The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R,and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.