Identification and Antifungal susceptibility profile of uncommon yeast species at Fattouma Bourguiba university hospital in Tunisia.

Despite severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba university hospital using phenotypic, molecular and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua and Candida guilliermondii. Ninety % of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool.

There is still little information on the epidemiology of uncommon emergent yeasts although their implication in severe diseases mainly invasive infections. Thus, the importance of an accurate identification and antifungal susceptibility testing for a better monitoring of related infections.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

The 'Erlenmeter': a low-cost, open-source turbidimeter for no-sampling phenotyping of microorganism growth.

This work presents a low-cost, open-source turbidimeter, the 'Erlenmeter', designed to monitor the growth of microorganisms in batch cultures. It is easy to build, based exclusively on inexpensive off-the-shelf electronic components and 3D-printed parts. The Erlenmeter allows measuring the optical density of cultures on standard Erlenmeyer flasks without the need to open the flasks to collect aliquots, ensuring speed, minimal use of consumables, and elimination of the risk of contamination. These features make it particularly well-suited not just for routine research assays but also for experimental teaching. Here we illustrate the use of the Erlenmeter turbidimeter to record the growth of the microalga Phaeodactylum tricornutum, of the bacterium Escherichia coli, and of the yeast Saccharomyces cerevisiae, model organisms that are widely used in research and teaching. The Erlenmeter allows a detailed characterization of the growth curves of all organisms, confirming its usefulness for studying microbial populations dynamics both for research purposes and in classroom settings.

Culturable yeast community associated with grape must and honey bees sampled from apiaries located in the vineyards.

AIM: In this study, we investigated culturable yeast community, present in grape must sampled from vineyards with apiaries on the borders, and in honey bees collected in these apiaries. METHODS AND RESULTS: To this aim, yeasts isolated from spontaneously fermented grapes randomly collected in two vineyards (P1 and P2) with apiaries on the borders (A1 and A2) were compared to those isolated from spontaneously fermented grapes collected from a vineyard without apiary (P4). At the same time, yeast community was analyzed on bees collected in each apiary placed in the vineyards, in comparison to yeasts isolated from an apiary (A3) located far from the vineyards. The analysis was performed for two consecutive years (2021 and 2022). The isolated yeasts were identified by restriction analysis of amplified ITS region, followed by sequencing of ITS fragment.Our research showed that the presence of apiaries seems to increase yeast counts of grape must, in particular of Saccharomyces cerevisiae; furthermore, the permanence of apiaries in the vineyards allowed the recovering of these yeasts also from bees. CONCLUSIONS: Our findings seem to corroborate the role of bees as vectors and reservoirs of oenologically relevant yeasts, such as a source of non-conventional yeasts with potential biotechnological applications.

The Effect of Sucrose and Yeast Extract on Total Phenolic, Flavonoid, and Anthocyanin of Lactic-Acid-Fermented Mangosteen Fruit Peel (Garcinia mangostana L.).

Objectives: This study aimed to determine the most suitable concentration of sucrose and yeast extract (SYE) and its impact on the levels of total phenol, flavonoid, and anthocyanin (TPFA) for lactic acid fermentation in mangosteen fruit peel. Materials and Methods: In this study, the primary components were mangosteen fruit peel, SYE, and lactic acid bacteria starter. The experimental design was conducted using the Factorial Design method. The colorimetric method was used to determine the total phenol (Folin-Ciocalteu reagent) and total flavonoid (AlCl3 reagent). In addition, the differential pH method was used to determine the total anthocyanins using KCl and the CH3COONa reagent. Results: The addition of SYE during the fermentation of mangosteen fruit peel significantly increased the concentrations of TPFA compared with the control (p value of 0.0001). The high sucrose concentration and low yeast extract produced the highest TPFA levels in mangosteen rind fermentation. Conclusion: The use of SYE affects the levels of TPFA in lactic acid-fermented mangosteen fruit peel, with the most suitable concentrations obtained using sucrose (45 g/L) and yeast extract (2.5 g/L).

