In vitro antivirulence activity of baicalin against Clostridioides difficile

Abstract

<b>Introduction.</b> <i>Clostridioides difficile</i> is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize <i>C. difficile</i> pathogenicity and disease outcome in affected individuals.<b>Aim.</b> This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in <i>C. difficile in vitro</i>.<b>Methodology.</b> Hypervirulent <i>C. difficile</i> isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining <i>C. difficile</i> toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, <i>C. difficile</i> spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on <i>C. difficile</i> toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR.<b>Results.</b> The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, <i>C. difficile</i> genes critical for pathogenesis were significantly down-regulated in the presence of baicalin.<b>Conclusion.</b> Our results suggest that baicalin could potentially be used to control <i>C. difficile</i>, and warrant future studies <i>in vivo</i>.