Otitis externa is common infection in dogs. Its most important aetiologic agent is Pseudomonas aeruginosa, which, besides its ability to form biofilm, can also be resistant to several antibiotics, leading to therapeutic failures in several cases. Promising therapeutic alternatives are constantly being sought to treat chronic, recurrent infections. Within the framework of this study, we investigated the antibacterial potential of essential oils against canine isolates of P. aeruginosa. Forty P. aeruginosa were isolated from individual dogs’ otitis externa. They were identified by MALDI-TOF MS, their antibiotic susceptibility was determined with disk diffusion, and their genetic relatedness was established using pulsed-field gel electrophoresis. Fifty-seven essential oils were screened for their antipseudomonas effects using the drop plate method, and six essential oils—cinnamon (bark and leaf), bay, rosemary, thyme, and bitter orange—were further tested for their mature-biofilm-degrading capacities at 30 °C and 37 °C. Even though the molecular relatedness of the forty P. aeruginosa isolates could be excluded, all isolates’ growth was inhibited and their biofilms degraded by a 2.5% concentration of cinnamon (bark and leaf), bay, rosemary, thyme, or bitter orange essential oil. Our results show that essential oils are potent alternatives in the treatment of otitis externa.

Background: Canine otitis externa is a clinically relevant niche where Pseudomonas aeruginosa may persist under inflammatory, antimicrobial, and biofilm-favoring conditions. This study characterized the virulome and population structure of canine otitis externa-derived P. aeruginosa isolates from Hungary. Methods: Of 110 isolates previously subjected to minimum inhibitory concentration testing, 70 phenotypically selected isolates underwent long-read whole-genome sequencing and were included in virulence-factor analysis. Virulence-associated genes were detected using the Virulence Factor Database with an 80% identity and 80% coverage threshold and were interpreted by functional systems, including motility, adhesion, biofilm formation, secretion systems, siderophores, quorum sensing, secreted enzymes, and type III secretion system effectors. Results: The isolates carried a broad and highly conserved virulence-associated backbone. Genes related to alginate production, type IV pili, flagellar motility, pyochelin, phenazine biosynthesis, Xcp secretion, type VI secretion, and proteolytic enzymes were widely distributed. Type III secretion effector genes were variable: exoT was detected in 68/70 isolates, exoY in 63/70, exoS in 50/70, and exoU in 15/70. MLST indicated a genetically diverse population, and virulome size showed no strong correlation with the modified multiple antimicrobial resistance index. Conclusions: These findings support integrated genomic surveillance of virulence and antimicrobial resistance in canine otitis-associated P. aeruginosa.

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.

Background/Objectives: Otitis externa is a common infection in dogs. The most important aetiological agent is Pseudomonas aeruginosa, and an increasing number of cases are proving difficult to treat due to this pathogen’s antibiotic resistance. Methods: We determined the composition of cinnamon (Cinnamomum verum J. Presl), petitgrain (Citrus x aurantium L.), rose geranium (Pelargonium graveolens L’Hér), and thyme (Thymus vulgaris L.) essential oils (EOs) using gas chromatography-mass spectrometry (GC-MS). We then used the bioautographic method to identify antibacterial components. Two-and-a-half- and five percent concentrations of the essential oils were added to four different ointment bases, such as hydrogelum methylcellulose, unguentum oleosum, unguentum stearini, and vaselinum cholesterinatum, and the in vitro antibacterial effects were tested on lawns of six P. aeruginosa isolates using the antibiotic-containing product Marbogen complex as a control. The keepability of the methylcellulose-based ointments was monitored at 4 °C and 27 °C for 12 months, with efficacy losses evaluated monthly. Results: Hydrogel methylcellulose (HM) was the most effective at supporting the therapeutic efficacy of each oil, reducing the bacterial count on the exposed area by 99.999%, 100%, 100%, and 100% in the case of 5% cinnamon, petitgrain, rose geranium, and thyme, respectively. EO-based HM ointments take effect within 15 min, with only slight improvement after 30, 45, and 60 min. Cinnamon was the most effective when applied in the HM matrix. In contrast to the HM and US gel matrices, the antibacterial supportive effects of Unguentum oleosum and Vazelinum cholestrinatum were drastically lower. The antimicrobial efficacy of essential oils in hydrogel formulation varied significantly depending on the type of oil (p < 0.001), its concentration (p < 0.001), the exposure time (p < 0.001), and the P. aeruginosa isolate used (p < 0.001). Cinnamon oil demonstrated the highest efficacy, achieving near-complete bacterial elimination across all isolates within 15 min. There was no significant difference between the 2.5% and 5% concentrations (EMM: 0.016 vs. 0.004; p = 0.904). Conclusions: Our results demonstrate that essential oil-based products can be effective alternatives to antibiotics when used in conjunction with an effective gel matrix.

Background: Canine otitis externa caused by Pseudomonas aeruginosa is frequently chronic and treatment refractory, yet integrated historical phenotype–genotype data remain limited. We characterized an archival Hungarian collection obtained in 2010 and 2017. Methods: Broth microdilution minimum inhibitory concentrations (MICs) were determined for 67 isolates against 13 antimicrobial agents and chlorhexidine, and 59 isolates had matched long-read whole-genome assemblies. Resistance-associated determinants were identified using the Comprehensive Antibiotic Resistance Database, and their predicted proximity to mobile genetic elements and plasmid-like contigs was evaluated. Results: Current canine Clinical and Laboratory Standards Institute breakpoints classified 59/67 isolates (88.1%) as enrofloxacin resistant and 43/67 (64.2%) as marbofloxacin resistant. European Committee on Antimicrobial Susceptibility Testing epidemiological cut-off values identified non-wild-type subsets for ciprofloxacin (7/67), tobramycin (4/67), imipenem (1/67), and piperacillin–tazobactam (51/67), whereas all isolates remained within the available wild-type distributions for ceftazidime, gentamicin, and amikacin. No continuous MIC distribution changed significantly between sampling years after false-discovery-rate correction, although categorical marbofloxacin resistance decreased from 87.5% in 2010 to 42.9% in 2017. Resistance-gene screening identified 64 unique determinants, including near-universal efflux-system components, PDC and OXA-50-like β-lactamases, APH(3′)-IIb, arnA, and basS. The fluoroquinolone-associated determinant crpP occurred in 37/59 genomes and was located on plasmid-predicted contigs in six isolates. No determinant–MIC association remained significant after correction for multiple testing. Conclusions: The discordance between conserved resistance-gene repertoires and heterogeneous MICs demonstrates that sequence-based detection alone is insufficient for therapeutic inference. Integrated phenotypic and genomic surveillance is therefore required to support evidence-based antimicrobial stewardship in canine pseudomonal otitis.