Exploring the Efficacy of Using Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus, Clay Minerals, and Walnut Nutshells for Mycotoxin Remediation.

The aim of this study was to evaluate the effectiveness of nine different biological compounds to reduce mycotoxins concentrations. The hypothesis of this study was that a static in vitro gastrointestinal tract model, as an initial screening tool, can be used to simulate the efficacy of Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus yeast cell walls and their polysaccharides, red and white clay minerals, and walnuts nutshells claiming to detoxify AFB1, ZEA, DON, and T-2 toxin mycotoxins. Mycotoxin concentrations were analyzed using high-performance liquid chromatography (HPLC) with fluorescent (FLD) and ultraviolet detectors (UV). The greatest effects on reducing mycotoxin concentrations were determined as follows: for AFB1, inserted G. fermentans cell wall polysaccharides and walnut nutshells; for ZEA, inserted R. rubra and G. fermentans cell walls and red clay minerals; for DON, R. rubra cell wall polysaccharides and red clay minerals; and for T-2 toxin, R. rubra cell walls, K. marxianus, and G. fermentans cell wall polysaccharides and walnut nutshells. The present study indicated that selected mycotoxin-detoxifying biological compounds can be used to decrease mycotoxin concentrations.

Enhancing Selenium Accumulation in Rhodotorula mucilaginosa Strain 6S Using a Proteomic Approach for Aquafeed Development.

It is known that selenium (Se) is an essential trace element, important for the growth and other biological functions of fish. One of its most important functions is to contribute to the preservation of certain biological components, such as DNA, proteins, and lipids, providing protection against free radicals resulting from normal metabolism. The objective of this study was to evaluate and optimize selenium accumulation in the native yeast Rhodotorula mucilaginosa 6S. Sodium selenite was evaluated at different concentrations (5-10-15-20-30-40 mg/L). Similarly, the effects of different concentrations of nitrogen sources and pH on cell growth and selenium accumulation in the yeast were analyzed. Subsequently, the best cultivation conditions were scaled up to a 2 L reactor with constant aeration, and the proteome of the yeast cultured with and without sodium selenite was evaluated. The optimal conditions for biomass generation and selenium accumulation were found with ammonium chloride and pH 5.5. Incorporating sodium selenite (30 mg/L) during the exponential phase in the bioreactor after 72 h of cultivation resulted in 10 g/L of biomass, with 0.25 mg total Se/g biomass, composed of 25% proteins, 15% lipids, and 0.850 mg total carotenoids/g biomass. The analysis of the proteomes associated with yeast cultivation with and without selenium revealed a total of 1871 proteins. The results obtained showed that the dynamic changes in the proteome, in response to selenium in the experimental medium, are directly related to catalytic activity and oxidoreductase activity in the yeast. R. mucilaginosa 6S could be an alternative for the generation of selenium-rich biomass with a composition of other nutritional compounds also of interest in aquaculture, such as proteins, lipids, and pigments.

Cultivation of yeasts on liquid digestate to remove organic pollutants and nutrients and for potential application as co-culture with microalgae.

In this study, the growth of yeast and yeast-like fungi in the liquid digestate from vegetable wastes was investigated in order to remove nutrients and organic pollutants, and for their application as co-culture members with green microalgae. The studied yeast strains were characterized for their assimilative and enzymatic profiles as well as temperature requirements. In the first experimental stage, the growth dynamics of each strain were determined, allowing to select the best yeasts for further studies. In the subsequent stage, the ability of selectants to remove organic pollutants was assessed. Different cultivation media containing respectively 1:3, 1:1, 3:1 vol ratio of liquid digestate and the basal minimal medium were used. Among all tested yeast strains, Rhodotorula mucilaginosa DSM 70825 showed the most promising results, demonstrating the highest potential for removing organic substrates and nutrients. Depending on the medium, this strain achieved 50-80% sCOD, 45-60% tVFAs, 21-45% TN, 33-52% PO43- reduction rates. Similar results were obtained for the strain Candida sp. OR687571. The high nutrient and organics removal efficiency by these yeasts could likely be linked to their ability to assimilate xylose (being the main source of carbon in the liquid digestate). In culture media containing liquid digestate, both yeast strains achieved good viability and proliferation potential. In the liquid digestate medium, R. mucilaginosa and Candida sp. showed vitality at the level of 51.5% and 45.0%, respectively. These strains seem to be a good starting material for developing effective digestate treatment strategies involving monocultures and/or consortia with other yeasts or green microalgae.

