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Identification by 16S rRNA sequencing showed substantial diversity among isolates, dominated by Staphylococcus hominis and Staphylococcus epidermidis. Co-culture with Caco-2 cells assessed cytotoxicity across tested concentrations, showing no noticeable cytotoxic effects. The strains modulated immune responses by regulating TNF-α–induced IL-8 production in a time-dependent manner, suggesting a role in colon inflammation modulation and innate immunity 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BMC Biotechnology (2026) 26:1 [https://doi.org/10.1186/s12896-025-01079-w](https://doi.org/10.1186/s12896-025-01079-w)  \nBMC Biotechnology  \nRESEARCH Open Access  \nStaphylococcus epidermidis MB614 and Staphylococcus hominis MB621 from human milk as up regulators ofTNF-α induced IL-8 production by Caco-2 cell line  \nAyesha Saeed 1,2*, Azra Yasmin2, Muhamad Arslan Ahmed3, Shomaila Zaib2, Muhammad Tabish4 and Bushra Rehman5  \nAbstract  \nBreast milk harbors many bacteria that shape the newborn microbiota. The present study aimed to investigate the immunomodulatory properties of bacteria from selected Pakistani mother’s breast milk samples. The probioticstrains studied are associated with immune modulation in intestinal epithelial cells, showing potential probiotic properties to be used in a simulated human milk formula. Identification was carried out through 16 S rRNA gene sequencing, which revealed the presence of 50% diversity of Staphylococcus hominis and 35% of Staphylococcus epidermidis among all the isolates. Probiotic bacteria are considered to protect the gastrointestinal tract (GIT) by adhering to the gut epithelial cells and reducing the population of pathogens. The cytotoxicity of Staphylococcus hominis MB614 and Staphylococcus epidermidis MB621 against epithelial cells was tested by co-culturing the strains with Caco-2 cells. Tested probiotic bacteria at concentrations, 1 × 106, to 1 × 102 CFU/ml for 24 h exhibited no noticeable cytotoxic effect on Caco-2 cells. Staphylococcus hominis MB614 and Staphylococcus epidermidis MB621 were examined for modulation of the immune system in human colon carcinoma cell lines (Caco-2) after the effect of the proinflammatory cytokine tumour necrosis factor α (TNF-α) by regulating the proinflammatory interleukin 8 (IL-8) . Staphylococcus hominis MB614 and Staphylococcus epidermidis MB621 regulate TNF-α induced IL-8 production in a time-dependent manner. In conclusion, Staphylococcus hominis MB614 and Staphylococcus epidermidis MB621, isolated from human milk, when administered in sufficient amounts and for a certain duration, may be used to modulate inflammation inside the colon by up-regulating the production of IL-8, potentially enhancing innate immunity. This study with these probiotics suggested the modulation of the immune system and disease prevention in the human body.  \nKeywords Staphylococcus hominis, Staphylococcus epidermidis, Interleukin 8, Caco-2, TNF-α, Human milk  \nClinical trial number  \nNot applicable.  \n*Correspondence: Ayesha Saeed  \n[ayeshas45@gmail.com](ayeshas45@gmail.com); [ayeshasaeed@fui.edu.pk](ayeshasaeed@fui.edu.pk)  \nFull list of author information is available at the end of the article  \n© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creati](http://creati)[vecommons.org/l](vecommons.org/l)icenses/by-nc-nd/4.0/.  \nGraphical Abstract  \nIntroduction  \nThe worldwide market for probiotic products is enormous, expected to range over $46 billion by 2020 [18, 32] but now according to allied market research, the 202","cbCaiqYbr6pSlvpp","https://ap.wps.com/l/cbCaiqYbr6pSlvpp","pdf",3883567,14,"English","# Abstract\n# Keywords\n# Introduction\n## Probiotic market context and challenges\n## Role of breast milk in infant immunity\n## Emerging omics approaches and immune defense","[{\"question\":\"What was the aim of the study regarding breast milk bacteria?\",\"answer\":\"To investigate the immunomodulatory properties and probiotic potential of bacteria isolated from selected Pakistani mothers’ breast milk samples.\"},{\"question\":\"How were the bacterial isolates identified in this work?\",\"answer\":\"Identification was performed using 16S rRNA gene sequencing.\"},{\"question\":\"Did the probiotic strains show cytotoxicity toward Caco-2 cells?\",\"answer\":\"No. At concentrations of 1 × 106 to 1 × 102 CFU/ml for 24 h, the strains exhibited no noticeable cytotoxic effect on Caco-2 cells.\"},{\"question\":\"How did the strains affect TNF-α induced IL-8 production?\",\"answer\":\"Staphylococcus hominis MB614 and Staphylococcus epidermidis MB621 regulated TNF-α–induced IL-8 production in a time-dependent manner.\"}]","Staphylococcus epidermidis MB614 and Staphylococcus hominis MB621 from human milk as up regulators of TNF-α induced IL-8 production by Caco-2 cell line | PDF",1790700697,35]