Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile.

Clostridioides difficile produces toxins that damage the colonic epithelium, causing colitis. Variation in disease severity is poorly understood and has been attributed to host factors and virulence differences between C. difficile strains. We test 23 epidemic ST1 C. difficile clinical isolates for their virulence in mice. All isolates encode a complete Tcd pathogenicity locus and achieve similar colonization densities. However, disease severity varies from lethal to avirulent infections. Genomic analysis of avirulent isolates reveals a 69-bp deletion in the cdtR gene, which encodes a response regulator for binary toxin expression. Deleting the 69-bp sequence in virulent R20291 strain renders it avirulent in mice with reduced toxin gene transcription. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile isolates without reducing colonization and persistence. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

Clostridioides difficile S-Layer Protein A (SlpA) Serves as a General Phage Receptor.

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ϕCD38-2, ϕCD111, and ϕCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ϕCD111 and SLCT-11 enabled infection by ϕCD38-2 and ϕCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ϕCD38-2 and ϕCD146 but not ϕCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile.

Clostridioides difficile (C. difficile) , a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88 -/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.