Easy-to-use imaging-cytometry assay to analyze chitin patterns in yeasts.

BACKGROUND & AIM: Pathogenic fungi are a major threat to public health, and fungal infections are becoming increasingly common and treatment resistant. Chitin, a component of the fungal cell wall, modifies host immunity and contributes to antifungal resistance. Moreover, chitin content is regulated by chitin synthases and chitinases. However, the specific roles and mechanisms remain unclear. In this study, we developed a cytometric imaging assay to quantify chitin content and identify the distribution of chitin in the yeast cell wall. METHODS: The Candida albicans SC5314 and Nakaseomyces glabratus (ex. C. glabrata) ATCC2001 reference strains, as well as 106 clinical isolates, were used. Chitin content, distribution, and morphological parameters were analysed in 12 yeast species. Moreover, machine learning statistical software was used to evaluate the ability of the cytometric imaging assay to predict yeast species using the values obtained for these parameters. RESULTS: Our imaging-cytometry assay was repeatable, reproducible, and sensitive to variations in chitin content in C. albicans mutants or after antifungal stimulation. The evaluated parameters classified the yeast species into the correct clade with an accuracy of 85 %. CONCLUSION: Our findings demonstrate that this easy-to-use assay is an effective tool for the exploration of chitin content in yeast species.

Exploring factors influencing the levels of biogenic amines in wine and microbiological strategies for controlling their occurrence in winemaking.

Fermented beverages, including wine, can accumulate high concentrations of biogenic amines (BAs), which can pose potential health risks. BAs are produced by various yeasts and lactic acid bacteria (LAB) during winemaking. LAB are the main contributors to the formation of histamine and tyramine, the most toxic and food safety relevant biogenic amines. Numerous factors, ranging from agricultural and oenological practices to sanitation conditions, can contribute to the formation of BAs in wines. Moreover, organic and biodynamic wines impose limitations on the use of common food additives employed to control the proliferation of native and spoilage microorganisms during vinification and storage. To mitigate histamine production, commercial starter cultures incapable of synthesising histamine have been effectively utilised to reduce wine histamine content. Alternative fermentative microorganisms are currently under investigation to enhance the safety, quality, and typicity of wines, including indigenous LAB, non-Saccharomyces yeasts, and BAs degrading strains. Furthermore, exploration of extracts from BAs-degrading microorganisms and their purified enzymes has been undertaken to reduce BAs levels in wines. This review highlights microbial contributors to BAs in wines, factors affecting their growth and BA production, and alternative microorganisms that can degrade or avoid BAs. The aim is to lessen reliance on additives, providing consumers with safer wine choices.

Sourdough fermentation for the valorization of sorghum flour: Microbiota characterization and metabolome profiling.

Due to a large adaptability to different cultivation conditions and limited input compared to other cereals, sorghum is considered an emerging crop. Its antioxidant properties, high fiber content and low glycemic index also make it a valuable addition to a healthy diet, nevertheless, the presence of antinutritional factors and the lack of gluten, hamper its use as food ingredient. This study investigated the impact of sourdough fermentation on sorghum nutritional quality. Lactic acid bacteria dominating sorghum flour and sourdough were identified by culture-dependent analysis revealing Lactiplantibacillus plantarum as the dominant species found in the mature sourdough, whereas Weissella cibaria and Weissella paramesenteroides were the species isolated the most after the first refreshment. Among yeasts, Saccharomyces cerevisiae was the most prevalent. Lactic acid bacteria pro-technological and functional performances as starter were evaluated in sorghum type-II sourdoughs through an integrated characterization combining chromatographic and NMR spectroscopic techniques. The metabolic profile of the strains mainly grouped together W. cibaria strains and W. paramesenteroides AI7 which distinguished for the intense proteolysis but also for the presence of compounds particularly interesting from a physiological perspective (allantoin, glutathione, γ-aminobutyric acid and 2-hydroxy-3-methylbutyric acid), whose concentration increased during fermentation in a species or strain specific matter.

