Gallbladder microbiota in healthy dogs and dogs with mucocele formation

PLOS ONE RESEARCH ARTICLE Gallbladder microbiota in healthy dogs and dogs with mucocele formation Jody L. Gookin ID1*, Ashley N. Hartley ID1¤a, Kathleen M. Aicher1¤b, Kyle G. Mathews1, Rachel Cullen1, John M. Cullen2, Benjamin J. Callahan2, Devorah M. Stowe2, Gabriela S. Seiler3, Megan E. Jacob2, Jason W. Arnold4, M. Andrea Azcarate-Peril4, Stephen H. Stauffer1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gookin JL, Hartley AN, Aicher KM, Mathews KG, Cullen R, Cullen JM, et al. (2023) Gallbladder microbiota in healthy dogs and dogs with mucocele formation. PLoS ONE 18(2): e0281432. https://doi.org/10.1371/journal. pone.0281432 Editor: Ricardo Santos, Universidade Lisboa, Instituto superior Técnico, PORTUGAL Received: September 30, 2021 Accepted: January 23, 2023 Published: February 10, 2023 Copyright: © 2023 Gookin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All sequence files are available from the NCBI Sequence Read Archive database (accession number PRJNA736462) https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA736462. Funding: These studies were supported by donations in remembrance of Netop Karch (Dr. Jody L. Gookin), a gift from the Edmonton Shetland Sheepdog club (Dr. Jody L. Gookin), and the Firestone Canine Research Endowment to the North Carolina Veterinary Medical Foundation (Dr. 1 Department of Clinical Sciences, College of Veterinary Medicine and Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America, 2 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America, 3 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America, 4 Department of Medicine, Division of Gastroenterology and Hepatology, and UNC Microbiome Core, Center for Gastrointestinal Biology and Disease, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America ¤a Current address: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, United States of America ¤b Current address: College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America * Abstract To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culturebased identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly PLOS ONE | https://doi.org/10.1371/journal.pone.0281432 February 10, 2023 1 / 22 PLOS ONE Ashley N. Hartley). The UNC Microbiome Core is supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases, Center for Gastrointestinal Biology and Disease (CGIBD) P30 DK034987 and the Nutrition Obesity Research Center (NORC), University of North Carolina P30 DK056350. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Gallbladder microbiota in canine bile and mucocele mucus contribute to the results of sequencing analysis when performed on samples with low bacterial biomass. Introduction Bile is synthesized by the liver and stored in the gallbladder. In response to ingestion of a meal, the gallbladder contracts and empties bile into the small intestine resulting in the delivery of bile salts that aid in the assimilation of dietary fat. Despite an intense interest in the influence of intestinal microbiota in health and disease, it is surprising that there are few studies investigating the culture-independent microbiome of bile [1–11], none of which has included dogs. In healthy dogs, bile is generally regarded as sterile [12]. What remains unknown is whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection, which is one of the most common and treatable causes of hepatobiliary disease in dogs [13–21]. Recently, a unique gallbladder disease in dogs has gained broad recognition [22–29]. The disorder, referred to as gallbladder mucocele formation, is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium [30]. This mucus can eventually lead to rupture of the gallbladder or obstruction of bile flow [30]. The cause of mucocele formation is unknown. In dogs undergoing surgery for removal of a gallbladder mucocele, routine aerobic and anaerobic culture of gallbladder content identifies the presence of common biliary pathogens such as E. coli and Enterococcus spp. in anywhere from 2.7 to 67% (median, 13%) of cases [22, 23, 25, 27, 28, 31–34]. What is unknown is whether uncultivable, and therefore unr (...truncated)