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 pathological conditions. In this work, we investigated 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 indicate 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.

Czech Centre for Phenogenomics Institute of Molecular Genetics Czech Academy of Sciences 252 50 Vestec Czech Republic

Faculty of Science Charles University Prague Czech Republic

Institute of Clinical Immunology and Allergology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Institute of Microbiology Czech Academy of Science Prague Czech Republic

Institute of Specific Prophylaxis and Tropical Medicine Centre for Pathophysiology Infectiology and Immunology Medical University of Vienna Vienna Austria

Zobrazit více v PubMed

Ramirez J, Guarner F, Bustos Fernandez L, Maruy A, Sdepanian VL, Cohen H (2020) Antibiotics as major disruptors of gut microbiota. Front Cell Infect Microbiol 10(24):572912 PubMed DOI PMC

Reyman M, van Houten MA, Watson RL, Chu MLJN, Arp K, de Waal WJ et al (2022) Effects of early-life antibiotics on the developing infant gut microbiome and resistome: a randomized trial. Nat Commun 13(1):893 PubMed DOI PMC

Duan H, Yu L, Tian F, Zhai Q, Fan L, Chen W (2022) Antibiotic-induced gut dysbiosis and barrier disruption and the potential protective strategies. Crit Rev Food Sci Nutr 62(6):1427–1452 PubMed DOI

Zhang X, Borbet TC, Fallegger A, Wipperman MF, Blaser MJ, Müller A (2021) An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses. MBio 12(1):e03335-e3420 PubMed DOI PMC

Vangoitsenhoven R, Cresci GAM (2020) Role of microbiome and antibiotics in autoimmune diseases. Nutr Clin Pract 35(3):406–416 PubMed DOI

Nguyen LH, Örtqvist AK, Cao Y, Simon TG, Roelstraete B, Song M et al (2020) Antibiotic use and the development of inflammatory bowel disease: a national case-control study in Sweden. Lancet Gastroenterol Hepatol 5(11):986–995 PubMed DOI PMC

Dar SH, Maniya MT, Merza N, Musheer A, Zahid M, Ahmed F et al (2023) The association of antibiotic exposure with new-onset inflammatory bowel disease: a systematic review and meta-analysis. Clin Res Hepatol Gastroenterol 47(6):102129 PubMed DOI

Baron R, Taye M, Besseling-van der Vaart I, Ujčič-Voortman J, Szajewska H, Seidell JC et al (2020) The relationship of prenatal and infant antibiotic exposure with childhood overweight and obesity: a systematic review. J Dev Orig Health Dis 4:335–349 DOI

Belizário JE, Faintuch J, Garay-Malpartida M (2018) Gut microbiome dysbiosis and immunometabolism: new frontiers for treatment of metabolic diseases. Mediators Inflamm 2018:2037838 PubMed DOI PMC

Flannigan KL, Ngo VL, Geem D, Harusato A, Hirota SA, Parkos CA et al (2017) IL-17A-mediated neutrophil recruitment limits expansion of segmented filamentous bacteria. Mucosal Immunol 10(3):673–684 PubMed DOI

Nguyen CT, Furuya H, Das D, Marusina AI, Merleev AA, Ravindran R et al (2022) Peripheral γδ T cells regulate neutrophil expansion and recruitment in experimental psoriatic arthritis. Arthritis Rheumatol 74(9):1524–1534 PubMed DOI PMC

Hall CHT, Campbell EL, Colgan SP (2017) Neutrophils as components of mucosal homeostasis. Cell Mol Gastroenterol Hepatol 4(3):329–337 PubMed DOI PMC

Zhang D, Frenette PS (2019) Cross talk between neutrophils and the microbiota. Blood 133(20):2168–2177 PubMed DOI PMC

Mondemé M, Zeroual Y, Soulard D, Hennart B, Beury D, Saliou JM et al (2024) Amoxicillin treatment of pneumococcal pneumonia impacts bone marrow neutrophil maturation and function. J Leukoc Biol 115(3):463–475 PubMed DOI

Watanabe K, Gilchrist CA, Uddin MJ, Burgess SL, Abhyankar MM, Moonah SN et al (2017) Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis. PLoS Pathog 13(8):e1006513 PubMed DOI PMC

Ganesh K, Joshi MB (2023) Neutrophil sub-types in maintaining immune homeostasis during steady state, infections and sterile inflammation. Inflamm Res 72(6):1175–1192 PubMed DOI PMC

Yousefi B, Eslami M, Ghasemian A, Kokhaei P, Farrokhi AS, Darabi N (2019) Probiotics importance and their immunomodulatory properties. J Cell Physiol 234(6):8008–8018 PubMed DOI

