Integrated Meta-omics Reveals a Fungus-Associated Bacteriome and Distinct Functional Pathways in Clostridioides difficile Infection.

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.

Response of Aquatic Bacterial Communities to Hydraulic Fracturing in Northwestern Pennsylvania: A Five-Year Study.

Horizontal drilling and hydraulic fracturing extraction procedures have become increasingly present in Pennsylvania where the Marcellus Shale play is largely located. The potential for long-term environmental impacts to nearby headwater stream ecosystems and aquatic bacterial assemblages is still incompletely understood. Here, we perform high-throughput sequencing of the 16 S rRNA gene to characterize the bacterial community structure of water, sediment, and other environmental samples (n = 189) from 31 headwater stream sites exhibiting different histories of fracking activity in northwestern Pennsylvania over five years (2012-2016). Stream pH was identified as a main driver of bacterial changes within the streams and fracking activity acted as an environmental selector for certain members at lower taxonomic levels within stream sediment. Methanotrophic and methanogenic bacteria (i.e. Methylocystaceae, Beijerinckiaceae, and Methanobacterium) were significantly enriched in sites exhibiting Marcellus shale activity (MSA+) compared to MSA- streams. This study highlighted potential sentinel taxa associated with nascent Marcellus shale activity and some of these taxa remained as stable biomarkers across this five-year study. Identifying the presence and functionality of specific microbial consortia within fracking-impacted streams will provide a clearer understanding of the natural microbial community's response to fracking and inform in situ remediation strategies.

Antibiotic Treatments for Clostridium difficile Infection Are Associated with Distinct Bacterial and Fungal Community Structures.

Clostridium difficile infection (CDI) is the most common nosocomial infection in the United States, being associated with high recurrence and persistence rates. Though the relationship between intestinal dysbiosis and CDI is well known, it is unclear whether different forms of dysbiosis may potentially affect the course of CDI. How this is further influenced by C. difficile-directed antibiotics is virtually uninvestigated. In this study, diarrheal stool samples were collected from 20 hospitalized patients, half of whom were confirmed to have CDI. Analyzing tissue ex vivo and in duplicate, CDI and non-CDI fecal samples (n = 176) were either not antibiotic treated or treated with metronidazole, vancomycin, or fidaxomicin, the three most common CDI therapies. The microbial community composition, interactions, and predicted metabolic functions were assessed by 16S rRNA gene and internal transcribed spacer sequencing, bipartite network analysis, and phylogenetic investigation of communities by reconstruction of unobserved states. Our results demonstrate that while all C. difficile-directed antibiotics were associated with similar reductions in alpha diversity, beta diversity significantly differed on the basis of the particular antibiotic, with differentiating relative abundances of bacterial and fungal assemblages. With the exception of fidaxomicin, each antibiotic was associated with the emergence of potentially pathogenic fungal operational taxonomic units, with predicted bacterial functions enriched for xenobiotic metabolism that could perpetuate the dysbiosis driving CDI. Toxin-independent mechanisms of colitis related to the relative abundance of pathogenic bacteria and fungi were also noted. This study suggests that a transkingdom interaction between fungi and bacteria may be important in CDI pathophysiology, including being a factor in the historically high persistence and recurrence rates associated with this disease. IMPORTANCE Using human fecal samples and including sequencing for both bacterial and fungal taxa, this study compared the conventional antibiotics used to treat C. difficile infection (CDI) from the perspective of the microbiome, which is particularly relevant, given the relationship between dysbiotic states and the development of CDI. Sequencing and imputed functional analyses suggest that C. difficile-directed antibiotics are associated with distinct forms of dysbiosis that may be influential in the course of CDI. Further, a role for fungal organisms in the perpetuation of the causal dysbiosis of CDI is discussed, suggesting a previously unappreciated, clinically relevant transkingdom interaction that warrants further study.

The Microbial Ecosystem Distinguishes Chronically Diseased Tissue from Adjacent Tissue in the Sigmoid Colon of Chronic, Recurrent Diverticulitis Patients.

Diverticular disease is commonly associated with the older population in the United States. As individual's age, diverticulae, or herniation of the mucosa through the colonic wall, develop. In 10-25% of individuals, the diverticulae become inflamed, resulting in diverticulitis. The gut ecosystem relies on the interaction of bacteria and fungi to maintain homeostasis. Although bacterial dysbiosis has been implicated in the pathogenesis of diverticulitis, associations between the microbial ecosystem and diverticulitis remain largely unstudied. This study investigated how the cooperative network of bacteria and fungi differ between a diseased area of the sigmoid colon chronically affected by diverticulitis and adjacent non-affected tissue. To identify mucosa-associated microbes, bacterial 16S rRNA and fungal ITS sequencing were performed on chronically diseased sigmoid colon tissue (DT) and adjacent tissue (AT) from the same colonic segment. We found that Pseudomonas and Basidiomycota OTUs were associated with AT while Microbacteriaceae and Ascomycota were enriched in DT. Bipartite co-occurrence networks were constructed for each tissue type. The DT and AT networks were distinct for each tissue type, with no microbial relationships maintained after intersection merge of the groups. Our findings indicate that the microbial ecosystem distinguishes chronically diseased tissue from adjacent tissue.