Pichia kudriavzevii as feed additive in Nile tilapia (Oreochromis niloticus) diet.

Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their nutritional benefits. Despite their potential, marine and mangrove yeast species used in the aquaculture industry have received little attention in the Philippines. Pichia kudriavzevii (A2B R1 ISO 3), sourced from bark samples, was selected and mass-produced due to its high protein content and amino acid profile. The dried biomass of P. kudriavzevii was incorporated into the diets of Nile tilapia (Oreochromis niloticus) juveniles at varying inclusion levels (0, 1, 2, and 4 g/kg diet) and its effect on their growth performance, body composition, and liver and intestinal morphology was assessed after 40 days of feeding. The groups that received P. kudriavzevii at a concentration of 2 g/kg diet exhibited higher final body weight, percent weight gain, and specific growth rate in comparison to the other treatment groups. Whole body proximate composition did not vary among the dietary groups. Intestinal and liver histopathology also indicated no abnormalities. These findings suggest the potential of ascomycetous P. kudriavzevii as a beneficial feed additive in Nile tilapia diets, warranting further investigation into its long-term effects and broader applications in fish culture.

Using yeasts for the studies of nonfunctional factors in protein evolution.

The evolution of protein sequence is driven not only by factors directly related to protein function and shape but also by nonfunctional factors. Such factors in protein evolution might be categorized as those connected to energetic costs, synthesis efficiency, and avoidance of misfolding and toxicity. A common approach to studying them is correlational analysis contrasting them with some characteristics of the protein, like amino acid composition, but these features are interdependent. To avoid possible bias, empirical studies are needed, and not enough work has been done to date. In this review, we describe the role of nonfunctional factors in protein evolution and present an experimental approach using yeast as a suitable model organism. The focus of the proposed approach is on the potential negative impact on the fitness of mutations that change protein properties not related to function and the frequency of mutations that change these properties. Experimental results of testing the misfolding avoidance hypothesis as an explanation for why highly expressed proteins evolve slowly are inconsistent with correlational research results. Therefore, more efforts should be made to empirically test the effects of nonfunctional factors in protein evolution and to contrast these results with the results of the correlational analysis approach.

Investigation of Freezing and Freeze-Drying for Preserving and Re-Using a Whole Microbial Cheese Community.

Preserving microbial ecosystems obtained from traditional cheese-making processes is crucial to safeguarding the biodiversity of microbial cheese communities and thus ensuring that the high flavor quality of traditional cheeses is maintained. Few protocols have been proposed for the long-term storage of microbial consortia. This work aimed to develop preservation methods to stabilize the entire microbial community in smear-ripened cheese without multiplication or isolation. A simplified microbial community, capable of reproducing the metabolic pattern of cheese maturation, was used in three independent cheese productions. Cheese samples were taken before and after the ripening step, mixed with maltodextrin or saline solution, and subjected to different stabilization conditions including freezing and freeze-drying, followed by 1 month of storage. Microbial survival was quantified using the colony-forming unit assay. Differential scanning calorimetry was used to relate the physical events occurring within the samples to the microbial storage stability. Freezing at -80 °C resulted in the lowest loss of culturability (<0.8 log unit), followed by freezing at -20 °C and freeze-drying. The ripening bacteria appeared as the most sensitive microorganisms within the community. Moreover, a successful cheese production using the best-stabilized community showed the possibility of preserving and re-using an entire microbial community of interest.

