Genomic diversity of the pathogenic fungus Aspergillus fumigatus in Japan reveals the complex genomic basis of azole resistance.

Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants.

Most Azole-Susceptible Isolates of Aspergillus fumigatus in Hokkaido, Japan are Clustered with Worldwide Strains That Do Not Have Tandem Repeats in Cyp51A Promoter.

In this study, we analyzed Aspergillus fumigatus short tandem repeat patterns of 106 strains isolated from the outdoor air, clinical specimens, and king penguins (Aptenodytes patagonicus) with aspergillosis in Japan, and compared them with those of 668 strains from AfumID (including six isolates from Japan). The results showed that the isolates were classified into three major groups. Group II contained most of the azole-resistant strains with 34- and 46-bp tandem repeats in cyp51A promoter. As in our previous study, OKH50 and Env1 strains were classified in Group II. Most of the azole-susceptible strains obtained in Japan were classified in Group III.

Usefulness of combining computed tomography and air sac fluid examination to rule out aspergillosis: case study in two gentoo penguins (Pygoscelis papua) with respiratory clinical signs.

Pulmonary aspergillosis occurring in captive penguins living in zoos and aquariums is a fatal disease owing to its high mortality rate. On conducting computed tomography, two gentoo penguins (Pygoscelis papua) with respiratory clinical signs that were housed at an aquarium in Hokkaido, Japan were found to have a certain amount of fluid in their air sacs. This fluid was collected and cultured for bacteria and fungi, the results of which both came back negative. These results enabled us to rule out bacterial infection or aspergillosis and supported our decision not to administer antibacterial and antifungal drugs. Overall, the combination of computed tomography and air sac fluid examination was useful for ruling out bacterial infection or aspergillosis in penguins with respiratory clinical signs.

Development of Loop-Mediated Isothermal Amplification for the Detection of Prototheca bovis Directly from Milk Samples of Dairy Cattle.

Prototheca bovis is an algal emerging pathogen in dairy farms causing refractory protothecal mastitis with increasing incidence worldwide and significant economic impact. P. bovis infects cows throughout the lactation cycle, including dry periods, and can persist in the udder and the environment for a long time. Since P. bovis does not respond to treatments with antibiotics, the suggested sanitary measure to restrict the spread is culling infected animals. A point-of-care test for early detection of the causative agent is critically needed to guide farm management and the appropriate treatment of mastitis. Loop-mediated isothermal amplification (LAMP) is a highly specific molecular method, time-saving, cost-effective and easy to perform in limited settings. This study aimed to develop a LAMP assay for P. bovis detection directly from milk samples; it was employed in conjunction with a commercial DNA extraction kit which was previously used to extract DNA from milk specimens containing microbes. The LAMP assay detected P. bovis DNA within 1 h in milk samples spiked with P. bovis at a concentration of 50 cells/µL, enabling on-farm disease monitoring and decision-making based on a reliable diagnosis. The LAMP method will contribute to the accurate and rapid identification of P. bovis in asymptomatic or recurrent mastitis cases and consequently aid the implementation of targeted control measures and the reduction of losses in milk production.

Clinical characteristics of allergic bronchopulmonary mycosis caused by Schizophyllum commune.

BACKGROUND: Allergic bronchopulmonary mycosis (ABPM) is an allergic disease caused by type I and type III hypersensitivity to environmental fungi. Schizophyllum commune, a basidiomycete fungus, is one of the most common fungi that causes non-Aspergillus ABPM. OBJECTIVE: Herein, we attempted to clarify the clinical characteristics of ABPM caused by S. commune (ABPM-Sc) compared with those of allergic bronchopulmonary aspergillosis (ABPA). METHODS: Patients with ABPM-Sc or ABPA were recruited from a nationwide survey in Japan, a multicenter cohort, and a fungal database at the Medical Mycology Research Center of Chiba University. The definition of culture-positive ABPM-Sc/ABPA is as follows: (1) fulfills five or more of the 10 diagnostic criteria for ABPM proposed by Asano et al., and (2) positive culture of S. commune/Aspergillus spp. in sputum, bronchial lavage fluid, or mucus plugs in the bronchi. RESULTS: Thirty patients with ABPM-Sc and 46 with ABPA were recruited. Patients with ABPM-Sc exhibited less severe asthma and presented with better pulmonary function than those with ABPA (p = 0.008-0.03). Central bronchiectasis was more common in ABPM-Sc than that in ABPA, whereas peripheral lung lesions, including infiltrates/ground-glass opacities or fibrotic/cystic changes, were less frequent in ABPM-Sc. Aspergillus fumigatus-specific immunoglobulin (Ig)E was negative in 10 patients (34%) with ABPM-Sc, who demonstrated a lower prevalence of asthma and levels of total serum IgE than those with ABPM-Sc positive for A. fumigatus-specific IgE or ABPA. CONCLUSIONS: Clinical characteristics of ABPM-Sc, especially those negative for A. fumigatus-specific IgE, differed from those of ABPA.

Nationwide Survey about the Occurrence of Aspergillosis in Captive Penguins in Zoos and Aquariums in Japan.

We surveyed the facilities that were members of the Japan Association of Zoos and Aquariums to clarify the incidence of aspergillosis, which is a major cause of death in captive penguins, and to discern effective preventive measures. Responses were obtained for 2910 penguins in 64 facilities; 73 penguins (2.5%) in 35 facilities had died from aspergillosis during the past 5 years from April 2016 to March 2021. Answers to questions about the rearing environment indicated that aspergillosis occurred significantly more often in facilities where penguins were reared outdoors, were in contact with soil, or were moved outside of the rearing enclosure. Answers to questions about their dead penguins indicated that 76% may have been at individual risk (e.g., young age, old age, molting period, and breeding season) and 54% were thought to be reared in uncomfortable environments (e.g., high temperature, high humidity). Aspergillosis may occur when individual risk factors and uncomfortable environmental factors are added to the risk factors of exposure to Aspergillus, such as the presence of soil. These conditions must be recognized as risk factors for aspergillosis, and appropriate preventive measures, such as avoiding penguin contact with the soil where Aspergillus is expected to be present, can minimize aspergillosis-related deaths.

Analysis of Prototheca and yeast species isolated from bulk tank milk collected in Tokachi District, Japan.

Bovine mastitis, a major infectious disease affecting milking cows, leads to reduced milk yield and quality, reduced animal welfare, and an increased need for culling. Although its major causative agents are bacteria, yeast species and achlorophyllous algae of the Prototheca genus are well known as causative agents of bovine refractory mastitis. Nevertheless, few studies have analyzed specific yeasts and Prototheca in this context. Herein, we present survey data of yeast species and Prototheca species isolated from bulk tank milk in the Tokachi district of Japan from April 2020 through March 2021. The species of 276 isolates were determined. Yeast species accounted for 184 isolates, of which Pichia kudriavzevii was the most prevalent species. Regarding Prototheca species, only Prototheca bovis was isolated (92 isolates). Prototheca bovis and Pichia kudriavzevii were detected throughout the year and were detected repeatedly on the same farm. Kluyveromyces marxianus was the second most frequently isolated yeast species after Pichia kudriavzevii. Candida parapsilosis, the fourth most frequently isolated yeast species, was found discontinuously. Analysis of monthly data indicated that Kluyveromyces marxianus and Candida parapsilosis were mainly found during the winter and summer months, respectively. Candida akabanensis and Pichia cactophila were the third and fifth most frequently isolated yeast species, respectively. They were detected repeatedly in bulk tank milk samples from the same farms. Results obtained from bulk tank milk underscore the prevalence of these species. These study results are expected to contribute to the elucidation of problematic yeast and Prototheca species.

Direct Rub Inoculation of Fungal Flora Changes Fatty Acid Composition and Volatile Flavors in Dry-Aged Beef: A Preliminary Study.

Here, we established a method to produce dry-aged beef (DAB) by rub inoculation with fungal flora on the prepared DAB surface. Portions of Holstein steers' rumps were prepared by direct rub inoculation of fungal flora or without treatment (conventional DAB) and dry-aged for 26 days in an aging room at 2.9 °C and 90% relative humidity. We compared the fungal covering and meat quality, including fatty acid composition and volatile aromatic compounds, of fungal-inoculated DAB with those of the conventional DAB. The fungal-inoculated DAB was almost entirely covered with white mold, in contrast to the conventional DAB. Moreover, the proportion of oleic acid and the concentration of nine volatile compounds significantly increased in the raw meat of fungal-inoculated DAB compared with those in the conventional DAB (p < 0.05). These results suggested that direct rub inoculation of fungal flora from prepared DAB may accelerate DAB production and efficiently enhance the "melt-in-the-mouth" feeling and flavors of DAB.

[Countermeasures Against Aspergillosis in Captive Penguins].

Aspergillosis is a major fungal infection in humans and animals. Penguins (Order Spheniscidae) are particularly susceptible to aspergillosis, and aspergillosis in captive penguins is presently a major problem. We were faced with the challenge of combating aspergillosis in an aquarium. As a solution, we organized a multidisciplinary aspergillosis control team, including a medical and veterinary mycologist. Since Aspergillus, including Aspergillus fumigatus, is abundant in soil, we thought it necessary to minimize contact between captive penguins and soil to prevent aspergillosis. As a countermeasure, we stopped using a route for outdoor penguin marches where the soil was exposed. Additionally, after outdoor penguin marches, the feet of penguins were washed with seawater to avoid bringing soil into the rearing facility for penguins. Furthermore, since A. fumigatus was detected on several spots in the environment of the rearing facility by swab analysis, we cleaned and sanitized the rearing facility with 0.02% sodium hypochlorite and hot water. As a result of the above measures, there has been no incidence of aspergillosis in captive penguins since 2016. These results show that our preventive measures are working well. As shown here, we presented an example of how the multidisciplinary control team, which included a mycologist, successfully implemented preventive measures against aspergillosis. Due to changes in the rearing environment and the impact of global warming on penguins, it is expected that the role of mycologists in aspergillosis control will expand in the future.

Evaluation of the air sac volume of penguins with respiratory diseases using computed tomography.

Captive penguins with respiratory diseases exhibit advanced pathological conditions upon the appearance of clinical signs. Therefore, the successful treatment of respiratory diseases remains difficult after the onset of clinical signs, leading to high mortality rates. In this study, we measured air sac volume using computed tomography (CT) to evaluate the respiratory condition of penguins. In a regular quarterly health checkup, blood samples were collected from 45 penguins housed at an aquarium in Hokkaido, Japan. A total of 12 penguins with abnormal blood parameters underwent CT. The air sac volumes were calculated in three-dimensional CT, and the scatter plots of the air sac volumes and body weights were analyzed. No correlation was found between the air sac volume and body weight in both the gentoo and king penguins. Two gentoo penguins with infiltration and one king penguin with multiple nodules on CT were tentatively diagnosed with aspergillosis and treated with oral administration of itraconazole. Follow-up CT examination was performed until the outcome: healed or died. The mean air sac volumes of the two gentoo penguins, which recovered after treatment, increased from 273.9 and 329.0 cm3 before healing to 449.0 and 424.6 cm3 after healing, respectively. Meanwhile, the air sac volume of the king penguin, which subsequently died, decreased from 1,556.9 to 920.6 cm3 despite treatment. Changes of the air sac volume in the same individual could be useful for evaluating the respiratory condition of penguins.

Azole-resistant Aspergillus fumigatus as an emerging worldwide pathogen.

Aspergillus fumigatus, a ubiquitous pathogen, causes aspergillosis in humans, especially in immunodeficient patients. Azoles are frontline antifungal drugs for treating aspergillosis. The recent global emergence of azole resistance in A. fumigatus has become a serious problem worldwide. It has arisen through two routes: long-term azole medical therapy, called the patient route, and the use of azole fungicides in its habitats especially for agricultural activities, called the environmental route. Resistant strains developed through the latter route show cross-resistance to medical azoles because of the identical molecular target Cyp51A between azole compounds used for medical treatment and agricultural disease control. In azole-resistant strains arising through the environmental route, A. fumigatus is observed frequently to possess mutations in the cyp51A gene linked to tandem repeats in the promoter region such as TR34 /L98H and TR46 /Y121F/T289A. The results of microsatellite genotyping analyses of resistant A. fumigatus strains have suggested a transboundary spread of this microorganism in many countries. Diverse actors are involved in the global highway of transmission. Therefore, the matter must be addressed as a "One Health" issue. This review presents a background of azole resistance in A. fumigatus and introduces newly discovered difficulties generated as this pathogen spreads worldwide.

In Vitro Assay of Translation Inhibition by Trichothecenes Using a Commercially Available System.

Trichothecenes are a family of major secondary metabolites produced by some common filamentous fungi, including plant pathogenic and entomopathogenic fungi. It may be considered difficult to conduct a comparison between the toxicities of trichothecenes with consideration of different conditions and cell lines. In the current study, we developed an in vitro assay based on a commercially available system to estimate the translation inhibition, that is, the main toxicity, of trichothecenes. The assay was applied to estimate the inhibition of protein synthesis by trichothecenes. Initially, we examined the assay using trichothecene dissolved in water followed by an assessment of trichothecene solutions dissolved in acetonitrile. The obtained data showed that the assay tolerated the small amount of acetonitrile. The assay examined in this study has the advantages of a short operation time (one day), ease of use, and data stability, as it is a non-cell-based assay whose components are commercially available. It is expected that this assay will contribute to the evaluation of the toxicity of a vast number of trichothecenes.

Identification of Volatile Sulfur Compounds Produced by Schizophyllum commune.

