The ability of probiotic strain Escherichia coli O83:K24:H31 to modulate gut homeostasis and immune function after antibiotic-induced dysbiosis

The ability of probiotic strain Escherichia coli O83:K24:H31 to modulate gut homeostasis and immune function after antibiotic-induced dysbiosis | 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 The ability of probiotic strain Escherichia coli O83:K24:H31 to modulate gut homeostasis and immune function after antibiotic-induced dysbiosis Eliška Miková, Eliška Krčmářová, Viktor Černý, Lydie Sklenářová, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6503496/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 12 You are reading this latest preprint version Abstract A healthy microbiome and a homeostatic interaction between the microbiome and the host immune system are essential for proper nutrition and overall health. Excessive use of antibiotics (ATB) can disrupt the healthy gut microenvironment, leading to dysbiosis, a condition linked to a wide range of disorders and diseases. Alterations in the composition and function of the microbiota have been associated with a broad spectrum of various pathological conditions In this work, we investigate the effect of ATB administration on microbiota composition and immune modulation, with a particular focus on neutrophil dynamics. We evaluated the capacity of the probiotic strain Escherichia coli O83:K24:H31 (EcO83) to mitigate ATB-induced dysbiosis and restore immune function. As expected, ATB treatment reduced microbiota diversity, which was partially restored by EcO83 supplementation. Furthermore, ATB administration affected the expression of tight junction proteins in the small intestine, an effect reversed by EcO83 treatment. Notably, our data indicates that ATB-induced dysbiosis accelerates neutrophil aging and reduces the release of neutrophils from the bone marrow. EcO83 supplementation counteracts these effects by promoting the influx of newly generated neutrophils into circulation. Overall, our findings confirm that ATB treatment disrupts gut microbiota homeostasis, adversely affecting immune function, including neutrophil turnover. However, probiotic supplementation with EcO83 can at least partially restore microbiome composition and immune homeostasis, highlighting its potential therapeutic application in mitigating ATB-induced dysbiosis. dysbiosis antibiotics probiotics E. coli neutrophils Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Excessive antibiotic (ATB) usage has a deleterious effect on microbiota ( 1 , 2 ). It is known that after ATB administration, a temporary dysbiosis occurs, possibly triggering immune system dysregulation ( 3 , 4 ). Upon steady state, commensal and beneficial microbes present in our physiological microbiota can prevent the overgrowth of pathobionts. But, during ATB treatments, these “good bugs” are also eliminated and pathobionts can easily overgrow and bias immune responses towards proinflammatory, leading to the development of diseases (e.g., inflammatory bowel diseases) ( 5 ). ATB intervention could negatively affect the long-term homeostatic interactions of the host's immune system with the microbiota; in the case of ATB-induced dysbiosis, chronic immunopathological responses may develop ( 6 , 7 ). Recently, early postnatal ATB administration limiting proper microbiota formation has been linked with obesity development ( 8 ). ATB administration has a profound effect on broad spectra of physiological functions, including immune system development and maintenance. The mucosal microbiota profoundly shapes the development and polarization of the mammalian immune system ( 9 ). Neutrophils, the most abundant population of leukocytes, present the first line of defense against pathogenic microorganisms. Upon encounter with a pathogen, neutrophils are immediately released from bone marrow to fight against infectious agents. Neutrophils are professional phagocytes, which can efficiently eliminate ingested microbes with an abundance of enzymes (e.g. NADPH oxidase, myeloperoxidase, neutrophil elastase) and antimicrobial peptides (α-defensins 1–4) present in their granules. Considering neutrophils high numbers and their antimicrobial capacity, they play an irreplaceable role in bacterial clearance in the tissue, and they are present in high numbers in the inflamed intestine. Neutrophil accumulation in lamina propria during intestinal inflammation is linked to the Th17 immune response. Flannigan and his team ( 10 ) showed in an experimental mouse model that IL-17A drives, through the engagement of CXCR2, the expansion, and migration of neutrophils to the ileum, where they control the numbers of segmented filamentous bacteria (SFB). They demonstrated that IL-23 is an important cytokine released after SFB colonization that induces the production of IL-22, which is linked to the secretion of antimicrobial peptides and, thus, the ability of neutrophils to control the numbers of bacteria in the intestine ( 10 ). Besides that, IL-17A induces the production of granulocyte colony-stimulating factor (G-CSF), a critical granulopoiesis growth factor ( 11 ). For a long time, it was thought that the enhanced presence of neutrophils was solely a hallmark of intestinal inflammation. Nevertheless, recent data show that neutrophils play a more complex role at the intestinal barrier and represent one of the important cell types maintaining mucosal homeostasis ( 12 ). Components of intestinal bacteria (e.g., lipopolysaccharide, peptidoglycan) promote the maturation and priming of circulating neutrophils. On the contrary, some bacterial products (e.g., short-chain fatty acids) can inhibit neutrophil maturation. Neutrophils can, in turn, limit the number of bacteria by producing reactive oxygen species (ROS), antimicrobial peptides (AMP), and enzymes. Extensive ROS production can prevent bacterial translocation and dissemination throughout the body ( 13 ). ATB treatment can affect neutrophil antimicrobial functions. In vitro experiments demonstrated that ATB administration can enhance antimicrobial killing by neutrophils ( 14 ) and delay their apoptosis during infection ( 15 ). Watanabe et al. showed that ATB administration can severely affect neutrophil trafficking to the gut and subsequently increase the degree of amebiasis ( 16 ). They also showed that this phenomenon was caused by the decreased presence of CXCR2 on the neutrophil surface ( 16 ). Importantly, it is established that neutrophils present a rather heterogeneous immune cell population with diverse functions ranging from pro-inflammatory to immunosuppressive and that the disruption of proportions of individual neutrophil subsets can lead to chronic inflammatory diseases. Distinct subpopulations of neutrophils are characterized according to the cell surface expression of typical markers. These include CD11b, Ly6G, Ly6C, CXCR2, and CD62L in mice and CD11b, CD11c, CD15, CD16, CD62L, and CD66b in humans ( 17 ). The beneficial effect of probiotics has been acknowledged, and the administration of probiotic strains is a rational way to promote the renewal of microbiota and mutual homeostatic interactions between the microbiota and the host immune system. Nevertheless, it is important to highlight that the beneficial effect of probiotics is highly strain-specific. Suitable strains used for the correction of ATB-disturbed microbial communities should thus be carefully selected. Notably, distinct probiotic strains have diverse capacities to modulate the immune system, ranging from pro-inflammatory and anti-infectious to immunoregulatory effects ( 18 – 20 ). Vong et al. showed that live probiotic bacteria Lacticaseibacillus rhamnosus (previously Lactobacillus rhamnosus ) can lower the production of ROS and phagocytic capacity and inhibit the formation of neutrophil extracellular traps (NETs) ( 21 ). Deregulated enhanced formation of NETs can be pathological, for example, in autoimmune diseases, such as rheumatoid arthritis ( 22 ), diabetes mellitus ( 23 ), or systemic lupus erythematosus ( 24 ). On the other hand, the probiotic Escherichia coli Nissle 1917 was a potent inducer of NETosis ( 21 ), suggesting that the inhibitory effect of probiotics on NET formation is strain-specific. Taken altogether, neutrophils play a key role in intestinal inflammation as well as its resolution. As one of the cell types mediating the immune response to intestinal bacteria, they are important for the maintenance of healthy and balanced microbiota. We thus aimed to elucidate how ATB-induced dysbiosis affects immune function with a special focus on neutrophils and how the introduction of probiotics can normalize this. To understand the effect of ATB administration on the relationship between the microbiota and the immune system, we used a mouse experimental model, where dysbiosis was induced by ATB administration. We tested the capacity of the probiotic strain Escherichia coli O83:K24:H31 (EcO83) to correct the dysbiosis and renew mutual homeostatic interactions between microbiota and host immune system, preventing the development of undesirable chronic pro-inflammatory responses. Originally, EcO83 was administered to neonates to prevent nosocomial infections ( 25 ). In addition to that, EcO83 has been shown to prevent allergy development ( 25 ) and to promote immunoregulatory responses, possibly limiting pro-allergic responses ( 26 ). Findings show that EcO83 contributes to immune system maturation together with setting regulatory responses ( 27 , 28 ). Based on previous results, we evaluated the capacity of EcO83 to dampen inflammation by inducing regulatory responses and promoting gut barrier function in mice with ATB-induced dysbiosis. 2. MATERIALS AND METHODS 2.1 Mice BALB/cAnNCrl were bred in the Czech Center for Phenogenomics (Vestec, Czech Republic) or purchased from Velaz and kept in sterilized, filter-topped cages and fed autoclaved food with free access to clean water. Female mice aged between 8–11 weeks were used in the experiments. Mice were sacrificed by cervical dislocation. All mouse experiments were approved by institutional review board of the First Faculty of Medicine, Charles University (IRB 1.LF-731) and the animal committee and executed according to good practice with animal models (MSMT-17298/2021-4). 2.2 Antibiotic and probiotic treatment Mice were treated with antibiotics introduced in sterile drinking water for two weeks. Control mice were given sterile water. The antibiotic mixture was added to the fresh drinking water and exchanged every 3 days. Ampicillin (1 g/L), neomycin sulphate (1 g/L), metronidazole (1 g/L), and vancomycin (0.5 g/L) (Sigma-Aldrich, USA) were diluted in sterile water to reach the final concentrations. Following the antibiotic treatment, mice were treated with a probiotic bacterial strain Escherichia coli O83:K24:H31 (EcO83) every day for 5 consecutive days by intragastric gavage, at 5x10 8 CFU in 200 µl of gavage buffer (PBS containing 200 mM NaHCO 3 and 2% glucose), with a stainless-steel mouse gavage needle. EcO83 was cultivated as previously described ( 23 ). Briefly, EcO83 was inoculated in Luria Bertani broth and cultivated for 6h (37°C upon agitation). After cultivation, bacterial cell culture was centrifuged, washed 2 times with PBS, and resuspended to a final concentration of 2.5x10 9 CFU/ml in the gavage buffer. 2.3 Blood, bone marrow, and tissue collection Tissue samples were collected from euthanized mice. Blood was drawn from the lateral saphenous vein into a tube containing heparin (10 USP units/ml of blood) to prevent coagulation and centrifuged to collect plasma for analysis of cytokine concentration. Bone marrow was obtained from the femur by flushing with sterile non-supplemented cell culture medium (RPMI1640), gently dissociated by pipetting up and down, and filtered through a cell strainer (70um) to eliminate clumps and remaining tissue to obtain single cell suspension for further analysis. Intestinal sections (ileum and colon) devoid of stool and single-cell suspensions from mesenteric lymph nodes (mLN) were stored in RNAprotect Tissue Reagent (QIAGEN, USA) until RNA extraction. 