Lacticaseibacillus rhamnosus LRa05 Mitigates DSS-Induced Colitis via Boosting Beneficial Gut Microbiota and Facilitating CD4⁺Foxp3⁺ Treg Differentiation

Lacticaseibacillus rhamnosus LRa05 Mitigates DSS-Induced Colitis via Boosting Beneficial Gut Microbiota and Facilitating CD4⁺Foxp3⁺ Treg Differentiation | 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 Lacticaseibacillus rhamnosus LRa05 Mitigates DSS-Induced Colitis via Boosting Beneficial Gut Microbiota and Facilitating CD4⁺Foxp3⁺ Treg Differentiation Jia Dong, Xuxiao Chen, Yuxin Zhang, Yixiang Zhang, Zehua He, Shengning Liang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7649694/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted 11 You are reading this latest preprint version Abstract Background Lacticaseibacillus rhamnosus LRa05, originally isolated from infant feces, has been widely used as a probiotic in the food industry. However, its potential as an orally administered probiotic therapeutic for inflammatory bowel disease (IBD) remains underexplored, particularly from an immune regulatory perspective. Results In this study, we demonstrated that administration of LRa05 conferred protective effects in a DSS-induced colitis mouse model. Such protective effects were reflected by attenuated weight loss, preserved colon length, reduced spleen index, and diminished histopathological damage in the colon. Mechanistically, while LRa05 administration exerted minimal impact on the overall microbiota diversity, it altered the species composition. Notably, the abundance of the beneficial bacterium Akkermansia muciniphila was 10-fold higher in LRa05 treated mice compared to controls. This change was thought to further modulate microbial metabolic pathways. More importantly, LRa05 promoted the expansion of CD4⁺Foxp3⁺ Treg cells, as evidenced by a remarkable increase in Treg frequency in both the spleen and mesenteric lymph nodes. Consequently, proinflammatory cytokine levels were reduced, thereby excessive colonic inflammation was suppressed. Conclusions Taken together, our findings identify LRa05 as a potential therapeutic agent for DSS-induced colitis, with its protective effects mediated through enhancing beneficial gut microbiota and facilitating Treg cell differentiation. LRa05 Colitis Treg Akkermansia muciniphila Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal disorder characterized by persistent inflammation, encompassing two primary subtypes-Crohn’s disease (CD) and ulcerative colitis (UC), with UC being one of the most prevalent variants( 1 , 2 ). Common clinical manifestations include diarrhea, abdominal pain, fatigue, anemia, reduced appetite, and unintended weight loss. Over the past five decades, global IBD prevalence has surged exponentially, reaching approximately 6.8 million documented cases in 2023( 3 ). This dramatic rise is driven by complex interactions between environmental factors (e.g., Western dietary patterns, excessive antibiotic use), genetic susceptibility, and gut microbiota dysbiosis( 4 ). T lymphocytes (T cells) constitute central players within the adaptive immune system, orchestrating cell mediated immune responses essential for maintaining host health and defending against a spectrum of threats. A delicate balance between proinflammatory subsets (e.g., T helper 1 (Th1), Th2, Th17) and T regulatory cell (Treg) is essential for maintaining immune homeostasis( 5 , 6 ). Dysregulation of this balance in gastrointestinal track, particularly the imbalance between pro-inflammatory Th1/Th17 cells and suppressive Tregs, serves as a key driver of IBD pathogenesis( 7 , 8 ). Treg, defined by the CD4⁺Foxp3⁺ phenotype, suppress pro-inflammatory activity through two main mechanisms: contact-dependent inhibition and secretion of anti-inflammatory cytokines( 5 ). In IBD patients, mucosal Treg depletion or impaired suppressive function is frequently observed, triggering uncontrolled inflammation that ultimately leads to irreversible damage to the intestinal mucosal barrier( 9 , 10 ). Modulation of gut microbiota has emerged as a promising therapeutic strategy for gastrointestinal inflammatory diseases, with probiotics supplementation playing a central role. Probiotics, which are live microorganisms that confer health benefits when administered in adequate amounts, exert therapeutic effects in IBD through multiple complementary pathways: 1) replenishing depleted beneficial taxa and inhibiting pathogenic bacteria via competition for nutrients and intestinal adhesion sites; 2) producing short-chain fatty acids (SCFAs), which act as an energy source for colonocytes, and can lower intestinal pH to inhibit pathogenic growth; 3) upregulating tight junction proteins to reduce intestinal permeability and stimulating goblet cells to secrete mucin, a key component of the protective barrier against luminal antigens( 11 – 13 ). However, a critical gap remains in our understanding: whether probiotics can directly modulate Treg cell differentiation, a process tightly linked to IBD pathogenesis. Lacticaseibacillus rhamnosus LRa05 (hereafter referred to as “LRa05”), a strain isolated from infant feces, has been demonstrated as a probiotics strain to alleviate inflammatory markers and restore gut microbiota balance in mouse models of obesity and type 2 diabetes, thereby conferring significant health benefits( 14 , 15 ). Building on these evidences, the present study aims to conduct a comprehensive investigation to evaluate the potential therapeutic efficacy of LRa05 as an orally administrable probiotic formulation for IBD. Moreover, we aim to explore whether this strain can directly modulate T cell immunity. Through these investigations, this study aims to provide novel insights into the role of probiotics in IBD management and pave the way for the development of targeted microbiota-based therapies. 2. Methods 2.1 Probiotic strain The probiotic strain used in this study was Lacticaseibacillus rhamnosus LRa05 (LRa05), isolated from the feces of healthy infants in Qinghai Province, China. It was provided by Microhealthcare (Suzhou) Co., Ltd. (Suzhou, China) in the form of lyophilized powder, with an activity of 90 billion CFU/g. For activation, transfer 100 mg of the powder into 20 mL MRS broth and incubate statically at 37°C for 24 h. Harvest the activated culture by centrifugation at 8000 rpm, 10°C for 20 min and discard the supernatant. Resuspend the pellet in 2 mL fresh MRS broth and mix this suspension with 50% glycerol at 1:1 ratio. Store this mixture in cryovials at -80°C for further experiments. 2.2 LRa05 growth curve determination Cultivate LRa05 by dissolving 20 µL frozen culture in 20 mL fresh MRS broth statically at 37°C. Sampling was conducted every 2 hours for the determination of viable bacterial counts by measuring the OD₆₀₀ of the culture with SpectraMax iD3/iD5 microplate reader (Molecular Devices). Plot the resulting OD₆₀₀ values against time to generate the growth curve and identify the culture phase with maximal bacterial activity. After 18 hours of cultivation, serially dilute the culture and determine the OD₆₀₀ of each dilution. In parallel, plate 100 µL aliquots of the serial dilutions onto MRS agar, incubate at 37°C for 24 hours and count the colonies. Calculate the viable bacterial concentration (CFU/mL) from the average colony counts across appropriate dilutions, and generate a standard curve relating OD₆₀₀ to bacterial counts. 2.3 Mice The mice used in this experiment were healthy 8-week-old C57BL/6J mice. All mice were purchased from Jiangsu GemPharmatech Co., Ltd (Nanjing, China) and were housed under specific pathogen-free conditions in the Animal Resource Center at the Shanghai Jiao Tong University under protocols approved by the Institutional Animal Care and Use Committee (IACUC# A2023233-005). Before all experiments, the mice were acclimated for 7 days. To ensure a painless and stress-free process, mice were rendered unconscious via anesthesia induced by exposure to 3% isoflurane in an induction chamber (RWD AIJI-IE, Shenzhen, China), where needed. At the end of the experiment, mice were humanely euthanized. The primary method employed was carbon dioxide (CO 2 ) inhalation, administered in a pre-filled chamber at a controlled displacement rate of 30–70% of the chamber volume per minute to minimize distress. Following the cessation of movement and respiration, death was confirmed by cervical dislocation. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University and is consistent with the AVMA Guidelines for the Euthanasia of Animals. 2.4 Antibodies Antibodies for flow cytometry including Brilliant Violet (BV) 605-conjugated anti-CD4 (dilution ratio, 1:100; RM4-5), BV 421-conjugated anti-CD3 (dilution ratio, 1:100; 145-2C11) were from BioLegend. Allophycocyanin (APC)-conjugated anti-Foxp3 (dilution ratio, 1:100; FJK-16s) was from Invitrogen. Fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (dilution ratio, 1:100; 30F11) was from Miltenyi Biotec. 2.5 Flow cytometry For surface staining, cells isolated from mice were directly stained with antibodies in PBS at 4°C for 15 min. For transcription factor Foxp3 staining, cells pre-stained with surface markers were fixed and permeabilized in TF Fix/Perm buffer (BD Biosciences) at 4°C for 20 min, washed once with TF Perm/Wash buffer, and stained with target markers in the TF Perm/Wash buffer at 4°C for 15 min. The expression of surface and intracellular markers was analyzed with a BD LSR Fortessa flow cytometer. 