Disseminated nocardiosis due to Nocardia terpenica.

We report the first case of disseminated nocardiosis due to trimethoprim/sulfamethoxazole-resistant Nocardia terpenica successfully treated with meropenem and clarithromycin. The patient travelled to Japan from Australia via Southeast Asia, which led to differential diagnoses of multiple lung nodules including miliary tuberculosis and melioidosis as well as nocardiosis. Because of variety of susceptibility depending on the Nocardia species, clinicians need to confirm the species and investigate its susceptibility.

Gordonia crocea sp. nov. and Gordonia spumicola sp. nov. isolated from sludge of a wastewater treatment plant.

Two novel actinobacteria, designated NBRC 107696T and NBRC 107697T, were isolated from sludge samples from a wastewater treatment plant and their taxonomic positions were investigated by a polyphasic approach. The cells of the strains were aerobic, rod-shaped, non-motile and non-endospore-forming. The strains contained glutamic acid, alanine and meso-diaminopimelic acid in the peptidoglycan. Galactose and arabinose were detected as cell-wall sugars. The predominant menaquinone was identified as MK-9(H2) and the major fatty acids were C16  :  0, C18 : 1ω9c and C16 : 1ω7c. The DNA G+C contents of NBRC 107696T and NBRC 107697T were 68.07 and 68.99 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that NBRC 107696T and NBRC 107697T were a clade with members of the genus Gordonia. The highest 16S rRNA gene sequence similarity values were obtained with Gordonia araii IFM 10211T (98.9 %) for NBRC 107697T, and Gordonia malaquae IMMIB WWCC-22T, Gordonia neofelifaecis AD-6T and Gordonia humi CC-12301T (98.1 %) for NBRC 107696T, respectively. The digital DNA-DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel separate species. The names proposed to accommodate these two strains are Gordonia spumicola sp. nov. and Gordonia crocea sp. nov., and the type strains are NBRC 107696T (=IFM 10067T=TBRC 11239T) and NBRC 107697T (=IFM 10881T=TBRC 11240T), respectively.

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses.

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.

Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation.

Aspergillus fumigatus is the major causative fungus of aspergillosis, and many studies have explored the relationship between A. fumigatus and pathogenicity. In the current study, we focused on a fucose-specific lectin, FleA, as a novel molecule which related to the pathogenicity of A. fumigatus. The disruption of the fleA gene did not lead to clear morphological changes compared to parental strain under several stress conditions in culture, but germination become earlier. In comparison with parental strain, the pathogenicity of disruptant was enhanced in a mouse infection model. The pattern of conidial phagocytosis and adhesion to cultured cells did not explain this enhanced pathogenicity. FleA was reported to contain six conserved fucose-binding sites; the analysis of constructed FleA point mutants revealed nonequivalent contribution of the fucose-binding sites to fucose binding. Based on the immune response induced in the cultured cells upon exposure to wild-type and mutant FleA, we propose a model of the FleA molecule in A. fumigatus infection.

Visible DNA Microarray System as an Adjunctive Molecular Test in Identification of Pathogenic Fungi Directly from a Blood Culture Bottle.

A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one Candida parapsilosis, two Candida tropicalis, and one Cryptococcus neoformans). Of note, the microarray detected Candida krusei DNA in two blood cultures from the same patient, whereas the automated system was only positive for Enterococcus faecium Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.

Global gene expression reveals stress-responsive genes in Aspergillus fumigatus mycelia.

BACKGROUND: Aspergillus fumigatus is a human fungal pathogen that causes aspergillosis in immunocompromised hosts. A. fumigatus is believed to be exposed to diverse environmental stresses in the host cells. The adaptation mechanisms are critical for infections in human bodies. Transcriptional networks in response to diverse environmental challenges remain to be elucidated. To gain insights into the adaptation to environmental stresses in A. fumigatus mycelia, we conducted time series transcriptome analyses. RESULTS: With the aid of RNA-seq, we explored the global gene expression profiles of mycelia in A. fumigatus upon exposure to diverse environmental changes, including heat, superoxide, and osmotic stresses. From the perspective of global transcriptomes, transient responses to superoxide and osmotic stresses were observed while responses to heat stresses were gradual. We identified the stress-responsive genes for particular stresses, and the 266 genes whose expression levels drastically fluctuated upon exposure to all tested stresses. Among these, the 77 environmental stress response genes are conserved in S. cerevisiae, suggesting that these genes might be more general prerequisites for adaptation to environmental stresses. Finally, we revealed the strong correlations among expression profiles of genes related to 'rRNA processing'. CONCLUSIONS: The time series transcriptome analysis revealed the stress-responsive genes underlying the adaptation mechanisms in A. fumigatus mycelia. These results will shed light on the regulatory networks underpinning the adaptation of the filamentous fungi.

Transcription factor Afmac1 controls copper import machinery in Aspergillus fumigatus.

Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in ∆Afmac1 cells, while those expressions were induced under copper depletion conditions in wild-type. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the ∆Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.

Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029T.

Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 µg/mL, respectively.

Zamamidine D, a Manzamine Alkaloid from an Okinawan Amphimedon sp. Marine Sponge.

