Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes

Abstract

Objective: The motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay.

Methods: A Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar.

Results: The highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d).

Conclusions: C. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

Keywords: Clonal complex; Clostridioides difficile; Hypervirulence; Motility; RFLP patterns; fliC and fliD genes.