2.4 Microbiome analysis from stool samples Stool samples were collected directly from the distant part of the colon right after the mouse sacrifice. Samples were snap-frozen in liquid nitrogen and kept at -80°C until further processing. Total DNA was extracted from the stool using the QIAmp Fast DNA Stool Mini Kit (Qiagen).. according to the manufacturer's protocol. DNA quality and concentration were determined using NanoDrop. 16S rDNA metagenomic NGS libraries from the DNA samples and subsequent sequencing using the Illumina MiSeq v3 was performed at the Institute of Applied Biotechnologies a.s. Only samples that passed the Quality Control (A260/280: 1.6–2.1) were used to prepare the NGS libraries, which were sequenced on NovaSeq X Plus using standard workflow. The microbial composition was analyzed using relative abundance, where the number of reads for each taxonomic level (phylum, class, order) was normalized to 100% of the total classified reads. Raw sequencing data are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA1226621 (cited 2025 Mar 10) available from: https://www.ncbi.nlm.nih.gov/sra/PRJNA1226621 . 2.5 Flow cytometry analysis Neutrophil subsets were analysed in BM single-cell suspensions using flow cytometry. Cell suspensions were stained for the following cell surface markers: anti-mouse CD11b (clone M1/70, EXBIO, CZ), Ly6G (clone 1A8, BioLegend, USA), Ly6C (clone HK1.4, BioLegend, USA), CXCR2 (clone SA044G4, BioLegend, USA), CD62L (clone MEL-14, EXBIO, CZ), incubated for 10 min at room temperature in the dark, followed by red blood cell lysis and acquired immediately using BD FACS Canto II (Becton Dickinson). The representative gating strategy for the identification of neutrophil phenotype is shown in Supplementary Fig. 1. 2.6 RNA isolation, cDNA library, and quantitative real-time PCR Total RNA was isolated from the mLN, ileum, and colon tissue using the RNeasy Mini Kit (QIAGEN, USA) according to the manufacturer’s instructions. The concentration and purity of isolated RNA was checked using Nanodrop (Thermo Fisher Scientific, USA) before following analyses. 0.5 µg of total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) to create a cDNA library. Relative gene expression was quantified as described previously ( 29 ). Briefly, TaqMan gene expression assay (Applied Biosystems, USA) was used to quantify the level of gene expression of target genes in tissue and cells. The list of TaqMan assays is provided in Table 1. Relative quantification of gene expression was related to the level of gene expression of beta-actin ( Actb ) used as a reference gene (housekeeping gene, endogenous control). The qPCR reactions were run in doublets using the Light Cycler 480 Real-Time PCR System (Roche, Switzerland). 2.8 Data analysis and statistics Flow cytometry results were analyzed using FlowJo v10 software (TreeStar, USA) using appropriate single stain compensation and FMO controls to set a proper gating strategy. Gene expression of the target gene was quantified relative to the housekeeping gene Actb using the 2 −ΔΔCt method ( 30 ). Data normality was assessed. One-way ANOVA was used for normally distributed data, while the Kruskal-Wallis test was applied for non-normal distributions. Outliers were identified using Dixon's test (p < 0.2). Analysis and graphical processing were performed using GraphPad Prism 8 (USA). Results are presented as mean ± SEM. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 3. RESULTS 3.1 Antibiotic treatment induces changes in microbiota composition To determine whether ATB treatment alters fecal microbiota composition and induces dysbiosis, adult mice were treated with a broad-spectrum antibiotic mixture (ampicillin, neomycin sulphate, vancomycin and metronidazole) for one week (ATB group). Another group received EcO83 (ATB + EcO83 group) via oral gavage for five consecutive days following ATB treatment. Control groups consisted of untreated mice (C group), and mice supplemented with EcO83 only (EcO83 group), according to the experimental setup (Fig. 1 A ). ATB treatment induced a drop in body weight, which was not statistically significant and normalized within one week regardless of EcO83 administration (data not shown). Although weight was restored, on the day of sacrifice, the caecum of ATB-treated mice appeared enlarged and dark (Fig. 1 B ) compared to the controls, a condition that was mitigated by EcO83 supplementation (Fig. 1 B ). Microbiota composition was analyzed using 16S rDNA gene sequencing from stool samples. Both EcO83 and ATB treatment induces minor nonsignificant changes in the diversity and abundance of bacterial species as assessed by the Shannon-Wiener index (Fig. 2 A ). The fecal microbiome was classified at the phylum, class, and order levels (Fig. 2 B ). Marked dysbiosis was evident immediately after the end of ATB treatment ( Figure S2 ). The significant impact of ATB and/or EcO83 administration on specific microbial phyla, classes, and genera is shown in Figure S3 . EcO83 supplementation significantly increased the abundance of Candidatus Saccharibacteria in both ATB-treated and non-treated mice ( Fig S3A) . The proportion of Deferribacteres/Deferribacteriales/Deferribacteres was reduced in ATB-treated mice, but EcO83 supplementation promoted their recovery ( Fig S3 ). Conversely, Flavobacteria were significantly underrepresented in the gut microbiota ATB-treated mice, and EcO83 had no effect on their restoration ( Fig S3B ). Similarly, Sphingobacteria levels were reduced following ATB-treatment, with EcO83 supplementation having no impact on their restoration ( Fig S3B, C ). In contrast, EcO83 significantly promoted the growth of Mollicutes , Anaeroplasmatales and Nautiliales as compared to control mice. Elevated levels of these taxa by EcO83 were also observed in ATB-treated mice (ATB + EcO83 group) ( Fig S3B, C ). 3.2 Antibiotics alter gene expression of tight junction proteins in the ileum, which is normalized with EcO83 administration We aimed to evaluate the impact of dysbiosis on intestinal tissue damage and investigate whether EcO83 administration could promote recovery. To achieve this, we performed qPCR analysis on tissue samples from the ileum and colon, targeting genes involved in gut barrier function, including tight junction proteins Claudin 1 ( Cldn1 ), Claudin 5 ( Cldn5 ), and Occludin ( Ocln ) as well as the regulatory cytokine IL-10 ( Il10 ). Our analysis revealed significant modulation of gene expression in the small intestine of mice with ATB-induced induced dysbiosis (Fig. 3). The expression of tight junction genes was increased, particularly for Cldn1 and Cldn5 in ATB-treated mice, compared to control groups. EcO83 supplementation promoted the Ocln gene expression and enhanced the expression of the Il10 gene . Importantly, EcO83 supplementation also induced Il10 expression in ATB-treated mice (Fig. 3 A ). Gene expression of tight junction proteins and Il10 was also measured in the colon, but no significant changes were detected (Fig. 3 B ). 3.3 Antibiotic treatment does not induce cytokine changes in the mesenteric lymph nodes Since we observed changes in the expression of ileal epithelial proteins, we investigated whether antibiotic treatment influenced cytokine expression in the mesenteric lymph nodes, which drain the gut epithelium. Interestingly, no significant changes were detected in any of the measured cytokines (Fig. 4), except for the normalization of IL-18 levels following EcO83 administration in ATB-treated mice (Fig. 4), a cytokine linked to neutrophil function and trafficking. To assess potential systemic immune alterations, we also measured inflammatory cytokine concentrations in serum. However, no significant changes in IL-1β serum levels were observed ( Supplementary Fig. 4 ), indicating that antibiotic treatment did not induce a systemic pro-inflammatory response. 3.4 Antibiotics induce an increase in the number of CD62L − CXCR2 − neutrophils, which is partially corrected by administration of EcO83 Finally, we sought to determine whether ATB treatment and EcO83 administration affected neutrophil phenotypes. Cell suspensions prepared from BM were analyzed for neutrophil surface markers, identifying neutrophils as CD11b + Ly6G + Ly6C − granulocytes (gating strategy, Supplementary Fig. 1 ). Although there was a trend towards an increased number of CD11b + cells in EcO83-treated mice, the overall mean fluorescence intensity (MFI) of CD11b remained comparable (Fig. 5 A ). Additionally, no significant changes were observed in the percentage of neutrophils (Ly6G + ), which constituted the largest proportion of CD11b + cells, or in the percentage of monocytes (Ly6C + ) (Fig. 5 B ). The most profound differences were observed in the expression of the adhesion molecule CD62L and the IL-8 receptor CXCR2 on Ly6G + neutrophils (Fig. 6). ATB treatment induced loss of CD62L expression, while EcO83 administration had the opposite effect. ATB-treated mice had a slightly increased number of CD62L − CXCR2 − double-negative cells, a change that was reversed to control levels upon probiotic administration (Fig. 6 A, bottom right ). When antibiotic treatment was followed by EcO83 intervention, the neutrophil phenotype was restored to its original state (Fig. 6 B ). 4. DISCUSSION The suitability of probiotic supplementation during ATB treatment remains an area of active debate. In this study, we investigated the ability of EcO83 to restore microbiota composition and immune function following ATB-induced dysbiosis in a murine model. The prolonged dysbiosis triggered by ATB administration could lead to altered immune responses characterized by low-grade chronic inflammation supporting inflammatory bowel disease development. Therefore, the potential of probiotics to promote the restoration of microbiota composition and mutual homeostatic interactions between microbiota and the host immune system will be beneficial for the host's health. EcO83 supplementation has no significant impact on microbiota composition 12 days after ATB administration. No effect or even adverse effect of probiotic supplementation has been reported previously when probiotic supplementation prolonged diarrhoea and dysbiosis documented by a lower number of microbial species detected in stool samples of patients ( 31 , 32 ). However, it is important to highlight that in these studies, a mixture of 11 bacterial species with a scarce probiotic effect documented by previous studies was used. In addition to that, according to the recommendation of ISAPP (International Scientific Association for Probiotics and Prebiotics), the probiotic strains should be reported unambiguously, i.e. including particular strain annotation. It is important to highlight that the probiotic effect is highly strain-specific and probiotic effect ranges from immunostimulatory to immunosuppressive/immunoregulatory ( 33 ). Therefore, the selection of appropriate probiotic strains for the effects desired in a particular context (i.e., the correction of a concrete pathology) is critical. In our study, EcO83 has been selected due to its immunoregulatory capacity ( 26 – 28 ) which is desirable to suppress pro-inflammatory responses occurring during dysbiosis. Nevertheless, we were not able to demonstrate a clear effect of EcO83 supplementation on higher microbiota diversity (Fig. 2 A ). To confirm that our model of ATB administration is able to cause dysbiosis, microbiota composition has been analyzed immediately after ATB administration; Figure S1 clearly demonstrates a huge dysbiosis in both ATB and ATB + EcO83 groups before EcO83 administration. In a healthy intestine, obligate anaerobes like Firmicutes and Bacteroidetes thrive in low-oxygen environments. Dysbiosis disrupts this delicate balance, leading to a decline in these anaerobes and a concurrent increase in facultative anaerobes such as Enterobacteriaceae , often associated with elevated ROS ( 34 ). Immediately after antibiotic treatment, we observed a marginal rise in Enterobacteriales , a characteristic sign of dysbiosis - a state associated with reduced microbial diversity and increased Proteobacteria , particularly within the Enterobacteriaceae family. Although Bacteroidetes and Firmicutes levels decreased following antibiotic exposure, they independently returned to baseline values, demonstrating their remarkable