Most human listeriosis is foodborne, and ready-to-eat (RET) foods contaminated by Listeria monocytogenes during processing are found to be common vehicles. In this study, a total of four L. monocytogens STs (ST5, ST121, ST120, and ST2) have been identified in two RTE food plants from 2019 to 2020 in Shanghai, China. The L. monocytogenes ST5 was predominant in one RTE food processing plant, and it persists in the RTE meat processing plant with continued clone transmission. The genetic features of the four STs isolates were different. ST5 and ST121 had the three genes clpL, mdrL, and lde; however, ST120 and ST2 had two genes except for clpL. SSI-1was present in ST5, ST121, and ST120. Additionally, SSI-2 was present only in the ST121 isolates. ST120 had all six biofilm-forming associated genes (actA, prfA, lmo0673, recO, lmo2504 and luxS). The ST2 isolate had only three biofilm-forming associated genes, which were prfA, lmo0673, and recO. The four ST isolates had different biofilm formation abilities at different stages. The biofilm formation ability of ST120 was significantly higher when grown for one day. However, the biofilm formation ability of ST120 reduced significantly after growing for four days. In contrast, the biofilm formation ability of ST5 and ST121 increased significantly. These results suggested that ST5 and ST121 had stronger ability to adapt to stressful environments. Biofilms formed by all four STs grown over four days can be sanitized entirely by a disinfectant concentration of 500 mg/L. Additionally, only ST5 and ST121 biofilm cells survived in sub-lethal concentrations of chlorine-containing disinfectant. These results suggested that ST5 and ST121 were more resistant to chlorine-containing disinfectants. These results indicated that the biofilm formation ability of L. monocytogenes isolates changed at different stages. Additionally, the persistence in food processing environments might be verified by the biofilm formation, stress resistance, etc. Alternatively, these results underlined that disinfectants should be used at lethal concentrations. More attention should be paid to ST5 and ST121, and stronger surveillance should be taken to prevent and control the clonal spread of L. monocytogenes isolates in food processing plants in Shanghai.

Salmonella, a major global foodborne pathogen, is a leading cause of salmonellosis. Quantitative detection of Salmonella provides a scientific basis for establishing microbiological criteria and conducting risk assessments. The plate count method remains the primary approach for bacterial quantification, whereas the most probable number (MPN) method is commonly used for detecting low levels of bacterial contamination. However, both methods are time-consuming and labor-intensive. Validated digital polymerase chain reaction (dPCR) techniques are emerging as promising alternatives because they enable rapid, absolute quantification with high specificity and sensitivity. Herein, we developed a novel droplet dPCR (ddPCR) assay for identifying and quantifying Salmonella using invA as the target. The assay demonstrated high specificity and sensitivity, with a limit of quantification of 1.1 × 10² colony-forming units/mL in meat samples. Furthermore, the log₁₀ values obtained via ddPCR and plate counting exhibited a strong linear relationship (R² > 0.99). Mathematical modeling of growth kinetics further confirmed a high correlation between plate count and ddPCR measurements (Pearson correlation coefficient: 0.996; calculated bias factor: 0.88). Collectively, these results indicate that ddPCR is a viable alternative to the MPN method and represents a powerful tool for the quantitative risk assessment of food safety.

Listeria monocytogenes is a Gram-positive bacterial species that causes listeriosis, a major foodborne disease worldwide. The virulence factor inlA facilitates the invasion of L. monocytogenes into intestinal epithelial cells expressing E-cadherin receptors. Naturally occurring premature stop codon (PMSC) mutations in inlA have been shown to result in the production of truncated proteins associated with attenuated virulence. Moreover, different L. monocytogenes strains contain distinct inlA variants. In this study, we first characterized inlA in 546 L. monocytogenes strains isolated from various foods in Shanghai. The results showed that 36.1% (95% Confidence Interval: 32.0~40.2%) of the food isolates harbored inlA with PMSC, which was found to be associated with clonal complex (CC) types, with the highest proportions observed in CC9 and CC121. To investigate the function of inlA, we first used the dominant CC87 isolated from patients as the test strain and constructed an inlA-deleted strain via homologous recombination. Resistance tests and virulence tests showed that while inlA did not affect the resistance of L. monocytogenes, it significantly influenced cell adhesion and invasiveness. To further explore the function of inlA, we performed virulence tests on five CC-type strains carrying inlA with PMSC and their corresponding strains with intact inlA. We found that the virulence of L. monocytogenes strains carrying inlA or inlA with PMSC was associated with their CC type. Our preliminary results showed that premature termination of inlA did not significantly affect the adhesion and invasion abilities of low-virulence CC-type L. monocytogenes strains in Caco-2 cells, but substantially promoted those of high-virulence strains such as CC8 and CC7. In summary, this study preliminarily evaluated the effects of inlA integrity and PMSC mutation variation on the virulence of L. monocytogenes, providing a foundation for further research on inlA-related pathogenic mechanisms.