In the food industry, bacterial cells usually adhere to equipment surfaces, forming biofilms that may cause persistent contamination. This study aimed to identify the key genes responsible for the stronger biofilm-forming capability of the Listeria monocytogenes LMB 33426 strain compared to that of the L. monocytogenes CICC 21662 strain through comparative genomics. Additionally, the expression of genes and related metabolic pathways of LMB 33426 and CICC 21662 strains were analyzed at the transcriptional level by high-throughput sequencing technology to uncover key differentially expressed genes between planktonic and biofilm cells of those two strains. Subsequently, the key genes found to present differences that were uncovered by those genome-wide and transcriptomic analyses were used to construct gene deletion strains. The crystalline violet assay and motility assay showed that GL002291, GL002712 and lmo1438 genes were involved in the regulation of biofilm formation as well as motility. The hydrophobicity and auto-aggregation ability assay results demonstrated an association between the clpB, lmo1438, and lmo0294 genes and bacterial adhesion. However, no significant differences were found regarding this association in the GL002291 and GL002712 genes. This study elucidates some potential regulatory genes associated with biofilm formation in L. monocytogenes, and laying a theoretical foundation for future research.

Listeria monocytogenes (L. monocytogenes) is a highly concerning food-borne pathogen, and its biofilm formation has a significant impact on food safety. Small RNAs (sRNAs) play a crucial role in the biofilm formation of L. monocytogenes. We identified three sRNAs potentially associated with biofilm formation: ssrS, sRNA003, and sRNA006. Notably, sRNA003 and sRNA006 were newly discovered. This study explored the effects of three sRNAs on biofilm formation in L. monocytogenes and their regulatory mechanisms. Deletion of these sRNAs resulted in a 40% reduction in biofilm formation, affecting all stages of the biofilm-forming process. Real-time quantitative PCR (RT-qPCR) results showed significant differences in the transcription levels of key genes in the quorum sensing and chemotaxis systems at various growth phases compared to the wild-type. These sRNAs alter gene expression by affecting promoter activity and bind to the 5' untranslated region (UTR) of key genes in quorum sensing and chemotaxis systems, thus influencing mRNA stability and regulating biofilm formation at the post-transcriptional level. Additionally, we predicted and validated the binding sites between these sRNAs and their target mRNAs. This study elucidated the pathways through which sRNAs regulate biofilm formation in L. monocytogenes at the post-transcriptional level. These findings highlight the potential of sRNAs as targets for novel antibacterial strategies, contributing to the control of L. monocytogenes and the reduction of biofilm-related issues in the food industry.

This work was undertaken to study the regulatory effects of the Agr and LuxS/AI-2 quorum sensing systems on biofilm formation by Listeria monocytogenes (Lm). The agrD and luxS genes of LMB33426 were knocked out by homologous recombination, and the biofilm formation characteristics of the wild-type and mutant strains were investigated comparatively. The results showed that the biofilm formation ability of ΔagrD and ΔluxS was reduced compared with that of the wild-type strain. The hydrophobicity of ΔagrD decreased significantly, and its swimming mobility was higher than that of the wild-type strain at 37 ℃. However, no significant difference was found in drug resistance between the wild-type and mutant strains. This study provides a basis for further research on the regulatory mechanism of quorum sensing systems on biofilm formation by Lm and more broadly, for the development of novel strategies for the prevention and control of Lm infections.