resilience ( 35 ). In our study, no delay of microbiota normalization has been observed in EcO83 supplemented group. Moreover, no difference among groups (C, EcO83, ATB, ATB + EcO83) has been demonstrated, confirming that probiotic administration has no impact on the microbial community ( 36 – 38 ). Nevertheless, we observed the capacity of EcO83 to increase the presence of Candidatus Saccharibacteria. This is important in the context of metabolomics since Candidatus Saccharibacteria was identified as the phylum with the most pronounced impact on metabolome associated with Alzheimer's disease ( 39 ). While EcO83 demonstrated the ability to support Candidatus Saccharibacteria colonization independently, the most pronounced effects were observed when mice received antibiotic treatment prior to probiotic supplementation. A comparable pattern emerged for the orders Coriobacteriales and Nautiliales , suggesting a potential synergistic interaction between antibiotic pretreatment and probiotic administration. EcO83 can elevate Deferribacteres , which belong to the phylum the most impacted by ATB administration in our study. Association of an increased proportion of Deferribacteres in piglets with improved antioxidant function was documented in a study supplementing mothers with probiotics ( 40 ). The potential of probiotics to promote Deferribacteres and limit inflammation has been demonstrated previously ( 41 , 42 ). On the other hand, Panpetch et al. demonstrated an increased presence of Deferribacteres in a mouse model of dysbiosis triggered by dextran sulphate solution together with Candida and Klebsiella pneumoniae administration ( 43 ) and IL-1β expression in the gut in a mouse model of Diet-Induced Obese Mice ( 44 ). Decreased levels of Flavobacteria were shown to be associated with IBD ( 45 ), but EcO83 supplementation was not able to restore diminished Flavobacteria after ATB treatment. In our study, only a single strain of probiotic EcO83 has been used, and possibly a mixture of carefully selected probiotic strains can be more beneficial and/or impact a complex microbial community in mice without ATB treatment. This observation agrees with a previous study of early postnatal EcO83 supplementation, where no change in the microbiota of ten-year-old children has been reported ( 28 ). However, even complex mixtures of probiotics have only a marginal effect on microbiota composition in healthy volunteers, as reviewed by Kristensen ( 37 ). On the other hand, several reports highlight the impact of probiotics on microbial communities ( 46 , 47 ). This discrepancy can be explained by various probiotic strains used, dose (CFU) of probiotics, single strain versus complex mixture of probiotic strains, duration of probiotic supplementation, and dietary, ethnic, and socioeconomic differences of probands among the studies. On the other hand, the beneficial effect of probiotics is not merely mediated by changes in microbiota composition measured by changes in alpha diversity, richness, or evenness. The beneficial effect of probiotics can be mediated by the production of SCFA directly by probiotic strains administered or the capacity of probiotic strains to trigger SCFA production ( 48 – 53 ). The other beneficial effect of probiotics involves the promotion of tight junction protein expression, leading to an increase in gut barrier function ( 54 – 56 ). We have demonstrated the capacity of EcO83 to promote tight junction protein expression in neonates supplemented by EcO83 ( 27 ). Previous studies have shown that ATB administration decreases the expression of various genes tight junction-associated proteins, namely Claudin-1, Occludin, and ZO-1 in the intestine (Feng et al., 2019) and Claudin 3,4 in the colon (Ran et al. al., 2020). Subsequent administration of probiotics normalized the expression of Zonulin-1 and Occludin (L. Huang et al., 2023). Previous research from our laboratory showed that early postnatal colonization of EcO83 increased the expression of the tight junction genes Cldn and Ocln as well as Il10 , thereby supporting intestinal barrier function ( 27 ). In the current study, the capacity of EcO83 to promote tight junction protein expression and restore gut barrier function in mice treated with ATB has been tested as well. In contrast to our hypothesis, the most elevated gene expression of all tight junction proteins has been detected in the ileum of mice treated with ATB, suggesting the increased gene expression is a compensatory effect for disturbed gut barrier function in the effort to renew the functionality of gut barrier. EcO83 was able to promote gene expression of tight junction proteins, documenting the capacity of probiotics to promote gut barrier function ( 57 , 58 ). Our hypothesis that the most prominent expression of tight junction proteins is a compensatory effect of ongoing inflammation is further supported by decreased gene expression of tight junction proteins in the ATB + EcO83 group. IL-10 represents a cytokine with immunoregulatory function preventing the development of pro-inflammatory responses. EcO83 was able to promote Il10 expression, which is in line with our previous observations ( 26 – 28 , 59 ). Importantly, mice treated with ATB and EcO83 exerted increased gene expression of Il10 as well, documenting the potential of EcO83 to limit inflammation triggered by ATB-induced dysbiosis. Surprisingly, neither ATB nor EcO83 modified the gene expression of tight junction proteins in the colon, Fig. 3B. The capacity of probiotics to modulate adaptive immune responses has been described previously ( 60 , 61 ). Therefore, we investigated the impact of ATB and/or EcO83 administration on the local immune system in MLN. To our surprise, no tremendous changes in gene expression of selected cytokines have been documented. Only IL-18 was lowered in the treated group, and EcO83 supplementation normalized the expression of IL-18 to the level of control mice, highlighting the role of EcO83 in triggering anti-infectious immune responses ( 62 , 63 ). On the other hand, IL-18 can contribute to inflammatory responses ( 64 ). Finally, IL-18 also plays an important role in gut microbiota monitoring and homeostasis, and its dysregulation could lead to microbiota dysbiosis and accelerate disease progression. Furthermore, the dysbiosis observed in Il18 −/− mice contributed to their increased susceptibility to Listeria infection ( 65 ). This highlights the importance of fine-tuning immune responses with emphasis on appropriate regulatory mechanisms. The other goal of the study was to characterize the impact of ATB-induced dysbiosis on the proportional and functional characteristics of neutrophils as cells of the first line of defense. Since the majority of neutrophils are located in the bone marrow, we investigated the proportion of particular neutrophil subsets there by flow cytometry. Both probiotic-treated groups (EcO83 alone and ATB + EcO83) possessed elevated levels of CD11b + in bone marrow. ATB-treated mice have a significantly increased proportion of CD62L − neutrophils associated with the typical phenotype of old senescent and exhausted cells. EcO83 supplementation was able to normalize the levels of CD62L − neutrophils to the values found in healthy control mice. Altered granulopoiesis triggered by ATB can be demonstrated by the levels of CXCR2, which was decreased in ATB-treated mice compared to control healthy mice. Diminished levels of CXCR2 in ATB-treated mice have already been published ( 16 ), and our study confirmed the potential effect of ATB on lowering the migration capacity of neutrophils to the site of infection/inflammation. Importantly, the effect of the probiotic strain Bacillus polyfermenticus in lowering the severity of colitis and promoting neoangiogenesis was dependent on CXCR2 and IL-8 signalling ( 66 ). Together, these results suggest that lower CD11b, along with decreased CD62L and CXCR2, indicate a reduced capacity for rolling, adhering to endothelial tissues, and migrating into target tissues, which might result in impaired local immune responses. Antibiotic treatment contributes to this altered neutrophil profile, potentially compromising the immune system's ability to effectively respond to infections. Interestingly, EcO83 was able to restore granulopoiesis in ATB-treated mice. These results highlight the capacity of EcO83 to normalize granulopoiesis. Conclusion While EcO83 did not significantly modify microbiota diversity, it played a critical role in restoring gut barrier integrity and immune balance. The probiotic counteracted ATB-induced changes in neutrophil subsets, promoted Il10 expression, and normalized Il18 expression levels. These findings emphasize the importance of strain-specific probiotic effects beyond microbiota composition. Further research is needed to elucidate the mechanisms by which probiotics modulate immune responses and their potential applications in ATB-associated dysbiosis. Declarations Conflict of Interest The authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest. JH obtained a collaborative grant with the company manufacturing probiotic vaccine called Colinfant Newborn. Company has no influence on study design and data interpretation. Author Contributions E.M. and J.H. conceptualized the study and designed the experiments. E.M., E. K. performed the most of experiments, E.M, E.K, J.H. and L.S. analyzed data, prepared figures, and wrote and edited the manuscript. E.M., E.K., V.C., L.S., P.A., J.V., O.N., P.P., A.K.Z and J.P. performed the experiments. E.M. and J.H. acquired funding. I.S. and V.C. edited the manuscript. Funding This project has been supported by the Grant Agency of Charles University (GA UK 6121), Charles University research project Cooperatio IMMU207032. Work was also supported by OP JAK project MSCA Fellowships CZ – UK2 (reg. n. CZ.02.01.01/00/22_010/0008115). OeAD-GmbH: CZ 04/2024 and CZ 07/2023, Austrian Science Fund: P 34867 Data Availability Statement Raw sequencing data are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA1226621 (cited 2025 Mar 10) available from: https://www.ncbi.nlm.nih.gov/sra/PRJNA1226621. References Ramirez J, Guarner F, Bustos Fernandez L, Maruy A, Sdepanian VL, Cohen H. Antibiotics as Major Disruptors of Gut Microbiota. Front Cell Infect Microbiol. 2020 Nov 24;10:572912. Reyman M, van Houten MA, Watson RL, Chu MLJN, Arp K, de Waal WJ, et al. Effects of early-life antibiotics on the developing infant gut microbiome and resistome: a randomized trial. Nat Commun. 2022 Feb 16;13(1):893. 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A Multi-Strain Probiotic Formulation Improves Intestinal Barrier Function by the Modulation of Tight and Adherent Junction Proteins. Cells. 2022 Aug 22;11(16):2617. Ulluwishewa D, Anderson RC, McNabb WC, Moughan PJ, Wells JM, Roy NC. Regulation of tight junction permeability by intestinal bacteria and dietary components. J Nutr. 2011 May;141(5):769–76. Zhao X, Zhou J, Liang W, Sheng Q, Lu L, Chen T, et al. Probiotics mixture reinforces barrier function to ameliorate necrotizing enterocolitis by regulating PXR-JNK pathway. Cell Biosci. 2021 Jan 19;11:20. Casula E, Pisano MB, Serreli G, Zodio S, Melis MP, Corona G, et al. Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation. Food Chem Toxicol Int J Publ Br Ind Biol Res Assoc. 2023 Feb;172:113558. Hrdý J, Kocourková I, Lodinová-Žádníková R, Kolářová L, Prokešová L. The effect of a probiotic Escherichia coli strain on regulatory T-cells in six year-old children. Benef Microbes. 2016 Nov 30;7(5):639–48. Konieczna P, Groeger D, Ziegler M, Frei R, Ferstl R, Shanahan F, et al. Bifidobacterium infantis 35624 administration induces Foxp3 T regulatory cells in human peripheral blood: potential role for myeloid and plasmacytoid dendritic cells. Gut. 2012 Mar;61(3):354–66. Tan TG, Sefik E, Geva-Zatorsky N, Kua L, Naskar D, Teng F, et al. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc Natl Acad Sci U S A. 2016 Dec 13;113(50):E8141–50. Rao A, Strauss O, Kokkinou E, Bruchard M, Tripathi KP, Schlums H, et al. Cytokines regulate the antigen-presenting characteristics of human circulating and tissue-resident intestinal ILCs. Nat Commun. 