Background -Multidrug-resistant pathogens present a major global challenge in antimicrobial therapy and frequently complicate otitis externa in dogs.Hypothesis/Objectives -In vitro efficacy of oregano oil, thyme oil and their main phenolic constituents against bacterial and fungal isolates associated with canine otitis externa were investigated. It was hypothesized that the main phenolic components would have greater antimicrobial activity compared to the relative essential oil.Methods and materials -Antimicrobial susceptibility testing was performed using broth microdilution with spot-plating technique to determine minimum inhibitory and bactericidal/fungicidal concentrations (MICs, MBCs and MFCs). A time-kill kinetics assay was performed to confirm the bactericidal and fungicidal activity of the oils and their phenolic constituents. One hundred bacterial and fungal isolates, including meticillin-susceptible Staphylococcus pseudintermedius (n = 10), meticillin-resistant S. pseudintermedius (n = 10), b-haemolytic Streptococcus spp. (n = 20), Pseudomonas aeruginosa (n = 20; including 10 isolates resistant to one or two antimicrobials), Proteus mirabilis (n = 20) and Malassezia pachydermatis (n = 20) from dogs with otitis externa were used.Results -Oregano oil, thyme oil, carvacrol and thymol exhibited antibacterial activity against all bacterial and fungal isolates tested. MIC 90 values ranged from 0.015 to 0.03% (146-292 lg/mL) for the Gram-positive bacteria and P. mirabilis. For P. aeruginosa and M. pachydermatis, MIC 90 values ranged from 0.09 to 0.25% (800-2,292 lg/mL).Conclusions and clinical significance -Oregano oil, thyme oil, carvacrol and thymol showed good in vitro bactericidal and fungicidal activity against 100 isolates from dogs with otitis externa, including some highly drug-resistant isolates. These essential oils and their main phenolic constituents have the potential to be further investigated in vivo for the treatment of canine otitis externa.

The emergence and global spread of antimicrobial resistance among bacterial pathogens demand alternative strategies to treat life-threatening infections. Combination drugs and repurposing of old compounds with known safety profiles that are not currently used in human medicine can address the problem of multidrug-resistant infections and promote antimicrobial stewardship in veterinary medicine. In this study, the antimicrobial activity of robenidine alone or in combination with ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) against Gram-negative bacterial pathogens, including those associated with canine otitis externa and human skin and soft tissue infection, was evaluated in vitro using microdilution susceptibility testing and the checkerboard method. Fractional inhibitory concentration indices (FICIs) and dose reduction indices (DRI) of the combinations against tested isolates were determined. Robenidine alone was bactericidal against Acinetobacter baumannii [minimum inhibitory concentrations (MIC) mode = 8 μg/ml] and Acinetobacter calcoaceticus (MIC mode = 2 μg/ml). Against Acinetobacter spp., an additivity/indifference of the combination of robenidine/EDTA (0.53 > FICIs > 1.06) and a synergistic effect of the combination of robenidine/PMBN (0.5 < FICI) were obtained. DRIs of robenidine were significantly increased in the presence of both EDTA and PMBN from 2- to 2048-fold. Robenidine exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae , and Pseudomonas aeruginosa , in the presence of sub-inhibitory concentrations of either EDTA or PMBN. Robenidine also demonstrated potent antibacterial activity against multidrug-resistant Gram-positive pathogens and all Gram-negative pathogens isolated from cases of canine otitis externa in the presence of EDTA. Robenidine did not demonstrate antibiofilm activity against Gram-positive and Gram-negative bacteria. EDTA facilitated biofilm biomass degradation for both Gram-positives and Gram-negatives. The addition of robenidine to EDTA was not associated with any change in the effect on biofilm biomass degradation. The combination of robenidine with EDTA or PMBN has potential for further exploration and pharmaceutical development, such as incorporation into topical and otic formulations for animal and human use.

Aims The antimicrobial activity of cinnamon essential oil and cinnamaldehyde against bacterial and fungal pathogens associated with canine otitis externa, as well as the effect of their combination with EDTA were investigated. Methods and Results Antimicrobial susceptibility testing was performed using the broth microdilution method while spot‐plating technique was used to determine their bactericidal activity. Time‐kill kinetics and checkerboard assays were performed to confirm the bactericidal activity and combination effects of the compounds. Cinnamon oil and cinnamaldehyde exhibited antimicrobial activity against Gram‐positive and Gram‐negative pathogens, as well as Malassezia pachydermatis. Synergistic interaction was shown when EDTA (672 μg ml−1) was combined with cinnamon oil (41 μg ml−1) and cinnamaldehyde (22 μg ml−1) against Pseudomonas aeruginosa. Cinnamaldehyde exhibited significantly stronger antimicrobial activity than cinnamon bark oil. Conclusions Cinnamon essential oil and cinnamaldehyde, either used alone or in combination with EDTA, were effective against the causative micro‐organisms of canine otitis externa. The data suggest that cinnamaldehyde could be a promising antimicrobial agent against canine otitis externa. Significance and Impact of the Study This study shows that cinnamon essential oil and cinnamaldehyde, especially the latter, could be used in combination with EDTA as novel treatment for sensitive and resistant bacterial and fungal pathogens involved in canine otitis externa.

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