Detection of Hanseniaspora opuntiae in anovaginal samples of pregnant women in Rio de Janeiro, Brazil-a case report.

In this study, we report the first isolation of Hanseniaspora opuntiae obtained from four pregnant women in Brazil. Clinical isolates were obtained from four samples taken between 35 and 37 gestational weeks, as part of the routine antenatal care for maternal colonization screening for Streptococcus agalactiae group B. The patients were immunocompetent, with two of them diagnosed with gestational diabetes mellitus. Species identification was performed by MALDI-TOF MS and rDNA sequencing. While Hanseniaspora species have not traditionally been considered a typical opportunist pathogen, our findings emphasize the importance of investigating and screening for Hanseniaspora in pregnant populations, highlighting H. opuntiae as a potential agent of human infections.

Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in Aureobasidium spp.

BACKGROUND: Non-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation. RESULTS: Here, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts. CONCLUSION: Here, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications.

Do flower-colonizing microbes influence floral evolution? A test with fast-cycling Brassica.

Pollinators are thought to be the main drivers of floral evolution. Flowers are also colonized by abundant communities of microbes that can affect the interaction between plants and their pollinators. Very little is known, however, about how flower-colonizing microbes influence floral evolution. Here we performed a six-generation experimental evolution study using fast-cycling Brassica rapa, in which we factorially manipulated the presence of pollinators and flower microbes to determine how pollinators and microbes interact in driving floral evolution. We measured the evolution of six morphological traits, as well as plant mating system and flower attractiveness. Only one of the six traits (flower number) evolved in response to pollinators, while microbes did not drive the evolution of any trait, nor did they interact with pollinators in driving evolution of morphological traits. Moreover, we did not find evidence that pollinators or microbes affected the evolution of flower attractiveness to pollinators. However, we found an interactive effect of pollinators and microbes on the evolution of autonomous selfing, a trait that is expected to evolve in response to pollinator limitation. Overall, we found only weak evidence that microbes mediate floral evolution. However, our ability to detect an interactive effect of pollinators and microbes might have been limited by weak pollinator-mediated selection in our experimental setting. Our results contrast with previous (similar) experimental evolution studies, highlighting the susceptibility of such experiments to drift and to experimental artefacts.

Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.

Inhibitory Effect and Potential Antagonistic Mechanism of Isolated Epiphytic Yeasts against Botrytis cinerea and Alternaria alternata in Postharvest Blueberry Fruits.

This study evaluated the biocontrol effect of isolated epiphytic yeasts (Papiliotrema terrestris, Hanseniaspora uvarum, and Rhodosporidium glutinis) against Botrytis cinerea and Alternaria alternata in blueberry fruits and its possible mechanisms. Our findings indicated that the three tested yeasts exerted a good biocontrol effect on postharvest diseases in blueberry, and that H. uvarum was the most effective. In addition, the three tested yeasts could improve the postharvest storage quality of blueberry fruits to some extent. H. uvarum demonstrated the strongest direct inhibitory effect on pathogens by suppressing spore germination, mycelial growth, and antifungal volatile organic compound (VOC) production. P. terrestris showed the highest extracellular lytic enzymes activities. It also had better adaptation to low temperature in fruit wounds at 4 °C. The biofilm formation capacity was suggested to be the main action mechanism of R. glutinis, which rapidly colonized fruit wounds at 20 °C. Several action mechanisms are employed by the superb biocontrol yeasts, while yeast strains possess distinctive characteristics and have substantially different action mechanisms.

A renal mycosis of roach (Rutilus rutilus) caused by the Aureobasidium pullulans.

Spontaneous mycosis caused by Aureobasidium pullulans is documented in roach in a cyprinid-prevalent water reservoir in Czechia. Gross pathological lesions included pale gills and splenomegaly, as revealed during necropsy examination. Histological examination showed extensive foci with fungal elements in the kidney. The isolated fungus was identified through phenotypic and molecular characterization, including phylogeny. This report represents the first case of A. pullulans infection in fish and cold-blooded vertebrates, to the best of our knowledge.