Clinical isolates from chronic wounds reveal strain-specific, alkyl-quinolone-independent competition in Pseudomonas aeruginosa–Staphylococcus aureus biofilms

<b>Introduction.</b> Chronic wounds are notoriously difficult to treat and are associated with decreased limb function, reduced quality of life and significant morbidity. Their recurrent nature, despite aggressive antibiotic therapy, is due in part to the presence of polymicrobial biofilms. <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> are two of the most frequently co-isolated pathogens in these infections and are known to form complex biofilms that hinder treatment.<b>Hypothesis.</b> We hypothesized that co-existence and competitive dynamics between <i>P. aeruginosa</i> and <i>S. aureus</i> in chronic wound infections are influenced by strain-specific interactions and may not rely solely on well-characterized inhibitory mechanisms such as 2-alkyl-4-quinolone (AQ) production by <i>P. aeruginosa</i> impacting on <i>S. aureus</i> fitness.<b>Aim.</b> To establish a polymicrobial chronic wound infection model and assess the contribution of AQ signalling and strain-specific interactions on co-existence.<b>Methodology.</b> We used a modified chronic wound biofilm model to co-culture matched and mismatched clinical isolate pairs of <i>P. aeruginosa</i> and <i>S. aureus</i>, collected from two different chronic wound patients. Viable bacterial counts (c.f.u.) were quantified over an 8-day period. AQ production by each <i>P. aeruginosa</i> strain was quantified using liquid chromatography-MS.<b>Results.</b> A stable culture of <i>P. aeruginosa</i> strains was achieved, but distinct behaviours between each <i>S. aureus</i> strain were seen. One matched clinical isolate pair maintained stable c.f.u. levels of both species throughout the 8-day model, indicating a compatible co-existence. In contrast, mismatched pairs showed early loss of <i>S. aureus</i> viability and the emergence of small colony variants after 4 days, not seen in matched pair growth. Interestingly, the most competitive <i>P. aeruginosa</i> strain exhibited undetectable levels of all AQs tested, indicating that its dominance was not due to AQ-mediated antagonism, as has previously been described.<b>Conclusion.</b> Our findings demonstrate that stable dual-species biofilm formation in chronic wounds is strain-dependent and that <i>P. aeruginosa</i> can impact on <i>S. aureus</i> fitness through AQ-independent mechanisms. These results highlight the importance of using clinical isolates in biofilm research and caution against generalizing findings from laboratory strains to complex clinical infections.