Novel insights into phage biology of the pathogen Clostridioides difficile based on the active virome.

The global pathogen Clostridioides difficile is a well-studied organism, and researchers work on unraveling its fundamental virulence mechanisms and biology. Prophages have been demonstrated to influence C. difficile toxin expression and contribute to the distribution of advantageous genes. All these underline the importance of prophages in C. difficile virulence. Although several C. difficile prophages were sequenced and characterized, investigations on the entire active virome of a strain are still missing. Phages were mainly isolated after mitomycin C-induction, which does not resemble a natural stressor for C. difficile. We examined active prophages from different C. difficile strains after cultivation in the absence of mitomycin C by sequencing and characterization of particle-protected DNA. Phage particles were collected after standard cultivation, or after cultivation in the presence of the secondary bile salt deoxycholate (DCA). DCA is a natural stressor for C. difficile and a potential prophage-inducing agent. We also investigated differences in prophage activity between clinical and non-clinical C. difficile strains. Our experiments demonstrated that spontaneous prophage release is common in C. difficile and that DCA presence induces prophages. Fourteen different, active phages were identified by this experimental procedure. We could not identify a definitive connection between clinical background and phage activity. However, one phage exhibited distinctively higher activity upon DCA induction in the clinical strain than in the corresponding non-clinical strain, although the phage is identical in both strains. We recorded that enveloped DNA mapped to genome regions with characteristics of mobile genetic elements other than prophages. This pointed to mechanisms of DNA mobility that are not well-studied in C. difficile so far. We also detected phage-mediated lateral transduction of bacterial DNA, which is the first described case in C. difficile. This study significantly contributes to our knowledge of prophage activity in C. difficile and reveals novel aspects of C. difficile (phage) biology.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

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 employing antibiotics. This suggests a significant bias among analyzed 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. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. 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 assessment of 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 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.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens 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 avoidance of 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 next-generation sequencing.

Genome sequence analysis of the temperate bacteriophage TBP2 of the solvent producer Clostridium saccharoperbutylacetonicum N1-4 (HMT, ATCC 27021).

The genus Clostridium consists of a diverse group of pathogenic and non-pathogenic bacteria. The non-pathogenic clostridia contain several solventogenic members of industrial importance, such as Clostridium acetobutylicum or C. beijerinckii. In the process of acetone-butanol-ethanol (ABE) fermentation, these strains are used in large scale fermentation plants since almost 100 years. Soon after establishment of the first plants, the fermentation processes suffered from different bacteriophage infections worldwide. A limited set of studies addressing bacteriophages in solventogenic clostridia have been conducted since then. In this study, we present the genome sequence of the temperate bacteriophage TBP2 of the solventogenic strain C. saccharoperbutylacetonicum N1-4 (HMT) that is used for ABE fermentation. The phage genome consists of 38 039 bp and includes 48 open reading frames. Sequence analysis indicates that the genome encloses random parts of the bacterial genome in addition to its own DNA. It represents the first fully sequenced genome of a temperate bacteriophage infecting solventogenic clostridia.

First Insights into the Genome Sequence of the Cellulolytic Bacterium Clostridium hungatei DSM 14427.

Clostridium hungatei is an obligate anaerobic and spore-forming bacterium, which was isolated from soil. It ferments carbohydrates, such as cellulose or d-glucose. C. hungatei is able to fix nitrogen. The draft genome consists of 1 chromosome (4.902 Mb) with 4,246 predicted protein-coding genes.