{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T12:13:23Z","timestamp":1788437603083,"version":"build-2803163510"},"reference-count":59,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Grant Piano Nazionale Complementare Salute","award":["PNC1221852F49EDDD"],"award-info":[{"award-number":["PNC1221852F49EDDD"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Recent advances in metagenomics have expanded our ability to detect low-abundance microbial communities. While the gut remains the most densely populated microbial habitat, emerging evidence has proposed that microorganisms might also inhabit anatomical sites once considered sterile, such as the biliary system. We apply next-generation DNA sequencing to characterize the bacterial community of bile in obese and non-obese patients with symptomatic gallstones. Bile samples were collected from 64 patients (32 obese, 32 non-obese) undergoing elective cholecystectomy. We incorporated negative (sterile tubes) and positive (mock microbial community standard) controls to evaluate contamination risks. We applied both 16S rRNA gene amplicon and shotgun metagenomic sequencing. Both sequencing methods detected extremely low bacterial biomass in bile. Specifically, shotgun metagenomic sequencing identified bacterial DNA traces in only eight samples, displaying minimal community similarity. In the positive controls, our measurements confirmed the expected microbial community composition, and in the negative controls, no bacterial DNA was detected. In contrast, 16S rRNA gene sequencing showed bacterial DNA in all bile samples as well as in negative controls, suggesting a higher susceptibility to contamination. Our findings suggest that bile may not be consistently colonized by bacterial communities in uncomplicated gallstone disease.<\/jats:p>","DOI":"10.3390\/life16091474","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:08:53Z","timestamp":1788433733000},"page":"1474","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9885-2855","authenticated-orcid":false,"given":"Claudia","family":"Carissimi","sequence":"first","affiliation":[{"name":"Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"De Angelis","sequence":"additional","affiliation":[{"name":"Department of Medico-Surgical Sciences and Biotechnologies, General Surgery Unit, ICOT Hospital Bariatric, 004100 Latina, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0990-3201","authenticated-orcid":false,"given":"Ilaria","family":"Laudadio","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1173-585X","authenticated-orcid":false,"given":"Valerio","family":"Fulci","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Stronati","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Manella","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences and Translational Medicine, Division of General Surgery, St. Andrea University Hospital, Sapienza University of Rome, 00185 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3771-9644","authenticated-orcid":false,"given":"Domenico","family":"Alvaro","sequence":"additional","affiliation":[{"name":"Department of Translational and Precision Medicine, Sapienza University of Rome, 00185 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giorgio","family":"D\u2019Andrea","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2356-0505","authenticated-orcid":false,"given":"Gianfranco","family":"Silecchia","sequence":"additional","affiliation":[{"name":"Department of Medico-Surgical Sciences and Biotechnologies, General Surgery Unit, ICOT Hospital Bariatric, 004100 Latina, Italy"},{"name":"Department of Medical and Surgical Sciences and Translational Medicine, Division of General Surgery, St. Andrea University Hospital, Sapienza University of Rome, 00185 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0234-3341","authenticated-orcid":false,"given":"Vincenzo","family":"Cardinale","sequence":"additional","affiliation":[{"name":"Department of Translational and Precision Medicine, Sapienza University of Rome, 00185 Rome, Italy"},{"name":"Department of Internal Medicine, Division of Gastroenterology, Saint Louis University School of Medicine-SSM SLUCare, SLU Hospital, 1008 S. Spring, St. Louis, MO 63110, USA"},{"name":"Department of Medicine, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1186\/s13054-025-05288-9","article-title":"Consistency between Metagenomic Next-Generation Sequencing versus Traditional Microbiological Tests for Infective Disease: Systemic Review and Meta-Analysis","volume":"29","author":"Liu","year":"2025","journal-title":"Crit. Care"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"155606","DOI":"10.1016\/j.prp.2024.155606","article-title":"Next Generation Sequencing: Latent Applications in Clinical Diagnostics with the Advent of Bioinformatic Frameworks","volume":"263","author":"Thapliyal","year":"2024","journal-title":"Pathol. Res. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1136\/ard-2024-225735","article-title":"Microbiome Research in Autoimmune and Immune-Mediated Inflammatory Diseases: Lessons, Advances and Unmet Needs","volume":"84","author":"Scher","year":"2025","journal-title":"Ann. Rheum. