Yeast species associated with diseased fish: Occurrence, identification, experimental challenges and antifungal susceptibility testing

The effort to characterize yeasts in fish is still meager, and identification of yeasts from aquacultured diseased fish could be a good strategy to identify those yeast species which have a role in overall fish health. Yet, knowledge on their identification using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is lacking. Hence, this study investigates the diversity of yeasts residing in the skin, gills, intestine and internal organs of three economically important diseased freshwater fish species. We carried out a comparative evaluation of the performance of the biochemical identification kit RapID Yeast Plus system and MALDI-TOF MS, with amplification/sequencing of Internal Transcribed Spacer (ITS1-ITS2) regions of rDNA as the reference method for yeast identification. Antifungal susceptibility pattern of yeast isolates was assessed using Vitek2 compact. Among 420 samples from Oreochromis species, Clarias gariepinus and Mugil cephalus, 215 yeast isolates (51.2%) were detected, with 7 genera: Candida, Cryptococcus, Debaryomyces, Rhodotorula, Trichosporon, Geotrichum and Pichia identified. Candida spp. accounted for 65.12% of the isolates and Rhodotorula spp. 28.84%. Overall, the yeast belonging to Ascomycota was higher than the Basidiomycota yeast (P < 0.001). There is statistically highly significant difference between incidences of yeast species in different fish. The intraperitoneal-challenged fish groups with Cryptococcus uniguttulatus, Candida lipolytica, Candida albicans and Trichosporon mucoides exhibited the highest cumulative mortality percentages (100, 80.95, 71.43 and 61.90%, respectively). C. uniguttulatus challenged group exhibited exophthalmia, opacity in the lens and yellow discoloration of the body and internal organs, meanwhile C. albicans group showed red mouth. The RapID Yeast Plus system identified the tested isolates at the species and genus level with a percentage of 30% for each. The MALDI-TOF MS accurately identified 75% of the isolates to the species level. Of the isolates (25%) not present in the MALDI-TOF MS database at the species level, 10% were ascribed to the genus level and an incorrect identification was given for 15%. Voriconazole, amphotericin B and caspofungin were the most active drugs against all the species tested, while fluconazole demonstrated poor activity. The diseased freshwater fish species may harbor potential clinically relevant yeast species, including C. albicans, non-albicans Candida species, Trichosporon, Cryptococcus and Rhodotorula species. MALDI-TOF MS offers an accurate and cost-effective alternative to the conventional and molecular methods for yeast identification. Subsequently, implementation of the VITEK MS with a more extended database is required.