2.6 Dextran sulfate sodium-induced colitis in mice Dextran sulfate sodium (DSS, Mw 36–50 kDa) was purchased from MP Biomedicals. Mice were divided into three groups, which is Control, DSS, and LRa05. In a 24-day experiment cycle, mice from Control group received normal water daily. Mice from DSS group received normal water daily for the first 10 days, but was gavaged with 200 uL PBS each mouse for 24 consecutive days. Mice from LRa05 group received normal water daily for the first 10 days, but were gavaged with 1 × 10⁹ CFU LRa05 in 200 uL PBS each mouse for 24 consecutive days. Beginning on day 11, the normal drinking water for DSS group and LRa05 group was switched to a DSS regimen, which is two cycles of 2% (w/v) DSS for 5 days followed by normal water for 2 days. On day 24, mice were euthanized and samples of spleen, mesenteric lymph nodes, colon, and feces were collected for experiments. Body weight and disease activity index (DAI) were measured daily to assess the health of the mice and to judge the success of the establishment of DSS model. The evaluation of the DAI is revised based on previous research( 16 ), and is shown in Fig. S1 . After the mice were euthanized, the spleen was weighed and the following formula was used to calculate the immune organ index: organ index = the weight of target organs (mg)/body weight (g). The length of the colon is measured as a physiological indicator. 2.7 Reverse transcription quantitative real-time PCR Total RNA of cells was extracted according to the manufacturer’s guide using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme). The first-strand cDNA synthesis was performed by reverse transcription using a HiScript III RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing, China). Subsequent qPCR was performed using ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in the CFX Connect Real-Time PCR System (Bio-Rad). The primers used for qPCR are listed in Table 1 . The amplification efficiency of all primers has been tested, and the optimized conditions were used in all qPCRs. Gene expression was calculated with the ∆∆ C t method normalized to the control gene encoding β-actin, and all measurements were performed in triplicate. Table 1 Primer sequences used for qPCR Gene Sequence Actin-F 5'-GGGAAATCGTGCGTGACAT-3' Actin-R 5'-GTCAGGCAGCTCGTAGCTCTT-3' TNF-α-F 5'-CCCTCACACTCAGATCATCTTCT-3' TNF-α-R 5'-GCTACGACGTGGGCTACAG-3' IFN-γ-F 5'-ATGAACGCTACACACTGCATC-3' IFN-γ-R 5'-CCATCCTTTTGCCAGTTCCTC-3' IL-6-F 5'-TAGTCCTTCCTACCCCAATTTCC-3' IL-6-R 5'-TTGGTCCTTAGCCACTCCTTC-3' 2.8 Histological evaluation of colon Colon specimens were first rinsed with PBS, fixed with paraformaldehyde (Servicebio, Wuhan, China) at a concentration of 4% (w/v), dehydrated and embedded by paraffin. Hematoxylin and eosin (H&E) were used to stain the sample after being sliced (4 µm slice). 2.9 Fecal metagenomic sequencing Fecal samples of 4 mice from each group were selected for metagenomic sequencing. Metagenomic DNA was extracted from the fecal samples using QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s protocol. Then, 0.8% agarose gel electrophoresis was used to measure the concentration and integrity of the DNA. The sequencing work was performed by BGI Group (Shenzhen, China). The raw data were processed by the sliding window method to remove low-quality sequences from the original sequence, resulting in high-quality clean reads, with host genomes removed. Finally, sickle software was used for tailoring and modifying the reads. 2.10 Data analysis Flow-cytometry data were processed and plotted using FlowJo 10.8.1. Colonic HE-stained colon sections were examined in SlideViewer. Other data were analyzed and graphed with GraphPad Prism 9.5. Statistical significance is indicated as ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001). 3. Results 3.1 LRa05 administration provided protection to mice in DSS-induced colitis model LRa05 was inoculated into MRS broth at a 0.1% (v/v) ratio and statically incubated at 37°C. OD₆₀₀ readings of the culture were recorded every 2 hours, and the resulting growth curve is presented in Fig. 1 A. According to the curve, LRa05 reached peak activity at approximately 18 hours post-inoculation. Therefore, all bacteria for gavage were collected at 18 hours post-cultivation. The DSS-induced colitis model was established as outlined in Fig. 1 B. During the adaptive gavage period, LRa05 exhibited an excellent safety profile in mice. Throughout 10 days continuous oral administration, no statistically significant changes in fecal consistency (data not shown) or body weight were observed compared to the Control group (Fig. 1 C). These results indicate that oral administration of LRa05 caused no adverse reactions or weight loss, confirming its safety and absence of harmful effects in this model. In the DSS-induced colitis model, LRa05 administration conferred significant protection to mice. As shown in Fig. 1 D, body weight of mice in the DSS group began to decline from day 15 and were significantly lower than those in the healthy Control group, confirming successful establishment of the colitis model. Importantly, LRa05 treatment markedly attenuated weight loss compared with the DSS group (Fig. 1 D). Furthermore, LRa05 administration resulted in a significant reduction in DAI scores in DSS-treated mice (Fig. 1 E), demonstrating its protective effect against colitis. 3.2 LRa05 mitigates colitis-associated colonic and systemic inflammation Colon length is a well-recognized tangible morphological marker of colitis severity, with shorter lengths directly reflecting more severe inflammatory damage. As expected, colons in the DSS group were significantly shorter compared to the Control group (Figs. 2 A-B). Notably, LRa05 administration effectively preserved colon length compared to the DSS group, a key indicator of its protective effect. As shown in Fig. 2 C, H&E-stained paraffin sections revealed extensive colonic tissue damage in the DSS group, including mucosal ulceration, dense inflammatory cell infiltration, structural disruption, and thick mucosal wall, which are pathological hallmarks of severe colitis. In contrast, LRa05 treatment significantly reduced the colonic histopathological disruption, clearly attenuating inflammatory damage. Moreover, DSS exposure induced a significant increase in spleen size and elevated spleen index, classic signs of systemic inflammatory activation (Figs. 2 D-E). Whereas, LRa05 administration markedly mitigated this splenomegaly, suggesting its ability to suppress excessive systemic immune activation. Taken together, these findings collectively demonstrate that LRa05 exerts robust protective efficacy against colitis-associated colon damage and systemic inflammation, highlighting its potential as a therapeutic agent for colitis. 3.3 LRa05 administration enhanced the abundance of beneficial gut microbiota Next, we investigated whether LRa05 administration altered the colonic microbiota. Microbial diversity was evaluated using metagenomic sequencing. α-diversity analysis (as measured by Chao1, Simpson, and Shannon index) revealed no significant differences between DSS and LRa05treated samples at both specie level (Fig. 3 A) and genus level (Fig. S2). Similarly, β-diversity (as assessed by PCoA) showed that samples from the different treatment groups did not separate, indicating no significant differences in species abundance (Fig. 3 B). These results suggest that the probiotic LRa05 has a minimal impact on the overall colonic microbiota structure. Although the most prevalent microbiota species were similar between DSS and LRa05 treated samples, including Duncaniella dubosii , Paramuribaculum intestinale , Muribaculum intestinale , Muribaculum gordoncarteri , Duncaniella sp.C9, and Akkermansia muciniphila , the composition of each species differed, as inllustrated in Fig. 3 C. Notably, the relative abundance of A. muciniphila was markedly increased in the LRa05 treated group, rising from 1.34% in the DSS group to 14.3% in the LRa05 group (Fig. 3 D and Fig. S2). This finding aligns with clinical evidence that A. muciniphila levels are consistently reduced in patients with inflammatory bowel disease (IBD). As a bacterium with robust beneficial effects, including rebalancing gut microbiota, mitigating systemic inflammation, and preserving the intestinal mucus layer, A. muciniphila has emerged as a promising candidate for next-generation probiotic therapy in colitis management( 17 ). Its mechanism of action is well characterized: it suppresses overactive proinflammatory signaling pathways and promotes the production of anti-inflammatory cytokines, thereby reducing intestinal inflammation and preventing colonic tissue damage. In our study, LRa05 administration significantly enhanced A. muciniphila colonization. This likely explains the alleviation of colitis symptoms observed in the treated group, linking the probiotic therapeutic effect to its ability to foster the growth of this key commensal bacterium. 3.4 LRa05 administration modified microbial metabolic pathways At the microbial gene level, we noticed that genes involved in metabolism were most identified (Fig. 4 A). We further analyzed the microbial metabolic pathways, and identified 7 pathways significantly enriched in abundance in the LRa05 treated gut microbiome, which are Streptomycin biosynthesis, Starch and sucrose metabolism, Galactose metabolism, Glycolysis, Pentose phosphate pathway, Peptidoglycan biosynthesis, and Aminoacyl-tRNA biosynthesis, as shown in Fig. 4 B. Among those microbiome enriched pathways, four out of seven were carbohydrate metabolism pathways, indicating that LRa05 supplementation may enhance the ability of the gut microbiota to degrade and utilize dietary carbohydrates. This shift likely promotes the production of beneficial metabolites such as SCFAs (e.g., acetate, propionate, and butyrate), which play critical roles in maintaining gut barrier integrity, modulating immune responses, and inhibiting the colonization of pathogenic bacteria. These findings imply that LRa05 restructures microbial metabolic function toward a more saccharolytic and anti-inflammatory profile, providing a plausible mechanism for its protective effects against colitis. 