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1821","DOI":"10.1016\/j.arth.2025.01.001","article-title":"Emerging Concepts in Periprosthetic Joint Infection Research: The Human Microbiome","volume":"40","author":"Heckmann","year":"2025","journal-title":"J. Arthroplast."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Gabrielli, M., Zileri Dal Verme, L., Zocco, M.A., Nista, E.C., Ojetti, V., and Gasbarrini, A. (2024). The Role of the Gastrointestinal Microbiota in Parkinson\u2019s Disease. Biomolecules, 15.","DOI":"10.3390\/biom15010026"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"102659","DOI":"10.1016\/j.arr.2025.102659","article-title":"Gut Microbiota in Alzheimer\u2019s Disease: Understanding Molecular Pathways and Potential Therapeutic Perspectives","volume":"104","author":"Lista","year":"2025","journal-title":"Ageing Res. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Kumari, S., Srilatha, M., and Nagaraju, G.P. (2024). Effect of Gut Dysbiosis on Onset of GI Cancers. Cancers, 17.","DOI":"10.3390\/cancers17010090"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/s13046-025-03313-x","article-title":"Dysbiosis and Extraintestinal Cancers","volume":"44","author":"He","year":"2025","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"8045646","DOI":"10.1155\/2020\/8045646","article-title":"The Human Microbiome and Its Impacts on Health","volume":"2020","author":"Ogunrinola","year":"2020","journal-title":"Int. J. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e14442","DOI":"10.1111\/1751-7915.14442","article-title":"Are There Any Completely Sterile Organs or Tissues in the Human Body? Is There Any Sacred Place?","volume":"17","year":"2024","journal-title":"Microb. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1038\/s41586-019-1451-5","article-title":"Human Placenta Has No Microbiome but Can Contain Potential Pathogens","volume":"572","author":"Lager","year":"2019","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2015","DOI":"10.1002\/hep.28829","article-title":"Changes in Blood Microbiota Profiles Associated with Liver Fibrosis in Obese Patients: A Pilot Analysis","volume":"64","author":"Lelouvier","year":"2016","journal-title":"Hepatology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s40168-015-0145-y","article-title":"Human Milk Microbiota Profiles in Relation to Birthing Method, Gestation and Infant Gender","volume":"4","author":"Urbaniak","year":"2016","journal-title":"Microbiome"},{"key":"ref_14","first-page":"1","article-title":"The Human Milk Microbiota: Origin and Potential Roles in Health and Disease","volume":"69","author":"Langa","year":"2013","journal-title":"Pharmacol. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"103376","DOI":"10.1016\/j.jaut.2025.103376","article-title":"Immunobiology of Bile and Cholangiocytes","volume":"151","author":"Li","year":"2025","journal-title":"J. Autoimmun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1007\/BF01296423","article-title":"Defense System in the Biliary Tract against Bacterial Infection","volume":"37","author":"Sung","year":"1992","journal-title":"Dig. Dis. Sci."},{"key":"ref_17","first-page":"2750","article-title":"Biliary Tract Microbiota: A New Kid on the Block of Liver Diseases?","volume":"24","author":"Nicoletti","year":"2020","journal-title":"Eur. Rev. Med. Pharmacol. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/s13755-023-00267-2","article-title":"Characterization of Biliary and Duodenal Microbiota in Patients with Primary and Recurrent Choledocholithiasis","volume":"12","author":"Liu","year":"2024","journal-title":"Health Inf. Sci. Syst."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Wirth, U., Jiang, T., Schardey, J., Kratz, K., Li, M., Schirren, M., K\u00fchn, F., Bazhin, A., Werner, J., and Guba, M. (2023). The Role of Microbiota in Liver Transplantation and Liver Transplantation-Related Biliary Complications. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24054841"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Chen, R., Li, X., Ding, J., Wan, J., Zhang, X., Jiang, X., Duan, S., Hu, X., Gao, Y., and Sun, B. (2023). Profiles of Biliary Microbiota in Biliary Obstruction Patients with Clonorchis Sinensis Infection. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1281745"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Ito, Z., Koido, S., Kato, K., Odamaki, T., Horiuchi, S., Akasu, T., Saruta, M., Hata, T., Kumagai, Y., and Fujioka, S. (2022). Dysbiosis of the Fecal and Biliary Microbiota in Biliary Tract Cancer. Cancers, 14.","DOI":"10.3390\/cancers14215379"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Saab, M., Mestivier, D., Sohrabi, M., Rodriguez, C., Khonsari, M.R., Faraji, A., and Sobhani, I. (2021). Characterization of Biliary Microbiota Dysbiosis in Extrahepatic Cholangiocarcinoma. