Listeria monocytogenes is a globally recognized foodborne pathogen. The ST87 L. monocytogenes strains have become the most prevalent clinical isolates in China and are closely associated with perinatal listeriosis. This study aims to investigate the essential roles of key virulence factors in the L. monocytogenes ST87 strain during placental infection, focusing on the clinically isolated epidemic ST87 strain from China and constructing gene deletion mutants of inlA, hly, inlP, and actA. The roles of these genes in placental infection were systematically investigated using 2D and 3D trophoblast cell models (HTR‑8/SVneo, JEG‑3), non‑pregnant and pregnant ICR mice, and transcriptomic analysis. The results showed that the Δhly mutant strain completely lost its virulence in mice models but showed a tendency of enhanced virulence in cell models; the ΔactA strain mutant exhibited reduced virulence in both animal models and placental cell models; the ΔinlA mutant strain displayed no significant change in virulence in animal models, yet its adhesion and invasion abilities decreased in cell models; the ΔinlP mutant strain only reduced the bacterial load in fetuses. By combining transcriptomic analysis with virulence phenotype analysis, it was found that the placental infection by ST87 L. monocytogenes depends on the synergistic effect of hly and actA, while inlA and inlP play auxiliary roles in the adhesion process. In addition, transcriptomic analysis identified potential genes associated with key virulence determinants of ST87 L. monocytogenes, including lytic polysaccharide monooxygenase, a MucBP domain-containing protein, and the alcohol dehydrogenase PduQ. This study provides a theoretical basis for elucidating the mechanism of gestational infection by the ST87 strain of L. monocytogenes and formulating intervention strategies.

L. monocytogenes is an important foodborne pathogen that first infects the intestinal epithelium. This study used human colon adenocarcinoma cells (Caco-2) to construct 2D monolayer models and 3D spheroid models. The results showed that Caco-2 cells could form stable 3D spheroids in low-adhesion culture dishes. Compared with the 2D model, the invasion efficiency of each strain in the 3D model was significantly reduced, tight junction protein ZO-1 remained intact, and excessive ROS bursts were not induced during infection. In both 2D and 3D models, ST87 and ST5 exhibited higher virulence levels, while ST9 and ST121 showed lower virulence levels. Adhesion and invasion rates of serogroup II strains were significantly higher than those of serogroups IIa and IIc, and lineage II strains were more virulent than lineage I strains. In the 2D model, clinical strains displayed higher virulence levels than food strains, whereas in the 3D model, no significant differences were observed between these two sources. Transcriptomic analysis showed that after L. monocytogenes infection, highly virulent strains were more likely to activate host cell feedback regulatory pathways; the 2D model mainly induced transcriptional suppression in host cells and activated related pathways including cell adhesion, glycosylation modification, and morphological remodeling; while the 3D model better preserved host transcriptome homeostasis, induced host defense mechanisms including oxidative stress regulation, protein folding, and DNA damage repair during the infection process. Overall, this study demonstrates that the 3D Caco-2 cell model has practical value for evaluating the virulence heterogeneity of L. monocytogenes and the corresponding host response in vitro.

Listeria monocytogenes is a significant foodborne pathogen posing substantial risks to specific populations. This study investigated the contamination status and sequence types (ST) distribution of L. monocytogenes in ready-to-eat cooked meat products in the Shanghai region, and conducted quantitative risk assessments for major ST based on their growth parameters and dose-response parameters. A total of 675 ready-to-eat cooked meat product samples were collected, with 33 positive samples detected. ST8, ST5, ST87, ST9, and ST121 exhibited high detection rates, representing the distribution of moderate-, low-, and high-virulence strains. Risk assessment results showed that consumption of cooked meat products contaminated with moderate-virulence (ST5/ST8) strains caused the highest number of illnesses. The number of cases per million population increased with age, with more female patients aged 15–44 years old than male patients, and slightly more male patients than female patients over 44 years old. Pregnant women also faced elevated risks. Sensitivity analysis identified the initial pollution level, transportation temperature, and household storage temperature as the most significant factors affecting the final disease risk. This study provides a scientific basis for precise risk assessment and control of L. monocytogenes.