndash;57.13%. In contrast, \u003cem\u003eFirmicutes\u003c/em\u003e decreased from 43.62\u0026ndash;36.82%, \u003cem\u003eProteobacteria\u003c/em\u003e dropped from 7.10\u0026ndash;4.31%, and \u003cem\u003eDeferribacteres\u003c/em\u003e showed a slight reduction from 1.98\u0026ndash;1.18%. \u003cem\u003eVerrucomicrobia\u003c/em\u003e, which was undetected before infection, emerged at 0.57% post-infection. In the \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 group, \u003cem\u003eBacteroidetes\u003c/em\u003e declined from 59.64\u0026ndash;53.66%, \u003cem\u003eFirmicutes\u003c/em\u003e increased from 31.97\u0026ndash;41.00%, and \u003cem\u003eProteobacteria\u003c/em\u003e decreased from 8.09\u0026ndash;4.71%. \u003cem\u003eVerrucomicrobia\u003c/em\u003e remained stable at 0.31%, and \u003cem\u003eDeferribacteres\u003c/em\u003e emerged at 0.32%. For the \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 group, \u003cem\u003eBacteroidetes\u003c/em\u003e decreased from 54.41\u0026ndash;42.24%, \u003cem\u003eFirmicutes\u003c/em\u003e rose from 34.80\u0026ndash;48.23%, and \u003cem\u003eProteobacteria\u003c/em\u003e slightly decreased from 8.67\u0026ndash;7.50%. \u003cem\u003eVerrucomicrobia\u003c/em\u003e dropped from 1.44\u0026ndash;0.70%, and \u003cem\u003eDeferribacteres\u003c/em\u003e doubled from 0.68\u0026ndash;1.34%. In the multi-strain group, \u003cem\u003eBacteroidetes\u003c/em\u003e increased from 50.34\u0026ndash;51.97%, \u003cem\u003eFirmicutes\u003c/em\u003e decreased from 44.59\u0026ndash;40.65%, and \u003cem\u003eProteobacteria\u003c/em\u003e showed a slight rise from 4.40\u0026ndash;5.73%. \u003cem\u003eDeferribacteres\u003c/em\u003e grew from 0.67\u0026ndash;1.31%, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e appeared at 0.34%. Post-infection, \u003cem\u003eFirmicutes\u003c/em\u003e became the most abundant phylum in the control and \u003cem\u003eS. cerevisiae\u003c/em\u003e TBRC-19857 groups, while \u003cem\u003eBacteroidetes\u003c/em\u003e remained dominant in the other groups. Single-strain probiotics caused a decrease in \u003cem\u003eProteobacteria\u003c/em\u003e, while the control group saw an increase in this phylum. \u003cem\u003eVerrucomicrobia\u003c/em\u003e and \u003cem\u003eDeferribacteres\u003c/em\u003e remained relatively stable across all groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAlpha diversity\u003c/h2\u003e \u003cp\u003eAlpha diversity, which assesses the richness and evenness of bacterial species within the gut ecosystem, was evaluated using the Shannon, Simpson, and Chao1 indices at the phylum and genus levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). No significant changes in bacterial diversity were observed before and after the \u003cem\u003eS.\u003c/em\u003e Typhimurium challenge in all groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBeta diversity\u003c/h2\u003e \u003cp\u003eBeta diversity, which measures differences in microbial composition between samples, was analyzed using Bray-Curtis and Jaccard indices at the phylum and genus levels, with results visualized through PCoA plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Statistical analysis using PERMANOVA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed no significant differences in microbial composition between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe increasing use of antimicrobial drugs in livestock contributes to the development of drug-resistant \u003cem\u003eSalmonella\u003c/em\u003e strains, which can pose significant risks for zoonotic transmission. Probiotics have emerged as a promising alternative to antibiotics, with potential to promote gut health and reduce reliance on antimicrobial treatments. This study demonstrates that probiotic supplementation, whether as a single strain or a multi-strain combination, offers protective effects against \u003cem\u003eS.\u003c/em\u003e Typhimurium infection and supports gut homeostasis in a mouse model.\u003c/p\u003e \u003cp\u003eIn this study, mice were pre-treated with \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e TBRC-15420, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e TBRC-15434, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, or a multi-strain combination for 30 days before being challenged with \u003cem\u003eS.\u003c/em\u003e Typhimurium SC2451-3. None of the infected mice succumbed to the infection. However, variations in clinical signs were noted, particularly in the control group, which exhibited mild diarrhea. Despite controlling for genetic background, environmental factors, and infection dose, the mice showed diverse physiological responses. This suggests that individual variations in immune function or microbial resilience could influence the response to infection.\u003c/p\u003e \u003cp\u003ePrevious research has shown that \u003cem\u003eS.