Combined Marbofloxacin-Geraniol-Tris-EDTA Exposure Delays Marbofloxacin Resistance Escalation in Pseudomonas aeruginosa While Early Genomic Changes Remain Limited

Background: Pseudomonas aeruginosa combines intrinsic resistance, adaptive plasticity, and biofilm formation, making treatment failure common in veterinary and human medicine. We examined whether geraniol and Tris-ethylenediaminetetraacetic acid (Tris-EDTA) modulate marbofloxacin activity during serial exposure and whether whole-genome sequencing detects genomic correlates of the observed phenotypic changes. Methods: A biofilm-producing reference P. aeruginosa ATCC 27853 strain was serially passaged for 10 days with marbofloxacin, geraniol, Tris-EDTA, and their double and triple combinations. Minimum inhibitory concentration (MIC) trajectories and fractional inhibitory concentration indices were determined. Efflux involvement was assessed by MIC testing with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in 22 canine isolates and the reference strain. Biofilm disruption was evaluated by crystal violet and MTS assays. Whole-genome sequencing was performed for day 0 controls and selected derivatives collected on days 2, 6, and 10. Results: Marbofloxacin alone showed a progressive MIC increase from 2 to 16 µg/mL, whereas its MIC remained low in the triple combination and in the marbofloxacin–Tris-EDTA combination. No antagonism was detected. The triple combination showed the most consistent interaction profile, meeting the prespecified synergy criterion on 7 of 10 passage days, compared with 5 of 10 days for marbofloxacin–Tris-EDTA, 2 of 10 days for geraniol–Tris-EDTA, and none for marbofloxacin–geraniol. CCCP caused at most a single twofold-dilution-step decrease in MIC, not supporting a dominant efflux-driven phenotype. In MTS assays, MTS-derived relative EC50 values approximated planktonic inhibitory concentrations, whereas crystal violet results were poorly reproducible. Draft genomes remained highly conserved, and only one candidate nonsynonymous sequence difference in oprN was observed in a day 2 marbofloxacin–Tris-EDTA derivative. Conclusions: Geraniol- and especially Tris-EDTA-containing combinations constrained in vitro marbofloxacin resistance escalation. The marked phenotypic separation despite minimal genomic divergence is consistent with a possible contribution from regulatory or transcriptional adaptation, although this mechanism requires direct experimental confirmation.