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

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 <i>Pseudomonas aeruginosa</i> PAO1 and methicillin-sensitive <i>Staphylococcus aureus</i> (MSSA). Compared to monocultures, MSSA exhibited reduced susceptibility to cephalexin, ampicillin, and cefazolin, while <i>P. aeruginosa</i> showed reduced susceptibility to cefazolin and nafcillin within dual-species biofilms. Using a set of <i>P. aeruginosa</i> 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 Δ<i>pslA pelF</i> mutant strain increased the survival of Δ<i>pslA pelF</i> 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.