Polystyrene microplastics facilitate Clostridioides difficile biofilm formation and attenuate antibiotic...

Clostridioides difficile infection (CDI) constitutes a critical global public health challenge, with its high recurrence rates intrinsically linked to spore germination, biofilm formation, and antibiotic resistance. Although microplastics are recognized as emerging foodborne contaminants, their potential to exacerbate the risk of CDI recurrence remains largely unexplored. This study systematically elucidates the impact of polystyrene microplastics (PS-MPs) on C. difficile (CD) pathogenicity, biofilm dynamics, and antibiotic resistance. Exposure to PS-MPs (0-400 μg/mL) induced intracellular oxidative stress, thereby facilitating bacterial proliferation and biofilm formation. Concurrently, PS-MPs upregulated the expression of quorum-sensing genes (agrD, luxS) and enhanced AI-2 secretion, which subsequently augmented bacterial motility. Furthermore, PS-MPs exposure significantly elevated the expression of virulence and sporulation genes (e.g., Spo0A), intensifying cytotoxicity toward intestinal epithelial cells (HT-29 and Caco-2). Critically, antimicrobial susceptibility testing demonstrated that both short-term (48 h) and long-term (20 d) PS-MPs exposure significantly increased the half-inhibitory concentration (IC₅₀) of CD against seven antibiotics. Notably, long-term exposure to 100 μg/mL PS-MPs resulted in a 2.42-fold increase in the IC₅₀ for vancomycin, concomitant with the upregulation of resistance genes (tetW, gyrA, and gyrB). Collectively, these findings indicate that PS-MPs exposure potentiates CD pathogenicity and antibiotic resistance by activating the quorum-sensing system and facilitating biofilm formation. This study provides novel evidence linking environmental pollutants to CDI epidemiology, suggesting that microplastic pollution may compound the clinical recurrence risk and therapeutic challenges associated with CDI.IMPORTANCEIt is well established that both microplastics and C. difficile (CD) can enter the human body through the food chain, where they pose significant health risks. However, the mechanistic interactions between these two factors remain poorly understood. In this study, we provide novel insights into this interaction by demonstrating that polystyrene microplastics induce intracellular oxidative stress in CD, thereby activating quorum-sensing pathways and promoting biofilm formation. These events collectively enhance bacterial proliferation, motility, and virulence expression. More importantly, microplastic exposure substantially increases the tolerance of CD to multiple clinically relevant antibiotics, including vancomycin, an effect that is closely associated with the upregulation of key antibiotic resistance genes. Collectively, these findings reveal that environmental microplastic pollution not only serves as a physical vector for pathogen dissemination but also exacerbates the therapeutic challenges and recurrence risk associated with CD infections through direct modulation of bacterial pathogenicity and antimicrobial resistance.