A new manzamine alkaloid, zamamidine D (1), was isolated from an Okinawan Amphimedon sp. marine sponge. The structure of zamamidine D (1) including the relative configuration was elucidated on the basis of spectroscopic data. Zamamidine D (1) is the first manzamine alkaloid possessing a 2,2'-methylenebistryptamine unit as the aromatic moiety instead of a ß-carboline unit. Zamamidine D (1) showed antimicrobial activity against several bacteria and fungi.

Discrepancy between Hepatitis C Virus Genotypes and NS4-Based Serotypes: Association with Their Subgenomic Sequences.

Determination of hepatitis C virus (HCV) genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4) region and 5'-untranslated region (5'-UTR), respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5'-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy.

BACKGROUND: Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae. RESULTS: We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy. CONCLUSIONS: A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Nocardia shinanonensis sp. nov., isolated from a patient with endophthalmitis.

A nocardioform strain IFM 11456T was isolated from the aqueous humor from a patient with endophthalmitis and was characterized to its taxonomic position. IFM 11456T contained arabinose, galactose and meso-diaminopimelic acid in whole-cell hydrolysates and mycolic acids that co-migrated with those from the type strain of Nocardiaasteroides. The acyl type of muramic acid was N-glycolyl. The diagnostic polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and two unidentified glycolipids and the predominant menaquinone was MK-8(H4, ω-cycl.). These characteristics are typical of members of the genus Nocardia. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate represented a novel species of the genus Nocardia and was most closely related to the type strains of Nocardia mikamii JCM 15508T (98.1 %) and Nocardiaaobensis IFM 0372T (98.1 %). However, analysis of partial gyrB sequences showed that strain IFM 11456T had 90.2 % similarity to Nocardia concava IFM 0354T and 90 % to Nocardianiigatensis IFM 0330T. The DNA-DNA relatedness values for strain IFM 11456T compared with N. mikamii JCM 15508T, N. aobensisIFM 0372T and N. concava IFM 0354T ranged from 24.4 to 39.9 %. Phenotypic characteristics that differentiated IFM 11456T from phylogenetically related species were growth at 45 °C, utilization of citrate and growth with inositol as a sole carbon source. On the basis of this polyphasic study, the isolate represents a novel species within the genus Nocardia, for which the name Nocardia shinanonensis sp. nov. is proposed. The type strain is IFM 11456T (=NBRC 109590T=TBRC 5149T).

Multi-azole resistant Aspergillus fumigatus harboring Cyp51A TR46/Y121F/T289A isolated in Japan.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent.

Hyrtinadines C and D, New Azepinoindole-Type Alkaloids from a Marine Sponge Hyrtios sp.

New bisindole alkaloids, hyrtinadines C (1) and D (2), have been isolated from an Okinawan marine sponge Hyrtios sp. The structures of hyrtinadines C (1) and D (2) were elucidated based on analyses of the spectral data. Hyrtinadines C (1) and D (2) were the relatively rare alkaloids possessing a 3,4-fused azepinoindole skeleton. Hyrtinadines C (1) and D (2) showed antimicrobial activity.

Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

Tyrokeradines G and H, new bromotyrosine alkaloids from an Okinawan Verongid sponge.

Two new bromotyrosine alkaloids, tyrokeradines G (1) and H (2), have been isolated from an Okinawan marine sponge of the order Verongida. The structures of 1 and 2 were elucidated on the basis of spectroscopic data. Tyrokeradine G (1) is the first bromotyrosine alkaloid possessing a ß-alanine unit, while tyrokeradine H (2) is a rare bromotyrosine alkaloid possessing a N-substituted pyridinium ring. Tyrokeradines G (1) and H (2) showed antifungal activity.

Prenylated benzophenones from Triadenum japonicum.

Six new prenylated benzophenones, (-)-nemorosonol (1) and trijapins A-E (2-6), were isolated from the aerial parts of Triadenum japonicum. (-)-Nemorosonol (1) and trijapins A-C (2-4) have a common tricyclo[4.3.1.0(3,7)]decane skeleton, while 1 is an enantiomer of (+)-nemorosonol previously isolated from Clusia nemorosa. The absolute configuration of (-)-nemorosonol (1) was assigned by ECD spectroscopy. Trijapins A-C (2-4) are analogues of 1 possessing an additional tetrahydrofuran ring. Trijapins D (5) and E (6) are prenylated benzophenones with a 1,2-dioxane moiety and a hydroperoxy group, respectively. (-)-Nemorosonol (1) exhibited antimicrobial activity against Escherichia coli (MIC, 8 µg/mL), Staphylococcus aureus (MIC, 16 µg/mL), Bacillus subtilis (MIC, 16 µg/mL), Micrococcus luteus (MIC, 32 µg/mL), Aspergillus niger (IC50, 16 µg/mL), Trichophyton mentagrophytes (IC50, 8 µg/mL), and Candida albicans (IC50, 32 µg/mL), while trijapin D (5) showed antimicrobial activity against C. albicans (IC50, 8 µg/mL).

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species.

BACKGROUND: Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology. RESULTS: Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text. CONCLUSION: We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Whole-genome comparison of Aspergillus fumigatus strains serially isolated from patients with aspergillosis.

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.