Synergistic biofilm formation by the coexistence of nontypeable Haemophilus influenzae and Moraxella catarrhalis reduces amoxicillin efficacy

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are recognized as the key pathogens that cause respiratory tract infections, and both are capable of forming biofilms. Although interspecies biofilm formation has been described, the impact of co-culture conditions on antimicrobial responsiveness remains incompletely defined. In this study, we examined biofilm formation by co-culturing NTHi and M. catarrhalis and evaluated their antimicrobial responsiveness to amoxicillin and alternative clinically relevant antibiotics, including sequential exposure following initial amoxicillin treatment, in an in vitro model. RESULTS: Biofilm formation was quantitatively evaluated using the crystal violet staining method with a 96-well pin replicator. Twenty clinical isolates of M. catarrhalis were first cultured for 24 h to assess their biofilm-forming capacities, which varied considerably among strains, with some producing robust biofilms. To further examine interspecies interactions, M. catarrhalis and NTHi were co-cultured at different CFU ratios, revealing that increasing the proportion of M. catarrhalis in the inoculum enhanced overall biofilm production compared with either species alone. Additionally, exposure to selected antibiotics showed that respiratory quinolones were associated with greater reductions in biofilm biomass and viable bacteria within co-culture-derived biofilms compared with amoxicillin, both during direct treatment and following prior amoxicillin exposure, under the experimental conditions tested. CONCLUSION: Co-culture of NTHi and M. catarrhalis was shown to significantly enhance biofilm formation compared with either culture species alone. In particular, a higher proportion of M. catarrhalis was associated with increased biofilm production. These findings suggest that polymicrobial coexistence may contribute to reduced antimicrobial responsiveness through enhanced biofilm formation.