US2

[US2-04] The genes in Streptococcus mutans that regulate biofilm formations of S. mutans and Staphylococcus aureus

〇Toshiki Uematsu¹, Hidenobu Senpuku¹ (1. Nihon Univ Sch Dent at Matsudo)

Keywords:バイオフィルム、クオラムセンシング、グルコシルトランスフェラーゼ

Purpose: Streptococcus mutans has a signal production mechanism called quorum sensing, which activates bacteriocin production, and extracellular gene uptake, and GtfB and GtfC production, in oral biofilm formation. Staphylococcus aureus, which is an intraoral opportunistic bacterium, is a salt-tolerant bacterium and also easily becomes a resistant bacterium to antibiotic medicine. Aspiration pneumonia and heart disease are associated with the infection of S. aureus in the oral cavity. Therefore, it is investigated whether a biofilm of S. aureus is formed by the presence of glucosyltransferases (GtfB, GtfC) that synthesize various polysaccharides of S. mutans and various molecules involved in quorum sensing (QS) in S. mutans. Material and Methods: Bacteria; Mutants to glucosyltransferase genes (gtfB, gtfC) and genes (comD, comR, comX, comY, luxS) associated with QS due to bacterial aggregation and mutants of various other genes (pknB, gbpC, sacB, SMU574, SMU833, SMU1009, SMU1013) were constructed. Biofilm formation assay; Bacteria were inoculated in tryptic soy broth with 0.25% sucrose (TSBs) with and without various concentrations of sonic extracts from various bacteria in 96-well polystyrene microtiter plates previously coated with human saliva. After incubation, the planktonic cells were removed by washing with distilled water (DW), and the adherent cells were stained with 0.25% safranin for 15 min. After washing with DW, safranin was extracted from biofilms with 70% (vol/vol) ethanol. Biofilm formation was quantified by measuring the absorbance of the extracted safranin at 492 nm. In order to observe dead and live bacteria, the biofilm was subjected to Live / Dead staining, and observed with a confocal laser scanning microscope. Results: The components from gtfB^- and gtfBC^- could not strongly induce biofilm formation in S. mutans gtfBC^-, which lacked biofilm-forming ability. Ingredients from mutants of the QS-related genes comD, comX, luxS and SMU833 involved in peptidoglycan synthesis also failed to induce biofilm formation. The components of the glucan synthetic gene mutant induced salt concentration (0.125M) -dependent biofilm formation of S. aureus. On the other hand, components of QS-related genes (comD, comX, comY, gbpC, luxS) and self-destroying autolysin-related genes (SMU574), fructan synthesis genes sacB and SMU833 mutants could not induce biofilm formation. Conclusion: Biofilm formation in S. aureus was not dependent on glucan formation by S. mutans and affected salinity and QS-controlled killing by S. mutans. Controlling salt intake rather than sugar inoculation, physical removal of oral biofilm by oral care and to block QS are important for blocking oral flora dysbiosis.