Characterization of flagellar and toxin phase variation in Clostridium difficile ribotype 012 isolates

Clostridium difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a \"flagellar switch\" upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting the strain is phase-locked. To determine whether a \"phase locked\" flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase OFF, non-motile, and attenuated for toxin production. We isolated low frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production, and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase ON derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains, and perhaps other ribotypes, which could impact disease progression and diagnosis.\n\nImportanceClostridium difficile causes diarrheal disease resulting in significant morbidity and mortality in many countries. C. difficile produces flagella that enhance bacterial motility, and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins are co-regulated via a flippable DNA element termed the \"flagellar switch\", which mediates the phase variable production of these factors. C. difficile can exist as flagellated, motile, toxigenic bacteria (\"flg ON\") or aflagellate, non-motile, nontoxigenic bacteria (\"flg OFF\") due in part to the flagellar switch orientation. Here we evaluate multiple isolates of C. difficile ribotype 012 strains and find them to be primarily flg OFF. Some, but not all, of these isolates showed the ability to switch between flg ON and OFF states. These findings suggest heterogeneity in the ability of C. difficile ribotype 012 strains to phase vary flagellum and toxin production, which may broadly apply to pathogenic C. difficile.