3.5 LRa05 administration upregulated the Treg cell while inhibited the expression of proinflammatory cytokines We next employed flow cytometry to analyze immune cells in differentially treated mice, focusing on CD4⁺Foxp3 + Treg cells, given the fact that dysfunction of Treg subset is a well-demonstrated major driver of IBD( 18 , 19 ). The gating strategy used in this study is shown in Fig. 5 A. While no significant difference was observed in total splenic CD4⁺ T cell percentages between DSS treated and LRa05 treated groups (Fig. 5 B), Foxp3 expression in CD4⁺ T cells was markedly upregulated after LRa05 administration. This upregulation was supported by two key findings: an increase in Treg frequency (Fig. 5 C) and elevated mean fluorescence intensity (MFI) of Foxp3 (Fig. 5 D), a measure of protein expression level. Furthermore, a remarkable increase in Treg frequency was also observed in mesenteric lymph nodes (mLN) (Fig. 5 E). We further analyzed proinflammatory cytokine levels, including interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α), in the spleen. Relative to the DSS control group, the LRa05 treated group exhibited reductions in such pro inflammatory cytokines at mRNA level (Fig. S3). Collectively, these data suggest that LRa05 mediated expansion of Tregs and consequent suppression of proinflammatory signaling accounts for the protective effects observed in DSS-induced colitis. 4. Discussion Lacticaseibacillus rhamnosus that detected in the digestive tracts of humans has been extensively studied. It has a variety of biological functions, such as the ability to regulate immune function, improve intestinal health, inhibit the growth of pathogenic microorganisms, and improve digestion, which has led to its being recognized as having wide applications in human health management( 20 , 21 ). LRa05 used in this study was isolated from the feces of infants. Previous studies systematically evaluated the safety of LRa05 by in vitro and in vivo assays, and found that it is sufficiently safe to be explored as a potential probiotic for use in the food industry( 22 , 23 ). Consistent with these findings, in our current study, LRa05 demonstrated an excellent safety profile in murine models. Throughout the continuous oral administration period, no statistically significant alterations in body weight were observed compared to the control group. In addition, normal fecal consistency and the absence of adverse clinical signs collectively indicate that oral gavage of LRa05 is non-toxic. Furthermore, LRa05 has been shown to improve the inflammatory parameters and gut microbiota of mice with obesity and mice with type 2 diabetes and thereby improve their health( 14 , 15 ). In a low-dose DSS chronic-colitis mouse model, LRa05 dynamically reversed dysbiosis, expanded Akkermansia and Bifidobacterium , and down-regulated colonic IL-6/TNF-α while up-regulating IL-10 and tight junction proteins( 24 ). In the current study, we used a medium-dose DSS to induce colitis and got the similar findings that LRa05 attenuated the progress of colitis and expanded Akkermansia. However, no changes in Bifidobacterium were found. IBD prevalence has surged exponentially in the past decades. Probiotics supplementation has emerged as key approach for alleviating this disease. Their effects are strain-specific and mediated through multiple interconnected mechanisms, including replenishing depleted beneficial microbiota and inhibiting pathogenic bacteria, producing SCFAs for energy source of colonocytes, lowering intestinal pH to inhibit pathogenic growth, and upregulating tight junction proteins to reduce intestinal permeability. However, the directly connection between probiotics supplementation and T cell immunity in mucosa is less explored so far. The core pathogenesis of IBD is rooted in immune dysregulation( 25 ). A breakdown in the balance between pro-inflammatory effector T cells (Th1, Th17) and suppressive Treg cells is a key driver of this disease. Treg dysfunction is common in IBD patients. This includes reduced Treg numbers in the mucosa, impaired suppressive capacity due to epigenetic modifications, as well as functional Tregs converting into pro-inflammatory Th1/Th17 cells in the inflamed mucosa. In DSS-induced mice model of IBD, it was also demonstrated that there is decrease expression of Treg cells and promoted inflammatory cytokines( 26 ). In our current study, we demonstrated that administration of LRa05 can increase the expression of Treg cells. This upregulation was evidenced by both increased Treg frequency and elevated MFI. As a result, the proinflammatory cytokines were inhibited. Probiotics ferment dietary fibers or host-derived substrates to produce bioactive metabolites, which may directly regulate T cell fate. The most well-studied metabolites include SCFAs, indole derivatives, polyamines and lactic acid( 27 – 31 ). Among them, butyrate and propionate inhibit histone deacetylases (HDACs) in naive T cells, increasing acetylation of the Foxp3 promoter. This enhances Foxp3 expression and drives differentiation into inducible Tregs (iTregs)( 32 ). Polyamines promote Treg survival and function by activating the mTOR pathway( 30 ). Building on these findings, our future work will focus on dissecting the specific mechanisms by which LRa05 promotes Treg differentiation in the context of colitis. 5. Conclusions LRa05, originally isolated from infant feces, is widely used as a probiotic in the food industry. In this study, we demonstrated that LRa05 administration exerted protective effects in a DSS-induced mouse colitis model. These protective effects were evidenced by attenuated weight loss, preserved colon length, reduced spleen organ index, and diminished histopathological damage in the colon. Mechanistically, the alleviation of colitis was partially mediated by LRa05 induced modulation of the gut microbiota, as a 10-fold increase in the abundance of the beneficial bacterium A. muciniphila was observed in the LRa05 treated group. More importantly, LRa05 promoted the expansion of Treg cells, evidenced by an increased frequency of Treg cells both in spleen and mesenteric lymph nodes. Consequently, proinflammatory cytokine levels were reduced, thereby excessive inflammation was suppressed. Further studies are warranted to dissect the crosstalk between LRa05 and T cell differentiation, which will shed light on the underlying mechanisms of its protective effects. Abbreviations LRa05 Lacticaseibacillus rhamnosus LRa05 IBD Inflammatory bowel disease CD Crohn’s disease UC Ulcerative colitis Th T helper cell Treg T regulatory cell SCFAs Short-chain fatty acids IACUC Institutional Animal Care and Use Committee BV Brilliant Violet APC Allophycocyanin FITC Fluorescein isothiocyanate DSS Dextran sulfate sodium DAI Disease activity index H&E Hematoxylin and eosin A. muciniphila Akkermansia muciniphila MFI Mean fluorescence intensity mLN Mesenteric lymph nodes IL-6 Interleukin-6 IFN-γ Interferon gamma TNF-α Tumor necrosis factor-alpha Declarations Ethics approval and consent to participate Not Applicable. Consent for publication Not Applicable. Availability of data The raw metagenomic sequencing data from all samples have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA1344417. Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Funding This work was funded by National Natural Science Foundation of China (82471776, 82373892) and the startup funding from Shanghai Jiao Tong University. Authors' contributions Jia Dong and Xuxiao Chen were responsible for investigation, methodology, data analysis and writing (original draft and review/editing). Yuxin Zhang, Yixiang Zhang and Zehua He were responsible for investigation and data analysis. Shengning Liang was responsible for writing and editing. Xu Cao and Yongquan Li was responsible for grammar checking and proof reading. Wencan Zhang was responsible for conceptualization, methodology, funding acquisition, resources and writing. Acknowledgements We appreciate the help from the Animal Resource Center and Biotechnology Core from Shanghai Jiao Tong University. Animals The mice used in this experiment were healthy 8-week-old C57BL/6J mice. All mice were purchased from Jiangsu GemPharmatech Co., Ltd (Nanjing, China) and were housed under specific pathogen-free conditions in the Animal Resource Center at the Shanghai Jiao Tong University under protocols approved by the Institutional Animal Care and Use Committee (IACUC# A2023233-005). Before all experiments, the mice were acclimated for 7 days. To ensure a painless and stress-free process, mice were rendered unconscious via anesthesia induced by exposure to 3% isoflurane in an induction chamber (RWD AIJI-IE, Shenzhen, China), where needed. At the end of the experiment, mice were humanely euthanized. 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Supplementary Files Supplementary20251011.pdf Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 09 Nov, 2025 Reviews received at journal 28 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 14 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Editor invited by journal 13 Oct, 2025 Submission checks completed at journal 11 Oct, 2025 First submitted to journal 11 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":660173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRa05 administration provided protection to mice in DSS-induced colitis model. A.\u003c/strong\u003eGrowth curve of LRa5 culture in MRS broth statically at 37 °C for 24 hours. \u003cstrong\u003eB.\u003c/strong\u003eOverview of the experimental settings used in this study. \u003cstrong\u003eC. \u003c/strong\u003eBody weight of \u003cem\u003emice \u003c/em\u003efrom Control group, DSS group and LRa05 group in the first 10 days (\u003cem\u003en\u003c/em\u003e ≥ 3 per group). \u003cstrong\u003eD.\u003c/strong\u003e Body weight of \u003cem\u003emice \u003c/em\u003efrom Control group, DSS group and LRa05 group from day 11 to day 24 (\u003cem\u003en\u003c/em\u003e ≥ 3 per group). \u003cstrong\u003eE.\u003c/strong\u003e Disease activity index (DAI) scores in mice from Control group, DSS group and LRa05 group from day 11 to day 24 (\u003cem\u003en\u003c/em\u003e ≥ 3 per group). Data are from three experiments (presented as means ± SEM). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ns, not significant (two-tailed Students’ t-test).