Antibiofilm mechanism of pyrrole-2-carboxylic acid by impairing flagellar motility of Listeria monocytogenes and its application in cheese

Listeria monocytogenes is a foodborne pathogen that forms biofilms on various surfaces in food processing environments, leading to significant safety issues. Pyrrole-2-carboxylic acid (PCA), a natural organic acid produced by a Bacillus cereus strain, has been reported to exhibit prominent antibacterial and antibiofilm activities against L. monocytogenes in our previous study. This study aimed to explore the underlying antibiofilm mechanism of PCA at the gene level, and assess its efficacy in controlling L. monocytogenes in cheese. The results showed that PCA specifically inhibited the early stage of biofilm development in L. monocytogenes , and remarkably reduced the cell surface hydrophobicity and auto-aggregation. Transcriptomic analysis revealed that PCA significantly altered the gene expression levels of L. monocytogenes , with 159 up-regulated and 360 down-regulated genes. The most representative dysregulated genes were enriched in flagellar assembly and bacterial chemotaxis pathways, which are associated with cell motility. Meanwhile, the inhibitory effects of PCA on the swimming and swarming motility of L. monocytogenes were verified. Transmission electron microscopic observation confirmed the impaired flagellar assembly of L. monocytogenes following PCA treatment. Additionally, PCA effectively inhibited the attachment of L. monocytogenes on eggshells and suppressed its proliferation in cheese without affecting the color and texture. These findings suggest that PCA inhibits L. monocytogenes biofilm formation by interfering with flagella-mediated motility and is a potential natural antimicrobial agent applied in the food industry.