A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ?CD08011, ?CD418, ?CD1801, and ?CD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ?CD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ?CD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI

CdtR (only) the regulator of binary toxin in Clostridium difficile

Clostridium difficile is a Gram-positive, endospore forming, anaerobic bacterium, responsible for most hospital acquired antibiotic induced diarrhoeal disease. The spores act as mean of transmission, while the disease itself is toxin mediated. Most strains produce two large glycosylating toxins, named toxin A and B, but about 20% of clinical isolates produce an additional binary toxin, called C. difficile toxin (CDT). CDT has been implicated in more severe infections and also in relapse, but little is known about its actual contribution to pathogenesis and disease outcome. Some evidence has been obtained that CDT might play a role in colonisation and there is also further data suggesting that it might act in concert with the large toxins. It is thought to be regulated by a LytTR response regulator, CdtR, encoded just upstream. Interestingly most strains that do not carry functional CDT genes have a ghost locus, encoding an intact copy of cdtR, but truncated cdt genes. Very few strains do not have cdtR; instead, these carry a conserved 68bp fragment.Here we are exploring the following two research questions: Is CdtR absolutely required for functional binary toxin? Has CdtR further functions (in addition to binary toxin regulation)? To address these, clean deletion mutants of cdtR have been generated in a ribotype 027 C. difficile strain (R20291, producing the large toxins A and B and also CDT) and also in CD630, containing the afore mentioned ghost locus. Furthermore the PaLoc (containing toxin A and B) has been deleted from R20291. Production of CDT is assessed in the R20291 mutants by Western blot and cytotoxicity. Effects of the CdtR deletion in a CDT negative strain are being investigated to potentially elucidate a role beyond binary toxin regulation. The here presented study will give an insight into the role(s) and regulation of the binary toxin regulator CdtR in C. difficile