Department of Clinical Sciences, College of Veterinary Medicine and Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.
Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
PLoS One. 2023 Feb 10;18(2):e0281432. doi: 10.1371/journal.pone.0281432. eCollection 2023.
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 culture-based 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 contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.
迄今为止，尚未有研究调查过犬胆汁的非培养微生物组，而在超声引导下无菌采集胆汁在该物种中是一种常规操作。尽管经常采集胆汁进行基于培养的细菌性胆囊炎诊断，但尚不清楚健康犬的胆汁是否存在不可培养的细菌或核心微生物群。这个问题的答案对于理解胆道感染的发病机制至关重要，并且是探索犬其他胆道疾病的基础，在这些疾病中不可培养的细菌可能发挥致病作用。这种疾病的一个紧迫例子就是犬胆囊粘液囊肿形成。这种普遍且致命的疾病的特征是胆囊上皮过度分泌异常粘液，最终可能导致胆囊破裂或胆汁流动受阻。粘液囊肿形成的原因尚不清楚，也不知道不可培养的、因此未被识别的细菌是否在发病机制中发挥任何系统性作用。在这项研究中，我们应用下一代 16S rRNA 基因测序来鉴定健康犬胆囊胆汁和患有粘液囊肿形成的犬胆囊粘液中的培养阴性细菌群落。我们研究的一个重要部分是在每个样本上使用两种不同的提取方法进行两次单独的 DNA 分离，并对阴性对照样本进行测序，从而能够识别和处理污染序列。通过同时进行基于培养的鉴定、胆汁细胞学检查和胆囊黏膜荧光原位杂交 (FISH)，对微生物组发现进行了验证。使用基于培养的、细胞学的、FISH 和 16S rRNA 测序方法，我们的研究结果不支持健康犬胆汁或患有粘液囊肿形成的犬胆囊粘液中存在核心微生物群。我们的研究结果进一步证明了在细菌生物量低的样本上进行测序分析时，污染序列如何会显著影响测序分析的结果。
