Direct and culture-independent detection of low-abundantClostridioides difficilein environmental DNA via PCR

Abstract

AbstractClostridioides difficilerepresents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examinedC. difficilestrains originated from clinical specimen and were isolated under selective conditions emplyoing antibiotics. This suggests a significant bias among analysedC. difficilestrains, which impedes a holistic view on this pathogen. In order to support extensive isolation ofC. difficilestrains from environmental samples, we designed a detection PCR that targets thehpdBCAoperon and thereby identifies low abundances ofC. difficilein environmental samples. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific forC. difficileand successfully detectedC. difficiledespite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of thehpdBCAdetection PCR sequence for initial phylogenetic classification, which allows assessingC. difficilediversity in environmental samples via amplicon sequencing. Our findings furthermore showed thatC. difficilestrains 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 inC. difficileisolation and research.ImportanceClostridioides difficileis 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 ofC. difficilefrom diverse environmental samples and avoiding antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from ourC. difficile-specific detection PCR, which rapidly verifiesC. difficilepresence in environmental samples and further allows estimation of theC. difficilediversity by using NGS.