Viable but non-culturable state: a strategy for Staphylococcus aureus survivable in dual-species biofilms with Pseudomonas aeruginosa

Abstract

While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Of particular interest is the ecological behaviour between P. aeruginosa-S. aureus, since microbial communities containing both pathogens can display enhanced virulence. Curiously, their interaction is often reported as competitive under laboratory conditions. Here we investigate the interaction between P. aeruginosa-S. aureus in a biofilm co-culture model. The growth of 24- and 48h-old dual-species biofilms was monitored by CFU and flow-cytometry counting, and the gene expression profile inspected by qPCR. Plating experiments showed that S. aureus growth significantly decreased from 24- up to 72-h of growth in dual-species biofilms with P. aeruginosa, suggesting a clear competitive advantage for P. aeruginosa after 24-h. The gene expression profile showed that P. aeruginosa PA14 significantly overexpressed several key virulence-related genes (pqsE, rhlR, pvdE and lasI) after 24-h of growth with S. aureus ATCC 25923, comparatively with singles-pecies biofilms, which could justify the rapid decline of S. aureus population after 24-h of co-culture with P. aeruginosa. Nevertheless, the isolated strain P. aeruginosa 362668 non-mucoid exhibited a reverse pattern by downregulated the expression of those genes during dual-species growth. As P. aeruginosa exoproducts in addition to cause S. aureus lysis or inhibition may also induce biofilm dispersion, the bulk-fluid of 48-h biofilms was analysed by culture counts and flow-cytometry. Interestingly, while no S. aureus cells were detected by CFU, flow-cytometry data revealed the presence of S. aureus in high numbers (~6 Log). The metabolic activity of S. aureus cells was confirmed by measure the expression levels of sodA gene. In conclusion, the presence of S. aureus in a viable but non-cultivable state within dual-species biofilms with P. aeruginosa underscores the impact of interspecies interactions on antibiotic efficacy in the context of polymicrobial infections.