Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that causes listeriosis in humans and other animals. Surface proteins with the LPXTG motif have important roles in the virulence of L. monocytogenes. Lmo0159 is one such protein, but little is known about its role in L. monocytogenes virulence, motility, and biofilm formation. Here, we constructed and characterized a deletion mutant of lmo0159 (∆lmo0159). We analyzed not only the capacity of biofilm formation, motility, attachment, and intracellular growth in different cell types but also LD₅₀; bacterial load in mice’s liver, spleen, and brain; expression of virulence genes; and survival time of mice after challenge. The results showed that the cross-linking density of the biofilm of ∆lmo0159 strain was lower than that of WT by microscopic examination. The expression of biofilm-formation and virulence genes also decreased in the biofilm state. Subsequently, the growth and motility of ∆lmo0159 in the culture medium were enhanced. Conversely, the growth and motility of L. monocytogenes were attenuated by ∆lmo0159 at both the cellular and mouse levels. At the cellular level, ∆lmo0159 reduced plaque size; accelerated scratch healing; and attenuated the efficiency of adhesion, invasion, and intracellular proliferation in swine intestinal epithelial cells (SIEC), RAW264.7, mouse-brain microvascular endothelial cells (mBMEC), and human-brain microvascular endothelial cells (hCMEC/D3). The expression of virulence genes was also inhibited. At the mouse level, the LD₅₀ of the ∆lmo0159 strain was 10^0.97 times higher than that of the WT strain. The bacterial load of the ∆lmo0159 strain in the liver and spleen was lower than that of the WT strain. In a mouse model of intraperitoneal infection, the deletion of the lmo0159 gene significantly prolonged the survival time of the mice, suggesting that the lmo0159 deletion mutant also exhibited reduced virulence. Thus, our study identified lmo0159 as a novel virulence factor among L. monocytogenes LPXTG proteins.

To explore the role of the membrane permease ⅡB (EⅡB) gene of Listeria pathogenicity island 4 (LIPI-4) in the virulence of Listeria monocytogenes, both an EⅡB deletion strain (∆EⅡB) and a complemented strain were constructed. In vitro experiments demonstrated that EⅡB deletion affected the biofilm formation ability of the wild-type strain (Lm928). Moreover, this deletion decreased the intracellular proliferation abilities of L. monocytogenes. Mice infected with ∆EⅡB survived longer and experienced less weight loss on days 1, 2, and 3 post-infection. The bacterial load in the liver tissue of ∆EⅡB-infected mice was significantly reduced, and a considerable decrease in the blood levels of inflammatory cytokines IL-β, IL-6, IL-10, and TNF-α were observed. Following EⅡB deletion, 65% (13/20) of genes were downregulated, 25% (5/20) were upregulated, and 10% (2/20) showed no change. These findings suggest that EⅡB deletion may reduce both the in vivo and in vitro virulence levels as well as the biofilm formation ability of Lm928 by downregulating the transcription levels of genes associated with virulence and biofilm formation. These findings provide a foundation for further examining the pathogenic mechanisms of LIPI-4 and EⅡB in L. monocytogenes.

Bovine-associated Klebsiella pneumoniae is an important bacterial species linking animal health, microbial ecology, and One Health-oriented antimicrobial resistance research. In this study, we performed a global genomic analysis of 1291 publicly available bovine-associated K. pneumoniae genomes collected from 18 countries between 2005 and 2024 using data retrieved from NCBI. MLST, core-genome phylogenetic analysis, pangenome analysis, CARD, VFDB, and PlasmidFinder were used to characterize sequence types, genomic diversity, antimicrobial resistance-associated genes, virulence-associated genes, and plasmid replicons. A total of 256 sequence types were identified, among which ST107 was the most common. Core-genome phylogenetic analysis revealed multiple genomic lineages, while pangenome analysis identified 46,325 gene clusters, including 1967 core genes and 40,595 cloud genes, indicating an open pangenome structure and substantial accessory gene diversity. Virulence-associated genes were unevenly distributed, with yagZ/ecpA being the most frequently detected determinant. In total, 138 antimicrobial resistance-associated genes or potential resistance determinants were detected across 16 antimicrobial categories, including clinically important β-lactamase- and carbapenemase-associated genes. IncF-family plasmid replicons, particularly IncFIB(K)_1_Kpn3, were frequently detected, suggesting widespread plasmid replicon-associated genomic backgrounds; however, physical co-localization between resistance genes and specific plasmid backbones could not be confirmed. Overall, this study reveals the genetic diversity, resistance-associated gene reservoir potential, heterogeneity of virulence-associated genes, and plasmid replicon backgrounds of bovine-associated K. pneumoniae. Importantly, the genome-predicted AMR potential identified in this study should not be interpreted as confirmed phenotypic resistance without further experimental validation. These findings provide genomic insights for risk surveillance, candidate control-target screening, and microbiota-oriented intervention research.