This study investigated how two major ciprofloxacin metabolites — 2-oxo ciprofloxacin (M3) and N-formyl ciprofloxacin (M4) — affect resistance development and biofilm characteristics in *P. aeruginosa* (PAO1) and *S. aureus* (ATCC 25923) after 30 days of subinhibitory co-culture exposure.
In *S. aureus*, M3 and M4 caused 8-fold and 4-fold increases in ciprofloxacin MIC, respectively. In *P. aeruginosa*, MICs were unchanged but both metabolites significantly enhanced biofilm formation (M4: P<0.0001; M3: P=0.0089); M3 exposure also selected a missense mutation (A290D) in *wspA*, activating cyclic-di-GMP biofilm pathways, and RNA-seq revealed 220 differentially expressed genes including upregulation of *rhlI*, *pqsH*, *nir*, *norCB*, and the *pel* operon.
- Only two reference strains (PAO1, ATCC 25923) were used — clinical isolate diversity is not represented. - The ecological relevance of metabolite concentrations used in vitro (subinhibitory, 30-day exposure) to real-world human or environmental exposures is not established. - The study is largely mechanistic and does not include in vivo validation of resistance or persistence outcomes.
Ciprofloxacin metabolites excreted after antibiotic use may independently drive resistance and biofilm adaptation in key pathogens, even at concentrations too low to kill bacteria. Clinicians and stewardship programs should consider the broader ecological footprint of fluoroquinolone metabolites, particularly in environments with chronic or recurrent ciprofloxacin exposure.
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