2020 Apr 27;11(1):2049. Kowalewicz-Kulbat M, Szpakowski P, Krawczyk KT, Kowalski ML, Kosinski S, Biet F, et al. Decrease of IL-5 Production by Naive T Cells Cocultured with IL-18-Producing BCG-Pulsed Dendritic Cells from Patients Allergic to House Dust Mite. Vaccines. 2021 Mar 18;9(3):277. Dinarello CA. Interleukin-18 and the pathogenesis of inflammatory diseases. Semin Nephrol. 2007 Jan;27(1):98–114. Zheng X, Liu L, Meng G, Zhu S, Zhou R, Jiang W. IL-18 maintains the homeostasis of mucosal immune system via inflammasome-independent but microbiota-dependent manner. Sci Bull. 2021 Oct 30;66(20):2115–23. Im E, Choi YJ, Kim CH, Fiocchi C, Pothoulakis C, Rhee SH. The angiogenic effect of probiotic Bacillus polyfermenticus on human intestinal microvascular endothelial cells is mediated by IL-8. Am J Physiol Gastrointest Liver Physiol. 2009 Nov;297(5):G999–1008. Table 1 Table 1. List of probes used for analysis of genes of interest. Gene symbol Assay ID Gene name Actb Mm00607939_s1 Actin beta Cldn1 Mm1342184_m1 Claudin 1 Cldn5 Mm00727012_s1 Claudin 5 Il2 Mm00434256_m1 Interleukin 2 Il4 Mm99999154_m1 Interleukin 4 Il10 Mm01288386_m1 Interleukin 10 Il18 Mm00434226_m1 Interleukin 18 Ifng Mm01168133_g1 Interferon gamma Ocln Mm00500910_m1 Occludin Clinical trial number: not applicable. Consent for publication: not applicable. Additional Declarations Competing interest reported. J.H. is co-investigator of grant EI22_002/0000879 together with Dyntec (company producing Colinfant Newborn). The other authors have no competing interests. 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10:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6503496/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6503496/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-025-10814-w","type":"published","date":"2025-11-19T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83108494,"identity":"3d13c400-6fb4-4ba2-ba33-ca713d9ffe60","added_by":"auto","created_at":"2025-05-20 06:49:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design and intestinal morphology.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of antibiotic-induced dysbiosis and subsequent probiotic treatment. BALB/c mice received a mixture of antibiotics (ampicillin 1 g/L, vancomycin 0.5 g/L, neomycin sulphate 1 g/L, and metronidazole 1 g/L) in drinking water for 2 weeks, with solution changes every 3 days, followed by oral gavage of probiotic EcO83 (5×10\u003csup\u003e8\u003c/sup\u003e CFU in PBS containing 200 mM NaHCO\u003csub\u003e3\u003c/sub\u003e and 2% glucose) for 5 consecutive days. Mice were sacrificed after the completion of probiotic treatment for sample collection. \u003cstrong\u003eAbbreviations:\u003c/strong\u003e C (control), ATB (antibiotic mixture), EcO83 (\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eO83:K24:H31). \u003cstrong\u003e(B)\u003c/strong\u003e Macroscopic images showing the morphology of the caecum and adjacent small intestine (ileum) and part of the colon.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/f70974df46f8101148fdb109.jpg"},{"id":83109382,"identity":"2941cc78-86f8-437a-8145-d51eb350952b","added_by":"auto","created_at":"2025-05-20 06:57:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe bacterial composition on the day of sacrifice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Alpha diversity of gut microbiota determined using the Shannon-Wiener index. \u003cstrong\u003e(B)\u003c/strong\u003e Stacked bar charts showing phylum, class, and order levels of bacterial composition. Groups are represented as follows: non-treated controls (C), EcO83-treated group (EcO83), antibiotic-treated group to induce dysbiosis (ATB), and combined antibiotic-treated group with subsequent EcO83 administration (ATB+EcO83).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/db7cdab7a94793197b43f9e3.jpg"},{"id":83108500,"identity":"e2378513-cd77-4a8c-bf3f-519783e834e3","added_by":"auto","created_at":"2025-05-20 06:49:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression analysis of markers associated with gut barrier function.\u003c/strong\u003e Relative quantification of genes encoding tight junction proteins (\u003cem\u003eCldn1\u003c/em\u003e, \u003cem\u003eCldn5\u003c/em\u003e, \u003cem\u003eOcln\u003c/em\u003e) and the regulatory cytokine \u003cem\u003eIl10\u003c/em\u003e was performed using qRT-PCR in the ileum \u003cstrong\u003e(A)\u003c/strong\u003e and colon \u003cstrong\u003e(B)\u003c/strong\u003e of mice treated with antibiotics and/or probiotic EcO83, compared to untreated controls. Data represent mean ± SEM. Statistical significance was determined using one-way ANOVA or Kruskal-Wallis test based on data distribution, where *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. Absence of significance indicators represents non-significant differences. C - non-treated controls, EcO83 – \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31-treated group, ATB - antibiotic-treated group to induce dysbiosis, ATB+EcO83 - combined antibiotic-treated group with subsequent \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 administration.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/ea31d3594762e272bf2b9258.jpg"},{"id":83109383,"identity":"f2150830-468e-4a6e-99c9-16700dcd59a5","added_by":"auto","created_at":"2025-05-20 06:57:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression of cytokines in mesenteric lymph nodes.\u003c/strong\u003e Relative quantification of \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eIl2\u003c/em\u003e, \u003cem\u003eIl4\u003c/em\u003e, \u003cem\u003eIl10\u003c/em\u003e, and \u003cem\u003eIfng\u003c/em\u003e, and \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eIl18 \u003c/em\u003ewas performed using qRT-PCR in mice treated with antibiotics and/or probiotic EcO83, compared to untreated controls. Data represent mean ± SEM. Statistical significance was determined using one-way ANOVA or Kruskal-Wallis test based on data distribution, where *p\u0026lt;0.05. Absence of significance indicators represents non-significant differences. C - non-treated controls, EcO83 – \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31-treated group, ATB - antibiotic-treated group to induce dysbiosis, ATB+EcO83 - combined antibiotic-treated group with subsequent \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 administration.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/3ec4ef51d030e7ebe00dca77.jpg"},{"id":83109619,"identity":"2c9fb168-539d-41df-ac4e-86aa525c7ab4","added_by":"auto","created_at":"2025-05-20 07:05:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of neutrophil phenotype in bone marrow.\u003c/strong\u003e Flow cytometric analysis of \u003cstrong\u003e(A)\u003c/strong\u003e CD11b\u003csup\u003e+\u003c/sup\u003e cells percentage and their mean of fluorescence intensity (MFI), and \u003cstrong\u003e(B)\u003c/strong\u003e percentage of Ly6G\u003csup\u003e+\u003c/sup\u003e granulocytes and Ly6C\u003csup\u003e+\u003c/sup\u003e monocytes/macrophages within CD11b\u003csup\u003e+\u003c/sup\u003e population in mice treated with antibiotics and/or probiotic EcO83, compared to untreated controls. Data represent mean ± SEM. Statistical significance was determined using one-way ANOVA or Kruskal-Wallis test based on data distribution, where *p\u0026lt;0.05. Absence of significance indicators represents non-significant differences. C - non-treated controls, EcO83 – \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31-treated group, ATB - antibiotic-treated group to induce dysbiosis, ATB+EcO83 - combined antibiotic-treated group with subsequent \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 administration.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/693bd264633839fcf87919b1.jpg"},{"id":83108501,"identity":"16d435d7-3221-4d02-b9a4-e1c22a392bdc","added_by":"auto","created_at":"2025-05-20 06:49:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of neutrophil subpopulations in bone marrow.\u003c/strong\u003e Flow cytometric analysis of \u003cstrong\u003e(A)\u003c/strong\u003e neutrophil subsets within Ly6G\u003csup\u003e+\u003c/sup\u003e population defined as CD62L\u003csup\u003e-\u003c/sup\u003eCXCR2\u003csup\u003e+\u003c/sup\u003e, CD62L\u003csup\u003e+\u003c/sup\u003eCXCR2\u003csup\u003e-\u003c/sup\u003e, CD62L\u003csup\u003e+\u003c/sup\u003eCXCR2\u003csup\u003e+\u003c/sup\u003e, and CD62L\u003csup\u003e-\u003c/sup\u003eCXCR2\u003csup\u003e-\u003c/sup\u003e, and \u003cstrong\u003e(B)\u003c/strong\u003e mean of fluorescence intensity (MFI) of CD62L and CXCR2 on Ly6G\u003csup\u003e+\u003c/sup\u003e cells in mice treated with antibiotics and/or probiotic EcO83, compared to untreated controls. Data represent mean ± SEM. Statistical significance was determined using one-way ANOVA or Kruskal-Wallis test based on data distribution, where *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. Absence of significance indicators represents non-significant differences. C - non-treated controls, EcO83 – \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31-treated group, ATB - antibiotic-treated group to induce dysbiosis, ATB+EcO83 - combined antibiotic-treated group with subsequent \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 administration.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/fe78388d28a5b03829f81b78.jpg"},{"id":96650074,"identity":"4ef0cbb1-dfba-454f-bcab-571e7cb9287a","added_by":"auto","created_at":"2025-11-24 16:06:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1563646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/3a5f61c2-9f60-4b9d-a65f-addf20c81889.pdf"},{"id":83108493,"identity":"49206983-96a9-49c3-86ad-2ccd71dba78d","added_by":"auto","created_at":"2025-05-20 06:49:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":796013,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6503496/v1/368428a8b6082795411d052a.docx"}],"financialInterests":"Competing interest reported. J.H. is co-investigator of grant EI22_002/0000879 together with Dyntec (company producing Colinfant Newborn). The other authors have no competing interests.","formattedTitle":"The ability of probiotic strain Escherichia coli O83:K24:H31 to modulate gut homeostasis and immune function after antibiotic-induced dysbiosis","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eExcessive antibiotic (ATB) usage has a deleterious effect on microbiota (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). It is known that after ATB administration, a temporary dysbiosis occurs, possibly triggering immune system dysregulation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Upon steady state, commensal and beneficial microbes present in our physiological microbiota can prevent the overgrowth of pathobionts. But, during ATB treatments, these \u0026ldquo;good bugs\u0026rdquo; are also eliminated and pathobionts can easily overgrow and bias immune responses towards proinflammatory, leading to the development of diseases (e.g., inflammatory bowel diseases) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). ATB intervention could negatively affect the long-term homeostatic interactions of the host's immune system with the microbiota; in the case of ATB-induced dysbiosis, chronic immunopathological responses may develop (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Recently, early postnatal ATB administration limiting proper microbiota formation has been linked with obesity development (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). ATB administration has a profound effect on broad spectra of physiological functions, including immune system development and maintenance.\u003c/p\u003e \u003cp\u003eThe mucosal microbiota profoundly shapes the development and polarization of the mammalian immune system (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Neutrophils, the most abundant population of leukocytes, present the first line of defense against pathogenic microorganisms. Upon encounter with a pathogen, neutrophils are immediately released from bone marrow to fight against infectious agents. Neutrophils are professional phagocytes, which can efficiently eliminate ingested microbes with an abundance of enzymes (e.g. NADPH oxidase, myeloperoxidase, neutrophil elastase) and antimicrobial peptides (α-defensins 1\u0026ndash;4) present in their granules.