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0247798"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Halimi, H., Hesami, Z., Asri, N., Khorsand, B., Rostami-Nejad, M., and Houri, H. (2025). Exploring the Biliary Microbiome in Hepatopancreatobiliary Disorders: A Comprehensive Systematic Review of Microbial Signatures and Diagnostic Potential. BMC Gastroenterol., 26.","DOI":"10.1186\/s12876-025-04551-1"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Meacci, D., Bruni, A., Cocquio, A., Dell\u2019Anna, G., Mandarino, F.V., Marasco, G., Cecinato, P., Barbara, G., and Zagari, R.M. (2025). Microbial Landscapes of the Gut\u2013Biliary Axis: Implications for Benign and Malignant Biliary Tract Diseases. Microorganisms, 13.","DOI":"10.3390\/microorganisms13091980"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.advms.2024.04.001","article-title":"Obesity\u2013Standards, Trends and Advances","volume":"69","year":"2024","journal-title":"Adv. Med. Sci."},{"key":"ref_26","unstructured":"Apovian, M.D., Caroline, M., Aronne, L., and Barenbaum, S.R. (2025). Clinical Management of Obesity, Professional Communications, Inc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8868","DOI":"10.1038\/s41598-021-88249-w","article-title":"A Computer Study of the Risk of Cholesterol Gallstone Associated with Obesity and Normal Weight","volume":"11","author":"Kubica","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e12983","DOI":"10.1111\/obr.12983","article-title":"Pathophysiological Connections between Gallstone Disease, Insulin Resistance, and Obesity","volume":"21","author":"Barrera","year":"2020","journal-title":"Obes. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3620","DOI":"10.2174\/0929867324666170530080636","article-title":"The Role of Diet in the Pathogenesis of Cholesterol Gallstones","volume":"26","author":"Garruti","year":"2019","journal-title":"CMC"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e71656","DOI":"10.1096\/fj.202503254RRRRRR","article-title":"Gut Microbiome Dysbiosis Promotes Gallstone Formation via Bile Acid Metabolic Disorder: A Multiomics Study","volume":"40","author":"Huang","year":"2026","journal-title":"FASEB J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"101493","DOI":"10.1016\/j.beem.2021.101493","article-title":"Gut Microbiome and Bile Acids in Obesity-Related Diseases","volume":"35","author":"Li","year":"2021","journal-title":"Best Pract. Res. Clin. Endocrinol. Metab."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"102766","DOI":"10.1016\/j.ebiom.2020.102766","article-title":"A Dysregulated Bile Acid-Gut Microbiota Axis Contributes to Obesity Susceptibility","volume":"55","author":"Wei","year":"2020","journal-title":"eBioMedicine"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1038","DOI":"10.14309\/ajg.0000000000002730","article-title":"Role of the Intestine and Its Gut Microbiota in Metabolic Syndrome and Obesity","volume":"119","author":"Busch","year":"2024","journal-title":"Am. J. Gastroenterol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1002\/jhbp.515","article-title":"Tokyo Guidelines 2018: Diagnostic Criteria and Severity Grading of Acute Cholecystitis (with Videos)","volume":"25","author":"Yokoe","year":"2018","journal-title":"J. Hepato Biliary Pancreat."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1007\/s00423-015-1300-4","article-title":"Laparoscopic Cholecystectomy: Consensus Conference-Based Guidelines","volume":"400","author":"Agresta","year":"2015","journal-title":"Langenbecks Arch. Surg."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/BF00187881","article-title":"Complications after Laparoscopic Cholecystectomy: Coordinated Radiologic, Endoscopic, and Surgical Treatment","volume":"9","author":"Bezzi","year":"1995","journal-title":"Surg. Endosc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1186\/s13059-019-1891-0","article-title":"Improved Metagenomic Analysis with Kraken 2","volume":"20","author":"Wood","year":"2019","journal-title":"Genome Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.jbiotec.2017.06.1198","article-title":"25 Years of Serving the Community with Ribosomal RNA Gene Reference Databases and Tools","volume":"261","author":"Yilmaz","year":"2017","journal-title":"J. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1633","DOI":"10.1038\/s41587-023-01688-w","article-title":"Extending and Improving Metagenomic Taxonomic Profiling with Uncharacterized Species Using MetaPhlAn 4","volume":"41","author":"Beghini","year":"2023","journal-title":"Nat. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"McMurdie, P.J., and Holmes, S. (2013). Phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0061217"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"783","DOI":"10.2307\/2531532","article-title":"Estimating the Population Size for Capture-Recapture Data with Unequal Catchability","volume":"43","author":"Chao","year":"1987","journal-title":"Biometrics"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/BF02289694","article-title":"Nonmetric Multidimensional Scaling: A Numerical Method","volume":"29","author":"Kruskal","year":"1964","journal-title":"Psychometrika"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"325","DOI":"10.2307\/1942268","article-title":"An Ordination of the Upland Forest Communities of Southern Wisconsin","volume":"27","author":"Bray","year":"1957","journal-title":"Ecol. Monogr."