Liévin-Le Moal V, Servin AL (2014) Anti-infective activities of Lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clin Microbiol Rev 27(2):167–199 PubMed DOI PMC

Cristofori F, Dargenio VN, Dargenio C, Miniello VL, Barone M, Francavilla R. Anti-Inflammatory and Immunomodulatory Effects of Probiotics in Gut Inflammation: A Door to the Body. Front Immunol [Internet]. 2021 Feb 26 [cited 2024 Nov 8];12. Available from: https://www.frontiersin.org/journals/immunology/articles/ https://doi.org/10.3389/fimmu.2021.578386/full

Szajewska H, Scott KP, de Meij T, Forslund-Startceva SK, Knight R, Koren O et al (2025) Antibiotic-perturbed microbiota and the role of probiotics. Nat Rev Gastroenterol Hepatol 22(3):155–172 PubMed DOI

Suez J, Zmora N, Zilberman-Schapira G, Mor U, Dori-Bachash M, Bashiardes S et al (2018) Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT. Cell 174(6):1406-1423.e16 PubMed DOI

Liu HY, Yuan P, Li S, Ogamune KJ, Shi X, Zhu C et al (2025) Lactobacillus johnsonii alleviates experimental colitis by restoring intestinal barrier function and reducing NET-mediated gut-liver inflammation. Commun Biol 8(1):1222 PubMed DOI PMC

Corsiero E, Pratesi F, Prediletto E, Bombardieri M, Migliorini P (2016) NETosis as source of autoantigens in rheumatoid arthritis. Front Immunol 7:485 PubMed DOI PMC

Parackova Z, Zentsova I, Vrabcova P, Klocperk A, Sumnik Z, Pruhova S et al (2020) Neutrophil extracellular trap induced dendritic cell activation leads to Th1 polarization in type 1 diabetes. Front Immunol 11:661 PubMed DOI PMC

Salemme R, Peralta LN, Meka SH, Pushpanathan N, Alexander JJ (2019) The role of NETosis in Systemic Lupus Erythematosus. J Cell Immunol 1(2):33–42 PubMed PMC

Vong L, Lorentz RJ, Assa A, Glogauer M, Sherman PM (2014) Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol Baltim Md 1950 192(4):1870–7.

Lodinová-Zádníková R, Cukrowska B, Tlaskalova-Hogenova H (2003) Oral administration of probiotic Escherichia coli after birth reduces frequency of allergies and repeated infections later in life (after 10 and 20 years). Int Arch Allergy Immunol 131(3):209–211 PubMed DOI

Hrdý J, Vlasáková K, Černý V, Súkeníková L, Novotná O, Petrásková P et al (2018) Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action. Eur J Immunol 48(12):2015–2030 PubMed DOI

Súkeníková L, Černý V, Věcek J, Petrásková P, Novotná O, Vobruba Š et al (2022) 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 11(10):1624 PubMed DOI PMC

Súkeníková L, Černý V, Thon T, Roubalová R, JiráskováZákostelská Z, Novotná O et al (2022) Effect of early postnatal supplementation of newborns with probiotic strain E. coli O83:K24:H31 on allergy incidence, dendritic cells, and microbiota. Front Immunol 13:1038328 PubMed DOI

Kennedy EA, King KY, Baldridge MT. Mouse Microbiota Models: Comparing Germ-Free Mice and Antibiotics Treatment as Tools for Modifying Gut Bacteria. Front Physiol [Internet]. 2018 Oct 31 [cited 2025 Oct 1];9. Available from: https://www.frontiersin.org/journals/physiology/articles/ https://doi.org/10.3389/fphys.2018.01534/full

Farzi A, Fröhlich EE, Holzer P (2018) Gut microbiota and the neuroendocrine system. Neurotherapeutics 15(1):5–22 PubMed DOI PMC

Keogh CE, Kim DHJ, Pusceddu MM, Knotts TA, Rabasa G, Sladek JA et al (2021) Myelin as a regulator of development of the microbiota-gut-brain axis. Brain Behav Immun 91:437–450 PubMed DOI

Jacob D, Deborde C, Lefebvre M, Maucourt M, Moing A (2017) NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics. Metabolomics 13(4):36 PubMed DOI PMC

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods San Diego Calif 25(4):402–408 PubMed DOI

Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S et al (2018) Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell 174(6):1388-1405.e21 PubMed DOI

Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B et al (2014) Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11(8):506–514 PubMed DOI

Rigottier-Gois L (2013) Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis. ISME J 7(7):1256–1261 PubMed DOI PMC

Zeng MY, Inohara N, Nuñez G (2017) Mechanisms of inflammation-driven bacterial dysbiosis in the gut. Mucosal Immunol 10(1):18–26 PubMed DOI

