Search

To clarify the terbinafine (TRF) resistance mechanism in highly TRF-resistant [minimum inhibitory concentration (MIC) >32 μg/mL] strains of Trichophyton indotineae (NUBS19006T and NUBS19007), we investigated the expression of squalene epoxidase (SQLE), pleiotropic drug resistance 1 (PDR1), multidrug resistance 2 (MDR2), and MDR4 genes by real-time quantitative PCR analysis, given the known interaction of the corresponding proteins with antifungals and the efflux blocker tacrolimus (FK506). SQLE, PDR1, MDR2, and MDR4 transcript levels in TRF-resistant strains cultured in SDB were not significantly higher than those of the respective genes in TRF-susceptible strains (1 and 10). By contrast, PDR1, MDR2, and MDR4 transcript levels in TRF-resistant and TRF-susceptible strains cultured in SDB containing 10 μg/mL TRF were 5-100 times higher than those of the respective genes in strains grown in the absence of TRF. However, no differences in PDR1, MDR2, and MDR4 transcript levels were found between TRF-resistant (NUBS19006T and NUBS19007) and TRF-susceptible strains cultured in SDB containing 10 μg/mL TRF. The interaction between TRF and FK506 on antifungal activity was not detected in TRF-resistant strains. These results indicate that ATP-dependent efflux pumps do not confer TRF-resistance mechanisms in TRF-resistant strains.

Ravuconazole (RVCZ) is a newly available human azole drug in Japan since 2018 and is a broad-spectrum antifungal agent that exhibits excellent activity against Candida albicans and Cryptococcus neoformans (formerly: Cryptococcus grubii). The drug is also highly active against isolates that are resistant to fluconazole (FLCZ). In the present study, the in vitro susceptibility to ravuconazole (RVCZ) of Japanese clinical isolates and multi-azole-resistant strains of C. neoformans was investigated using the Clinical & Laboratory Standards Institute (CLSI) M27-A3 test. The minimum inhibitory concentrations for the 14 clinical isolates and the multi-azole-resistant strains were 0.003125-0.125 mg/L and 0.25-0.5 mg/L for RVCZ, respectively. RVCZ is as effective as ITCZ and VRCZ for treating clinical isolates from cats and humans. Moreover, RVCZ is highly effective against multi-azole-resistant strains that encode a protein with a G344S substitution in ERG11. Consequently, RVCZ has considerable potential for use as a therapeutic agent for multi-azole resistant cryptococcosis.

Prototheca zopfii is an achlorophyllic algae that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. The aim of the present study was to evaluate the bacterial flora in the udder environment in protothecal mastitis. We used metagenomic next-generation sequencing (NGS) analysis to identify 16S rRNA genes from bacterial flora present in milk samples from protothecal mastitic dairy cows.Seven clinical strains of P. zopfii genotype 2 were isolated from 7 milk samples from 7 cases (Holstein cow) of protothecal mastitis; another 9 milk samples were obtained from 9 normal Holstein cows. The samples were collected in 2017 from cows in one dairy located in the Kushiro region in Hokkaido, Japan, which had a history of protothecal mastitis infection.The NGS produced 10,000 to 15,000 sequences in each DNA sample. To facilitate comparison, we grouped the sequencing results according to the culture-based protothecal mastitis diagnosis; sequences derived from the milk samples obtained from healthy cows were grouped separately.Sequences classified as Streptococcus spp., Pseudomonas spp. and Sphingomonas spp., Caulobacter segnis, Macrococcus caseolyticus, Methylobacterium tarhaniae, and Sphingomonas leidyi were the main sequences detected in the groups of samples from cows characterized by culture as having protothecal mastitis. Notably, Calothrix desertica (a cyanobacterium) sequences showed higher prevalence in these samples.To our knowledge, this is the first study to report that C. desertica sequences, effectively absent in the samples derived from healthy cows, are detected at high prevalence in samples from protothecal mastitic animals.