\u003c/p\u003e \u003cp\u003eConsidering neutrophils high numbers and their antimicrobial capacity, they play an irreplaceable role in bacterial clearance in the tissue, and they are present in high numbers in the inflamed intestine. Neutrophil accumulation in lamina propria during intestinal inflammation is linked to the Th17 immune response. Flannigan and his team (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) showed in an experimental mouse model that IL-17A drives, through the engagement of CXCR2, the expansion, and migration of neutrophils to the ileum, where they control the numbers of segmented filamentous bacteria (SFB). They demonstrated that IL-23 is an important cytokine released after SFB colonization that induces the production of IL-22, which is linked to the secretion of antimicrobial peptides and, thus, the ability of neutrophils to control the numbers of bacteria in the intestine (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Besides that, IL-17A induces the production of granulocyte colony-stimulating factor (G-CSF), a critical granulopoiesis growth factor (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For a long time, it was thought that the enhanced presence of neutrophils was solely a hallmark of intestinal inflammation. Nevertheless, recent data show that neutrophils play a more complex role at the intestinal barrier and represent one of the important cell types maintaining mucosal homeostasis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Components of intestinal bacteria (e.g., lipopolysaccharide, peptidoglycan) promote the maturation and priming of circulating neutrophils. On the contrary, some bacterial products (e.g., short-chain fatty acids) can inhibit neutrophil maturation. Neutrophils can, in turn, limit the number of bacteria by producing reactive oxygen species (ROS), antimicrobial peptides (AMP), and enzymes. Extensive ROS production can prevent bacterial translocation and dissemination throughout the body (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eATB treatment can affect neutrophil antimicrobial functions. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that ATB administration can enhance antimicrobial killing by neutrophils (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and delay their apoptosis during infection (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Watanabe et al. showed that ATB administration can severely affect neutrophil trafficking to the gut and subsequently increase the degree of amebiasis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). They also showed that this phenomenon was caused by the decreased presence of CXCR2 on the neutrophil surface (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, it is established that neutrophils present a rather heterogeneous immune cell population with diverse functions ranging from pro-inflammatory to immunosuppressive and that the disruption of proportions of individual neutrophil subsets can lead to chronic inflammatory diseases. Distinct subpopulations of neutrophils are characterized according to the cell surface expression of typical markers. These include CD11b, Ly6G, Ly6C, CXCR2, and CD62L in mice and CD11b, CD11c, CD15, CD16, CD62L, and CD66b in humans (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe beneficial effect of probiotics has been acknowledged, and the administration of probiotic strains is a rational way to promote the renewal of microbiota and mutual homeostatic interactions between the microbiota and the host immune system. Nevertheless, it is important to highlight that the beneficial effect of probiotics is highly strain-specific. Suitable strains used for the correction of ATB-disturbed microbial communities should thus be carefully selected. Notably, distinct probiotic strains have diverse capacities to modulate the immune system, ranging from pro-inflammatory and anti-infectious to immunoregulatory effects (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVong et al. showed that live probiotic bacteria \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e (previously \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e) can lower the production of ROS and phagocytic capacity and inhibit the formation of neutrophil extracellular traps (NETs) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Deregulated enhanced formation of NETs can be pathological, for example, in autoimmune diseases, such as rheumatoid arthritis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), diabetes mellitus (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), or systemic lupus erythematosus (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). On the other hand, the probiotic \u003cem\u003eEscherichia coli\u003c/em\u003e Nissle 1917 was a potent inducer of NETosis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), suggesting that the inhibitory effect of probiotics on NET formation is strain-specific.\u003c/p\u003e \u003cp\u003eTaken altogether, neutrophils play a key role in intestinal inflammation as well as its resolution. As one of the cell types mediating the immune response to intestinal bacteria, they are important for the maintenance of healthy and balanced microbiota. We thus aimed to elucidate how ATB-induced dysbiosis affects immune function with a special focus on neutrophils and how the introduction of probiotics can normalize this. To understand the effect of ATB administration on the relationship between the microbiota and the immune system, we used a mouse experimental model, where dysbiosis was induced by ATB administration. We tested the capacity of the probiotic strain \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 (EcO83) to correct the dysbiosis and renew mutual homeostatic interactions between microbiota and host immune system, preventing the development of undesirable chronic pro-inflammatory responses. Originally, EcO83 was administered to neonates to prevent nosocomial infections (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition to that, EcO83 has been shown to prevent allergy development (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and to promote immunoregulatory responses, possibly limiting pro-allergic responses (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Findings show that EcO83 contributes to immune system maturation together with setting regulatory responses (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Based on previous results, we evaluated the capacity of EcO83 to dampen inflammation by inducing regulatory responses and promoting gut barrier function in mice with ATB-induced dysbiosis.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Mice\u003c/h2\u003e \u003cp\u003eBALB/cAnNCrl were bred in the Czech Center for Phenogenomics (Vestec, Czech Republic) or purchased from Velaz and kept in sterilized, filter-topped cages and fed autoclaved food with free access to clean water. Female mice aged between 8\u0026ndash;11 weeks were used in the experiments. Mice were sacrificed by cervical dislocation. All mouse experiments were approved by institutional review board of the First Faculty of Medicine, Charles University (IRB 1.LF-731) and the animal committee and executed according to good practice with animal models (MSMT-17298/2021-4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Antibiotic and probiotic treatment\u003c/h2\u003e \u003cp\u003eMice were treated with antibiotics introduced in sterile drinking water for two weeks. Control mice were given sterile water. The antibiotic mixture was added to the fresh drinking water and exchanged every 3 days. Ampicillin (1 g/L), neomycin sulphate (1 g/L), metronidazole (1 g/L), and vancomycin (0.5 g/L) (Sigma-Aldrich, USA) were diluted in sterile water to reach the final concentrations. Following the antibiotic treatment, mice were treated with a probiotic bacterial strain \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 (EcO83) every day for 5 consecutive days by intragastric gavage, at 5x10\u003csup\u003e8\u003c/sup\u003e CFU in 200 \u0026micro;l of gavage buffer (PBS containing 200 mM NaHCO\u003csub\u003e3\u003c/sub\u003e and 2% glucose), with a stainless-steel mouse gavage needle. EcO83 was cultivated as previously described (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Briefly, EcO83 was inoculated in Luria Bertani broth and cultivated for 6h (37\u0026deg;C upon agitation). After cultivation, bacterial cell culture was centrifuged, washed 2 times with PBS, and resuspended to a final concentration of 2.5x10\u003csup\u003e9\u003c/sup\u003e CFU/ml in the gavage buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Blood, bone marrow, and tissue collection\u003c/h2\u003e \u003cp\u003eTissue samples were collected from euthanized mice. Blood was drawn from the lateral saphenous vein into a tube containing heparin (10 USP units/ml of blood) to prevent coagulation and centrifuged to collect plasma for analysis of cytokine concentration. Bone marrow was obtained from the femur by flushing with sterile non-supplemented cell culture medium (RPMI1640), gently dissociated by pipetting up and down, and filtered through a cell strainer (70um) to eliminate clumps and remaining tissue to obtain single cell suspension for further analysis. Intestinal sections (ileum and colon) devoid of stool and single-cell suspensions from mesenteric lymph nodes (mLN) were stored in RNAprotect Tissue Reagent (QIAGEN, USA) until RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Microbiome analysis from stool samples\u003c/h2\u003e \u003cp\u003eStool samples were collected directly from the distant part of the colon right after the mouse sacrifice. Samples were snap-frozen in liquid nitrogen and kept at -80\u0026deg;C until further processing. Total DNA was extracted from the stool using the QIAmp Fast DNA Stool Mini Kit (Qiagen).. according to the manufacturer's protocol. DNA quality and concentration were determined using NanoDrop. 16S rDNA metagenomic NGS libraries from the DNA samples and subsequent sequencing using the Illumina MiSeq v3 was performed at the Institute of Applied Biotechnologies a.s. Only samples that passed the Quality Control (A260/280: 1.6\u0026ndash;2.1) were used to prepare the NGS libraries, which were sequenced on NovaSeq X Plus using standard workflow. The microbial composition was analyzed using relative abundance, where the number of reads for each taxonomic level (phylum, class, order) was normalized to 100% of the total classified reads. Raw sequencing data are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA1226621 (cited 2025 Mar 10) available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/PRJNA1226621\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra/PRJNA1226621\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Flow cytometry analysis\u003c/h2\u003e \u003cp\u003eNeutrophil subsets were analysed in BM single-cell suspensions using flow cytometry. Cell suspensions were stained for the following cell surface markers: anti-mouse CD11b (clone M1/70, EXBIO, CZ), Ly6G (clone 1A8, BioLegend, USA), Ly6C (clone HK1.4, BioLegend, USA), CXCR2 (clone SA044G4, BioLegend, USA), CD62L (clone MEL-14, EXBIO, CZ), incubated for 10 min at room temperature in the dark, followed by red blood cell lysis and acquired immediately using BD FACS Canto II (Becton Dickinson). The representative gating strategy for the identification of neutrophil phenotype is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;1.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RNA isolation, cDNA library, and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the mLN, ileum, and colon tissue using the RNeasy Mini Kit (QIAGEN, USA) according to the manufacturer\u0026rsquo;s instructions. The concentration and purity of isolated RNA was checked using Nanodrop (Thermo Fisher Scientific, USA) before following analyses. 