Kristensen NB, Bryrup T, Allin KH, Nielsen T, Hansen TH, Pedersen O (2016) Alterations in fecal microbiota composition by probiotic supplementation in healthy adults: a systematic review of randomized controlled trials. Genome Med 8(1):52 PubMed DOI PMC

Washburn RL, Sandberg D, GazdikStofer MA (2022) Supplementation of a single species probiotic does not affect diversity and composition of the healthy adult gastrointestinal microbiome. Hum Nutr Metab 1(28):200148 DOI

He J, Jin Y, He C, Li Z, Yu W, Zhou J, et al. Danggui Shaoyao San: comprehensive modulation of the microbiota-gut-brain axis for attenuating Alzheimer’s disease-related pathology. Front Pharmacol [Internet]. 2024 Jan 12 [cited 2024 Nov 25];14. Available from: https://www.frontiersin.org/journals/pharmacology/articles/ https://doi.org/10.3389/fphar.2023.1338804/full

Starr EP, Shi S, Blazewicz SJ, Probst AJ, Herman DJ, Firestone MK et al (2018) Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon. Microbiome 6(1):122 PubMed DOI PMC

Hendrickson EL, Bor B, Kerns KA, Lamont EI, Chang Y, Liu J et al (2022) Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis. J Bacteriol. 204(9):e0011222 PubMed DOI

Spring S, Rohde M, Bunk B, Spröer C, Will SE, Neumann-Schaal M (2022) New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat. Environ Microbiol 24(5):2543–2575 PubMed DOI

Slobodkin A, Slobodkina G, Allioux M, Alain K, Jebbar M, Shadrin V et al (2019) Genomic Insights into the Carbon and Energy Metabolism of a Thermophilic Deep-Sea Bacterium Deferribacter autotrophicus Revealed New Metabolic Traits in the Phylum Deferribacteres. Genes 10(11):849 PubMed DOI PMC

Vasconcelos JA, Mota AS, Olímpio F, Rosa PC, Damaceno-Rodrigues N, de Paula Vieira R, et al (2023) Lactobacillus rhamnosus Modulates Lung Inflammation and Mitigates Gut Dysbiosis in a Murine Model of Asthma-COPD Overlap Syndrome. Probiotics Antimicrob Proteins

Tian X, Yu Z, Feng P, Ye Z, Li R, Liu J, et al. Lactobacillus plantarum TW1–1 Alleviates Diethylhexylphthalate-Induced Testicular Damage in Mice by Modulating Gut Microbiota and Decreasing Inflammation. Front Cell Infect Microbiol [Internet]. 2019 June 26 [cited 2024 Nov 25];9. Available from: https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/ https://doi.org/10.3389/fcimb.2019.00221/full

Panpetch W, Phuengmaung P, Hiengrach P, Issara-Amphorn J, Cheibchalard T, Somboonna N et al (2022) Candida Worsens Klebsiella pneumoniae Induced-Sepsis in a Mouse Model with Low Dose Dextran Sulfate Solution through Gut Dysbiosis and Enhanced Inflammation. Int J Mol Sci 23(13):7050 PubMed DOI PMC

León IC, Quesada-Vázquez S, Sáinz N, Guruceaga E, Escoté X, Moreno-Aliaga MJ (2020) Effects of Maresin 1 (MaR1) on Colonic Inflammation and Gut Dysbiosis in Diet-Induced Obese Mice. Microorganisms 8(8):1156 PubMed DOI PMC

Ng SC, Lam EFC, Lam TTY, Chan Y, Law W, Tse PCH et al (2013) Effect of probiotic bacteria on the intestinal microbiota in irritable bowel syndrome. J Gastroenterol Hepatol 28(10):1624–1631 PubMed DOI

O’Toole PW, Cooney JC (2008) Probiotic bacteria influence the composition and function of the intestinal microbiota. Interdiscip Perspect Infect Dis 2008:175285 PubMed PMC

Bloemendaal M, Szopinska-Tokov J, Belzer C, Boverhoff D, Papalini S, Michels F et al (2021) Probiotics-induced changes in gut microbial composition and its effects on cognitive performance after stress: exploratory analyses. Transl Psychiatry 11(1):300 PubMed DOI PMC

Savilahti E, Härkönen T, Savilahti EM, Kukkonen K, Kuitunen M, Knip M (2018) Probiotic intervention in infancy is not associated with development of beta cell autoimmunity and type 1 diabetes. Diabetologia 61(12):2668–2670 PubMed DOI

Zhang S, Dogan B, Guo C, Herlekar D, Stewart K, Scherl EJ et al (2020) Short Chain Fatty Acids Modulate the Growth and Virulence of Pathosymbiont Escherichia coli and Host Response. Antibiotics 9(8):462 PubMed DOI PMC