\u003c/em\u003e Typhimurium infection is typically associated with significant weight loss in livestock [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, probiotic supplementation, especially the multi-strain treatment, notably reduced weight loss in infected mice, indicating a possible synergistic interaction between the bacterial and yeast strains that enhances immune modulation and inhibits pathogen growth. Among the single-strain treatments, \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 consistently showed the greatest protective effect, likely due to its specific probiotic properties. These findings align with previous studies, which reported that probiotics reduce weight loss during bacterial infections. For example, pre-treatment with \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e or \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e alleviated weight loss and improved survival rates in \u003cem\u003eS.\u003c/em\u003e Typhimurium-infected mice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther investigation into the impact of probiotics on infection and translocation of \u003cem\u003eS.\u003c/em\u003e Typhimurium revealed that probiotic supplementation significantly reduced bacterial loads in intestinal fluid, liver, and spleen across different time points post-infection. On day 1 post-infection, bacterial levels were high in all groups, including the liver and spleen of the control group. However, probiotic-treated groups exhibited reduced bacterial loads, with \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 showing early systemic protection against \u003cem\u003eS.\u003c/em\u003e Typhimurium. By day 3, these probiotic groups demonstrated significant reduction in bacterial loads in the gut, with no detectable bacteria in the liver or spleen, indicating effective pathogen clearance. By day 5, all probiotic-treated groups had cleared \u003cem\u003eS.\u003c/em\u003e Typhimurium from the intestinal fluid, liver, and spleen, whereas the control group continued to show bacterial presence. The immune response in the liver, particularly through Kupffer cells, may have contributed to the reduced pathogen levels observed in probiotic-treated groups [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The spleen, which plays a key role in pathogen storage and filtration, had higher bacterial loads than the liver, further emphasizing the importance of the immune system in controlling pathogen spread [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These results align with previous studies that have shown probiotics' ability to reduce bacterial loads and prevent systemic spread of pathogens. For example, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e HN001 has been shown to reduce \u003cem\u003eS.\u003c/em\u003e Typhimurium loads by 100-fold in the spleen and liver [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], Similarly, \u003cem\u003eLactobacillus casei\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e have been demonstrated to prevent bacterial translocation and significantly reduce \u003cem\u003eS.\u003c/em\u003e Typhimurium infections [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to bacterial load reduction, we examined the intestinal histology for signs of enteritis, typically characterized by neutrophil infiltration, goblet cell depletion, and epithelial damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Interestingly, despite a high bacterial load in the intestine, no evidence of enteritis was observed, and the small intestine remained structurally intact. This aligns with previous studies showing that while \u003cem\u003eS.\u003c/em\u003e Typhimurium-infected mice often develop systemic infections, gastrointestinal symptoms such as enteritis may not always occur [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo confirm the effect of probiotics in preventing pathogen translocation, histological analysis of the liver and spleen was performed. The liver, which plays a crucial role in pathogen elimination [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], showed normal histology in probiotic-treated mice, suggesting that probiotics help prevent liver inflammation and damage. The spleen complements liver function by filtering blood and coordinating adaptive immunity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It consists of the red pulp, which houses macrophages that remove pathogens, and the white pulp, which facilitates immune responses [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After \u003cem\u003eS.\u003c/em\u003e Typhimurium infection, control mice displayed severe splenic tissue damage, whereas probiotic-treated mice maintained normal morphology, with active macrophages combating infection. These findings suggest that probiotics help preserve splenic function and reduce inflammation. Consistent with previous studies, our results demonstrate that probiotics reduce bacterial translocation to systemic sites. \u003cem\u003eLactobacillus casei\u003c/em\u003e CRL431 and \u003cem\u003eLactobacillus paracasei\u003c/em\u003e CNCMI-1518 significantly lowered \u003cem\u003eS.\u003c/em\u003e Typhimurium loads in the spleen and liver [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], while \u003cem\u003eBifidobacterium lactis\u003c/em\u003e INL1 prevented bacterial dissemination [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings underscore the potential of probiotics in mitigating systemic complications from enteric infections.