Strain Variation in Clostridioides difficile Cytotoxicity Associated with Genomic Variation at Both Pathogenic and Nonpathogenic Loci.

Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes located within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the cytotoxicity of C. difficile may be modulated by genomic variants outside the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between cytotoxicity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model cytotoxicity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci and changes in cytotoxicity. Combined, these data support a model of C. difficile disease wherein cytotoxicity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections, including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Finally, we highlight the intersection of loci identified by the GWAS for different phenotypes and clinical severity. This strategy to identify overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCE Clostridioides difficile has two major disease-mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study, we demonstrate via multiple approaches that genomic variants outside the PaLoc are associated with changes in cytotoxicity. These genomic variants may provide new avenues of exploration in the hunt for novel disease-modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical for clinical infection, such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and, as such, vary in their appropriateness for certain bacterial genome-wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important for both bacterial function and human clinical disease.

Hogwash: three methods for genome-wide association studies in bacteria.

Bacterial genome-wide association studies (bGWAS) capture associations between genomic variation and phenotypic variation. Convergence-based bGWAS methods identify genomic mutations that occur independently multiple times on the phylogenetic tree in the presence of phenotypic variation more often than is expected by chance. This work introduces hogwash, an open source R package that implements three algorithms for convergence-based bGWAS. Hogwash additionally contains two burden testing approaches to perform gene or pathway analysis to improve power and increase convergence detection for related but weakly penetrant genotypes. To identify optimal use cases, we applied hogwash to data simulated with a variety of phylogenetic signals and convergence distributions. These simulated data are publicly available and contain the relevant metadata regarding convergence and phylogenetic signal for each phenotype and genotype. Hogwash is available for download from GitHub.

prewas: data pre-processing for more informative bacterial GWAS.

While variant identification pipelines are becoming increasingly standardized, less attention has been paid to the pre-processing of variants prior to their use in bacterial genome-wide association studies (bGWAS). Three nuances of variant pre-processing that impact downstream identification of genetic associations include the separation of variants at multiallelic sites, separation of variants in overlapping genes, and referencing of variants relative to ancestral alleles. Here we demonstrate the importance of these variant pre-processing steps on diverse bacterial genomic datasets and present prewas, an R package, that standardizes the pre-processing of multiallelic sites, overlapping genes, and reference alleles before bGWAS. This package facilitates improved reproducibility and interpretability of bGWAS results. prewas enables users to extract maximal information from bGWAS by implementing multi-line representation for multiallelic sites and variants in overlapping genes. prewas outputs a binary SNP matrix that can be used for SNP-based bGWAS and will prevent the masking of minor alleles during bGWAS analysis. The optional binary gene matrix output can be used for gene-based bGWAS, which will enable users to maximize the power and evolutionary interpretability of their bGWAS studies. prewas is available for download from GitHub.

Genetic Determinants of Trehalose Utilization Are Not Associated With Severe Clostridium difficile Infection Outcome.

In a case-control study of patients with Clostridium difficile infection, we found no statistically significant association between the presence of trehalose utilization variants in infecting C. difficile strains and development of severe infection outcome. These results do not support trehalose utilization conferring enhanced virulence in the context of human C. difficile infections.

Outbreak of Murine Infection with Clostridium difficile Associated with the Administration of a Pre- and Perinatal Methyl Donor Diet.

Between October 2016 and June 2017, a C57BL/6J mouse colony that was undergoing a pre- and perinatal methyl donor supplementation diet intervention to study the impact of parental nutrition on offspring susceptibility to disease was found to suffer from an epizootic of unexpected deaths. Necropsy revealed the presence of severe colitis, and further investigation linked these outbreak deaths to a Clostridium difficile strain of ribotype 027 that we term 16N203. C. difficile infection (CDI) is associated with antibiotic use in humans. Current murine models of CDI rely on antibiotic pretreatment to establish clinical phenotypes. In this report, the C. difficile outbreak occurs in F1 mice linked to alterations in the parental diet. The diagnosis of CDI in the affected mice was confirmed by cecal/colonic histopathology, the presence of C. difficile bacteria in fecal/colonic culture, and detection of C. difficile toxins. F1 mice from parents fed the methyl supplementation diet also had significantly reduced survival (P < 0.0001) compared with F1 mice from parents fed the control diet. When we tested the 16N203 outbreak strain in an established mouse model of antibiotic-induced CDI, we confirmed that this strain is pathogenic. Our serendipitous observations from this spontaneous outbreak of C. difficile in association with a pre- and perinatal methyl donor diet suggest the important role that diet may play in host defense and CDI risk factors.IMPORTANCEClostridium difficile infection (CDI) has become the leading cause of infectious diarrhea in hospitals worldwide, owing its preeminence to the emergence of hyperendemic strains, such as ribotype 027 (RT027). A major CDI risk factor is antibiotic exposure, which alters gut microbiota, resulting in the loss of colonization resistance. Current murine models of CDI also depend on pretreatment of animals with antibiotics to establish disease. The outbreak that we report here is unique in that the CDI occurred in mice with no antibiotic exposure and is associated with a pre- and perinatal methyl supplementation donor diet intervention study. Our investigation subsequently reveals that the outbreak strain that we term 16N203 is an RT027 strain, and this isolated strain is also pathogenic in an established murine model of CDI (with antibiotics). Our report of this spontaneous outbreak offers additional insight into the importance of environmental factors, such as diet, and CDI susceptibility.