PROJECT SUMMARY ABSTRACTAntibiotic-associated diarrhea (AAD) is a very common problem; approximately 50% of hospitalized patientsreceive antibiotics, and up to 25% of those patients develop AAD. While many AAD infectious etiologies, suchas Clostridium difficile infection (CDI), have been well described, a recent study from Taiwan characterizedClostridium innocuum (CI) as a previously unrecognized cause of AAD with multidrug-resistance, including tovancomycin. This study suggested that CI causes gastrointestinal illness indistinguishable from CDI, which caninclude severe and pseudomembranous colitis, in both humans and an intestinal model of infection in mice.This recognition of the pathogenic potential of CI is in contrast to its prior classification as a clinically innocuousintestinal commensal. This was based on a single 1962 study that failed to demonstrate CI pathogenicity in anon-intestinal mouse model. Our preliminary clinical, microbiologic, and genomic data support potentialreclassification of CI as an emerging pathogen. We have recently isolated CI from more than 20 childrendiagnosed with CDI (in whom the contribution of CI to illness is unclear) and one adult with recurrent AAD (inwhom CI caused AAD). Further analysis suggests that many of these children were colonized with C. difficileand had another unidentified diarrheal etiology. In addition, several of our CI strains were noted to be positivefor C. difficile toxins A/B by enzyme immunoassay (EIA). Thus, our preliminary data suggest a model wherebysome CI strains have the ability to produce a protein that is recognized by C. difficile anti-toxin antibodies, andthat CI may cause some cases of AAD, potentially related to this uncharacterized protein. To bettercharacterize the epidemiologic significance and microbiologic characteristics of this emerging pathogen in USadults and children, we propose to: (1) Determine the clinical and molecular epidemiology of CI in symptomaticand asymptomatic adults and children; (2a) Identify the CI protein responsible for C. difficile toxin A/B EIAcross-reactivity, and (2b) Determine the association between this protein and CI pathogenicity. Becausevancomycin use for CDI is increasing in the US, selective pressure may favor a rise in clinical infections withmultidrug-resistant CI. Because of widespread use of C. difficile-specific diagnostic tests for CDI, diagnosis ofclinically indistinguishable CI AAD will require substantial clinical microbiology practice change. For thesereasons, it is imperative to understand the scope of CI AAD in the US. Successful completion of the proposedstudies will guide future investigation of the pathogenesis, diagnosis, and clinical characteristics of CI infection.Our team and the Northwestern University academic environment possess the skills and resources necessaryfor successful completion of the proposed studies, including assembly of patient cohorts, microbiology,bacterial pathogenesis, proteomics, genomics, and bioinformatics.