0.5 \u0026micro;g of total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) to create a cDNA library. Relative gene expression was quantified as described previously (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Briefly, TaqMan gene expression assay (Applied Biosystems, USA) was used to quantify the level of gene expression of target genes in tissue and cells. The list of TaqMan assays is provided in Table\u0026nbsp;1. Relative quantification of gene expression was related to the level of gene expression of beta-actin (\u003cem\u003eActb\u003c/em\u003e) used as a reference gene (housekeeping gene, endogenous control). The qPCR reactions were run in doublets using the Light Cycler 480 Real-Time PCR System (Roche, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Data analysis and statistics\u003c/h2\u003e \u003cp\u003eFlow cytometry results were analyzed using FlowJo v10 software (TreeStar, USA) using appropriate single stain compensation and FMO controls to set a proper gating strategy. Gene expression of the target gene was quantified relative to the housekeeping gene \u003cem\u003eActb\u003c/em\u003e using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Data normality was assessed. One-way ANOVA was used for normally distributed data, while the Kruskal-Wallis test was applied for non-normal distributions. Outliers were identified using Dixon's test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.2). Analysis and graphical processing were performed using GraphPad Prism 8 (USA). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Antibiotic treatment induces changes in microbiota composition\u003c/h2\u003e \u003cp\u003eTo determine whether ATB treatment alters fecal microbiota composition and induces dysbiosis, adult mice were treated with a broad-spectrum antibiotic mixture (ampicillin, neomycin sulphate, vancomycin and metronidazole) for one week (ATB group). Another group received EcO83 (ATB\u0026thinsp;+\u0026thinsp;EcO83 group) via oral gavage for five consecutive days following ATB treatment. Control groups consisted of untreated mice (C group), and mice supplemented with EcO83 only (EcO83 group), according to the experimental setup (Fig.\u0026nbsp;1\u003cb\u003eA\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eATB treatment induced a drop in body weight, which was not statistically significant and normalized within one week regardless of EcO83 administration (data not shown). Although weight was restored, on the day of sacrifice, the caecum of ATB-treated mice appeared enlarged and dark (Fig.\u0026nbsp;1\u003cb\u003eB\u003c/b\u003e) compared to the controls, a condition that was mitigated by EcO83 supplementation (Fig.\u0026nbsp;1\u003cb\u003eB\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eMicrobiota composition was analyzed using 16S rDNA gene sequencing from stool samples. Both EcO83 and ATB treatment induces minor nonsignificant changes in the diversity and abundance of bacterial species as assessed by the Shannon-Wiener index (Fig.\u0026nbsp;2\u003cb\u003eA\u003c/b\u003e). The fecal microbiome was classified at the phylum, class, and order levels (Fig.\u0026nbsp;2\u003cb\u003eB\u003c/b\u003e). Marked dysbiosis was evident immediately after the end of ATB treatment (\u003cb\u003eFigure S2\u003c/b\u003e). The significant impact of ATB and/or EcO83 administration on specific microbial phyla, classes, and genera is shown in \u003cb\u003eFigure S3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eEcO83 supplementation significantly increased the abundance of \u003cem\u003eCandidatus\u003c/em\u003e Saccharibacteria in both ATB-treated and non-treated mice (\u003cb\u003eFig S3A)\u003c/b\u003e. The proportion of \u003cem\u003eDeferribacteres/Deferribacteriales/Deferribacteres\u003c/em\u003e was reduced in ATB-treated mice, but EcO83 supplementation promoted their recovery (\u003cb\u003eFig S3\u003c/b\u003e). Conversely, \u003cem\u003eFlavobacteria\u003c/em\u003e were significantly underrepresented in the gut microbiota ATB-treated mice, and EcO83 had no effect on their restoration (\u003cb\u003eFig S3B\u003c/b\u003e). Similarly, \u003cem\u003eSphingobacteria\u003c/em\u003e levels were reduced following ATB-treatment, with EcO83 supplementation having no impact on their restoration (\u003cb\u003eFig S3B, C\u003c/b\u003e). In contrast, EcO83 significantly promoted the growth of \u003cem\u003eMollicutes\u003c/em\u003e, \u003cem\u003eAnaeroplasmatales\u003c/em\u003e and \u003cem\u003eNautiliales\u003c/em\u003e as compared to control mice. Elevated levels of these taxa by EcO83 were also observed in ATB-treated mice (ATB\u0026thinsp;+\u0026thinsp;EcO83 group) (\u003cb\u003eFig S3B, C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Antibiotics alter gene expression of tight junction proteins in the ileum, which is normalized with EcO83 administration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe aimed to evaluate the impact of dysbiosis on intestinal tissue damage and investigate whether EcO83 administration could promote recovery. To achieve this, we performed qPCR analysis on tissue samples from the ileum and colon, targeting genes involved in gut barrier function, including tight junction proteins Claudin 1 (\u003cem\u003eCldn1\u003c/em\u003e), Claudin 5 (\u003cem\u003eCldn5\u003c/em\u003e), and Occludin (\u003cem\u003eOcln\u003c/em\u003e) as well as the regulatory cytokine IL-10 (\u003cem\u003eIl10\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eOur analysis revealed significant modulation of gene expression in the small intestine of mice with ATB-induced induced dysbiosis (Fig.\u0026nbsp;3). The expression of tight junction genes was increased, particularly for \u003cem\u003eCldn1\u003c/em\u003e and \u003cem\u003eCldn5\u003c/em\u003e in ATB-treated mice, compared to control groups. EcO83 supplementation promoted the \u003cem\u003eOcln\u003c/em\u003e gene expression and enhanced the expression of the \u003cem\u003eIl10 gene\u003c/em\u003e. Importantly, EcO83 supplementation also induced \u003cem\u003eIl10\u003c/em\u003e expression in ATB-treated mice (Fig.\u0026nbsp;3\u003cb\u003eA\u003c/b\u003e). Gene expression of tight junction proteins and \u003cem\u003eIl10\u003c/em\u003e was also measured in the colon, but no significant changes were detected (Fig.\u0026nbsp;3\u003cb\u003eB\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Antibiotic treatment does not induce cytokine changes in the mesenteric lymph nodes\u003c/h2\u003e \u003cp\u003eSince we observed changes in the expression of ileal epithelial proteins, we investigated whether antibiotic treatment influenced cytokine expression in the mesenteric lymph nodes, which drain the gut epithelium. Interestingly, no significant changes were detected in any of the measured cytokines (Fig.\u0026nbsp;4), except for the normalization of IL-18 levels following EcO83 administration in ATB-treated mice (Fig.\u0026nbsp;4), a cytokine linked to neutrophil function and trafficking.\u003c/p\u003e \u003cp\u003eTo assess potential systemic immune alterations, we also measured inflammatory cytokine concentrations in serum. However, no significant changes in IL-1β serum levels were observed (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e), indicating that antibiotic treatment did not induce a systemic pro-inflammatory response.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Antibiotics induce an increase in the number of CD62L\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eCXCR2\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eneutrophils, which is partially corrected by administration of EcO83\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFinally, we sought to determine whether ATB treatment and EcO83 administration affected neutrophil phenotypes. Cell suspensions prepared from BM were analyzed for neutrophil surface markers, identifying neutrophils as CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e\u0026minus;\u003c/sup\u003e granulocytes (gating strategy, \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAlthough there was a trend towards an increased number of CD11b\u003csup\u003e+\u003c/sup\u003e cells in EcO83-treated mice, the overall mean fluorescence intensity (MFI) of CD11b remained comparable (Fig.\u0026nbsp;5\u003cb\u003eA\u003c/b\u003e). Additionally, no significant changes were observed in the percentage of neutrophils (Ly6G\u003csup\u003e+\u003c/sup\u003e), which constituted the largest proportion of CD11b\u003csup\u003e+\u003c/sup\u003e cells, or in the percentage of monocytes (Ly6C\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;5\u003cb\u003eB\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe most profound differences were observed in the expression of the adhesion molecule CD62L and the IL-8 receptor CXCR2 on Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;6). ATB treatment induced loss of CD62L expression, while EcO83 administration had the opposite effect. ATB-treated mice had a slightly increased number of CD62L\u003csup\u003e\u0026minus;\u003c/sup\u003eCXCR2\u003csup\u003e\u0026minus;\u003c/sup\u003e double-negative cells, a change that was reversed to control levels upon probiotic administration (Fig.\u0026nbsp;6\u003cb\u003eA, bottom right\u003c/b\u003e). When antibiotic treatment was followed by EcO83 intervention, the neutrophil phenotype was restored to its original state (Fig.\u0026nbsp;6\u003cb\u003eB\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe suitability of probiotic supplementation during ATB treatment remains an area of active debate. In this study, we investigated the ability of EcO83 to restore microbiota composition and immune function following ATB-induced dysbiosis in a murine model.\u003c/p\u003e \u003cp\u003eThe prolonged dysbiosis triggered by ATB administration could lead to altered immune responses characterized by low-grade chronic inflammation supporting inflammatory bowel disease development. Therefore, the potential of probiotics to promote the restoration of microbiota composition and mutual homeostatic interactions between microbiota and the host immune system will be beneficial for the host's health. EcO83 supplementation has no significant impact on microbiota composition 12 days after ATB administration. No effect or even adverse effect of probiotic supplementation has been reported previously when probiotic supplementation prolonged diarrhoea and dysbiosis documented by a lower number of microbial species detected in stool samples of patients (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, it is important to highlight that in these studies, a mixture of 11 bacterial species with a scarce probiotic effect documented by previous studies was used. In addition to that, according to the recommendation of ISAPP (International Scientific Association for Probiotics and Prebiotics), the probiotic strains should be reported unambiguously, i.e. including particular strain annotation. It is important to highlight that the probiotic effect is highly strain-specific and probiotic effect ranges from immunostimulatory to immunosuppressive/immunoregulatory (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Therefore, the selection of appropriate probiotic strains for the effects desired in a particular context (i.e., the correction of a concrete pathology) is critical. In our study, EcO83 has been selected due to its immunoregulatory capacity (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) which is desirable to suppress pro-inflammatory responses occurring during dysbiosis. Nevertheless, we were not able to demonstrate a clear effect of EcO83 supplementation on higher microbiota diversity (Fig.\u0026nbsp;2\u003cb\u003eA\u003c/b\u003e). To confirm that our model of ATB administration is able to cause dysbiosis, microbiota composition has been analyzed immediately after ATB administration; \u003cb\u003eFigure S1\u003c/b\u003e clearly demonstrates a huge dysbiosis in both ATB and ATB + EcO83 groups before EcO83 administration. In a healthy intestine, obligate anaerobes like \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e thrive in low-oxygen environments. Dysbiosis disrupts this delicate balance, leading to a decline in these anaerobes and a concurrent increase in facultative anaerobes such as \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, often associated with elevated ROS (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Immediately after antibiotic treatment, we observed a marginal rise in \u003cem\u003eEnterobacteriales\u003c/em\u003e, a characteristic sign of dysbiosis - a state associated with reduced microbial diversity and increased \u003cem\u003eProteobacteria\u003c/em\u003e, particularly within the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family. Although \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e levels decreased following antibiotic exposure, they independently returned to baseline values, demonstrating their remarkable resilience (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In our study, no delay of microbiota normalization has been observed in EcO83 supplemented group. Moreover, no difference among groups (C, EcO83, ATB, ATB + EcO83) has been demonstrated, confirming that probiotic administration has no impact on the microbial community (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e–\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNevertheless, we observed the capacity of EcO83 to increase the presence of \u003cem\u003eCandidatus\u003c/em\u003e Saccharibacteria. This is important in the context of metabolomics since \u003cem\u003eCandidatus\u003c/em\u003e Saccharibacteria was identified as the phylum with the most pronounced impact on metabolome associated with Alzheimer's disease (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). While EcO83 demonstrated the ability to support \u003cem\u003eCandidatus\u003c/em\u003e Saccharibacteria colonization independently, the most pronounced effects were observed when mice received antibiotic treatment prior to probiotic supplementation. A comparable pattern emerged for the orders \u003cem\u003eCoriobacteriales\u003c/em\u003e and \u003cem\u003eNautiliales\u003c/em\u003e, suggesting a potential synergistic interaction between antibiotic pretreatment and probiotic administration. EcO83 can elevate \u003cem\u003eDeferribacteres\u003c/em\u003e, which belong to the phylum the most impacted by ATB administration in our study. Association of an increased proportion of \u003cem\u003eDeferribacteres\u003c/em\u003e in piglets with improved antioxidant function was documented in a study supplementing mothers with probiotics (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The potential of probiotics to promote \u003cem\u003eDeferribacteres\u003c/em\u003e and limit inflammation has been demonstrated previously (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). On the other hand, Panpetch et al. demonstrated an increased presence of \u003cem\u003eDeferribacteres\u003c/em\u003e in a mouse model of dysbiosis triggered by dextran sulphate solution together with \u003cem\u003eCandida\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e administration (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) and IL-1β expression in the gut in a mouse model of Diet-Induced Obese Mice (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Decreased levels of \u003cem\u003eFlavobacteria\u003c/em\u003e were shown to be associated with IBD (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), but EcO83 supplementation was not able to restore diminished \u003cem\u003eFlavobacteria\u003c/em\u003e after ATB treatment.\u003c/p\u003e \u003cp\u003eIn our study, only a single strain of probiotic EcO83 has been used, and possibly a mixture of carefully selected probiotic strains can be more beneficial and/or impact a complex microbial community in mice without ATB treatment. This observation agrees with a previous study of early postnatal EcO83 supplementation, where no change in the microbiota of ten-year-old children has been reported (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, even complex mixtures of probiotics have only a marginal effect on microbiota composition in healthy volunteers, as reviewed by Kristensen (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). On the other hand, several reports highlight the impact of probiotics on microbial communities (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). This discrepancy can be explained by various probiotic strains used, dose (CFU) of probiotics, single strain versus complex mixture of probiotic strains, duration of probiotic supplementation, and dietary, ethnic, and socioeconomic differences of probands among the studies.\u003c/p\u003e \u003cp\u003eOn the other hand, the beneficial effect of probiotics is not merely mediated by changes in microbiota composition measured by changes in alpha diversity, richness, or evenness. The beneficial effect of probiotics can be mediated by the production of SCFA directly by probiotic strains administered or the capacity of probiotic strains to trigger SCFA production (\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e–\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The other beneficial effect of probiotics involves the promotion of tight junction protein expression, leading to an increase in gut barrier function (\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e–\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). We have demonstrated the capacity of EcO83 to promote tight junction protein expression in neonates supplemented by EcO83 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Previous studies have shown that ATB administration decreases the expression of various genes tight junction-associated proteins, namely Claudin-1, Occludin, and ZO-1 in the intestine (Feng et al., 2019) and Claudin 3,4 in the colon (Ran et al. al., 2020). Subsequent administration of probiotics normalized the expression of Zonulin-1 and Occludin (L. Huang et al., 2023). Previous research from our laboratory showed that early postnatal colonization of EcO83 increased the expression of the tight junction genes \u003cem\u003eCldn\u003c/em\u003e and \u003cem\u003eOcln\u003c/em\u003e as well as \u003cem\u003eIl10\u003c/em\u003e, thereby supporting intestinal barrier function (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In the current study, the capacity of EcO83 to promote tight junction protein expression and restore gut barrier function in mice treated with ATB has been tested as well. In contrast to our hypothesis, the most elevated gene expression of all tight junction proteins has been detected in the ileum of mice treated with ATB, suggesting the increased gene expression is a compensatory effect for disturbed gut barrier function in the effort to renew the functionality of gut barrier. EcO83 was able to promote gene expression of tight junction proteins, documenting the capacity of probiotics to promote gut barrier function (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Our hypothesis that the most prominent expression of tight junction proteins is a compensatory effect of ongoing inflammation is further supported by decreased gene expression of tight junction proteins in the ATB + EcO83 group.\u003c/p\u003e \u003cp\u003eIL-10 represents a cytokine with immunoregulatory function preventing the development of pro-inflammatory responses. EcO83 was able to promote \u003cem\u003eIl10\u003c/em\u003e expression, which is in line with our previous observations (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Importantly, mice treated with ATB and EcO83 exerted increased gene expression of \u003cem\u003eIl10\u003c/em\u003e as well, documenting the potential of EcO83 to limit inflammation triggered by ATB-induced dysbiosis. Surprisingly, neither ATB nor EcO83 modified the gene expression of tight junction proteins in the colon, Fig.\u0026nbsp;3B.\u003c/p\u003e \u003cp\u003eThe capacity of probiotics to modulate adaptive immune responses has been described previously (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Therefore, we investigated the impact of ATB and/or EcO83 administration on the local immune system in MLN. To our surprise, no tremendous changes in gene expression of selected cytokines have been documented. Only IL-18 was lowered in the treated group, and EcO83 supplementation normalized the expression of IL-18 to the level of control mice, highlighting the role of EcO83 in triggering anti-infectious immune responses (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). On the other hand, IL-18 can contribute to inflammatory responses (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Finally, IL-18 also plays an important role in gut microbiota monitoring and homeostasis, and its dysregulation could lead to microbiota dysbiosis and accelerate disease progression. Furthermore, the dysbiosis observed in \u003cem\u003eIl18\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice contributed to their increased susceptibility to \u003cem\u003eListeria\u003c/em\u003e infection (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). This highlights the importance of fine-tuning immune responses with emphasis on appropriate regulatory mechanisms.\u003c/p\u003e \u003cp\u003eThe other goal of the study was to characterize the impact of ATB-induced dysbiosis on the proportional and functional characteristics of neutrophils as cells of the first line of defense. Since the majority of neutrophils are located in the bone marrow, we investigated the proportion of particular neutrophil subsets there by flow cytometry. Both probiotic-treated groups (EcO83 alone and ATB + EcO83) possessed elevated levels of CD11b\u003csup\u003e+\u003c/sup\u003e in bone marrow. ATB-treated mice have a significantly increased proportion of CD62L\u003csup\u003e−\u003c/sup\u003e neutrophils associated with the typical phenotype of old senescent and exhausted cells. EcO83 supplementation was able to normalize the levels of CD62L\u003csup\u003e−\u003c/sup\u003e neutrophils to the values found in healthy control mice. Altered granulopoiesis triggered by ATB can be demonstrated by the levels of CXCR2, which was decreased in ATB-treated mice compared to control healthy mice. Diminished levels of CXCR2 in ATB-treated mice have already been published (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and our study confirmed the potential effect of ATB on lowering the migration capacity of neutrophils to the site of infection/inflammation. Importantly, the effect of the probiotic strain \u003cem\u003eBacillus polyfermenticus\u003c/em\u003e in lowering the severity of colitis and promoting neoangiogenesis was dependent on CXCR2 and IL-8 signalling (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Together, these results suggest that lower CD11b, along with decreased CD62L and CXCR2, indicate a reduced capacity for rolling, adhering to endothelial tissues, and migrating into target tissues, which might result in impaired local immune responses. Antibiotic treatment contributes to this altered neutrophil profile, potentially compromising the immune system's ability to effectively respond to infections. Interestingly, EcO83 was able to restore granulopoiesis in ATB-treated mice. These results highlight the capacity of EcO83 to normalize granulopoiesis.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eWhile EcO83 did not significantly modify microbiota diversity, it played a critical role in restoring gut barrier integrity and immune balance. The probiotic counteracted ATB-induced changes in neutrophil subsets, promoted \u003cem\u003eIl10\u003c/em\u003e expression, and normalized \u003cem\u003eIl18\u003c/em\u003e expression levels. These findings emphasize the importance of strain-specific probiotic effects beyond microbiota composition. Further research is needed to elucidate the mechanisms by which probiotics modulate immune responses and their potential applications in ATB-associated dysbiosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest. JH obtained a collaborative grant with the company manufacturing probiotic vaccine called Colinfant Newborn. Company has no influence on study design and data interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.M. and J.H. conceptualized the study and designed the experiments. E.M., E. K. performed the most of experiments, E.M, E.K, J.H. and L.S. analyzed data, prepared figures, and wrote and edited the manuscript. E.M., E.K., V.C., L.S., P.A., J.V., O.N., P.P., A.K.Z and J.P. performed the experiments. E.M. and J.H. acquired funding. I.S. and V.C. edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has been supported by the Grant Agency of Charles University (GA UK 6121), Charles University research project Cooperatio IMMU207032. Work was also supported by OP JAK project MSCA Fellowships CZ \u0026ndash; UK2 (reg. n. CZ.02.01.01/00/22_010/0008115). OeAD-GmbH: CZ 04/2024 and CZ 07/2023, Austrian Science Fund: P 34867\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequencing data are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA1226621 (cited 2025 Mar 10) available from: https://www.ncbi.nlm.nih.gov/sra/PRJNA1226621.