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.

Diversity and Evolution in the Genome of Clostridium difficile

“…It is currently among the top three most frequently encountered RTs in European hospitals (92,93,176). Furthermore, RT078 shares similar genetic features (binary toxin and tcdC mutations) and disease phenotypes (increased mortality and morbidity) with RT027 (28,109,176,177).…”

“…This is interesting, as it shows that RT is indicative of not only differences in the amplified 16S-23S rRNA gene ISR but also specific differences in the nucleotide sequences of a number of conserved genes. Similarly, gene variations in hypervirulent RT027 and RT078 are MLST lineage specific (109).…”

Clostridium difficile PCR ribotypes 001 and 176 – the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014

“…Ribotype 176 is closely related to 027 [6,7] and can be misidentified by commercial tests targeting a singlebase-pair deletion at nucleotide 117 in the C. difficiletcdC gene [8]. In 2009, shortly afterward the 2008 study, the occurrence of ribotype 176 was reported in certain areas of the country (Eastern Bohemia and Moravia) [9].…”

Clostridium difficile Infection

“…lineages can be discriminated and several different PCR ribotypes have been shown to be closely related to the epidemic types using these sequence-based methods 98,230 . WGS is also used to study transmission and outbreaks 25,231 , although this approach can be costly and is predominantly performed retrospectively.…”