\u003c/p\u003e \u003cp\u003eProbiotic supplementation also increased the production of secretory IgA (sIgA), which plays a crucial role in mucosal immunity by binding and preventing the adhesion of enteric pathogens, and facilitating their clearance via mucus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Among the probiotic-treated groups, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 and \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 were most effective in enhancing sIgA production. The increased sIgA levels observed in probiotic-treated groups likely contributed to bacterial clearance by preventing adhesion of \u003cem\u003eS.\u003c/em\u003e Typhimurium to the intestinal epithelium, thereby reducing its ability to colonize and translocate to systemic sites. These findings highlight the potential of probiotics to boost mucosal immune defenses during infection, as supported by previous studies demonstrating that probiotic supplementation elevates sIgA levels and modulates immune responses. For instance, \u003cem\u003eL. casei\u003c/em\u003e and \u003cem\u003eBifidobacterium animalis\u003c/em\u003e supplementation led to significant increases in IgA\u003csup\u003e+\u003c/sup\u003e cells in the small intestine and higher sIgA levels in the intestines following \u003cem\u003eS.\u003c/em\u003e Typhimurium challenge [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, total sIgA levels increased in the intestinal fluid of mice fed with \u003cem\u003eL. casei\u003c/em\u003e CRL 431 following a challenge with \u003cem\u003eS.\u003c/em\u003e Typhimurium [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to immune modulation, probiotic pre-treatment in this study led to shifts in gut microbiota composition. Notably, probiotics reduced the abundance of \u003cem\u003eProteobacteria\u003c/em\u003e, which includes pathogens like \u003cem\u003eSalmonella\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while maintaining \u003cem\u003eBacteroidetes\u003c/em\u003e, which are beneficial for gut health. \u003cem\u003eBacteroidetes\u003c/em\u003e play a critical role in immune modulation and short-chain fatty acid (SCFA) production, supporting gut homeostasis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The reduction in \u003cem\u003eProteobacteria\u003c/em\u003e and maintenance of \u003cem\u003eBacteroidetes\u003c/em\u003e suggest that probiotics contribute to gut stability during infection. However, alpha and beta diversity analyses showed no significant differences between pre- and post-infection groups, indicating that the gut microbiome composition remained stable despite S. Typhimurium challenge. This stability may reflect microbiome resilience or the protective effects of probiotics in maintaining gut homeostasis. Although overall microbial diversity was not significantly altered, the observed reduction in Proteobacteria and the maintenance of Bacteroidetes, particularly in single-strain probiotic groups, suggest that probiotics may support gut health by modulating specific bacterial taxa. Further research should investigate how probiotic strain selection, dosage, and host factors influence microbial diversity and gut health outcomes.\u003c/p\u003e \u003cp\u003eOur findings provide further support for probiotics as a viable strategy to mitigate \u003cem\u003eS.\u003c/em\u003e Typhimurium infection without relying on antibiotics. This is particularly relevant in livestock production, where reducing antibiotic use is a global priority to combat antimicrobial resistance. Further studies should assess the long-term stability of probiotic colonization and its implications for pathogen resistance over extended periods.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that probiotics from native swine feces, including \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e TBRC-15420, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e TBRC-15434, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, and their combination, provide significant protection against \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium infection in mice. Both single-strain and multi-strain treatments effectively reduced weight loss, with the most notable effects in the multi-strain combination and TBRC-15420. These probiotics reduced pathogen burden, prevented systemic dissemination, and maintained the integrity of intestinal and systemic organs, including the liver and spleen. They also enhanced secretory IgA levels, particularly in TBRC-15434 and TBRC-15420. Additionally, they promoted gut microbiome stability by increasing beneficial bacteria and reducing harmful ones. These findings highlight the potential of probiotics as an effective, antibiotic-free approach to improving livestock health and resilience to infections. Future studies should explore the long-term effects of these probiotics in different animal models and their potential for broader applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments received approval from the Ethics and Research Standardization Section, Srinakharinwirot University (Approval number: COA/AE-003-2566).