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/b0de6eb0af3f57e3d6bae850.jpg"},{"id":94639952,"identity":"dae29c8e-3eb1-4b8b-9783-f3a5b32fa421","added_by":"auto","created_at":"2025-10-29 07:46:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1136094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRa05 mitigates colitis-associated colonic and systemic inflammation. A.\u003c/strong\u003eRepresentative image of colons from Control group, DSS group and LRa05 group. \u003cstrong\u003eB. \u003c/strong\u003eColon length (cm) from Control group, DSS group and LRa05 group (\u003cem\u003en\u003c/em\u003e ≥ 3 per group). \u003cstrong\u003eC. \u003c/strong\u003eSection of hematoxylin and eosin (H\u0026amp;E)–stained colon from Control group, DSS group and LRa05 group.\u003cstrong\u003e D.\u003c/strong\u003e Representative image of mice spleens from Control group, DSS group and LRa05 group. \u003cstrong\u003eE.\u003c/strong\u003e Spleen index from Control group, DSS group and LRa05 group (\u003cem\u003en\u003c/em\u003e ≥ 3 per group). Data are from three experiments (presented as means ± SD). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005; ns, not significant (two-tailed Students’ t-test).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/6c3729a851279dac553eb0af.jpg"},{"id":94640088,"identity":"ce1b1012-eaeb-4ed2-88af-2e604f5ac4f1","added_by":"auto","created_at":"2025-10-29 07:48:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1140058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRa05 administration enhanced the abundance of beneficial gut microbiota. A. \u003c/strong\u003eBox plots of α-diversity in colon microbiota at the specie level from DSS group and LRa05 group. \u003cstrong\u003eB. \u003c/strong\u003eThe PCoA plots of β-diversity between rectum microbiota from DSS group and LRa05 group using Bray-Curtis distance.\u003cstrong\u003e C. \u003c/strong\u003ePie charts of the specie-level composition in gut microbiota from DSS group and LRa05 group; top 16 most abundant species were shown. \u003cstrong\u003eD. \u003c/strong\u003eBar plot of specie-level relative frequency in gut microbiota from DSS group and LRa05 group.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/8a20accb070f0d81fdd346e6.jpg"},{"id":94625860,"identity":"966cfa74-1861-4fba-8511-6f8dd5d7e082","added_by":"auto","created_at":"2025-10-29 04:46:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1357027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRa05 administration modified microbial metabolic pathways.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Microbial gene enrichment based on KEGG classification. \u003cstrong\u003eB. \u003c/strong\u003eThe relative frequencies for significantly altered microbial metabolic pathways in gut microbiota from DSS group and LRa05 group. Pathways with a minimum absolute value of log\u003csub\u003e2\u003c/sub\u003e fold change (log\u003csub\u003e2\u003c/sub\u003eFC) of 1.65 were included in the plot.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/b0e397f673e6836a37d9939b.jpg"},{"id":94640027,"identity":"0a3571c3-ed9f-4a48-a722-76c982eaa961","added_by":"auto","created_at":"2025-10-29 07:47:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1110417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLRa05 administration upregulated the Treg cell while inhibited the expression of proinflammatory cytokines. A. \u003c/strong\u003eGating strategy for Fig. 5B-E. \u003cstrong\u003eB. \u003c/strong\u003eRepresentative flow cytometric analysis (left panel) and percentage (right panel) of CD4\u003csup\u003e+\u003c/sup\u003e cells among CD3\u003csup\u003e+\u003c/sup\u003e cells in the mice spleen from DSS group and LRa05 group (n≥4 per group). \u003cstrong\u003eC.\u003c/strong\u003e Representative flow cytometric analysis (left panel) and percentage (right panel) of Foxp3\u003csup\u003e+\u003c/sup\u003e cells among CD4\u003csup\u003e+\u003c/sup\u003e cells in the mice spleen from DSS group and LRa05 group (n≥4 per group). \u003cstrong\u003eD.\u003c/strong\u003e Representative flow cytometric analysis (left panel) and the MFI (right panel) for Foxp3 in Foxp3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells in the mice spleen from DSS group and LRa05 group (\u003cem\u003en\u003c/em\u003e\u0026nbsp;≥ 4 per group).\u0026nbsp;\u003cstrong\u003eE.\u003c/strong\u003e Representative flow cytometric analysis (left panel) and percentage (right panel) of Foxp3\u003csup\u003e+\u003c/sup\u003e cells among CD4\u003csup\u003e+\u003c/sup\u003e cells in the mice mesenteric lymph nodes (mLN) from DSS group and LRa05 group (n≥4 per group). Boxed area: Cell population of interest. Data are from three experiments (presented as means ± SD). *\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01; ns, not significant (two-tailed Students’ t-test).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/65ea440df6e463af613c1fad.jpg"},{"id":101152556,"identity":"649705e3-99f3-416a-8701-3f0cfe5ede9e","added_by":"auto","created_at":"2026-01-26 16:12:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6490889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/4018267d-8299-4682-b562-0ee27380eb63.pdf"},{"id":94640447,"identity":"a93ac93b-6b0e-46cb-9fe1-def39ad8e77f","added_by":"auto","created_at":"2025-10-29 07:49:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":412504,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary20251011.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7649694/v1/0aaec3164db0b17a35ebdd22.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lacticaseibacillus rhamnosus LRa05 Mitigates DSS-Induced Colitis via Boosting Beneficial Gut Microbiota and Facilitating CD4⁺Foxp3⁺ Treg Differentiation","fulltext":[{"header":"1. Background","content":"\u003cp\u003eInflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal disorder characterized by persistent inflammation, encompassing two primary subtypes-Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), with UC being one of the most prevalent variants(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Common clinical manifestations include diarrhea, abdominal pain, fatigue, anemia, reduced appetite, and unintended weight loss. Over the past five decades, global IBD prevalence has surged exponentially, reaching approximately 6.8\u0026nbsp;million documented cases in 2023(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). This dramatic rise is driven by complex interactions between environmental factors (e.g., Western dietary patterns, excessive antibiotic use), genetic susceptibility, and gut microbiota dysbiosis(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eT lymphocytes (T cells) constitute central players within the adaptive immune system, orchestrating cell mediated immune responses essential for maintaining host health and defending against a spectrum of threats. A delicate balance between proinflammatory subsets (e.g., T helper 1 (Th1), Th2, Th17) and T regulatory cell (Treg) is essential for maintaining immune homeostasis(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Dysregulation of this balance in gastrointestinal track, particularly the imbalance between pro-inflammatory Th1/Th17 cells and suppressive Tregs, serves as a key driver of IBD pathogenesis(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Treg, defined by the CD4⁺Foxp3⁺ phenotype, suppress pro-inflammatory activity through two main mechanisms: contact-dependent inhibition and secretion of anti-inflammatory cytokines(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In IBD patients, mucosal Treg depletion or impaired suppressive function is frequently observed, triggering uncontrolled inflammation that ultimately leads to irreversible damage to the intestinal mucosal barrier(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eModulation of gut microbiota has emerged as a promising therapeutic strategy for gastrointestinal inflammatory diseases, with probiotics supplementation playing a central role. Probiotics, which are live microorganisms that confer health benefits when administered in adequate amounts, exert therapeutic effects in IBD through multiple complementary pathways: 1) replenishing depleted beneficial taxa and inhibiting pathogenic bacteria via competition for nutrients and intestinal adhesion sites; 2) producing short-chain fatty acids (SCFAs), which act as an energy source for colonocytes, and can lower intestinal pH to inhibit pathogenic growth; 3) upregulating tight junction proteins to reduce intestinal permeability and stimulating goblet cells to secrete mucin, a key component of the protective barrier against luminal antigens(\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, a critical gap remains in our understanding: whether probiotics can directly modulate Treg cell differentiation, a process tightly linked to IBD pathogenesis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e LRa05 (hereafter referred to as \u0026ldquo;LRa05\u0026rdquo;), a strain isolated from infant feces, has been demonstrated as a probiotics strain to alleviate inflammatory markers and restore gut microbiota balance in mouse models of obesity and type 2 diabetes, thereby conferring significant health benefits(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Building on these evidences, the present study aims to conduct a comprehensive investigation to evaluate the potential therapeutic efficacy of LRa05 as an orally administrable probiotic formulation for IBD. Moreover, we aim to explore whether this strain can directly modulate T cell immunity. Through these investigations, this study aims to provide novel insights into the role of probiotics in IBD management and pave the way for the development of targeted microbiota-based therapies.