Symbiotic biofilms formed by <i>Clostridioides difficile</i> and <i>bacteroides thetaiotaomicron</i> in the presence of vancomycin

Vancomycin (VAN) treatment in <i>Clostridioides difficile</i> infection (CDI) suffers from a relatively high rate of recurrence, with a variety of reasons behind this, including biofilm-induced recurrent infections. <i>C. difficile</i> can form monophyletic or symbiotic biofilms with other microbes in the gut, and these biofilms protect <i>C. difficile</i> from being killed by antibiotics. In this study, we analyzed the ecological relationship between <i>Bacteroides thetaiotaomicron</i> and <i>C. difficile</i> and their formation of symbiotic biofilm in the VAN environment. The production of symbiotic biofilm formed by <i>C. difficile</i> and <i>B. thetaiotaomicron</i> was higher than that of <i>C. difficile</i> and <i>B. thetaiotaomicron</i> alone in the VAN environment. In symbiotic biofilms, <i>C. difficile</i> was characterized by increased production of the toxin protein TcdA and TcdB, up-regulation of the expression levels of the virulence genes <i>tcdA</i> and <i>tcdB</i>, enhanced bacterial cell swimming motility and c-di-GMP content, and increased adhesion to Caco-2 cells. The scanning electron microscope (SEM) combined with confocal laser scanning microscopy (CLSM) results indicated that the symbiotic biofilm was elevated in thickness, dense, and had an increased amount of mixed bacteria, while the fluorescence in situ hybridization (FISH) probe and plate colony counting results further indicated that the symbiotic biofilm had a significant increase in the amount of <i>C. difficile</i> cells, and was able to better tolerate the killing of the simulated intestinal fluid. Taken together, <i>C. difficile</i> and <i>B. thetaiotaomicron</i> become collaborative in the VAN environment, and targeted deletion or attenuation of host gut <i>B. thetaiotaomicron</i> content may improve the actual efficacy of VAN in CDI treatment.