Antibiotics · Published 2026-08-05 · DOI 10.3390/antibiotics15080754
Mercédesz Adrienn Veres, Barbara Bodnár, Luca Takáts, Eszter Kaszab, Enikő Fehér, Ádám Kerek, Ákos Jerzsele
<b>Background</b>: <i>Pseudomonas aeruginosa</i> 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. <b>Methods</b>: A biofilm-producing reference <i>P. aeruginosa</i> 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. <b>Results</b>: 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 EC<sub>50</sub> 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 <i>oprN</i> was observed in a day 2 marbofloxacin–Tris-EDTA derivative. <b>Conclusions</b>: 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.
Abstract from DOAJ. Public domain (CC0 1.0).
Read the article at the publisher →
Veres, M., Bodnár, B., Takáts, L., et al. (2026). Combined Marbofloxacin-Geraniol-Tris-EDTA Exposure Delays Marbofloxacin Resistance Escalation in Pseudomonas aeruginosa While Early Genomic Changes Remain Limited. Antibiotics. https://doi.org/10.3390/antibiotics15080754