In mixed infections, the bacterial susceptibility differs significantly compared to monocultures of bacteria, and generally the concentrations of antibiotics required for the treatment increases drastically. For S. aureus and P. aeruginosa dual species biofilms, it has been numerously reported that P. aeruginosa decreases S. aureus susceptibility to a broad range of antibiotics, including beta-lactams, glycopeptides, aminoglycosides, macrolides, while sensitizes to quinolones via secretion of various metabolites. Here we show that S. aureus also modulates the susceptibility of P. aeruginosa to antibiotics in mixed cultures. Thus, S. aureus—P. aeruginosa consortium was characterized by tenfold increase in susceptibility to ciprofloxacin and aminoglycosides compared to monocultures. The same effect could be also achieved by the addition of cell-free culture of S. aureus to P. aeruginosa biofilm. Moreover, similar increase in antibiotics efficacy could be observed following addition of S. aureus suspension to the P. aeruginosa mature biofilm, compared to P. aeruginosa monoculture, and vice versa. These findings open promising perspectives to increase the antimicrobial treatment efficacy of the wounds infected with nosocomial pathogens by the transplantation of the skin residential microflora.

16While in biofilms bacteria are embedded into an extracellular matrix which forms 17 inaccessible barrier for antimicrobials thereby drastically increasing the concentrations 18 of antibiotics required for treatment. Here we show that the susceptibility of S. aureus 19 and P. aeruginosa to antibiotics in mixed biofilms significantly differs from monoculture 20 biofilms depending on both conditions and chosen antimicrobial agents. While S. aureus 21 could completely avoid vancomycin, ampicillin and ceftriaxone by embedding into the 22 biofilm of P. aeruginosa, the very same consortium was characterized by 10-fold 23 increase in susceptibility to broad-spectrum antimicrobials like ciprofloxacin and 24 aminoglycosides compared to monocultures. These data clearly indicate that efficient 25 treatment of biofilm-associated mixed infections requires antimicrobials active against 26 both pathogens, since the interbacterial antagonism would enhance the efficacy of 27 treatment. Moreover, similar increase in antibiotics efficacy was observed when 28 P. aeruginosa suspension was added to the mature S. aureus biofilm, compared to 29 S. aureus monoculture, and vice versa. These findings open promising perspectives to 30 increase the antimicrobial treatment efficacy of the wounds infected with nosocomial 31 pathogens by the transplantation of the skin residential microflora.32 42 polymicrobial biofilms 11,12 which drastically reduce their susceptibility to both antimicrobials 43 and the immune system of the host 13,14 . Current data suggests that bacterial pathogenicity is 44 promoted during polymicrobial infections and recovery is delayed in comparison with 45 monoculture infections 15-17 . Accordingly, interspecies interactions between S. aureus and 46 P. aeruginosa within mixed biofilms attracted major attention in recent years including both in 47 vitro 15 and in vivo studies 16 . 48 P. aeruginosa is known as a common dominator in polymicrobial biofilm-associated 49 infections due to multiple mechanisms allowing its rapid adaptation to the specific conditions of 50 the host. In particular, P. aeruginosa produces multiple molecules to compete with other 51 microorganisms for space and nutrients. The main anti-staphylococcal tools of P. aeruginosa are 52 siderophores and 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), the inhibitor of the electron 53 transport chain of S. aureus. Their presence shifts S. aureus to a fermentative mode of growth, 54 eventually leading to reduced S. aureus viability 18-22 , forces increased S. aureus biofilm 55 formation 23,24 and transition of S. aureus into small-colony variants (SCVs) 25 . SCV is a well-56 characterized phenotype detected in various diseases, including cystic fibrosis and device-related 57 infections 23-26 . SCVs appear as small, smooth colonies on a culture plate and grow significantly 58 slower compared to wild type colonies. Remarkably, switch to the SCV phenotype improves the 59 survival of S. aureus under unfavorable conditions, as it exhibits increased vancomycin and...