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe probiotic strains have been deposited at the Thailand Bioresources Research Center (TBRC) under the following accession numbers: \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e LC5.2 (TBRC-15420), \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e NL1.2 (TBRC-15434), and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e YH14 (TBRC-19857). All the sequencing data presented in this study is available at the Sequence Read Archive (SRA), under Bioproject accession number ID PRJNA1190112.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by the National Research Council of Thailand (NRCT): NRCT5-RGJ63005-085.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKK and ML contributed to the conceptualization and methodology of the study. KK, ML, and MT conducted the mouse model experiments and interpreted the results. KK, VS, and SP performed the gut microbiome analysis and interpreted the data. KK was responsible for the original draft preparation, while ML, MT, VS, and SP contributed to the review and editing of the manuscript by critically evaluating and revising it. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank the Central Equipment Center and the Laboratory Animal Center of the Research and International Affairs, Faculty of Medicine, Srinakharinwirot University, for providing research facilities, equipment, and laboratory support for animal experiments. We also extend our special thanks to Dr. Akkanee Pewhom, Department of Biological Science, Faculty of Science and Digital Innovation, Thaksin University, for his expertise in histology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKuang X, Hao H, Dai M, et al. 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Microbiol. 2011;2:93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2011.00093\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2011.00093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotics, Lactiplantibacillus plantarum, Bacillus amyloliquefaciens, Saccharomyces cerevisiae, Protective effect, Salmonella Typhimurium, Immune response, Gut microbiome","lastPublishedDoi":"10.21203/rs.3.rs-6367837/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6367837/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotics confer strain-specific health benefits, including protection against pathogenic infections. This study evaluated the protective efficacy of single- and multi-strain probiotics against \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium in a mouse model. Mice were administered \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e TBRC-15420, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e TBRC-15434, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e TBRC-19857, or their combination for 30 days prior to \u003cem\u003eS.\u003c/em\u003e Typhimurium challenge. Protective effects were assessed through survival rates, clinical symptoms, weight changes, pathogen clearance, histopathology, secretory IgA levels, and gut microbiota shifts using \u003cem\u003e16S rRNA\u003c/em\u003e sequencing. No mortality was observed; however, mice exhibited varying symptoms post-infection, with some recovering by day five. Probiotics mitigated weight loss, with the multi-strain combination being most effective, while \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 provided the strongest single-strain protection. Probiotics enhanced secretory IgA levels, with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e TBRC-15434 and \u003cem\u003eL. plantarum\u003c/em\u003e TBRC-15420 eliciting robust immune responses. All strains effectively reduced \u003cem\u003eS.\u003c/em\u003e Typhimurium levels in the small intestine and prevented its translocation to the liver and spleen, achieving complete bacterial clearance by day five. Probiotic pretreatment preserved the structural integrity of the intestine, liver, and spleen. It also promoted beneficial bacterial phyla such as \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e while suppressing \u003cem\u003eProteobacteria\u003c/em\u003e, thereby maintaining gut microbiome homeostasis during infection. These findings support probiotics as antibiotic alternatives for \u003cem\u003eSalmonella\u003c/em\u003e infection management, emphasizing their role in immune modulation and microbiota stability.\u003c/p\u003e","manuscriptTitle":"Harnessing Native Swine Probiotics: Unveiling Their Protective Shield Against Salmonella Typhimurium – Insights from an Immune and Gut Microbiome Perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 13:11:36","doi":"10.21203/rs.3.rs-6367837/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"643758b3-c7ff-48cd-9af4-d2ed1b34c68c","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-14T13:25:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 13:11:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6367837","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6367837","identity":"rs-6367837","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}