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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

Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes encoded within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the toxin activity of C. difficile may be modulated by genomic variants outside of the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between toxin activity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model toxin activity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci with changes in toxin activity. Combined, these data support a model of C. difficile disease wherein toxin activity 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. Lastly, we highlight the intersection of loci identified by GWAS for different phenotypes and clinical severity. This strategy to identify the overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes.IMPORTANCEClostridioides 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 of the PaLoc are associated with changes in toxin activity. 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 to 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 to both bacterial function and human clinical disease.

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.

Background The composition of the human vaginal microbiota is related to many aspects of health from infection susceptibility to preterm birth. Factors that influence human vaginal microbiota composition, including its source, are not well understood. Objective The goal of this study was to determine if vaginal microbiota transmission from mother to daughter at birth influences the human vaginal microbiota composition in adolescence. Study Design Weekly vaginal swab samples from 13 adolescents and their mothers were collected for up to 4 weeks. After DNA was isolated from the swabs, the V4 region of the bacterial 16S rRNA genes were amplified, sequenced and analyzed. We calculated distances between the bacterial communities in different samples to investigate the relationship between the vaginal microbiota of the mother/daughter pairs and the daughter’s birth mode. We also cultivated Lactobacillus crispatus from the mother and daughter of 1 pair. To investigate the possibility of direct transmission and persistence of one member of the vaginal microbiota, we isolated DNA from the L. crispatus isolates and compared their genomes with each other and other publicly available L. crispatus genome sequences. Results The vaginal microbiotas of mother/daughter pairs were more similar to each other if the daughter was born by vaginal delivery rather than by C-section. Additionally, genome sequences from an important member of the vaginal microbiota, L. crispatus, isolated from one mother/daughter pair in which the daughter was born by vaginal delivery, were highly similar. Conclusion Both community-level analysis and isolate genome sequence analysis are consistent with birth-mode dependent transmission and persistence of at least some members of the vaginal microbiota.

Clostridium 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., 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. IMPORTANCE Clostridium 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.