Evaluation of CRISPR Spacers Diversity in Clostridium Difficile Clinical Isolates

Antibiotic-associated colitis caused by enteropathogen Clostridium difficile represents a great challenge through multiple antibiotic resistance these bacteria possess¹. Recently, bacteriophages have been reported as a promising alternative to antimicrobial agents^2. Regarding C. difficile infection, the phage treatment has the potential to become a biologically solid and highly efficient approach³, though its usage might be limited by active CRISPR-Cas defense systems of this pathogen⁴. Along with cas genes, CRISPR-Cas system of C. difficile contains up to ten CRISPR arrays. By targeting complementary sequences in mobile genomic elements, such as phages and plasmids, spacers provide immunity to host cells and their analysis can reveal a history of host-phage interactions and inform on the choice of therapeutic phages. Our study aims to develop a straightforward analysis of CRISPR spacers of C. difficile in clinical isolates and whole communities. The approach relies on PCR with oligonucleotides complementary to C. difficile CRISPR repeats, followed by high throughput sequencing and bioinformatics reconstruction of whole arrays. This procedure should provide information necessary for fast assessment of a phage-sensitivity of C. difficile strains in patients’ samples. Based on this information individual bacteriophage cocktail treatment can be designed.

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