title: Genetic markers for Clostridium difficile lineages linked to hypervirulence.
abstract: Rapid identification of hypervirulent Clostridium difficile strains is essential for preventing their spread. Recent completion of several full-length C. difficile genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between C. difficile strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase (thyX) gene and replaces it with an equivalent, catalytically more efficient, thyA gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within C. difficile that are highly related to hypervirulent strains.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Cornelis W
authors.initials: CW
authors.lastname: Knetsch
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Marjolein P M
authors.initials: MPM
authors.lastname: Hensgens
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Céline
authors.initials: C
authors.lastname: Harmanus
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Madelon W
authors.initials: MW
authors.lastname: van der Bijl
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Paul H M
authors.initials: PHM
authors.lastname: Savelkoul
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Ed J
authors.initials: EJ
authors.lastname: Kuijper
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Corver
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Hans C
authors.initials: HC
authors.lastname: van Leeuwen
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D055028:Comparative Genomic Hybridization; D004269:DNA, Bacterial / Q000235:genetics; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology / Q000401:mortality; D005819:Genetic Markers; D016680:Genome, Bacterial; D006801:Humans; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D021521:Ribotyping; D017422:Sequence Analysis, DNA; D015996:Survival Rate; D014774:Virulence
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers