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Ciprofloxacin metabolites drive resistance development and biofilm changes in Pseudomonas aeruginosa and

Mairéad Gallagher1, Lyuboslava G Harkova2, Dominika Krawiel2

  • 1Centre for AMR and One Health Research, TU Dublin Tallaght Campus, Dublin, Ireland.

Journal of Global Antimicrobial Resistance
|July 29, 2026
PubMed
Abstract

Insights

Ciprofloxacin metabolites, 2-oxo ciprofloxacin (M3) and N-formyl ciprofloxacin (M4), drive bacterial adaptation. These metabolites promote antibiotic resistance and biofilm formation in bacteria, highlighting their clinical significance in antimicrobial resistance.

Area of Science:

  • Microbiology
  • Pharmacology
  • Genomics

Background:

  • Ciprofloxacin is a widely used antibiotic.
  • Antibiotic metabolism can lead to the formation of active or inactive compounds.
  • The impact of ciprofloxacin metabolites on bacterial adaptation remains largely unexplored.

Purpose of the Study:

  • To investigate the influence of two major ciprofloxacin metabolites, 2-oxo ciprofloxacin (M3) and N-formyl ciprofloxacin (M4), on the resistance profiles and biofilm characteristics of *Pseudomonas aeruginosa* and *Staphylococcus aureus*.
  • To elucidate the molecular mechanisms underlying these adaptive responses.

Main Methods:

  • Synthesis and structural validation of ciprofloxacin metabolites M3 and M4.
  • Co-culture of bacterial strains (*P. aeruginosa* PAO1 and *S. aureus* ATCC 25923) with metabolites at sub-inhibitory concentrations (SICs) for 30 days.
  • Phenotypic analysis using MIC assays and crystal violet biofilm quantification.
  • Molecular analysis using whole genome and differential RNA sequencing.

Main Results:

  • Exposure to M3 and M4 induced significant increases in ciprofloxacin Minimum Inhibitory Concentration (MIC) values in *S. aureus*.
  • Prolonged exposure to M3 and M4 enhanced biofilm formation in *P. aeruginosa*.
  • Genomic analysis of *P. aeruginosa* revealed a mutation in the *wspA* gene associated with M3 exposure, activating biofilm-promoting pathways.
  • Transcriptomic analysis identified differential expression of genes involved in quorum sensing, nitric oxide metabolism, and biofilm formation.

Conclusions:

  • Ciprofloxacin metabolites possess the capacity to induce adaptive responses in bacteria, contributing to antimicrobial resistance (AMR) and persistence.
  • These metabolites can drive stable genetic and transcriptomic shifts, underscoring their potential ecological and clinical significance.
  • The study highlights the importance of considering antibiotic metabolites as factors in AMR development.
Keywords:
AMRAntibiotic metabolitesBiofilmsCiprofloxacinPseudomonas aeruginosaStaphylococcus aureus

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