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1093\/biomet\/53.3-4.325","article-title":"Some Distance Properties of Latent Root and Vector Methods Used in Multivariate Analysis","volume":"53","author":"Gower","year":"1966","journal-title":"Biometrika"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"4027","DOI":"10.1093\/bioinformatics\/btac438","article-title":"microbiomeMarker: An R\/Bioconductor Package for Microbiome Marker Identification and Visualization","volume":"38","author":"Cao","year":"2022","journal-title":"Bioinformatics"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1016\/j.bbrc.2015.12.083","article-title":"Analysis of the Microbiome: Advantages of Whole Genome Shotgun versus 16S Amplicon Sequencing","volume":"469","author":"Ranjan","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1089\/omi.2018.0013","article-title":"Quantitative Assessment of Shotgun Metagenomics and 16S rDNA Amplicon Sequencing in the Study of Human Gut Microbiome","volume":"22","author":"Laudadio","year":"2018","journal-title":"OMICS J. Integr. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.tim.2018.11.003","article-title":"Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations","volume":"27","author":"Eisenhofer","year":"2019","journal-title":"Trends Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Olomu, I.N., Pena-Cortes, L.C., Long, R.A., Vyas, A., Krichevskiy, O., Luellwitz, R., Singh, P., and Mulks, M.H. (2020). Elimination of \u201cKitome\u201d and \u201cSplashome\u201d Contamination Results in Lack of Detection of a Unique Placental Microbiome. BMC Microbiol., 20.","DOI":"10.1186\/s12866-020-01839-y"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1038\/s41564-018-0202-y","article-title":"Recognizing the Reagent Microbiome","volume":"3","author":"Lager","year":"2018","journal-title":"Nat. Microbiol."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Effenberger, M., Al-Zoairy, R., Gstir, R., Graziadei, I., Schwaighofer, H., Tilg, H., and Zoller, H. (2023). Transmission of Oral Microbiota to the Biliary Tract during Endoscopic Retrograde Cholangiography. BMC Gastroenterol., 23.","DOI":"10.1186\/s12876-023-02721-7"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1038\/s41579-018-0029-9","article-title":"Best Practices for Analysing Microbiomes","volume":"16","author":"Knight","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2239","DOI":"10.1111\/1462-2920.12668","article-title":"The Effects of Variable Sample Biomass on Comparative Metagenomics","volume":"17","author":"Chafee","year":"2015","journal-title":"Environ. Microbiol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"e00598-21","DOI":"10.1128\/mBio.00598-21","article-title":"Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers","volume":"12","author":"Dyrhovden","year":"2021","journal-title":"mBio"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Bragazzi, M.C., Venere, R., Vignone, A., Alvaro, D., and Cardinale, V. (2023). Role of the Gut-Liver Axis in the Pathobiology of Cholangiopathies: Basic and Clinical Evidence. Int. J. Mol. Sci., 24.","DOI":"10.20944\/preprints202303.0164.v1"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2005751","DOI":"10.1080\/19490976.2021.2005751","article-title":"When to Suspect Contamination Rather than Colonization\u2013Lessons from a Putative Fetal Sheep Microbiome","volume":"14","author":"Bihl","year":"2022","journal-title":"Gut Microbes"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1038\/s41586-022-05546-8","article-title":"Questioning the Fetal Microbiome Illustrates Pitfalls of Low-Biomass Microbial Studies","volume":"613","author":"Kennedy","year":"2023","journal-title":"Nature"},{"key":"ref_58","doi-asserted-by":"crossref","unstructured":"D\u2019Amico, F., Bertacco, A., Finotti, M., Di Renzo, C., Rodriguez-Davalos, M.I., Gondolesi, G.E., Cillo, U., Mulligan, D., and Geibel, J. (2021). Bile Microbiota in Liver Transplantation: Proof of Concept Using Gene Amplification in a Heterogeneous Clinical Scenario. Front. Surg., 8.","DOI":"10.3389\/fsurg.2021.621525"},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Gookin, J.L., Hartley, A.N., Aicher, K.M., Mathews, K.G., Cullen, R., Cullen, J.M., Callahan, B.J., Stowe, D.M., Seiler, G.S., and Jacob, M.E. (2023). Gallbladder Microbiota in Healthy Dogs and Dogs with Mucocele Formation. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0281432"}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/9\/1474\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:19:34Z","timestamp":1788434374000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/9\/1474"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":59,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["life16091474"],"URL":"https:\/\/doi.org\/10.3390\/life16091474","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}