Direct and culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR

Abstract Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions emplyoing antibiotics. This suggests a significant bias among analysed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA operon and thereby identifies low abundances of C. difficile in environmental samples. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessing C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to detection PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research. Importance Clostridioides difficile is mainly responsible for hospital-acquired infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence focused on bacterial isolation from clinical specimen under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies however require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoiding antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile -specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using NGS.