A FISCHER¹, M GIRARD¹, P FRANÇOIS¹, J SCHRENZEL<sup>1

1</sup>Genomic Research Laboratory, Department of Medical Specialties, Geneva University Hospitals, 1211 Geneva 14, Switzerland.

Biofilm is the most common mode of bacterial growth on medical devices and has also been reported on human tissues, e.g. during lung infection by Pseudomonas aeruginosa. Some antibiotics are inefficient against susceptible bacteria growing in biofilm, even in the presence of very high drug concentration, either because molecules are too large to penetrate the biofilm matrix or the antibiotic has higher affinity for matrix components (e.g. vancomycin and extracellular DNA, eDNA), but other mechanisms likely remain to be deciphered. Matrix is composed of proteins, glucids (PIA for Staphylococcus aureus) and eDNA. eDNA provides structuration and stability in mature biofilms and is degraded by DNase. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. We tested wt and mutant biofilm susceptibility to antibiotics using a similar protocol as the Calgary biofilm device. Involvement of rpiRc in Staphylococus aureus virulence was assessed ex vivo by internalization experiments in HEK293 cells and in vivo in a mouse model of subcutaneous catheter infection. RpiRc is a transcription factor from the pentose phosphate pathway whose product is a PIA precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB® (a commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon regulating PIA production). While MICs of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. Our results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.