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Molecular typing and antifungal drug susceptivity profile of Rhodotorula mucilaginosa from canine skin and ear canal

Rhodotorula mucilaginosa are saprophytic yeast, and opportunistic infections known as human rhodotorulosis are increasing in immunocompromised patients. In this study, we isolated R. mucilaginosa from pet dogs in Japan and determined the minimum inhibitory concentrations (MICs) of antifungal drugs on these isolates to investigate the drug susceptibility pattern. All 10 isolates according to the broth microdilution (BM) assay of the Clinical and Laboratory Standards Institute (CLSI) M27-A2 were resistance to azoles and genetically close to fluconazole (FLZ)-resistant human isolates of R. mucilaginosa. Due to resistance, it is expected that treatment will be difficult if they infect humans.

of denaturation for 30 sec at 94°C, primer annealing for 30 sec at 55°C, and extension for 60 sec at 72°C. Final extension was performed at 72°C for 2 min. For each strain/reaction, the amplicon was electrophoresed in agarose, and an approximately 550-bp long DNA band was purified using the ExoSAP-IT ® kit (USB Corp., Cleveland, OH, USA), and sequenced on an ABI PRISM 3130 DNA Analyzer (Thermo Fisher Scientific, Inc., Tokyo, Japan) using the PCR primers for sequencing.
Comparative sequence analyses were carried out using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI).
Comparative nucleotide sequence analysis using the BLAST algorithm on the NCBI website showed that the ITS sequences amplified from all 10 isolates were 99 to 100% identical to R. mucilaginosa type strain CBS 316 T (GenBank accession No. NR_073296). The region sequences were also 87 to 98% identical to other Rhodotorula species. Therefore, all isolates were identified as R. mucilaginosa.
The ITS sequences determined in this study have been deposited in the GenBank database: R. mucilaginosa strains NUBS21001 to 21010 (Table 1).
The resulting sequences of the ITS region were compared with those of the Rhodotorula species type strains, FLZ-resistance strains of R. mucilaginosa (CMRP3462 and CMRP3463), and 10 isolates using the ClustalW multiple sequence alignment program [10]. The phylogenetic tree was constructed with the neighbor-joining method using the GENETYX ver.15 program (GENETYX Corp., Tokyo, Japan) [7]. Bootstrap analysis was performed on 1,000 replicates of random samples and analyzed with the ClustalW program [4].
Phylogenetic analysis of the ITS region sequences revealed that the isolates (NUBS21001 to NUBS21010) were grouped in a cluster of R. mucilaginosa type strain CBS 316 T and FLZ-resistance human isolates of R. mucilaginosa (CMRP3462 and CMRP3463) [6], and were independent of the cluster of the other type strains of Rhodotorula species (Fig. 1).
Rhodotorula species were classified as being resistant to FLZ, ITZ, and VRZ according to the clinical breakpoints outlined in the M27-A3 guidelines prepared by the CLSI [2]. The MICs of FLZ, ITZ, and VRZ in the resistant strains were determined to be >64 mg/l, >1 mg/l, and >4 mg/l, respectively [2].
To our knowledge, this is the first report of antifungal-resistant profiles for R. mucilaginosa isolates from canine skin and ear canals. All isolates were resistant to azoles and genetically close to FLZ-resistance human isolates of R. mucilaginosa (Table 1 and Fig. 1) [6]. Due to resistance, it is expected that treatment will be difficult if it infects humans. However, the prevalence of resistant strains of R. mucilaginosa in dogs was unclear in this study, but we found more isolates than expected (the original purpose of this study was the isolation of M. pachydermatis). Unfortunately, there are few reports of drug susceptibility testing of R. mucilaginosa 1545-1548, 2021 isolated in the environment or from the skin of healthy humans and dogs, so it is unknown whether it was resistant from the beginning or acquired resistance by antifungal treatments.
Tang et al. investigated canine skin and ear microbiomes using next-generation sequencing assays and did not detect Rhodotorula spp. genes from healthy and clinically affected dogs [9]. From their report, R. mucilaginosa is not expected to be a major microbe of the skin and ear canal [9]. However, we isolated R. mucilaginosa easily.
The history of antifungal treatment may have led to microbial replacement by R. mucilaginosa, that is resistant to antifungal agents. The desired isolation of M. pachydermatis was not possible from these cases. Only R. mucilaginosa was isolated from all the growing colonies on mDixon, which is the isolation medium for Malassezia species from canine skin [5]. We speculate that treatment with antifungal agents may be due to fungal alternation, in which M. pachydermatis on the skin surface, which was originally desired to be isolated, was reduced and R. mucilaginosa is increased.
If artificial changes in the microbial flora to drug-resistant yeasts occur in the skin and ear canals of Japanese pet dogs, there is concern that the risk of zoonotic diseases will increase. In future research, it is necessary to investigate the microbial flora of the skin and ear canals of Japanese pet dogs by microbiome analysis using next-generation sequence analysis.