There is increasing incidence of food spoilage and health hazards caused by heat-resistant fungi belonging to the genera Byssochlamys, Thermoascus, and Neosartorya, among others. Their ascospores ...cannot be sterilized by heating the food. The microbiological risk assessment studies of these fungi during the production of food and beverages indicated that these fungal species or genera in food are associated with different health risks. Therefore, it is necessary to distinguish Byssochlamys, Thermoascus, and Neosartorya from other fungi in the food industry. These genera can be identified by sequence analysis of housekeeping genes such as β-tubulin, but the process is costly and time-consuming. Therefore, rapid and simple PCR-based methods have been developed using specific primer sets for genus- or species-level identification. PCR amplification products are observed to be specific for each of these genera or species and do not cross-react with other fungi associated with food spoilage and environmental contamination. These identification methods are simple, rapid, and highly specific, making them feasible for use in the quality management of food production plants.
Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of ...patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all
clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor
gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu
, Phe
, Phe
, and His
) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous
gene of a terbinafine-sensitive
(formerly
) strain. All of the generated
transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in
transformants expressing mutant forms of
SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.
Fludioxonil and iprodione are effective fungicides widely used for crop protection and are essential for controlling plant pathogenic fungi. The emergence of fungicide-resistant strains of targeted ...pathogens is regularly monitored, and several cases have been reported. Non-targeted fungi may also be exposed to the fungicide residues in agricultural fields. However, there are no comprehensive reports on fungicide-resistant strains of non-targeted fungi. Here, we surveyed 99 strains, representing 12 Penicillium species, that were isolated from a variety of environments, including foods, dead bodies, and clinical samples. Among the Penicillium strains, including non-pathogenic P. chrysogenum and P. camembertii, as well as postharvest pathogens P. expansum and P. digitatum, 14 and 20 showed resistance to fludioxonil and iprodione, respectively, and 6 showed multi-drug resistance to the fungicides. Sequence analyses revealed that some strains of P. chrysogenum and Penicillium oxalicum had mutations in NikA, a group III histidine kinase of the high-osmolarity glycerol pathway, which is the mode of action for fludioxonil and iprodione. The single nucleotide polymorphisms of G693D and T1318P in P. chrysogenum and T960S in P. oxalicum were only present in the fludioxonil- or iprodione-resistant strains. These strains also exhibited resistance to pyrrolnitrin, which is the lead compound in fludioxonil and is naturally produced by some Pseudomonas species. This study demonstrated that non-targeted Penicillium strains distributed throughout the environment possess fungicide resistance.
In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates ...that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.
Aspergillus fumigatus-related species are responsible for causing aspergillosis, which is a fatal infectious disease. Recently, there has been a series of reports of A. fumigatus-related species that ...are resistant to azole drugs used in clinical practice for the treatment of fungal infections. Some of these species have been isolated from outdoor environments. Testing the drug susceptibility of the strains from outdoor environments, therefore, is important. In this study, we isolated and cultured 72 strains of A. fumigatus-related species from the outdoor environment in Japan. The isolates identified via morphological observation and molecular phylogenetic analysis were Aspergillus felis, Aspergillus lentulus, Aspergillus pseudoviridinutans, Aspergillus udagawae, and Aspergillus wyomingensis. The results of the drug susceptibility testing revealed that A. felis (6 of 14 strains) and A. pseudoviridinutans (13 of 17 strains) were resistant to itraconazole (ITCZ), with 4 mg/L or higher minimum inhibitory concentrations (MICs). The voriconazole (VRCZ)-resistant strains with 4 mg/L or higher MICs were A. felis (14 of 14), A. lentulus (4 of 4), A. pseudoviridinutans (15 of 17), A. udagawae (23 of 34), A. wyomingensis (1 of 3), and A. pseudoviridinutans (1 of 3). Among them, A. felis (1 of 14) and A. pseudoviridinutans (7 of 17) demonstrated 8 mg/L or higher MICs for ITCZ and VRCZ. These results indicate that A. fumigatus-related species resistant to ITCZ and VRCZ are widely distributed in outdoor environments in Japan.
The taxon Aspergillus section Fumigati comprises several causative agents of aspergillosis. Here, the distribution of Aspergillus sect. Fumigati in outdoor environments of Izu and Ogasawara Islands ...was investigated. Different strains were isolated from soil samples collected from 68 sites on 9 islands (Izu-oshima, Toshima, Shikinejima, Kozushima, Miyakejima, Hachijojima, Mukojima, Chichijima, and Hahajima), including different landscapes, and identified using morphological characteristics and calmodulin (CaM) sequences. Seven Aspergillus sect. Fumigati species were identified. The occurrence frequency of Aspergillus fumigatus was higher in forest sites on the islands, except for Ogasawara Islands, whereas that of species other than A. fumigatus was higher in bare land and grassland sites on all islands. The occurrence frequency of A. fumigatus was more than 50% on islands between Izu-oshima and Toshima, decreased on islands between Shikinejima and Hachijojima, and was zero on Ogasawara Islands. Considering other Aspergillus species, Aspergillus felis showed high occurrence frequency on islands between Izu-oshima and Shikinejima, Aspergillus pseudoviridinutans on islands between Kozushima and Hachijojima, and Aspergillus udagawae on Ogasawara Islands. At two study sites (grassland and forest sites), the soil was sampled throughout the year to evaluate whether the occurrence frequency of each fungal species was affected by sampling season. At the grassland site, A. pseudoviridinutans was isolated at more than 90% frequency, regardless of the sampling season. A. fumigatus occurrence frequency at the forest site ranged from 0% to 60% and greatly varied among sampling seasons. Thus, differences in island location and landscape affected the distribution of Aspergillus sect. Fumigati.
Background
Filamentous fungi of the genus
Madurella
are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a ...morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of
Madurella
spp. (
M
.
mycetomatis
,
M
.
pseudomycetomatis
,
M
.
tropicana
, and
M
.
fahalii
), the common causative organisms of eumycetoma.
Principal findings
We successfully designed a primer pair targeting the rDNAs of three
Madurella
spp. excluding
M
.
fahalii
, and detected up to 100 fg of genomic DNA extracted from isolates of
M
.
mycetomatis
and 1 pg of
M
.
pseudomycetomatis
and
M
.
tropicana
, within one hour. Second, a primer pair specific to
M
.
mycetomatis
, the most common causative species, or
M
.
fahalii
, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus
Madurella
from various fungal species known to cause mycetomas.
Conclusion
In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies
Madurella
isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.
Background
Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced ...sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up‐regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC.
Objectives
We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092.
Methods
The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3‐lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively.
Results
The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3‐lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092.
Conclusions
Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum.
The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and also occur in animal intestines; occasionally, infections in humans and animals are observed. In this ...study, we conducted tests of assimilative abilities and thermotolerance in comparison with morphological characteristics of six opportunistic species (Prototheca blaschkeae, Prototheca bovis, Prototheca ciferrii, Prototheca cutis, Prototheca miyajii, and Prototheca wickerhamii) along with Prototheca paracutis. Five of the seven species could be differentiated by physiological characteristics, but P. wickerhamii and P. cutis had identical profiles. Of the cattle-associated species, only P. bovis was able to grow at 42°C. Both type strains of P. cutis and P. miyajii were most susceptible to ravuconazole compared with the other azoles.
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