Flagellar switch inverted repeat sequence variation impactsClostridioides difficileRT027/MLST1 virulence

ABSTRACT The impact of Clostridioides difficile strain diversity on the severity of intestinal infection and diarrhea remains unclear. The RT027/ST1 strain is highly prevalent and has been associated with more severe disease and increased toxin production. Previous studies using antibiotic-treated mice, however, have demonstrated that the severity of diarrhea and colonic inflammation caused by clinical RT027/ST1 C. difficile isolates cultured from different patients is isolate-specific and varies from avirulent to lethal. Herein we demonstrate that increased flagellar expression is associated with increased in vivo virulence. While sequencing the flagellar operon did not identify differences between high and low virulence isolates, high virulence was associated with strains encoding higher proportions of the phase variable flagellar switch in the ON mode. We found that the proportion of bacteria with the switch in the ON mode correlates with maximum weight loss, a readout for virulence in mice. Further analysis of flagellar switch sequences identified variant inverted repeat (IR) sequences, with 40% of isolates having lost one A or T in the IR compared to the genome of C. difficile R20291. The isolates with the R20291 IR sequence have increased virulence compared to those with variant IR (p = 3.2e-05) and these isolates have markedly reduced ability to invert the flagellar switch. Our results suggest that a restricted capacity to invert the flagellar switch during infection is associated with reduced virulence of C. difficile RT027/ST1 strains and may account for some of the inconsistent associations between C. difficile infection and disease severity in patients. IMPORTANCE C. difficile RT027/ST1 has been associated with high virulence. However, variable virulence among ST1 isolates has also been observed. Recent studies have shed light on the impacts of the gut microbiota and host immunity during Clostridioides difficile infection (CDI). Beyond that, determining the genetic factors of C. difficile regulating virulence is essential for a successful CDI diagnosis and treatment. Here, we report a strong association between the flexibility of the flagellar switch and the virulence of C. difficile . Specifically, the inverted repeats of the flagellar switch correlate with switch flexibility. Overall, our study explores variations in C. difficile virulence and highlights the potential of using flagellar switch inverted repeats as an easily accessible genetic trait to predict pathogen virulence.