For example, synthetic AI-2 directly stimulates Escherichia coli biofilm formation and controls biofilm architecture by stimulating bacterial motility [31]. Subsequently, several studies also indicated that AI-2 indeed controls biofilm formation [32–34]. In contrast,

some researchers reported that addition of AI-2 failed to restore biofilm phenotype of the parental Temozolomide solubility dmso strain [35–40], owing to the central metabolic effect of LuxS or difficulty in complementation of AI-2 Vadimezan concentration [41]. There exists a conserved luxS gene in S. aureus, and it has been proved to be functional for generating AI-2 [42]. Previous work indicated that AI-2-mediated QS modulated capsular polysaccharide synthesis and virulence in S. aureus[43], deletion of the luxS gene led to increased biofilm formation in Staphylococcus epidermis[20], and biofilm enhancement due to luxS repression was manifested by an increase in PIA [44]. In this study, we provide evidence that S. aureus ΔluxS strain formed stronger biofilms than the WT strain RN6390B, and that the luxS mutation was complemented by adding chemically synthesized DPD, the exogenous precursor of AI-2. AI-2 activated the transcription of icaR, and subsequently Caspase Inhibitor VI concentration led to decreased icaA transcription,

as determined by real-time RT-PCR analysis. Furthermore, the differences in biofilm-forming ability of S. aureus RN6911, ΔluxS strain, and the ΔagrΔluxS strain were also investigated. Our data suggest that Carnitine palmitoyltransferase II AI-2 could inhibit biofilm formation in S. aureus RN6390B through the IcaR-dependent regulation of the ica operon. Methods Bacterial strains, plasmids and DNA manipulations The bacterial strains and plasmids used in this study are described in Table 1. E. coli cells were grown in Luria-Bertani (LB) medium (Oxoid) with appropriate antibiotics for cloning selection. S. aureus strain RN4220, a cloning intermediate, was used for propagation of plasmids prior to transformation into other S. aureus strains.

S. aureus cells were grown at 37°C in tryptic soy broth containing 0.25% dextrose (TSBg) (Difco No. 211825). In the flow cell assay, biofilm bacteria were grown in tryptic soy broth without dextrose (TSB) (Difco No. 286220). Medium was supplemented when appropriate with ampicillin (150 μg/ml), kanamycin (50 μg/ml), erythromycin (2.5 μg/ml) and chloramphenicol (15 μg/ml). Table 1 Strains and plasmids used in this study Strain or plasmid Description Reference or source RN6390B Standard laboratory strain NARSAa RN4220 8325-4 r- NARSA ΔluxS RN6390B luxS::ermB This study RN6911 RN6390B derivative; agr locus replaced with tetM cassette NARSA ΔagrΔluxS RN6911 luxS::ermB, agr/luxS double mutant This study ΔluxSpluxS Complemented strain of ΔluxS; Apr Cmr This study RN6390BG RN6390B/pgfp This study ΔluxSG ΔluxS/pgfp This study RN6911G RN6911/pgfp This study ΔagrΔluxSG ΔagrΔluxS/pgfp This study NCTC8325 Standard Laboratory strain NARSA NCTC8325ΔluxS NCTC8325 luxS::ermB 60 E.