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Type VI secretion system 2 (T6SS2) of Vibrio parahaemolyticus is required for cell adhesion and autophagy in macrophages; however, other phenotypes conferred by this T6SS have not been thoroughly investigated. We deleted TssL2, a key component of T6SS2 assembly, to explore the role of the T6SS2 in environmental adaptation and virulence. TssL2 deletion reduced Hcp2 secretion, suggesting that TssL2 played an important role in activity of functional T6SS2. We found that TssL2 was necessary for cell aggregation, wrinkly phenotype formation, and participates in motility and biofilm formation by regulating related genes, suggesting that TssL2 was essential for V. parahaemolyticus to adapt changing environments. In addition, this study demonstrated TssL2 significantly affected adhesion, cytotoxicity, bacterial colonization ability, and mortality in mice, even the levels of the proinflammatory cytokines IL-6 and IL-8, suggesting that TssL2 was involved in bacterial virulence and immunity. Proteome analysis revealed that TssL2 significantly affected the expression of 163 proteins related to ABC transporter systems, flagellar assembly, biofilm formation, and multiple microbial metabolism pathways, some of which supported the effect of TssL2 on the different phenotypes of V. parahaemolyticus. Among them, the decreased expression of the T3SS1 and T2SS proteins was confirmed by the results of gene transcription, which may be the main reason for the decrease in cytotoxicity. Altogether, these findings further our understanding of T6SS2 components on environmental adaption and virulence during bacterial infection. Key points: • The role of T6SS2 in V. parahaemolyticus was far from clear. • TssL2 participates in cell aggregation, wrinkly phenotype formation, motility, and biofilm formation. • TssL2 is essential for cell bacterial colonization, cytotoxicity, virulence, and proinflammatory cytokine production. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA‐LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Methods and results: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony‐forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA‐LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross‐reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA‐LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7‐fold more sensitive than that of the conventional polymerase chain reaction method. When RPA‐LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. Conclusions: The newly developed IMS‐RPA‐LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. Significance and Impact of the Study: The newly developed IMS‐lateral flow RPA‐LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS‐RPA‐LF assay will have important implications for the rapid detection of S. aureus in contaminated food. [ABSTRACT FROM AUTHOR]