Human and animal dermatophytoses are most commonly treated with systemic antifungal drugs such as itraconazole (ITZ) and terbinafine (TRF). The antifungal susceptibility of Nannizia fulva, however, remains poorly documented. In the present study, we investigated the in vitro susceptibility of N. fulva to ITZ and TRF using the CLSI M38-A2 test. The mean MICs for the 12 tested strains were 0.6542 mg/L (range: 0.0625-1 mg/L) for ITZ and 0.15625 mg/L (range

Morphology and molecular characteristics of Microsporum gypseum clinical isolates obtained from the fur of a normal rabbit (n=1) and the soil from 10 different rabbit hutches in six elementary schools (n=10) were examined. Isolates were also identified by sequence analysis of the internal transcribed spacer (ITS) region. All 11 isolates demonstrated homology with the Arthroderma fulvum ITS sequence. Furthermore, PCR analysis for the presence of mating type genes detected positivity for MAT1-1 (n=10) and MAT1-2 (n=1). However, no mating reaction was detected between A. fulvum reference strains and the clinical isolates.

A 26-year-old female homemaker presented with an approximately 2-month history of an erythematous lesion with agminated seropapules in the right popliteal fossa associated with scales and crusts. The lesion was initially treated as contact dermatitis, but there was no improvement. KOH examination revealed filamentous fungi. The fungal culture was positive, and the morphological characteristics were identical to those of Trichophyton mentagrophytes complex. The fungus was identified as T. erinacei based on genetic analysis. This is the second case report of human tinea corporis due to this fungus in Japan.

A 36-year-old female elementary schoolteacher presented with aggregated serous papules surrounded by mild erythema, extending from both nasal wings/nostrils down to the upper lip. No improvement was seen following treatment of the lesions with topical antibiotics for impetigo. Potassium hydroxide (KOH) direct microscopy confirmed the presence of mycelia, and the infection was diagnosed as tinea faciei. The isolate was identified as Trichophyton mentagrophytes using morphological analysis and as Arthroderma benhamiae using genetic analysis. Here we describe that case and summarize the clinical features of other cases of A. benhamiae infection in Japan that have been reported in the literature.

In this study, we report the first isolation of Aspergillus allahabadii from a Japanese cormorant with pulmonary aspergillosis. We performed molecular identification and antifungal susceptibility testing with the E-test. A 7-month-old male cormorant died because of uric acid deposition secondary to dehydration. Whitish nodular lesions were present on the caudal thoracic air sac in the right thoracic cavity. Histopathology revealed multifocal pyogranulomatous necrotic lesions with numerous fungal hyphae in the thoracic air sac. Identification of the etiologic agent was confirmed by comparative analyses of the sequences of the internal transcribed spacer (ITS) region and β-tubulin-encoding genes. According to the E-test, the minimum inhibitory concentrations of the isolate to amphotericin B, fluconazole, itraconazole, and voriconazole were 0.75 μg/ml, >256 μg/ml, 0.38 μg/ml, and 0.38 μg/ml, respectively.

D. Gruby (1842-1844) detected the fungus in tinea as a causative agent and C.P. Robin (1853) described Microsporum mentagrophytes that was transferred to Trichophyton by Blanchard (1896). Sabouraud (1910) established a group of ectothrix microïde which was divided into gypseum type (6 species: T. asteroids, T. granulosum, T. lacticolor and 3 species) and niveum type (T. radians and T. denticulatum). Thereafter, Epidermophyton simii Pinoy, 1912 and T. interdigitale Priestly, 1917 were reported. These species were classified as T. mentagrophytes by C.W. Emmons (1934 and 1940). Arthroderma simii Stockdale et al., 1965, A. benhamiae, Ajello and Cheng, 1967 and A. vanbreuseghemii Takashio, 1973 were discovered as teleomorphs of T. simii, T. mentagrophytes var. granulosum and T. mentagrophytes (mainly granulosum-asteroides form), respectively. Makimura et al., (1998) reported phylogenetic classification of T. mentagrophytes complex strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1) regions, indicating that A. vanbreuseghemii and T. interdigitale belong to the same clade that was later named T. interdigatale by Gräser et al.(1999). This naming has confused medical and veterinary doctors since anthropophilic isolates (T. interdigitale) and zoophilic isolates (A. vanbreuseghemii) were included as the same species.