Markowiak-Kopeć P, Śliżewska K (2020) The Effect of Probiotics on the Production of Short-Chain Fatty Acids by Human Intestinal Microbiome. Nutrients 12(4):1107 PubMed DOI PMC

Ranjbar R, Vahdati SN, Tavakoli S, Khodaie R, Behboudi H (2021) Immunomodulatory roles of microbiota-derived short-chain fatty acids in bacterial infections. Biomed Pharmacother 141:111817 PubMed DOI

Rooks MG, Garrett WS (2016) Gut microbiota, metabolites and host immunity. Nat Rev Immunol 16(6):341–352 PubMed DOI PMC

Rose EC, Odle J, Blikslager AT, Ziegler AL (2021) Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function. Int J Mol Sci 22(13):6729 PubMed DOI PMC

di Vito R, Conte C, Traina G (2022) A Multi-Strain Probiotic Formulation Improves Intestinal Barrier Function by the Modulation of Tight and Adherent Junction Proteins. Cells 11(16):2617 PubMed DOI PMC

Feng Y, Huang Y, Wang Y, Wang P, Song H, Wang F (2019) Antibiotics induced intestinal tight junction barrier dysfunction is associated with microbiota dysbiosis, activated NLRP3 inflammasome and autophagy. PLOS ONE. 6;14(6):e0218384

Ran Y, Fukui H, Xu X, Wang X, Ebisutani N, Tanaka Y et al (2020) Alteration of Colonic Mucosal Permeability during Antibiotic-Induced Dysbiosis. Int J Mol Sci 21(17):6108 PubMed DOI PMC

Li W, Zhang S, Wang Y, Bian H, Yu S, Huang L, et al. Complex probiotics alleviate ampicillin-induced antibiotic-associated diarrhea in mice. Front Microbiol [Internet]. 2023 Apr 14 [cited 2025 Oct 1];14. Available from: https://www.frontiersin.org/journals/microbiology/articles/ https://doi.org/10.3389/fmicb.2023.1156058/full

Zhao X, Zhou J, Liang W, Sheng Q, Lu L, Chen T et al (2021) Probiotics mixture reinforces barrier function to ameliorate necrotizing enterocolitis by regulating PXR-JNK pathway. Cell Biosci 19(11):20 DOI

Casula E, Pisano MB, Serreli G, Zodio S, Melis MP, Corona G et al (2023) Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: focus on tight junction modulation. Food Chem Toxicol 172:113558 PubMed DOI

Volynets V, Reichold A, Bárdos G, Rings A, Bleich A, Bischoff SC (2016) Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice. Dig Dis Sci 61(3):737–746 PubMed DOI

Hrdý J, Kocourková I, Lodinová-Žádníková R, Kolářová L, Prokešová L (2016) The effect of a probiotic Escherichia coli strain on regulatory T-cells in six year-old children. Benef Microbes 7(5):639–648 PubMed DOI

Konieczna P, Groeger D, Ziegler M, Frei R, Ferstl R, Shanahan F et al (2012) Bifidobacterium infantis 35624 administration induces Foxp3 T regulatory cells in human peripheral blood: potential role for myeloid and plasmacytoid dendritic cells. Gut 61(3):354–366 PubMed DOI

Tan TG, Sefik E, Geva-Zatorsky N, Kua L, Naskar D, Teng F et al (2016) 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 113(50):E8141–E8150 PubMed DOI PMC

Rao A, Strauss O, Kokkinou E, Bruchard M, Tripathi KP, Schlums H et al (2020) Cytokines regulate the antigen-presenting characteristics of human circulating and tissue-resident intestinal ILCs. Nat Commun 11(1):2049 PubMed DOI PMC

Kowalewicz-Kulbat M, Szpakowski P, Krawczyk KT, Kowalski ML, Kosinski S, Biet F et al (2021) 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 9(3):277 PubMed DOI PMC

Johnson AF, Sands JS, Trivedi KM, Russell R, LaRock DL, LaRock CN (2023) Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection. PLoS Pathog 19(4):e1011321 PubMed DOI PMC

Zheng X, Liu L, Meng G, Zhu S, Zhou R, Jiang W (2021) IL-18 maintains the homeostasis of mucosal immune system via inflammasome-independent but microbiota-dependent manner. Sci Bull 66(20):2115–2123 DOI

Im E, Choi YJ, Kim CH, Fiocchi C, Pothoulakis C, Rhee SH (2009) The angiogenic effect of probiotic Bacillus polyfermenticus on human intestinal microvascular endothelial cells is mediated by IL-8. Am J Physiol Gastrointest Liver Physiol 297(5):G999-1008 PubMed DOI PMC