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Probiotic strain\u003c/h2\u003e\u003cp\u003eThe probiotic strain used in this study was \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e LRa05 (LRa05), isolated from the feces of healthy infants in Qinghai Province, China. It was provided by Microhealthcare (Suzhou) Co., Ltd. (Suzhou, China) in the form of lyophilized powder, with an activity of 90\u0026nbsp;billion CFU/g. For activation, transfer 100 mg of the powder into 20 mL MRS broth and incubate statically at 37\u0026deg;C for 24 h. Harvest the activated culture by centrifugation at 8000 rpm, 10\u0026deg;C for 20 min and discard the supernatant. Resuspend the pellet in 2 mL fresh MRS broth and mix this suspension with 50% glycerol at 1:1 ratio. Store this mixture in cryovials at -80\u0026deg;C for further experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 LRa05 growth curve determination\u003c/h2\u003e\u003cp\u003eCultivate LRa05 by dissolving 20 \u0026micro;L frozen culture in 20 mL fresh MRS broth statically at 37\u0026deg;C. Sampling was conducted every 2 hours for the determination of viable bacterial counts by measuring the OD₆₀₀ of the culture with SpectraMax iD3/iD5 microplate reader (Molecular Devices). Plot the resulting OD₆₀₀ values against time to generate the growth curve and identify the culture phase with maximal bacterial activity. After 18 hours of cultivation, serially dilute the culture and determine the OD₆₀₀ of each dilution. In parallel, plate 100 \u0026micro;L aliquots of the serial dilutions onto MRS agar, incubate at 37\u0026deg;C for 24 hours and count the colonies. Calculate the viable bacterial concentration (CFU/mL) from the average colony counts across appropriate dilutions, and generate a standard curve relating OD₆₀₀ to bacterial counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Mice\u003c/h2\u003e\u003cp\u003eThe mice used in this experiment were healthy 8-week-old C57BL/6J mice. All mice were purchased from Jiangsu GemPharmatech Co., Ltd (Nanjing, China) and were housed under specific pathogen-free conditions in the Animal Resource Center at the Shanghai Jiao Tong University under protocols approved by the Institutional Animal Care and Use Committee (IACUC# A2023233-005). Before all experiments, the mice were acclimated for 7 days. To ensure a painless and stress-free process, mice were rendered unconscious via anesthesia induced by exposure to 3% isoflurane in an induction chamber (RWD AIJI-IE, Shenzhen, China), where needed.\u003c/p\u003e\u003cp\u003eAt the end of the experiment, mice were humanely euthanized. The primary method employed was carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) inhalation, administered in a pre-filled chamber at a controlled displacement rate of 30\u0026ndash;70% of the chamber volume per minute to minimize distress. Following the cessation of movement and respiration, death was confirmed by cervical dislocation. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University and is consistent with the AVMA Guidelines for the Euthanasia of Animals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Antibodies\u003c/h2\u003e\u003cp\u003eAntibodies for flow cytometry including Brilliant Violet (BV) 605-conjugated anti-CD4 (dilution ratio, 1:100; RM4-5), BV 421-conjugated anti-CD3 (dilution ratio, 1:100; 145-2C11) were from BioLegend. Allophycocyanin (APC)-conjugated anti-Foxp3 (dilution ratio, 1:100; FJK-16s) was from Invitrogen. Fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (dilution ratio, 1:100; 30F11) was from Miltenyi Biotec.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Flow cytometry\u003c/h2\u003e\u003cp\u003eFor surface staining, cells isolated from mice were directly stained with antibodies in PBS at 4\u0026deg;C for 15 min. For transcription factor Foxp3 staining, cells pre-stained with surface markers were fixed and permeabilized in TF Fix/Perm buffer (BD Biosciences) at 4\u0026deg;C for 20 min, washed once with TF Perm/Wash buffer, and stained with target markers in the TF Perm/Wash buffer at 4\u0026deg;C for 15 min. The expression of surface and intracellular markers was analyzed with a BD LSR Fortessa flow cytometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Dextran sulfate sodium-induced colitis in mice\u003c/h2\u003e\u003cp\u003eDextran sulfate sodium (DSS, Mw 36\u0026ndash;50 kDa) was purchased from MP Biomedicals. Mice were divided into three groups, which is Control, DSS, and LRa05. In a 24-day experiment cycle, mice from Control group received normal water daily. Mice from DSS group received normal water daily for the first 10 days, but was gavaged with 200 uL PBS each mouse for 24 consecutive days. Mice from LRa05 group received normal water daily for the first 10 days, but were gavaged with 1 \u0026times; 10⁹ CFU LRa05 in 200 uL PBS each mouse for 24 consecutive days. Beginning on day 11, the normal drinking water for DSS group and LRa05 group was switched to a DSS regimen, which is two cycles of 2% (w/v) DSS for 5 days followed by normal water for 2 days. On day 24, mice were euthanized and samples of spleen, mesenteric lymph nodes, colon, and feces were collected for experiments.\u003c/p\u003e\u003cp\u003eBody weight and disease activity index (DAI) were measured daily to assess the health of the mice and to judge the success of the establishment of DSS model. The evaluation of the DAI is revised based on previous research(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and is shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. After the mice were euthanized, the spleen was weighed and the following formula was used to calculate the immune organ index: organ index\u0026thinsp;=\u0026thinsp;the weight of target organs (mg)/body weight (g). The length of the colon is measured as a physiological indicator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Reverse transcription quantitative real-time PCR\u003c/h2\u003e\u003cp\u003eTotal RNA of cells was extracted according to the manufacturer\u0026rsquo;s guide using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme). The first-strand cDNA synthesis was performed by reverse transcription using a HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) kit (Vazyme, Nanjing, China). Subsequent qPCR was performed using ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in the CFX Connect Real-Time PCR System (Bio-Rad). The primers used for qPCR are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The amplification efficiency of all primers has been tested, and the optimized conditions were used in all qPCRs. Gene expression was calculated with the ∆∆\u003cem\u003eC\u003c/em\u003et method normalized to the control gene encoding β-actin, and all measurements were performed in triplicate.\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\u003ePrimer sequences used for qPCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eActin-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-GGGAAATCGTGCGTGACAT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eActin-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-GTCAGGCAGCTCGTAGCTCTT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNF-α-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-CCCTCACACTCAGATCATCTTCT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNF-α-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-GCTACGACGTGGGCTACAG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIFN-γ-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-ATGAACGCTACACACTGCATC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIFN-γ-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-CCATCCTTTTGCCAGTTCCTC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-6-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-TAGTCCTTCCTACCCCAATTTCC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-6-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-TTGGTCCTTAGCCACTCCTTC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.8 Histological evaluation of colon\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eColon specimens were first rinsed with PBS, fixed with paraformaldehyde (Servicebio, Wuhan, China) at a concentration of 4% (w/v), dehydrated and embedded by paraffin. Hematoxylin and eosin (H\u0026amp;E) were used to stain the sample after being sliced (4 \u0026micro;m slice).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Fecal metagenomic sequencing\u003c/h2\u003e\u003cp\u003eFecal samples of 4 mice from each group were selected for metagenomic sequencing. Metagenomic DNA was extracted from the fecal samples using QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer\u0026rsquo;s protocol. Then, 0.8% agarose gel electrophoresis was used to measure the concentration and integrity of the DNA. The sequencing work was performed by BGI Group (Shenzhen, China). The raw data were processed by the sliding window method to remove low-quality sequences from the original sequence, resulting in high-quality clean reads, with host genomes removed. Finally, sickle software was used for tailoring and modifying the reads.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Data analysis\u003c/h2\u003e\u003cp\u003eFlow-cytometry data were processed and plotted using FlowJo 10.8.1. Colonic HE-stained colon sections were examined in SlideViewer. Other data were analyzed and graphed with GraphPad Prism 9.5. Statistical significance is indicated as ns (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), * (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ** (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and *** (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 LRa05 administration provided protection to mice in DSS-induced colitis model\u003c/h2\u003e\u003cp\u003eLRa05 was inoculated into MRS broth at a 0.1% (v/v) ratio and statically incubated at 37\u0026deg;C. OD₆₀₀ readings of the culture were recorded every 2 hours, and the resulting growth curve is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. According to the curve, LRa05 reached peak activity at approximately 18 hours post-inoculation. Therefore, all bacteria for gavage were collected at 18 hours post-cultivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe DSS-induced colitis model was established as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. During the adaptive gavage period, LRa05 exhibited an excellent safety profile in mice. Throughout 10 days continuous oral administration, no statistically significant changes in fecal consistency (data not shown) or body weight were observed compared to the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicate that oral administration of LRa05 caused no adverse reactions or weight loss, confirming its safety and absence of harmful effects in this model.