Ciprofloxacin Metabolites Drive Resistance Development and Biofilm Changes in Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

Objectives: To investigate how two major ciprofloxacin metabolites, 2-oxo ciprofloxacin (M3) and N-formyl ciprofloxacin (M4), influence the resistance profiles and biofilm characteristics of Pseudomonas aeruginosa (PAO1) and Staphylococcus aureus (ATCC 25923). Methods: Both metabolites were synthesised, structurally validated, and co-cultured with bacterial strains at sub-inhibitory concentrations (SICs) over a 30-day period. Minimum inhibitory concentration (MIC) assays, crystal violet biofilm quantification, and genomic and transcriptomic analyses (whole genome and differential RNA sequencing) were employed to assess phenotypic and molecular adaptations. Results: Exposure to ciprofloxacin metabolites altered bacterial behaviour despite their weak intrinsic antimicrobial activity. In S. aureus, continuous exposure to M3 and M4 resulted in an eight and four-fold increase, respectively, in ciprofloxacin MIC values. In P. aeruginosa, although MIC values remained unchanged, prolonged exposure to both metabolites enhanced biofilm formation, M4 (p<0.0001) and M3 (p<0.0089)). Genomic sequencing of P. aeruginosa revealed a missense mutation (A290D) in the wspA gene following M3 exposure, which activates biofilm-promoting pathways via cyclic-di-GMP signalling. RNA sequencing identified 220 differentially expressed genes, including upregulation of quorum sensing regulators (rhlI, pqsH), nitric oxide cycle genes (nir, norCB), and the pel operon. Conclusions: Ciprofloxacin metabolites, though less potent than the parent antibiotic, can drive adaptive responses linked to resistance and persistence. Their capacity to induce stable genetic and transcriptomic shifts underscores their potential ecological and clinical significance as underexplored factors in AMR.