Characterization of β-lactam resistance in dual-species biofilms of Pseudomonas aeruginosa and methicillin-sensitive Staphylococcus aureus

Polymicrobial biofilms are frequently associated with chronic infections and are highly tolerant to antibiotic treatment. Given that β-lactam antibiotics are the most prescribed antibiotics worldwide, we investigated β-lactam resistance in dual-species biofilms formed by Pseudomonas aeruginosa PAO1 and methicillin-sensitive Staphylococcus aureus (MSSA). Compared to monocultures, MSSA exhibited reduced susceptibility to cephalexin, ampicillin, and cefazolin, while P. aeruginosa showed reduced susceptibility to cefazolin and nafcillin within dual-species biofilms. Using a set of P. aeruginosa mutants, we demonstrate that the PAO1 AmpC β-lactamase, the exopolysaccharides Psl and Pel, and the quorum sensing regulators LasR and RhlR each play significant roles in protecting MSSA from β-lactam treatment. Interestingly, co-cultures of MSSA with the ΔpslA pelF mutant strain increased the survival of ΔpslA pelF under β-lactam exposure. Overall, these findings advance our understanding of how interdependent bacterial interactions compensate for the loss of matrix components and thereby contribute to antimicrobial tolerance in polymicrobial biofilms.