\u003c/p\u003e\u003cp\u003eIn the DSS-induced colitis model, LRa05 administration conferred significant protection to mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, body weight of mice in the DSS group began to decline from day 15 and were significantly lower than those in the healthy Control group, confirming successful establishment of the colitis model. Importantly, LRa05 treatment markedly attenuated weight loss compared with the DSS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, LRa05 administration resulted in a significant reduction in DAI scores in DSS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), demonstrating its protective effect against colitis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 LRa05 mitigates colitis-associated colonic and systemic inflammation\u003c/h2\u003e\u003cp\u003eColon length is a well-recognized tangible morphological marker of colitis severity, with shorter lengths directly reflecting more severe inflammatory damage. As expected, colons in the DSS group were significantly shorter compared to the Control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Notably, LRa05 administration effectively preserved colon length compared to the DSS group, a key indicator of its protective effect. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, H\u0026amp;E-stained paraffin sections revealed extensive colonic tissue damage in the DSS group, including mucosal ulceration, dense inflammatory cell infiltration, structural disruption, and thick mucosal wall, which are pathological hallmarks of severe colitis. In contrast, LRa05 treatment significantly reduced the colonic histopathological disruption, clearly attenuating inflammatory damage. Moreover, DSS exposure induced a significant increase in spleen size and elevated spleen index, classic signs of systemic inflammatory activation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). Whereas, LRa05 administration markedly mitigated this splenomegaly, suggesting its ability to suppress excessive systemic immune activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaken together, these findings collectively demonstrate that LRa05 exerts robust protective efficacy against colitis-associated colon damage and systemic inflammation, highlighting its potential as a therapeutic agent for colitis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 LRa05 administration enhanced the abundance of beneficial gut microbiota\u003c/h2\u003e\u003cp\u003eNext, we investigated whether LRa05 administration altered the colonic microbiota. Microbial diversity was evaluated using metagenomic sequencing. α-diversity analysis (as measured by Chao1, Simpson, and Shannon index) revealed no significant differences between DSS and LRa05treated samples at both specie level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and genus level (Fig. S2). Similarly, β-diversity (as assessed by PCoA) showed that samples from the different treatment groups did not separate, indicating no significant differences in species abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results suggest that the probiotic LRa05 has a minimal impact on the overall colonic microbiota structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the most prevalent microbiota species were similar between DSS and LRa05 treated samples, including \u003cem\u003eDuncaniella dubosii\u003c/em\u003e, \u003cem\u003eParamuribaculum intestinale\u003c/em\u003e, \u003cem\u003eMuribaculum intestinale\u003c/em\u003e, \u003cem\u003eMuribaculum gordoncarteri\u003c/em\u003e, \u003cem\u003eDuncaniella\u003c/em\u003e sp.C9, and \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, the composition of each species differed, as inllustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Notably, the relative abundance of \u003cem\u003eA. muciniphila\u003c/em\u003e was markedly increased in the LRa05 treated group, rising from 1.34% in the DSS group to 14.3% in the LRa05 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Fig. S2). This finding aligns with clinical evidence that \u003cem\u003eA. muciniphila\u003c/em\u003e levels are consistently reduced in patients with inflammatory bowel disease (IBD). As a bacterium with robust beneficial effects, including rebalancing gut microbiota, mitigating systemic inflammation, and preserving the intestinal mucus layer, \u003cem\u003eA. muciniphila\u003c/em\u003e has emerged as a promising candidate for next-generation probiotic therapy in colitis management(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Its mechanism of action is well characterized: it suppresses overactive proinflammatory signaling pathways and promotes the production of anti-inflammatory cytokines, thereby reducing intestinal inflammation and preventing colonic tissue damage. In our study, LRa05 administration significantly enhanced \u003cem\u003eA. muciniphila\u003c/em\u003e colonization. This likely explains the alleviation of colitis symptoms observed in the treated group, linking the probiotic therapeutic effect to its ability to foster the growth of this key commensal bacterium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 LRa05 administration modified microbial metabolic pathways\u003c/h2\u003e\u003cp\u003eAt the microbial gene level, we noticed that genes involved in metabolism were most identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We further analyzed the microbial metabolic pathways, and identified 7 pathways significantly enriched in abundance in the LRa05 treated gut microbiome, which are Streptomycin biosynthesis, Starch and sucrose metabolism, Galactose metabolism, Glycolysis, Pentose phosphate pathway, Peptidoglycan biosynthesis, and Aminoacyl-tRNA biosynthesis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Among those microbiome enriched pathways, four out of seven were carbohydrate metabolism pathways, indicating that LRa05 supplementation may enhance the ability of the gut microbiota to degrade and utilize dietary carbohydrates. This shift likely promotes the production of beneficial metabolites such as SCFAs (e.g., acetate, propionate, and butyrate), which play critical roles in maintaining gut barrier integrity, modulating immune responses, and inhibiting the colonization of pathogenic bacteria. These findings imply that LRa05 restructures microbial metabolic function toward a more saccharolytic and anti-inflammatory profile, providing a plausible mechanism for its protective effects against colitis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 LRa05 administration upregulated the Treg cell while inhibited the expression of proinflammatory cytokines\u003c/h2\u003e\u003cp\u003eWe next employed flow cytometry to analyze immune cells in differentially treated mice, focusing on CD4⁺Foxp3\u003csup\u003e+\u003c/sup\u003e Treg cells, given the fact that dysfunction of Treg subset is a well-demonstrated major driver of IBD(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The gating strategy used in this study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. While no significant difference was observed in total splenic CD4⁺ T cell percentages between DSS treated and LRa05 treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), Foxp3 expression in CD4⁺ T cells was markedly upregulated after LRa05 administration. This upregulation was supported by two key findings: an increase in Treg frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and elevated mean fluorescence intensity (MFI) of Foxp3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), a measure of protein expression level. Furthermore, a remarkable increase in Treg frequency was also observed in mesenteric lymph nodes (mLN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further analyzed proinflammatory cytokine levels, including interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α), in the spleen. Relative to the DSS control group, the LRa05 treated group exhibited reductions in such pro inflammatory cytokines at mRNA level (Fig. S3).\u003c/p\u003e\u003cp\u003eCollectively, these data suggest that LRa05 mediated expansion of Tregs and consequent suppression of proinflammatory signaling accounts for the protective effects observed in DSS-induced colitis.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e that detected in the digestive tracts of humans has been extensively studied. It has a variety of biological functions, such as the ability to regulate immune function, improve intestinal health, inhibit the growth of pathogenic microorganisms, and improve digestion, which has led to its being recognized as having wide applications in human health management(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). LRa05 used in this study was isolated from the feces of infants. Previous studies systematically evaluated the safety of LRa05 by \u003cem\u003ein vitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e assays, and found that it is sufficiently safe to be explored as a potential probiotic for use in the food industry(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Consistent with these findings, in our current study, LRa05 demonstrated an excellent safety profile in murine models. Throughout the continuous oral administration period, no statistically significant alterations in body weight were observed compared to the control group. In addition, normal fecal consistency and the absence of adverse clinical signs collectively indicate that oral gavage of LRa05 is non-toxic.