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRamirez J, Guarner F, Bustos Fernandez L, Maruy A, Sdepanian VL, Cohen H. Antibiotics as Major Disruptors of Gut Microbiota. Front Cell Infect Microbiol. 2020 Nov 24;10:572912. \u003c/li\u003e\n\u003cli\u003eReyman M, van Houten MA, Watson RL, Chu MLJN, Arp K, de Waal WJ, et al. Effects of early-life antibiotics on the developing infant gut microbiome and resistome: a randomized trial. Nat Commun. 2022 Feb 16;13(1):893. \u003c/li\u003e\n\u003cli\u003eDuan H, Yu L, Tian F, Zhai Q, Fan L, Chen W. 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Int Arch Allergy Immunol. 2003 Jul;131(3):209\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eHrd\u0026yacute; J, Vlas\u0026aacute;kov\u0026aacute; K, Čern\u0026yacute; V, S\u0026uacute;ken\u0026iacute;kov\u0026aacute; L, Novotn\u0026aacute; O, Petr\u0026aacute;skov\u0026aacute; P, et al. Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action. Eur J Immunol. 2018 Dec;48(12):2015\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eS\u0026uacute;ken\u0026iacute;kov\u0026aacute; L, Čern\u0026yacute; V, Věcek J, Petr\u0026aacute;skov\u0026aacute; P, Novotn\u0026aacute; O, Vobruba \u0026Scaron;, et al. The Impact of Escherichia coli Probiotic Strain O83:K24:H31 on the Maturation of Dendritic Cells and Immunoregulatory Functions In Vitro and In Vivo. Cells. 2022 May 12;11(10):1624. \u003c/li\u003e\n\u003cli\u003eS\u0026uacute;ken\u0026iacute;kov\u0026aacute; L, Čern\u0026yacute; V, Thon T, Roubalov\u0026aacute; R, Jir\u0026aacute;skov\u0026aacute; Z\u0026aacute;kostelsk\u0026aacute; Z, Novotn\u0026aacute; O, et al. Effect of early postnatal supplementation of newborns with probiotic strain E. coli O83:K24:H31 on allergy incidence, dendritic cells, and microbiota. Front Immunol. 2022;13:1038328. \u003c/li\u003e\n\u003cli\u003eHrd\u0026yacute; J, Zanvit P, Novotn\u0026aacute; O, Kocourkov\u0026aacute; I, Žižka J, Proke\u0026scaron;ov\u0026aacute; L. Cytokine expression in cord blood cells of children of healthy and allergic mothers. Folia Microbiol (Praha). 2010 Sep 1;55(5):515\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 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Int J Mol Sci. 2022 Jun 24;23(13):7050. \u003c/li\u003e\n\u003cli\u003eLe\u0026oacute;n IC, Quesada-V\u0026aacute;zquez S, S\u0026aacute;inz N, Guruceaga E, Escot\u0026eacute; X, Moreno-Aliaga MJ. Effects of Maresin 1 (MaR1) on Colonic Inflammation and Gut Dysbiosis in Diet-Induced Obese Mice. Microorganisms. 2020 Jul 30;8(8):1156. \u003c/li\u003e\n\u003cli\u003eNg SC, Lam EFC, Lam TTY, Chan Y, Law W, Tse PCH, et al. Effect of probiotic bacteria on the intestinal microbiota in irritable bowel syndrome. J Gastroenterol Hepatol. 2013 Oct;28(10):1624\u0026ndash;31. \u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Toole PW, Cooney JC. Probiotic bacteria influence the composition and function of the intestinal microbiota. Interdiscip Perspect Infect Dis. 2008;2008:175285. \u003c/li\u003e\n\u003cli\u003eBloemendaal M, Szopinska-Tokov J, Belzer C, Boverhoff D, Papalini S, Michels F, et al. 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Food Chem Toxicol Int J Publ Br Ind Biol Res Assoc. 2023 Feb;172:113558. \u003c/li\u003e\n\u003cli\u003eHrd\u0026yacute; J, Kocourkov\u0026aacute; I, Lodinov\u0026aacute;-Ž\u0026aacute;dn\u0026iacute;kov\u0026aacute; R, Kol\u0026aacute;řov\u0026aacute; L, Proke\u0026scaron;ov\u0026aacute; L. The effect of a probiotic Escherichia coli strain on regulatory T-cells in six year-old children. Benef Microbes. 2016 Nov 30;7(5):639\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003eKonieczna P, Groeger D, Ziegler M, Frei R, Ferstl R, Shanahan F, et al. Bifidobacterium infantis 35624 administration induces Foxp3 T regulatory cells in human peripheral blood: potential role for myeloid and plasmacytoid dendritic cells. Gut. 2012 Mar;61(3):354\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eTan TG, Sefik E, Geva-Zatorsky N, Kua L, Naskar D, Teng F, et al. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc Natl Acad Sci U S A. 2016 Dec 13;113(50):E8141\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eRao A, Strauss O, Kokkinou E, Bruchard M, Tripathi KP, Schlums H, et al. Cytokines regulate the antigen-presenting characteristics of human circulating and tissue-resident intestinal ILCs. Nat Commun. 2020 Apr 27;11(1):2049. \u003c/li\u003e\n\u003cli\u003eKowalewicz-Kulbat M, Szpakowski P, Krawczyk KT, Kowalski ML, Kosinski S, Biet F, et al. Decrease of IL-5 Production by Naive T Cells Cocultured with IL-18-Producing BCG-Pulsed Dendritic Cells from Patients Allergic to House Dust Mite. Vaccines. 2021 Mar 18;9(3):277. \u003c/li\u003e\n\u003cli\u003eDinarello CA. Interleukin-18 and the pathogenesis of inflammatory diseases. Semin Nephrol. 2007 Jan;27(1):98\u0026ndash;114. \u003c/li\u003e\n\u003cli\u003eZheng X, Liu L, Meng G, Zhu S, Zhou R, Jiang W. IL-18 maintains the homeostasis of mucosal immune system via inflammasome-independent but microbiota-dependent manner. Sci Bull. 2021 Oct 30;66(20):2115\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eIm E, Choi YJ, Kim CH, Fiocchi C, Pothoulakis C, Rhee SH. The angiogenic effect of probiotic Bacillus polyfermenticus on human intestinal microvascular endothelial cells is mediated by IL-8. Am J Physiol Gastrointest Liver Physiol. 2009 Nov;297(5):G999\u0026ndash;1008.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1. List of probes used for analysis of genes of interest.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"427\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003eGene symbol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eAssay ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.4736%;\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eActb\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm00607939_s1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.4736%;\"\u003e\n \u003cp\u003eActin beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eCldn1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm1342184_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.4736%;\"\u003e\n \u003cp\u003eClaudin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eCldn5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm00727012_s1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33.4736%;\"\u003e\n \u003cp\u003eClaudin 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eIl2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm00434256_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.1019%;\"\u003e\n \u003cp\u003eInterleukin 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eIl4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm99999154_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.1019%;\"\u003e\n \u003cp\u003eInterleukin 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eIl10\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm01288386_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5044%;\"\u003e\n \u003cp\u003eInterleukin 10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eIl18\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm00434226_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5044%;\"\u003e\n \u003cp\u003eInterleukin 18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eIfng\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm01168133_g1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5044%;\"\u003e\n \u003cp\u003eInterferon gamma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.9467%;\"\u003e\n \u003cp\u003e\u003cem\u003eOcln\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.8294%;\"\u003e\n \u003cp\u003eMm00500910_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5044%;\"\u003e\n \u003cp\u003eOccludin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication: not applicable.\u003c/p\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":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dysbiosis, antibiotics, probiotics, E. coli, neutrophils","lastPublishedDoi":"10.21203/rs.3.rs-6503496/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6503496/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA healthy microbiome and a homeostatic interaction between the microbiome and the host immune system are essential for proper nutrition and overall health. Excessive use of antibiotics (ATB) can disrupt the healthy gut microenvironment, leading to dysbiosis, a condition linked to a wide range of disorders and diseases. Alterations in the composition and function of the microbiota have been associated with a broad spectrum of various pathological conditions\u003c/p\u003e \u003cp\u003eIn this work, we investigate the effect of ATB administration on microbiota composition and immune modulation, with a particular focus on neutrophil dynamics. We evaluated the capacity of the probiotic strain \u003cem\u003eEscherichia coli\u003c/em\u003e O83:K24:H31 (EcO83) to mitigate ATB-induced dysbiosis and restore immune function. As expected, ATB treatment reduced microbiota diversity, which was partially restored by EcO83 supplementation. Furthermore, ATB administration affected the expression of tight junction proteins in the small intestine, an effect reversed by EcO83 treatment. Notably, our data indicates that ATB-induced dysbiosis accelerates neutrophil aging and reduces the release of neutrophils from the bone marrow. EcO83 supplementation counteracts these effects by promoting the influx of newly generated neutrophils into circulation.\u003c/p\u003e \u003cp\u003eOverall, our findings confirm that ATB treatment disrupts gut microbiota homeostasis, adversely affecting immune function, including neutrophil turnover. However, probiotic supplementation with EcO83 can at least partially restore microbiome composition and immune homeostasis, highlighting its potential therapeutic application in mitigating ATB-induced dysbiosis.\u003c/p\u003e","manuscriptTitle":"The ability of probiotic strain Escherichia coli O83:K24:H31 to modulate gut homeostasis and immune function after antibiotic-induced dysbiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 06:49:15","doi":"10.21203/rs.3.rs-6503496/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-10T15:28:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T15:23:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-02T16:47:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T21:16:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286462371847931196669187881796504213968","date":"2025-05-18T17:35:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287648221523228858324851676299538431248","date":"2025-05-16T13:39:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297230673262667376560110106237687727902","date":"2025-05-16T02:28:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260779020913110489877870782847649073788","date":"2025-05-16T01:38:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-16T01:28:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-23T07:37:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-23T07:34:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-04-22T10:51:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0bb0b61d-6907-4cd5-9500-db7c2bdce3e0","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:00:40+00:00","versionOfRecord":{"articleIdentity":"rs-6503496","link":"https://doi.org/10.1007/s12602-025-10814-w","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2025-11-19 15:57:26","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-05-20 06:49:15","video":"","vorDoi":"10.1007/s12602-025-10814-w","vorDoiUrl":"https://doi.org/10.1007/s12602-025-10814-w","workflowStages":[]},"version":"v1","identity":"rs-6503496","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6503496","identity":"rs-6503496","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}