Data_Sheet_1_Clostridioides difficile infection is associated with differences in transcriptionally active microbial communities.docx

Clostridioides difficile infection (CDI) is responsible for around 300,000 hospitalizations yearly in the United States, with the associated monetary cost being billions of dollars. Gut microbiome dysbiosis is known to be important to CDI. To the best of our knowledge, metatranscriptomics (MT) has only been used to characterize gut microbiome composition and function in one prior study involving CDI patients. Therefore, we utilized MT to investigate differences in active community diversity and composition between CDI+ (n = 20) and CDI− (n = 19) samples with respect to microbial taxa and expressed genes. No significant (Kruskal-Wallis, p > 0.05) differences were detected for richness or evenness based on CDI status. However, clustering based on CDI status was significant for both active microbial taxa and expressed genes datasets (PERMANOVA, p ≤ 0.05). Furthermore, differential feature analysis revealed greater expression of the opportunistic pathogens Enterocloster bolteae and Ruminococcus gnavus in CDI+ compared to CDI− samples. When only fungal sequences were considered, the family Saccharomycetaceae expressed more genes in CDI−, while 31 other fungal taxa were identified as significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) associated with CDI+. We also detected a variety of genes and pathways that differed significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) based on CDI status. Notably, differential genes associated with biofilm formation were expressed by C. difficile. This provides evidence of another possible contributor to C. difficile’s resistance to antibiotics and frequent recurrence in vivo. Furthermore, the greater number of CDI+ associated fungal taxa constitute additional evidence that the mycobiome is important to CDI pathogenesis. Future work will focus on establishing if C. difficile is actively producing biofilms during infection and if any specific fungal taxa are particularly influential in CDI.

艰难梭菌感染（Clostridioides difficile infection，简称CDI）每年在美国导致约30万次住院，相关经济成本高达数十亿美元。肠道菌群失调已被证实与CDI密切相关。据我们所知，元转录组学（metatranscriptomics，简称MT）仅在一项涉及CDI患者的研究中用于描述肠道菌群的组成和功能。因此，我们采用MT技术，旨在探究CDI阳性（CDI+，n=20）与CDI阴性（CDI−，n=19）样本在活性微生物群落的多样性和组成上是否存在差异，并针对微生物类群和表达基因进行分析。基于CDI状态，在丰富度和均匀度方面未检测到显著差异（Kruskal-Wallis检验，p>0.05）。然而，基于CDI状态的聚类分析在活跃微生物类群和表达基因数据集中均显示出显著性（PERMANOVA检验，p≤0.05）。此外，差异特征分析揭示了机会性病原体肠球菌Enterocloster bolteae和瘤胃球菌Ruminococcus gnavus在CDI+样本中的表达显著高于CDI−样本。当仅考虑真菌序列时，在CDI−样本中，酵母科Saccharomycetaceae表达更多基因，而31种其他真菌类群被认定为与CDI+显著相关（Kruskal-Wallis检验，p≤0.05，log(LDA)≥2）。我们还检测到一系列基于CDI状态差异显著（Kruskal-Wallis检验，p≤0.05，log(LDA)≥2）的基因和通路。值得注意的是，与生物膜形成相关的差异基因由艰难梭菌表达，这为艰难梭菌对抗生素的耐药性和体内频繁复发提供了另一可能的证据。此外，与CDI+相关的真菌类群数量更多，这也构成了真菌群在CDI发病机制中具有重要作用的额外证据。未来的研究将聚焦于确定艰难梭菌在感染过程中是否主动产生生物膜，以及是否存在特定真菌类群在CDI中具有特别显著的影响。