Contribution of viable but non culturable cells and small colony variants in antibiotic insusceptibility and therapeutic failure against S. aureus and P. aeruginosa biofilm co-infections

aeruginosa and S. aureus are often co-isolated from biofilm-associated infections, such as those afflicting cystic fibrosis (CF) patients. Biofilms, along with the interspecies interactions, play a significant role in fostering antibiotic insusceptibility, contributing to infection chronicity. Previously, we showed that S. aureus adopts a viable but non-culturable (VBNC) state in biofilms with P. aeruginosa . Here, we aimed to gain insight into the impact of VBNC and phenomena such as phenotypic switching on antimicrobial treatment and vice-versa. Single- and dual-species biofilms of two isolates from each species were characterised in terms of viability, culturability, clonal diversification, and pathogenic potential, upon treatment with ciprofloxacin and vancomycin. Data show that S. aureus became less susceptible to antibiotics in its VBNC state induced by P. aeruginosa and by the treatments. P. aeruginosa ’s susceptibility to ciprofloxacin diminished in dual-species biofilms, suggesting mutual benefits. Following treatment, S. aureus persisted as VBNC in the dual-species biofilm and its tolerance to ciprofloxacin endured after planktonic regrowth. P. aeruginosa triggered S. aureus ’s small colony variants (SCV), but P. aeruginosa’s rugose SCV probably explains S. aureus ’s protection due to enhanced biofilm formation. This work sheds light on P. aeruginosa and S. aureus ’ co-increased tolerance to antibiotics, with cooperative interactions, phenotypic diversification, and VBNC underpinning this and the persistence of S. aureus within P. aeruginosa biofilms. This work is the first relating S. aureus ’s decreased susceptibility in dual-species biofilms to its VBNC state. Findings highlight the importance of microbial ecology, viability and colony morphotyping studies when designing treatments for multispecies infections. • S. aureus loses cultivability but increases its prevalence after treatment. • Bidirectional protection of both species was observed during biofilm growth. • S. aureus changes to VBNC because of P. aeruginosa and of antibiotic treatment. • S. aureus CIP insusceptibility remains after biofilm disruption. • VBC selection may explain antibiotic failure and synergic relationship.