Schizophyllum commune is a causative agent of allergic bronchopulmonary mycosis, allergic fungal rhinosinusitis, and basidiomycosis. Diagnosis of these diseases remains difficult because no commercially available tool exists to identify the pathogen. Unique volatile organic compounds produced by a pathogen might be useful for non-invasive diagnosis. Here, we explored microbial volatile organic compounds produced by S. commune. Volatile sulfur compounds, dimethyl disulfide (48 of 49 strains) and methyl ethyl disulfide (49 of 49 strains), diethyl disulfide (34 of 49 strains), dimethyl trisulfide (40 of 49 strains), and dimethyl tetrasulfide (32 of 49 strains) were detected from headspace air in S. commune cultured vials. Every S. commune strain produced at least one volatile sulfur compound analyzed in this study. Those volatile sulfur compounds were not detected from the cultures of Aspergillus spp. (A. fumigatus, A. flavus, A. niger, and A. terreus), which are other major causative agents of allergic bronchopulmonary mycosis. The last, we examined H2S detection using lead acetate paper. Headspace air from S. commune rapidly turned the lead acetate paper black. These results suggest that those volatile sulfur compounds are potent targets for the diagnosis of S. commune and infectious diseases.

Identification of Novel Mutations Contributing to Azole Tolerance of Aspergillus fumigatus through In Vitro Exposure to Tebuconazole.

Azole resistance of Aspergillus fumigatus is a global problem. The major resistance mechanism is through cytochrome P450 14-α sterol demethylase Cyp51A alterations such as a mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerance and resistance mechanisms, such as the hmg1 (a 3-hydroxy-3-methylglutaryl coenzyme-A reductase gene) mutation, are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three rounds of selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the isolates obtained and the parental strain revealed a nonsynonymous mutation in MfsD, a major facilitator superfamily protein (Afu1g11820; R337L mutation [a change of R to L at position 337]), in all isolates. Furthermore, nonsynonymous mutations in AgcA, a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220; E535Stop mutation), UbcD, a ubiquitin-conjugating enzyme E2 (Afu3g06030; T98K mutation), AbcJ, an ABC transporter (Afu3g12220; G297E mutation), and RttA, a putative protein responsible for tebuconazole tolerance (Afu7g04740; A83T mutation), were found in at least one isolate. Disruption of the agcA gene led to decreased susceptibility to azoles. Reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. Reversion of the T98K mutation in UbcD to the wild type led to decreased susceptibility to azoles. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and agricultural azole fungicides.

Dry-aged beef manufactured in Japan: Microbiota identification and their effects on product characteristics.

We aimed to determine the mold, yeast, and bacterial distributions in dry-aged beef (DAB) manufactured in Hokkaido, Japan, and to study their effects on meat quality compared to wet-aged beef (WAB). Two rump blocks from Holstein steer were dry- and wet-aged for 35 days at 2.9 °C and 90% RH. The psychrophilic molds Mucor flavus and Helicostylum pulchrum and other fungi (Penicillium sp. and Debaryomyces sp.) appeared on the crust of DAB, while lactic acid bacteria and coliforms were suppressed in the inner part of the meat. The composition of C16:0, C18:0, and C18:1 fatty acids did not differ between DAB and WAB, while more C17:0 fatty acids were detected in DAB. Dry aging suppressed acids and increased the production of various aroma compounds with mushroom-like, nutty, and other pleasant flavors. The meat quality and free amino acid (FAA) contents of DAB and WAB did not differ significantly. In this study, we identified major molds on DAB, which might contribute to an increase in aroma. Keywords: dry-aged beef; Mucor flavus; Helicostylum pulchrum; psychrophilic mold; meat quality; volatile aroma compounds.

Adiaspore development and morphological characteristics in a mouse adiaspiromycosis model.

Lesions of adiaspiromycosis, a respiratory disease affecting wild animals, have been found mainly in dead mammals and free-living mammals captured for surveillance. No report has described an investigation of adiaspore formation progress in the lung. After establishing an experimental mouse model of intratracheal adiaspiromycosis infection with the causative agent Emmonsia crescens, we observed adiaspore development. The spores grew and reached a plateau of growth at 70 days post-infection. The median adiaspore diameter showed a plateau of around 40 µm. The characteristic three-layer cell-wall structure of adiaspores was observed in the lung at 70 days post-infection. We examined infection with a few spores, which revealed that adiaspores in the mouse lung progressed from intratracheal infection of at least 400 spores. Moreover, we developed adiaspores in vitro by culture in fetal bovine serum. Although most spores broke, some large spores were intact. They reached about 50 µm diameter. Thick cell walls and dense granules were found as common points between in vitro adiaspores and in vivo adiaspores. These models are expected to be useful for additional investigations of E. crescens adiaspores and adiaspiromycosis.

