Volatile essential oils as a non-antibiotic strategy against bacteria causing urinary tract infections in companion animals

Abstract

Urinary tract infections (UTI) in companion animals are increasingly associated with multidrug-resistant (MDR) bacteria, highlighting the need to explore alternative antimicrobial approaches. This study aimed to evaluate the in vitro antimicrobial activity of three essential oils (EO): Tea Tree - Melaleuca alternifolia (TTEO), Lavender - Lavandula angustifolia (LEO), and bergamot - Citrus bergamia (BEO) against MDR bacterial isolates obtained from urine samples of dogs and cats. A total of twelve clinical isolates, comprising Gram-positive and Gram-negative bacteria, were characterized for antimicrobial resistance profiles and biofilm-forming capacity. Antimicrobial susceptibility testing revealed widespread resistance to multiple antibiotic classes, particularly among Gram-negative isolates, including isolates exhibiting extended-spectrum β-lactamases (ESBL) phenotypes. All isolates exhibited MDR phenotypes, and the majority demonstrated strong biofilm-forming ability. The antimicrobial activity of EO was assessed using an adapted disk diffusion method and two volatile exposure assays (M1 and M2). TTEO demonstrated the highest antimicrobial activity across all assays, producing significantly larger inhibition zones compared with LEO and bergamot EO (BEO) ( p < 0.001). LEO exhibited high activity, particularly against Gram-positive isolates, while BEO showed limited and inconsistent effects. Although direct contact assays resulted in greater inhibition than volatile exposure, TTEO and LEO maintained measurable antimicrobial activity in the vapour phase. No significant differences were observed between the two volatile methods for most comparisons. Minimum inhibitory concentrations were determined using serial two-fold dilutions starting at 1%. Gram-positive bacteria showed generally higher inhibition, particularly to TTEO and LEO, whereas Gram-negative isolates exhibited higher inhibition to TTEO compared with the other EO tested. Dose - response analysis indicated lower half-maximal Inhibitory Concentration (IC₅₀) values for TTEO in Gram-negative isolates whereas LEO showed the lowest IC₅₀ for Gram-positive isolates. Notably, TTEO and LEO showed strong inhibition at higher concentrations, followed by a marked decrease with increasing dilutions, while BEO displayed a more uniform but weaker inhibitory profile. These findings suggest that selected EO, particularly TTEO, exhibit promising in vitro antimicrobial activity against MDR uropathogens of veterinary origin. However, given the exploratory nature of this study and its in vitro design, further research is required to evaluate their safety, pharmacokinetics, and clinical applicability in companion animals.