title: CRISPR Diversity and Microevolution in Clostridium difficile.
abstract: Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.
authors: Department of Food, Processing and Nutritional Sciences, North Carolina State University, NC.
authors: Joakim M
authors: JM
authors: Andersen
authors: Department of Microbiology and Molecular Genetics, Michigan State University, MI.
authors: Madelyn
authors: M
authors: Shoup
authors: Department of Microbiology and Molecular Genetics, Michigan State University, MI.
authors: Cathy
authors: C
authors: Robinson
authors: Department of Molecular Virology and Microbiology, Center for Metagenomics and Microbiome Research, Baylor College of Medicine, TX.
authors: Robert
authors: R
authors: Britton
authors: Microbial Competence Centre, Novo Nordisk, Bagsværd, Denmark (Former Employment: Department of Microbiology & Infection Control, Statens Serum Institut, Copenhagen, Denmark).
authors: Katharina E P
authors: KE
authors: Olsen
authors: Department of Food, Processing and Nutritional Sciences, North Carolina State University, NC rbarran@ncsu.edu.
authors: Rodolphe
authors: R
authors: Barrangou
mesh_terms: D064113:CRISPR-Cas Systems; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019143:Evolution, Molecular; D010802:Phylogeny; D011110:Polymorphism, Genetic
keywords: BI/NAP1/RT027/ST1Type-IB; CRISPR phylogeny; CRISPR typing; CRISPR-Cas