There has been no prior application of matched metagenomics and metatranscriptomics in Clostridioides difficile infection (CDI) evaluating the role of fungi in CDI or identifying community functions that contribute to the development of this disease. We collected diarrheal stools from 49 inpatients (18 of whom tested positive for CDI) under stringent inclusion criteria. We utilized a tiered sequencing approach to identify enriched bacterial and fungal taxa, using 16S and internal transcribed spacer (ITS) rRNA gene amplicon sequencing, with matched metagenomics and metatranscriptomics performed on a subset of the population. Distinct bacterial and fungal compositions distinguished CDI-positive and -negative patients, with the greatest differentiation between the cohorts observed based on bacterial metatranscriptomics. Bipartite network analyses demonstrated that Aspergillus and Penicillium taxa shared a strong positive relationship in CDI patients and together formed negative cooccurring relationships with several bacterial taxa, including the Oscillospira, Comamonadaceae, Microbacteriaceae, and Cytophagaceae. Metatranscriptomics revealed enriched pathways in CDI patients associated with biofilm production primarily driven by Escherichia coli and Pseudomonas, quorum-sensing proteins, and two-component systems related to functions such as osmotic regulation, linoleic acid metabolism, and flagellar assembly. Differential expression of functional pathways unveiled a mechanism by which the causal dysbiosis of CDI may self-perpetuate, potentially contributing to treatment failures. We propose that CDI has a distinct fungus-associated bacteriome, and this first description of metatranscriptomics in human subjects with CDI demonstrates that inflammation, osmotic changes, and biofilm production are key elements of CDI pathophysiology. IMPORTANCE Our data suggest a potential role for fungi in the most common nosocomial bacterial infection in the United States, introducing the concept of a transkingdom interaction between bacteria and fungi in this disease. We also provide the first direct measure of microbial community function in Clostridioides difficile infection using patient-derived tissue samples, revealing antibiotic-independent mechanisms by which C. difficile infection may resist a return to a healthy gut microbiome.

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Clostridium difficile is a bacterial pathogen of the gut that causes nearly 500,000 infections per year in the United States, with 20 to 30 percent of cases re‐occurring. Little is known about how C. difficile modulates the gut's fungal community and how this dysbiosis may perpetuate its reocurrence. This study aimed to contribute to the understanding of the disease's mechanism by identifying bacterial and fungal community structures and the bacterial‐fungal interactions in C. difficile infected (CDI) patients. Forty‐nine diarrheal stool samples, 18 CDI and 31 non‐CDI, were collected from hospitalized patients. The taxonomic marker, or “thumbprint,” regions of DNA, 16S rRNA for bacteria and ITS for fungi, were isolated and sequenced to determine the microbial communities. Metagenomic and metatranscriptomic analyses were also preformed to further characterize gut microbial structure and function. Bioinformatic analysis of the 16S rRNA and ITS data revealed a greater number of fungal taxa enriched in CDI samples than non‐CDI samples. Further, co‐occurrence network analysis using the program CoNet displayed negative correlations between the fungal taxon Candida and several bacterial taxa in CDI samples. These findings indicate that CDI infections may create an environment that allows fungal communities to bloom and possibly suppress commensal bacteria. Additionally, bioinformatic analysis of the metatrancriptomic and metagenomic data revealed that pathways involving biofilm formation, inflammation, flagellar assembly, and two‐component systems involving osmotic regulation were enriched in CDI samples. These functionalities may allow C. difficile to persist in the gut, and therefore may lead to failed treatments and reoccurrences. Our data suggest that the differential bacterial and fungal communities in CDI and non‐CDI patients may lead to high recurrence rates. The enriched fungal community in CDI patients, may be perpetuating the dysbiosis, thus leading to the development of new therapeutic approaches.Support or Funding InformationThis research was supported by a grant from the American Society of Colon & Rectal Surgeons in 2017 (ASCRS Research Foundation Benign Colorectal Disease Grant RFP‐002). This research was also supported by a grant to Juniata College from the Howard Hughes Medical Institute (http://www.hhmi.org) through the Precollege and Undergraduate Science Education Program, as well as by the National Science Foundation (http://www.nsf.gov) through NSF award DBI‐1248096.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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