\u003c/p\u003e\u003cp\u003eFurthermore, LRa05 has been shown to improve the inflammatory parameters and gut microbiota of mice with obesity and mice with type 2 diabetes and thereby improve their health(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In a low-dose DSS chronic-colitis mouse model, LRa05 dynamically reversed dysbiosis, expanded \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, and down-regulated colonic IL-6/TNF-α while up-regulating IL-10 and tight junction proteins(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In the current study, we used a medium-dose DSS to induce colitis and got the similar findings that LRa05 attenuated the progress of colitis and expanded \u003cem\u003eAkkermansia.\u003c/em\u003e However, no changes in \u003cem\u003eBifidobacterium\u003c/em\u003e were found.\u003c/p\u003e\u003cp\u003eIBD prevalence has surged exponentially in the past decades. Probiotics supplementation has emerged as key approach for alleviating this disease. Their effects are strain-specific and mediated through multiple interconnected mechanisms, including replenishing depleted beneficial microbiota and inhibiting pathogenic bacteria, producing SCFAs for energy source of colonocytes, lowering intestinal pH to inhibit pathogenic growth, and upregulating tight junction proteins to reduce intestinal permeability. However, the directly connection between probiotics supplementation and T cell immunity in mucosa is less explored so far.\u003c/p\u003e\u003cp\u003eThe core pathogenesis of IBD is rooted in immune dysregulation(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). A breakdown in the balance between pro-inflammatory effector T cells (Th1, Th17) and suppressive Treg cells is a key driver of this disease. Treg dysfunction is common in IBD patients. This includes reduced Treg numbers in the mucosa, impaired suppressive capacity due to epigenetic modifications, as well as functional Tregs converting into pro-inflammatory Th1/Th17 cells in the inflamed mucosa. In DSS-induced mice model of IBD, it was also demonstrated that there is decrease expression of Treg cells and promoted inflammatory cytokines(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In our current study, we demonstrated that administration of LRa05 can increase the expression of Treg cells. This upregulation was evidenced by both increased Treg frequency and elevated MFI. As a result, the proinflammatory cytokines were inhibited.\u003c/p\u003e\u003cp\u003eProbiotics ferment dietary fibers or host-derived substrates to produce bioactive metabolites, which may directly regulate T cell fate. The most well-studied metabolites include SCFAs, indole derivatives, polyamines and lactic acid(\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Among them, butyrate and propionate inhibit histone deacetylases (HDACs) in naive T cells, increasing acetylation of the Foxp3 promoter. This enhances Foxp3 expression and drives differentiation into inducible Tregs (iTregs)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Polyamines promote Treg survival and function by activating the mTOR pathway(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Building on these findings, our future work will focus on dissecting the specific mechanisms by which LRa05 promotes Treg differentiation in the context of colitis.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eLRa05, originally isolated from infant feces, is widely used as a probiotic in the food industry. In this study, we demonstrated that LRa05 administration exerted protective effects in a DSS-induced mouse colitis model. These protective effects were evidenced by attenuated weight loss, preserved colon length, reduced spleen organ index, and diminished histopathological damage in the colon. Mechanistically, the alleviation of colitis was partially mediated by LRa05 induced modulation of the gut microbiota, as a 10-fold increase in the abundance of the beneficial bacterium \u003cem\u003eA. muciniphila\u003c/em\u003e was observed in the LRa05 treated group. More importantly, LRa05 promoted the expansion of Treg cells, evidenced by an increased frequency of Treg cells both in spleen and mesenteric lymph nodes. Consequently, proinflammatory cytokine levels were reduced, thereby excessive inflammation was suppressed. Further studies are warranted to dissect the crosstalk between LRa05 and T cell differentiation, which will shed light on the underlying mechanisms of its protective effects.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLRa05\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e LRa05\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIBD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInflammatory bowel disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCrohn\u0026rsquo;s disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUlcerative colitis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTh\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eT helper cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTreg\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eT regulatory cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSCFAs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eShort-chain fatty acids\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIACUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInstitutional Animal Care and Use Committee\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBrilliant Violet\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAllophycocyanin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFITC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFluorescein isothiocyanate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDSS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDextran sulfate sodium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDisease activity index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHematoxylin and eosin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eA. \u003cem\u003emuciniphila\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eAkkermansia muciniphila\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMFI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMean fluorescence intensity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emLN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMesenteric lymph nodes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-6\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-6\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIFN-γ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon gamma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor necrosis factor-alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cbr\u003eNot Applicable.\u003cbr\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not Applicable.\u003cbr\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The raw metagenomic sequencing data from all samples have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA1344417.\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cbr\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest\u003cbr\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;This work was funded by National Natural Science Foundation of China (82471776, 82373892) and the startup funding from Shanghai Jiao Tong University.\u003cbr\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003cbr\u003eJia Dong and Xuxiao Chen were responsible for investigation, methodology, data analysis and writing (original draft and review/editing). Yuxin Zhang, Yixiang Zhang\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand Zehua He were responsible for investigation and data analysis. Shengning Liang was responsible for writing and editing. Xu Cao and Yongquan Li was responsible for grammar checking and proof reading. Wencan Zhang was responsible for conceptualization, methodology, funding acquisition, resources and writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the help from the Animal Resource Center and Biotechnology Core from Shanghai Jiao Tong University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The mice used in this experiment were healthy 8-week-old C57BL/6J mice. All mice were purchased from Jiangsu GemPharmatech Co., Ltd (Nanjing, China) and were housed under specific pathogen-free conditions in the Animal Resource Center at the Shanghai Jiao Tong University under protocols approved by the Institutional Animal Care and Use Committee (IACUC# A2023233-005). Before all experiments, the mice were acclimated for 7 days. To ensure a painless and stress-free process, mice were rendered unconscious via anesthesia induced by exposure to 3% isoflurane in an induction chamber (RWD AIJI-IE, Shenzhen, China), where needed.\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, mice were humanely euthanized. The primary method employed was carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) inhalation, administered in a pre-filled chamber at a controlled displacement rate of 30-70% of the chamber volume per minute to minimize distress. Following the cessation of movement and respiration, death was confirmed by cervical dislocation. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University and is consistent with the AVMA Guidelines for the Euthanasia of Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerative AI statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that no Generative AI was used in the creation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuan Q. A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Disease. J Immunol Res. 2019;2019:7247238.\u003c/li\u003e\n\u003cli\u003ePlevris N, Lees CW. Disease Monitoring in Inflammatory Bowel Disease: Evolving Principles and Possibilities. Gastroenterology. 2022;162(5):1456\u0026ndash;75.e1.\u003c/li\u003e\n\u003cli\u003eKaplan GG. The global burden of inflammatory bowel disease: from 2025 to 2045. Nat Rev Gastroenterol Hepatol. 2025.\u003c/li\u003e\n\u003cli\u003eHodson R. Inflammatory bowel disease. Nature. 2016;540(7634):S97.