In the flow-cell assay, as shown in Figure 6A, the Δagr ΔluxS strain formed stronger biofilms than RN6911, as shown by CLSM, indicating that mutation of luxS indeed influences biofilm formation and that the two systems seem to play a cumulative effect. Moreover, similar results were obtained in the microtitre plate assay and the anaerobic jar assay under anaerobic conditions (Figure 6B and D). Figure 6 Additive
effect played by the LuxS/AI-2 QS system and the agr -mediated QS system. (A) The ΔagrΔluxSG and RN6911G grew biofilms in the flow cell, and the representative images were measured by CLSM at the 3rd and 5th day of biofilm formation. Strains are indicated in the figure. (B) Overnight cultures of WT (RN6390B), Δagr (RN6911), ΔluxS and Δagr ΔluxS were inoculated in 24-well plate and formed biofilms under anaerobic conditions. (C) WT, Δagr, ΔluxS and Δagr ΔluxS formed 5 days biofilms in a flow cell on the upper
Angiogenesis inhibitor surface of the coverslips, which were cut and examined by scanning electron microscopy. (D) The anaerobic jar was used for monitoring the biofilm formation of the WT, Δagr, ΔluxS and Δagr ΔluxS, OD560 was measured after crystal violet staining. To accurately describe the distinct biofilm formation resulting from luxS deletion, SEM was used for evaluating the structure and surface appearance of the mature biofilm. Therefore, the coverslips of the flow-cell chamber on which 5 days biofilms of WT and the ΔluxS strain grew were cut out. SEM GF120918 molecular weight analysis showed that the ΔluxS strain produced a compact GDC-0449 cost Selleck Ibrutinib biofilm structure with increased coverage than that of the WT strain (Figure 6C). On closer inspection, we found that the ΔluxS strain displayed stronger intercellular adhesion and this was also reflected in the Δagr ΔluxS strain. The Δagr ΔluxS strain showed stronger intercellular
adhesion ability than RN6911 (Figure 6C), indicating a possible result of elevated expression of PIA. Interestingly, microscopic analysis of the biofilm structure revealed that the agr mutation led to biofilms that adopted a “”ridged”" appearance with many channels, rather than the relatively smooth, confluent layer normally detected in the WT and ΔluxS strains, presumably because the thicker biofilms with a dense compact structure restrict the growth of bacteria inside. Based on these results, we speculate that the LuxS/AI-2 QS system and the agr-mediated QS system play a cumulative effect on the regulation of biofilm formation in S. aureus. It has been reported that induction of the agr system in established S. aureus biofilms detaches cells in an ica-independent manner and they also demonstrate that the dispersal mechanism requires extracellular protease activity [60]. Therefore, it seems that the influences of the LuxS/AI-2 QS system and the agr-mediated QS system on biofilm formation are through different pathways in S. aureus.