Comparative RNA-Seq reveals conserved and species-specific biofilm-associated pathways across clinically relevant airway bacteria

Introduction Biofilms are structured microbial communities associated with chronic airway disease and increased tolerance to antimicrobial treatments. While biofilm-associated physiology has been studied in individual species, the extent to which transcriptional responses are conserved across diverse airway pathogens remains poorly defined. Methods We performed comparative RNA sequencing across eight clinically relevant bacterial species under three conditions: planktonic growth, biofilm formation, and perturbation of established biofilms using doxycycline or a biocidal agent ( Zoono GermFree24 ). Ortholog clustering and fold-change–based prioritisation were integrated to identify conserved transcriptional responses across species. Directional conservation was quantified to assess the extent of shared regulation within orthologous gene clusters. Results Biofilm formation induced widespread but modest transcriptional changes across species, with partial conservation observed in functional categories related to metabolism, transport and stress response. However, gene-level conservation was limited, with only a subset of ortholog clusters showing consistent directional change. In contrast, transcriptional responses to perturbation were highly heterogeneous, with substantial variation in both magnitude and direction across species. While doxycycline preferentially affected information processing pathways and Zoono induced broader functional shifts, both perturbations exhibited limited cross-species conservation. Quantitative analysis confirmed that most ortholog clusters displayed intermediate or inconsistent directional agreement, with strongly conserved responses representing a minority. Conclusions These findings demonstrate that biofilm formation is associated with partially conserved functional adaptation across airway pathogens, whereas responses to antimicrobial perturbation are largely species-specific. This distinction highlights the importance of functional constraints during biofilm establishment and regulatory flexibility under stress, with implications for the development of broadly effective strategies to target biofilm-associated infections.