Characteristic imaging findings of the respiratory system in penguins with suspected aspergillosis in an aquarium.

Twenty penguins, including the King penguin (Aptenodytes patagonicus), Gentoo penguin (Pygoscelis papua), and African penguin (Spheniscus demersus), housed at an aquarium in Hokkaido, Japan, underwent regular health screening via blood test, and five penguins with suspected aspergillosis were extracted. In cases 1 and 2, a thickened membrane and/or fluid level and/or calcification in the air sac were observed on both radiography and computed tomography (CT). These two penguins died after 19 and 43 days, respectively. At the time the radiographic changes were observed, the disease had likely progressed to a point at which it was too late for recovery. Aspergillus fumigatus infection was confirmed by nucleotide sequence analysis in case 1. In case 3, infiltration in the pulmonary parenchyma was observed on CT, and the infiltration disappeared following oral administration of itraconazole as diagnostic therapy for 8 months. In case 4, defects in the pulmonary parenchyma were observed only on CT. These defects remained unchanged in size for 7 months despite the lack of any treatment, and were not considered clinically significant. However, the blood antigen level in case 5 was increased, both radiography and CT were unremarkable. The combination of a screening blood test and CT examination could be useful clues for an early diagnosis of aspergillosis as well as for initiating treatment.

Detection of Substitutions at 98th, 121st, and 289th Amino Acid Residues in Cyp51A using Cycling Probes.

Azole-resistant Aspergillus fumigatus containing unique mutation(s) of cyp51A with tandem repeats in the promoter region has emerged and has become dispersed in environments worldwide. For this study, we designed primers and cycling probes to detect mutations associated with tandem repeats. Substitutions at the 293rd nucleotide (leucine or histidine at the 98th amino acid residue), at the 362nd nucleotide (tyrosine or phenylalanine at the 121st amino acid residue), and at the 865th nucleotide (threonine or alanine at the 289th amino acid residue) in cyp51A were detected using these primers and probes. These results suggest that the primer and probe sets are helpful in detecting these mutations and in differentiating the types of tandem repeats in cyp51A.

Autopsy imaging for aspergillosis in King Penguin, an economically valuable zoo animal.

Autopsy imaging (Ai) was performed for a King Penguin. Ai-computed tomography (CT) revealed air sac membrane thickening, multiple nodules in the cranial air sac, suspected abscess, lung infiltration, and air sac contraction. Based on these findings, respiratory disorder was concerned. Aspergillosis, which is the highly observed in penguins, was considered as the primary differential diagnosis. The cultured sample showed characteristic conidial head of Aspergillus spp., the DNA of which was 100% identical to that of A. fumigatus. The cause of death was determined to respiratory failure due to aspergillosis. Ai-CT findings facilitated the dissection workflow and alerted the pathologist to potential hazards during the autopsy. Ai is useful to determine the cause of death and for readiness and safe pathological dissection.

Prospective survey of Aspergillus species isolated from clinical specimens and their antifungal susceptibility: A five-year single-center study in Japan.

Aspergillus fumigatus is the most prevalent species that causes aspergillosis. A. fumigatus strains with tandem repeats in the cyp51A promoter have emerged in the environment. Aspergillus species other than A. fumigatus have also been recognized as causative agents of aspergillosis; however, they show lower susceptibility to antifungals compared with A. fumigatus. Therefore, it is important to precisely identify Aspergillus species and determine their antifungal susceptibility. Herein, we collected 119 mold strains isolated from clinical specimens collected at a hospital between November 2013 and December 2018. The collected strains were identified by sequencing several regions, including internal transcribed spacers, and determined their susceptibility to the antifungals itraconazole, voriconazole, and amphotericin B. Of 119 strains, 107 were Aspergillus species, which were identified as A. fumigatus (67), Aspergillus section Nigri (21), A. flavus (7), A. terreus (6), and A. nidulans (6). In Aspergillus section Nigri, the number of A. niger was less than the number of A. welwitschiae and A. tubingensis. Two azole-resistant A. fumigatus samples were included among the isolates. Four of the eight A. tubingensis isolates showed less susceptibility to voriconazole; however, all isolates of A. niger and A. welwitschiae were susceptible to itraconazole and voriconazole. Because of lack of susceptibility data for non-fumigatus Aspergillus and an increasing frequency of antifungal resistance among A. fumigatus, our data along with further surveillance may contribute to determining the frequency and susceptibility of Aspergillus spp. clinical isolates in Japan.