Trichophyton mentagrophytes is epidemiologically divided into two distinct forms, zoophilic and anthropophilic. The zoophilic isolates of T.mentagrophytes have generally been identified by morphological and biochemical examination as well as through mating experiments. The confirmed teleomorphs of the zoophilic isolates of the T.mentagrophytes complex are Arthroderma benhamiae, A. simii and A. vanbreuseghemii. On the other hand, no teleomorph has been identified in an anthropophilic isolate of T. mentagrophytes, such as T. mentagrophytes var. interdigitale (T. interdigitale) or in the other anthropophilic strains. In the present study, the mating type (MAT) (-)-specific gene of the MAT1-1 (alpha-box) and the MAT (+)-specific gene of the MAT1-2 (high-mobility-group : HMG) DNA binding domain were confirmed in zoophilic dermatophytes of A. benhamiae, A. simii and A. vanbreuseghemii. The sequence of the MAT1-1 was about 1.3 kbp, containing 2 exons in the A. benhamiae, A. simii and A. vanbreuseghemii (-) mating type strain. The sequence of the MAT1-2 was 1.9 kbp, containing 2 exons in the A. benhamiae, A. simii and A. vanbreuseghemii (+) mating type strain. Of 15 animal isolates and 72 human isolates examined, the MAT1-1 was detected in 5 of the animal isolates and in none of the human isolates, while the MAT1-2 was detected in the other 10 of the animal isolates and in all of the human isolates. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (+) mating strain.

Human cases of dermatophytoses are occasionally transmitted from animals, and suffered from tinea corporis and sometimes Kerion celsi. The most frequent causative agent of these diseases is Microsporum canis. The other dermatophyte, Arthroderma benhamiae is now prevailing in rabbits, rodents and hedgehogs that are popular household pets in Japan. Therefore, some human cases of A. benhamiae infection were reported and the transmission of this infection from rabbits and rodents was confirmed.Cryptococcosis is regarded as dangerous zoonosis, but its transmission from animal to peoples has not been documented in Japan. Animal cases of cryptococcosis are possible to increase in number by developing immunosuppressive animals as well as by spreading of newly introduced C. gattii to Japan.Animal cases of sporotrichosis are rarely reported in Japan. However, feline sporotrichosis should be prevented and promptly treated since it easily transmitted to people from cat lesions and the exudates where copious numbers of organisms are found in tissues.

Animal mycosis, particularly deep mycosis, is one of the most challenging conditions encountered by veterinarians. Pathogens causing mycotic infections in animals include fungi such as Cryptococcus neoformans, Candida spp., and Aspergillus spp. The antifungal drugs used for the treatment of deep mycoses in animals as well as humans are polyenes and azoles. However, the sensitivity of clinical isolates obtained from animals toward these drugs has rarely been assayed. In this study, the antifungal activities of itraconazole and voriconazole against clinical isolates of C. neoformans, Candida spp., and A. fumigatus isolated from animals with mycoses were examined using the broth microdilution method performed according to the guidelines provided by the Clinical and Laboratory Standards Institute. The minimum inhibitory concentrations (MICs) of itraconazole toward the C. neoformans, Candida spp., and A. fumigatus isolates were 0.125 - 1, 0.125 - 2, and 0.25 - 2 microg/ml, respectively, and those of voriconazole were 0.0625 - 0.5, < or =0.0313 - 0.0625, and 0.0625 - 1 microg/ml, respectively. The results of the MIC analyses implied that the fungal isolates obtained from infected animals exhibit an equivalent degree of susceptibility to itraconazole and voriconazole, as is observed in the case of isolates obtained from humans. The appropriate antifungal therapeutic strategy for the treatment of mycoses in animals must be selected taking into consideration the host immune status and organ function as well as the in vitro sensitivity of the pathogens to antifungal drugs.

Malassezia species have been recognized as members of the microbiological flora of human and animal skin; they are also considered to play an important role in the pathogenesis of folliculitis, atopic dermatitis and otitis externa. Therefore, the molecular characteristics were investigated to clarify the epidemiology and the pathogenesis of diseases associated with Malassezia species in human and animals. Molecular investigation was made of 105 clinical isolates of M. pachydermatis from dogs and cats by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD analysis and CHS2 gene analysis indicated that clinical isolates of M. pachydermatis were divided into four distinct genetic types (A, B, C and D). Type A was isolated from lesions of atopic dermatitis, flea allergic dermatitis, otitis externa, pyoderma and seborrheic (dermatitidis) in dogs and cats, and might be predominant on this. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of standard strains of 11 Malassezia species showed 11 distinct clusters of this species.

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.