\u003c/li\u003e\n\u003cli\u003eDikiy S, Rudensky AY. Principles of regulatory T cell function. Immunity. 2023;56(2):240\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eWu C, Jiang ML, Pang T, Zhang CJ. T Cell Subsets and Immune Homeostasis. Methods Mol Biol. 2024;2782:39\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eJiang P, Zheng C, Xiang Y, Malik S, Su D, Xu G, et al. The involvement of TH17 cells in the pathogenesis of IBD. Cytokine Growth Factor Rev. 2023;69:28\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eJia L, Jiang Y, Wu L, Fu J, Du J, Luo Z, et al. Porphyromonas gingivalis aggravates colitis via a gut microbiota-linoleic acid metabolism-Th17/Treg cell balance axis. Nat Commun. 2024;15(1):1617.\u003c/li\u003e\n\u003cli\u003eWang H, Hu D, Cheng Y, Gao Q, Liu K, Mani NL, et al. Succinate drives gut inflammation by promoting FOXP3 degradation through a molecular switch. Nat Immunol. 2025;26(6):866\u0026ndash;80.\u003c/li\u003e\n\u003cli\u003ePedros C, Duguet F, Saoudi A, Chabod M. Disrupted regulatory T cell homeostasis in inflammatory bowel diseases. World J Gastroenterol. 2016;22(3):974\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eJakubczyk D, Leszczyńska K, G\u0026oacute;rska S. The Effectiveness of Probiotics in the Treatment of Inflammatory Bowel Disease (IBD)-A Critical Review. Nutrients. 2020;12(7).\u003c/li\u003e\n\u003cli\u003eZhou J, Li M, Chen Q, Li X, Chen L, Dong Z, et al. Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery. Nat Commun. 2022;13(1):3432.\u003c/li\u003e\n\u003cli\u003eRondanelli M, Faliva MA, Perna S, Giacosa A, Peroni G, Castellazzi AM. Using probiotics in clinical practice: Where are we now? A review of existing meta-analyses. Gut Microbes. 2017;8(6):521\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eWu T, Zhang Y, Li W, Zhao Y, Long H, Muhindo EM, et al. Lactobacillus rhamnosus LRa05 Ameliorate Hyperglycemia through a Regulating Glucagon-Mediated Signaling Pathway and Gut Microbiota in Type 2 Diabetic Mice. J Agric Food Chem. 2021;69(31):8797\u0026ndash;806.\u003c/li\u003e\n\u003cli\u003eSun M, Wu T, Zhang G, Liu R, Sui W, Zhang M, et al. Lactobacillus rhamnosus LRa05 improves lipid accumulation in mice fed with a high fat diet via regulating the intestinal microbiota, reducing glucose content and promoting liver carbohydrate metabolism. Food Funct. 2020;11(11):9514\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003ePark YH, Kim N, Shim YK, Choi YJ, Nam RH, Choi YJ, et al. Adequate Dextran Sodium Sulfate-induced Colitis Model in Mice and Effective Outcome Measurement Method. J Cancer Prev. 2015;20(4):260\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eZheng M, Han R, Yuan Y, Xing Y, Zhang W, Sun Z, et al. The role of Akkermansia muciniphila in inflammatory bowel disease: Current knowledge and perspectives. Front Immunol. 2022;13:1089600.\u003c/li\u003e\n\u003cli\u003eKosinsky RL, Gonzalez MM, Saul D, Barros LL, Sagstetter MR, Fedyshyn Y, et al. The FOXP3(+) Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn\u0026apos;s Disease. Gastroenterology. 2024;166(4):631\u0026ndash;44.e17.\u003c/li\u003e\n\u003cli\u003eTian M, Hao F, Wang X, Zheng X, Wang H, Li J, et al. OGG1 augments the transcriptional activation of Foxp3 to promote iTreg differentiation for IBD alleviation. Proc Natl Acad Sci U S A. 2025;122(30):e2424733122.\u003c/li\u003e\n\u003cli\u003eXavier-Santos D, Scharlack NK, Pena FL, Antunes AEC. Effects of Lacticaseibacillus rhamnosus GG supplementation, via food and non-food matrices, on children\u0026apos;s health promotion: A scoping review. Food Res Int. 2022;158:111518.\u003c/li\u003e\n\u003cli\u003eZhu M, Yang L, Kong S, Bai Y, Zhao B. Lacticaseibacillus rhamnosus LRa05 alleviates cyclophosphamide-induced immunosuppression and intestinal microbiota disorder in mice. J Food Sci. 2024;89(12):10003\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eChen T, Shao Y, Zhang Y, Zhao Y, Han M, Gai Z. In vitro and in vivo genome-based safety evaluation of Lacticaseibacillus rhamnosus LRa05. Food Chem Toxicol. 2024;186:114600.\u003c/li\u003e\n\u003cli\u003eWu J, Zhou L, He S, Tang X, Wang J, Li Y. Longitudinal Analysis of the Immunostimulatory Properties and Safety Profile of Lacticaseibacillus rhamnosus LRa05 as a Dietary Supplement. J Microbiol Biotechnol. 2025;35:e2502053.\u003c/li\u003e\n\u003cli\u003eGu J, Chen Y, Wang J, Gao Y, Gai Z, Zhao Y, et al. Lacticaseibacillus rhamnosus LRa05 alleviated liver injury in mice with alcoholic fatty liver disease by improving intestinal permeability and balancing gut microbiota. Benef Microbes. 2024;15(5):481\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003ede Souza HS, Fiocchi C. Immunopathogenesis of IBD: current state of the art. Nat Rev Gastroenterol Hepatol. 2016;13(1):13\u0026ndash;27.\u003c/li\u003e\n\u003cli\u003eYan JB, Luo MM, Chen ZY, He BH. The Function and Role of the Th17/Treg Cell Balance in Inflammatory Bowel Disease. J Immunol Res. 2020;2020:8813558.\u003c/li\u003e\n\u003cli\u003eKrause FF, Mangold KI, Ruppert AL, Leister H, Hellhund-Zingel A, Lopez Krol A, et al. Clostridium sporogenes-derived metabolites protect mice against colonic inflammation. Gut Microbes. 2024;16(1):2412669.\u003c/li\u003e\n\u003cli\u003eBhaskaran N, Quigley C, Paw C, Butala S, Schneider E, Pandiyan P. Role of Short Chain Fatty Acids in Controlling T(regs) and Immunopathology During Mucosal Infection. Front Microbiol. 2018;9:1995.\u003c/li\u003e\n\u003cli\u003eFong W, Li Q, Ji F, Liang W, Lau HCH, Kang X, et al. Lactobacillus gallinarum-derived metabolites boost anti-PD1 efficacy in colorectal cancer by inhibiting regulatory T cells through modulating IDO1/Kyn/AHR axis. Gut. 2023;72(12):2272\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eCarriche GM, Almeida L, St\u0026uuml;ve P, Velasquez L, Dhillon-LaBrooy A, Roy U, et al. Regulating T-cell differentiation through the polyamine spermidine. J Allergy Clin Immunol. 2021;147(1):335\u0026ndash;48.e11.\u003c/li\u003e\n\u003cli\u003eLopez Krol A, Nehring HP, Krause FF, Wempe A, Raifer H, Nist A, et al. Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells. EMBO Rep. 2022;23(12):e54685.\u003c/li\u003e\n\u003cli\u003eMcBride DA, Dorn NC, Yao M, Johnson WT, Wang W, Bottini N, et al. Short-chain fatty acid-mediated epigenetic modulation of inflammatory T cells in vitro. Drug Deliv Transl Res. 2023;13(7):1912\u0026ndash;24.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LRa05, Colitis, Treg, Akkermansia muciniphila, Inflammation","lastPublishedDoi":"10.21203/rs.3.rs-7649694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7649694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e LRa05, originally isolated from infant feces, has been widely used as a probiotic in the food industry. However, its potential as an orally administered probiotic therapeutic for inflammatory bowel disease (IBD) remains underexplored, particularly from an immune regulatory perspective.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we demonstrated that administration of LRa05 conferred protective effects in a DSS-induced colitis mouse model. Such protective effects were reflected by attenuated weight loss, preserved colon length, reduced spleen index, and diminished histopathological damage in the colon. Mechanistically, while LRa05 administration exerted minimal impact on the overall microbiota diversity, it altered the species composition. Notably, the abundance of the beneficial bacterium \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e was 10-fold higher in LRa05 treated mice compared to controls. This change was thought to further modulate microbial metabolic pathways. More importantly, LRa05 promoted the expansion of CD4⁺Foxp3⁺ Treg cells, as evidenced by a remarkable increase in Treg frequency in both the spleen and mesenteric lymph nodes. Consequently, proinflammatory cytokine levels were reduced, thereby excessive colonic inflammation was suppressed.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eTaken together, our findings identify LRa05 as a potential therapeutic agent for DSS-induced colitis, with its protective effects mediated through enhancing beneficial gut microbiota and facilitating Treg cell differentiation.\u003c/p\u003e","manuscriptTitle":"Lacticaseibacillus rhamnosus LRa05 Mitigates DSS-Induced Colitis via Boosting Beneficial Gut Microbiota and Facilitating CD4⁺Foxp3⁺ Treg Differentiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 04:46:12","doi":"10.21203/rs.3.rs-7649694/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-10T04:44:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T05:04:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T13:31:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194166356171543395415819246776913192545","date":"2025-10-15T15:16:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166236940766665133357064631538647946537","date":"2025-10-15T13:53:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25420470006366906277946563597624566770","date":"2025-10-15T03:00:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T00:21:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T00:19:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-13T06:02:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-11T11:16:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-10-11T11:12:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9ac07a8-9731-41c0-b223-9f3bd1e06f04","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:08:07+00:00","versionOfRecord":{"articleIdentity":"rs-7649694","link":"https://doi.org/10.1186/s12866-026-04745-x","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-20 15:57:02","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-10-29 04:46:12","video":"","vorDoi":"10.1186/s12866-026-04745-x","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04745-x","workflowStages":[]},"version":"v1","identity":"rs-7649694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7649694","identity":"rs-7649694","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}