Ciprofloxacin resistance enhances biofilm formation and modulates virulence in Acinetobacter baumannii: Insights into the role of efflux pumps and quorum sensing.

Acinetobacter baumannii is a critical priority pathogen due to its capacity to develop resistance to last-resort antibiotics and to form persistent biofilms. Both contribute to treatment failure. This study investigated co-regulatory mechanisms of ciprofloxacin resistance and biofilm formation in A. baumannii ATCC 19606. Stepwise antibiotic exposure yielded a ciprofloxacin-resistant isogenic strain (CipR), with a minimum inhibitory concentration (MIC) of 128 µg/mL. CipR exhibited significantly enhanced biofilm formation compared to the susceptible strain (P ≤ 0.0001), with a strong positive correlation (Rs = 0.9818) between MIC values and biofilm biomass. Minimum biofilm eradication concentrations (MBEC) increased markedly in the CipR strain. Whole-genome sequencing identified CipR-mutations in gyrB (DNA gyrase subunit B), a multidrug efflux transporter, and a hypothetical protein. Transcriptomic analyses revealed overexpression of the quorum sensing (QS) system AbaI/AbaR in CipR biofilm cells. Inhibition of QS with sub-inhibitory streptomycin concentrations reduced biofilm formation without altering ciprofloxacin MIC, suggesting the existence of other co-regulatory pathways. Efflux pump inhibition with CCCP did not impact either biofilm biomass or resistance levels. Ciprofloxacin resistance acquisition incurred a metabolic cost, evidenced by XTT assays and reduced bacterial growth. Resistance downregulated the expression of adhesion-related genes and diminished functional adhesion/invasion of human lung epithelial cells. Overall, the findings suggest that ciprofloxacin resistance, biofilm formation, and virulence may be co-regulated in A. baumannii, possibly through QS and other yet-to-be-identified regulatory networks. These results provide novel insights into the adaptive mechanisms of A. baumannii and highlight potential targets for therapeutic intervention.