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Volume 16
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10.3390/life16091474
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Article

Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing

by
Claudia Carissimi
1,*,†,
Francesco De Angelis
2,†,
Ilaria Laudadio
1,
Valerio Fulci
1,
Laura Stronati
1,
Sara Manella
3,
Domenico Alvaro
4,
Giorgio D’Andrea
1,
Gianfranco Silecchia
2,3,‡ and
Vincenzo Cardinale
4,5,6,‡
1
Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy
2
Department of Medico-Surgical Sciences and Biotechnologies, General Surgery Unit, ICOT Hospital Bariatric, 004100 Latina, Italy
3
Department of Medical and Surgical Sciences and Translational Medicine, Division of General Surgery, St. Andrea University Hospital, Sapienza University of Rome, 00185 Rome, Italy
4
Department of Translational and Precision Medicine, Sapienza University of Rome, 00185 Rome, Italy
5
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
6
Department of Medicine, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
The authors share the position as last co-authors.
Life 2026, 16(9), 1474; https://doi.org/10.3390/life16091474
Submission received: 11 May 2026 / Revised: 17 August 2026 / Accepted: 25 August 2026 / Published: 3 September 2026
(This article belongs to the Special Issue The Emerging Role of Microbiota in Health and Diseases)
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Abstract

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.
Keywords: microbiome; bile; gallbladder; obesity; metagenomics; shotgun metagenomic sequencing and 16S rRNA gene amplicon sequencing microbiome; bile; gallbladder; obesity; metagenomics; shotgun metagenomic sequencing and 16S rRNA gene amplicon sequencing

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MDPI and ACS Style

Carissimi, C.; De Angelis, F.; Laudadio, I.; Fulci, V.; Stronati, L.; Manella, S.; Alvaro, D.; D’Andrea, G.; Silecchia, G.; Cardinale, V. Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing. Life 2026, 16, 1474. https://doi.org/10.3390/life16091474

AMA Style

Carissimi C, De Angelis F, Laudadio I, Fulci V, Stronati L, Manella S, Alvaro D, D’Andrea G, Silecchia G, Cardinale V. Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing. Life. 2026; 16(9):1474. https://doi.org/10.3390/life16091474

Chicago/Turabian Style

Carissimi, Claudia, Francesco De Angelis, Ilaria Laudadio, Valerio Fulci, Laura Stronati, Sara Manella, Domenico Alvaro, Giorgio D’Andrea, Gianfranco Silecchia, and Vincenzo Cardinale. 2026. "Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing" Life 16, no. 9: 1474. https://doi.org/10.3390/life16091474

APA Style

Carissimi, C., De Angelis, F., Laudadio, I., Fulci, V., Stronati, L., Manella, S., Alvaro, D., D’Andrea, G., Silecchia, G., & Cardinale, V. (2026). Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing. Life, 16(9), 1474. https://doi.org/10.3390/life16091474

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