Transient sub-MIC ciprofloxacin exposure produces a stress-conditioned Pseudomonas aeruginosa phenotype that enhances NF-κB activation and inflammatory injury in lung epithelial cells
摘要
Pseudomonas aeruginosa is a major opportunistic pathogen in healthcare-associated infections, and its management is increasingly complicated by antimicrobial resistance. In clinical settings, bacteria may be exposed to sub-minimum inhibitory concentration (sub-MIC) antibiotic gradients because of pharmacokinetic variation and uneven drug distribution. Although sub-MIC exposure can alter bacterial phenotypes and host–pathogen interactions, it remains unclear whether fluoroquinolone-induced bacterial stress phenotypes persist after antibiotic removal, whether they are reversible over time, and whether residual antibiotic carryover contributes to the observed host response. This study investigated whether transient sub-MIC ciprofloxacin exposure produces a stress-conditioned P. aeruginosa phenotype that enhances epithelial inflammatory signaling and injury, and whether key findings are retained in an additional lung epithelial cell model and after exposure to another fluoroquinolone. The minimum inhibitory concentration of ciprofloxacin was determined by broth microdilution. Mid-log phase cultures were exposed for 4 h to ¼ MIC or ½ MIC ciprofloxacin, extensively washed, allowed to recover in antibiotic-free medium for defined post-wash intervals, normalized by colony-forming units, and used to infect A549 lung epithelial cells at a multiplicity of infection of 10. Key inflammatory and viability readouts were further validated in BEAS-2B bronchial epithelial cells, and a limited second-antibiotic validation was performed using levofloxacin stress-conditioning. Final wash supernatant was also tested as a carryover control. Early bacterial interaction was assessed at 2 h, NF-κB activation at 45 min by Western blotting, inflammatory gene expression at 6 h by quantitative PCR, and epithelial viability at 24 h by MTT assay. Stress-conditioned bacteria remained viable and, after normalization, showed significantly increased total epithelial association without changes in extracellular bacterial counts. Gentamicin protection analysis further indicated that this increase was driven predominantly by enhanced surface adherence, with only limited recovery of intracellular/gentamicin-protected bacteria. They induced stronger NF-κB activation, reflected by increased p-p65 and reduced IκBα, together with elevated IL-8, TNF-α, IL-6, and COX-2 transcript expression. This inflammatory amplification was further supported at the protein level by increased IL-8 secretion, while parallel 6 h CFU enumeration showed comparable cell-associated bacterial burdens among infected groups at the time of RNA harvest. Targeted bacterial qPCR further showed a dose-dependent induction of SOS-response and surface-interaction-associated genes, including recA, lexA, fliC, pilA, and algD, with stronger responses after ½ MIC exposure than ¼ MIC exposure. The enhanced IL-8-inducing phenotype was greatest immediately after antibiotic removal and declined during antibiotic-free recovery, whereas final wash supernatant did not induce IL-8. In addition, comparable 6 h cell-associated CFU across infected groups indicated that the amplified cytokine response was not simply explained by higher bacterial burden at RNA harvest. Functionally, ciprofloxacin stress-conditioned bacteria caused a graded reduction in epithelial viability, whereas ciprofloxacin alone showed no direct epithelial effect. The graded IL-8 induction and viability loss were also observed in BEAS-2B cells, supporting that the phenotype was not restricted to the A549 model. In addition, levofloxacin stress-conditioning reproduced similar IL-8 and viability patterns in A549 cells, suggesting that the response was not strictly ciprofloxacin-specific within the fluoroquinolone exposure context. Transient sub-MIC ciprofloxacin exposure therefore generates a reversible, post-wash stress-conditioned P. aeruginosa phenotype that enhances epithelial surface adherence, amplifies inflammatory transcript and IL-8 protein responses, and worsens epithelial injury after antibiotic removal, independent of detectable ciprofloxacin carryover or increased bacterial burden at the 6 h inflammatory readout. The stronger ½ MIC response was supported by a graded bacterial stress-response and surface-interaction gene-expression signature, while BEAS-2B and levofloxacin validation experiments strengthened the relevance of the phenotype beyond a single epithelial model and a single fluoroquinolone.