CHAMPAIGN, Ill. — Mutations in a key protein make a yeast found in dogs with common outer ear infections more resistant to the topical antifungals used to treat it, veterinarians and pathobiologists at the University of Illinois Urbana-Champaign found in a new study.
By isolating clinical yeast samples from dogs with recurrent ear infections and studying their DNA sequences and protein structures, "we gained clarity regarding the reasons why yeast are showing decreased susceptibility to antifungal drugs," said Lois Hoyer , a professor of pathobiology at Illinois. "This information allows us to best use existing drugs in a way that maintains their efficacy."
Hoyer designed the study with Dr. Clarissa Pimentel de Souza , a clinician and professor of dermatology and otology in the U. of I. College of Veterinary Medicine, during a conversation in which Souza mentioned the increasing prevalence of persistent fungal otitis externa cases she was seeing in the clinic, and rising concerns about resistance to topical antifungals.
"A common situation seen in practice is the use of an antifungal to treat recurrent yeast otitis externa, without addressing the underlying allergies driving the infection. If the primary allergic disease is not concurrently worked up, the dog will develop another infection shortly after the discontinuation of the topical antifungal. This situation leads to the chronic use of antifungals in the ears, which can be a risk factor for developing resistance," Souza said.
For the study, Souza collected samples from patients with fungal ear infections, with some having infections in both ears. The samples were sent to clinical professor Dr. Chien-Che Hung , a veterinary microbiologist, who isolated the yeast in pure culture. Then Hoyer and graduate student Cole Belcher analyzed the cultured samples, seeking to classify them.
"We first wanted to understand better how the yeasts are related to each other. We did some DNA sequencing, and we made a tree. We found they cluster into three groups," Hoyer said.
To put the classifications into the context of antifungal resistance, the team then focused on the ERG11 gene. The gene encodes the Erg11 protein, a critical component of the pathway for making the yeast cell membrane — the target of azoles, the most commonly prescribed antifungals for otitis externa.
"We got the DNA sequences of the ERG11 genes in the samples, translated them into amino acids and compared them back to the reference strain. We noted amino acids that were changed in these different yeast isolates clustering in the same way the yeast did on the tree," Hoyer said.
Many of the mutations in the ERG11 gene were not related to antifungal sensitivity, but rather how these isolates are related to each other. So the Illinois team partnered with pharmacy professor Joel Tyndall and Elyas McGuire at the University of Otago in New Zealand to investigate how the structure of the Erg11 protein affected how it interacts with various antifungal drugs.
"The most common topical antifungal treatments prescribed are azoles, and miconazole is the first line of defense," Souza said. "If the resistance is caused by a gene mutation altering the structure of the target protein, the antifungal cannot bind to it and will not kill the yeast."
The researchers found a key amino acid that was substituted in the strains that were resistant to miconazole. Samples that did not clinically respond to miconazole were more effectively treated with posaconazole, an azole with a longer molecular tail that has more contact points with the Erg11 protein.
However, that does not mean veterinarians should jump straight to treating all fungal otitis externa infections with the "stronger," longer-tailed azoles, Souza cautioned.
"Veterinarians hope that by using an all-in-one product, it will address any type of infection. This practice may lead to unnecessary use of antifungals and in consequence contribute to inducing resistance," Souza said. "Based on our findings, it is recommended to use a topical short-tailed azole such as miconazole or clotrimazole as an initial therapy for dogs with yeast ear infections. The medium- and longer-tailed azoles such as ketoconazole and posaconazole, respectively, should be reserved for recurrent or nonresponsive cases."
Next, the researchers plan to study further the miconazole-resistant yeast isolates interacting with the longer-tailed azoles. They also plan to collect and culture more yeast samples that are likely to be resistant to treatment.
"We want to test these isolates against multiple drugs using the same assay and then start to associate the DNA sequence changes we see with the results of the drug assays," Hoyer said. "We want to learn how best to steward the antimicrobial agents we have."
The American College of Veterinary Dermatology Research Foundation and the U. of I. College of Veterinary Medicine supported this work.
