Regulatory plasticity of the rdar biofilm morphotype in clinical uropathogenic Escherichia coli and its modulation by ciprofloxacin

Red, dry and rough (rdar) biofilm formation in Escherichia coli is characterized by the coordinated production of extracellular cellulose and amyloid curli fimbriae. Although rdar biofilm formation has been extensively characterized in laboratory strains, its clinical relevance and implications for antimicrobial treatment remain poorly understood. Here, we systematically investigate rdar biofilm formation of 150 consecutively isolated E. coli strains recovered from patients with urinary tract infections and correlate it to antimicrobial resistance. Genetic analysis of rdar regulation revealed distinct nucleotide signatures within the csgD promoter region that discriminate semi-constitutive rdar expression from temperature-dependent phenotypes, highlighting regulatory plasticity among clinical isolates. Whole-genome sequencing-based phylogenetic analysis further demonstrated that rdar-positive isolates are distributed across diverse E. coli phylogroups and sequence types, indicating that rdar biofilm formation is not restricted to specific clonal lineages. Strikingly, phenotypic assays revealed that the fluoroquinolone antibiotic ciprofloxacin suppresses rdar biofilm formation and associated extracellular matrix architecture in ciprofloxacin-resistant isolates at subinhibitory concentrations, suggesting that ciprofloxacin modulates biofilm-associated pathways beyond its canonical bactericidal targets. Together, our findings establish the rdar morphotype as a clinically relevant biofilm phenotype in uropathogenic E. coli and reveal an antibiofilm activity of ciprofloxacin that is uncoupled from antibiotic resistance. These results underscore the importance of considering antibiotic-mediated modulation of biofilm behavior when interpreting treatment responses and designing strategies to combat persistent urinary tract infections.