In Vitro Efficacy of Ionophores and Antimicrobial Adjuvants Against Planktonic and Biofilm Producing Pathogens Associated with Canine Otitis Externa

Abstract

Antimicrobial resistance is an emerging global concern in human and veterinary medicine. Antibiotics that are critical for human health are also used in veterinary medicine, including for treatment of non-life threatening conditions such as otitis externa. Otitis externa is one of the most frequently diagnosed dermatological conditions in dogs with a prevalence of up to 20%, and it is traditionally treated with products containing antibiotics and antifungal agents. Though the use of antimicrobials can potentially contribute to the development of antimicrobial resistance, antimicrobials are an essential component to treat, manage and improve the welfare of dogs suffering from otitis externa. However, this may not represent good antimicrobial stewarship. One approach to minimising the use of critical or new antibiotics is to repurpose old drugs for new purposes. This approach has not yet been explored in the field of veterinary otology. The experiments in this thesis explored the efficacy of polyether ionophores and antimicrobial adjuvants against pathogens associated with canine otitis externa. Staphylococcus pseudintermedius, multidrug-resistant S. pseudintermedius, β-haemolytic Streptococcus spp., Pseudomonas aeruginosa, Proteus mirabilis and Malassezia pachydermatis clinical otic isolates, as well as Gram-positive and Gram-negative American Type Culture Collection (ATCC) reference strains, were used in minimal inhibitory concentration (MIC) testing, checkerboard assays and biofilm assays in vitro. Narasin and monensin are polyether ionophores conventionally used as rumen modifiers in ruminants and as anticoccidial agents in avian and mammalian production animals. Both narasin and monensin were found to be effective against Gram-positive otic and ATCC strains, including the multidrug-resistant staphylococci. Gram-positive pathogens in the planktonic state were more susceptible to narasin than monensin, whereby the MIC50 and MIC90 of narasin were 32 times lower than those of monensin. Also, narasin had anti-yeast activity against M. pachydermatis otic isolates at higher concentration. However, both ionophores lacked activity against Gram-negative pathogens which would preclude their use as sole antimicrobial agents in cases of otitis involving rods or mixed infections. The non-antibiotic mucolytic agent, N-acetylcysteine (NAC), and two metal chelating agents, Tris-EDTA and disodium EDTA, were found to be promising adjuvants that had both intrinsic antimicrobial activity and the ability to enhance the efficacy of ionophores against Gram-negative bacterial infections. The combination of narasin with either Tris-EDTA or disodium EDTA produced additive effects against P. aeruginosa ATCC strains. An additive effect was also found against the Staphylococcus aureus ATCC strain when narasin or monensin was combined with NAC. The ability of otic pathogens to produce j is an increasing concern in chronic and relapsing cases of otitis externa in dogs. In our experiments, all S. pseudintermedius otic isolates (n=20) formed biofilms with an 80:20 ratio between weak and moderate biofilm production. 95% of P. aeruginosa otic isolates (n=20) were biofilm producers, evenly distributed amongst weak (30%), moderate (35%) and strong (30%) production. The presence of biofilms may play an essential role in the resistance of otic pathogens to antimicrobial agents. S. pseudintermedius otic isolates which were inherently susceptible to narasin and monensin in the planktonic state became resistant to them in the biofilm state. NAC was demonstrated to be an effective antibiofilm agent that eradicated preformed biofilms of S. pseudintermedius and P. aeruginosa otic isolates. The presence of Tris enhances the ability of disodium EDTA (Tris- EDTA) to eradicate preformed P. aeruginosa biofilms whereby disodium EDTA only reduced biofilms when used alone. In conclusion, the use of ionophores could represent a future strategy for the treatment of Gram-positive bacterial and Malassezia infections in canine otitis externa, and when combined with adjuvants, could be effective in Gram-negative infections. In addition, NAC and Tris-EDTA are potential antibiofilm agents for the treatment of biofilm-associated otitis externa. The study results have provided the basis for further development of these repurposed drugs which would represent an example of good antimicrobial stewardship.