The genus Blastobotrys, which now includes species previously assigned to the synonymous genera Arxula and Sympodiomyces, represents the anamorph of the ascosporogenous genus Trichomonascus. Six novel species are proposed for assignment to Blastobotrys. They were detected from their unique nucleotide sequences in large-subunit rDNA, ITS1-5.8S-ITS2 rDNA, mitochondrial small-subunit rDNA and the cytochrome oxidase II gene. The proposed novel species are Blastobotrys americana sp. nov. (type strain NRRL Y-6844(T)=CBS 10337(T); substrate unknown; Kansas, USA), Blastobotrys illinoisensis sp. nov. (type strain NRRL YB-1343(T)=CBS 10339(T); from forest debris; Illinois, USA), Blastobotrys malaysiensis sp. nov. (type strain NRRL Y-6417(T)=CBS 10336(T); from soil; Malaysia), Blastobotrys muscicola sp. nov. (type strain NRRL Y-7993(T)=CBS 10338(T); from moss; Louisiana, USA), Blastobotrys peoriensis sp. nov. (type strain NRRL YB-2290(T)=CBS 10340(T); from a fungus; Peoria, IL, USA) and Blastobotrys raffinosifermentans sp. nov. (type strain NRRL Y-27150(T)=CBS 6800(T); substrate unknown).
Psidium guajava wilt is known from South Africa, Malaysia and Taiwan. The fungus causing this disease, Myxosporium psidii, forms dry chains of conidia on surfaces of pseudoparenchymatous sporodochia, which develop in blisters on bark. Similar sporodochia are characteristic of Nalanthamala madreeya, the type species of Nalanthamala. Nalanthamala, therefore, is the appropriate anamorph genus for Myxosporium psidii, while Myxosporium is a nomen nudum (based on M. croceum). For M. psidii the combination Nalanthamala psidii is proposed. Nalanthamala psidii, the palm pathogen Gliocladium (Penicillium) vermoesenii, another undescribed anamorphic species from palm, two species of Rubrinectria and the persimmon pathogen Acremonium diospyri are monophyletic and belong to the Nectriaceae (Hypocreales) based on partial nuclear large subunit ribosomal DNA (LSU rDNA) analyses. Rubrinectria, therefore, is the teleomorph of Nalanthamala, in which the anamorphs are classified as N. vermoesenii, N. diospyri or Nalanthamala sp. Nalanthamala squamicola, the only other Nalanthamala species, has affinities with the Bionectriaceae and is excluded from this group. Rubrinectria/Nalanthamala species form dimorphic conidiophores and conidia in culture. Fusiform, cylindrical, or allantoid conidia arise in colorless liquid heads on acremonium-like conidiophores; ovoidal conidia with somewhat truncated ends arise in long, persistent, dry chains on penicillate conidiophores. No penicillate but irregularly branched conidiophores were observed in N. diospyri. Conidia of N. psidii that are held in chains are shorter than those of N. madreeya, of which no living material is available. Nalanthamala psidii and N. diospyri are pathogenic specifically to their hosts. They form pale yellow to pale orange or brownish orange colonies, respectively, and more or less white conidial masses. Most strains of Rubrinectria sp., Nalanthamala sp. and N. vermoesenii originate from palm hosts, form mostly greenish or olive-brown colonies and white-to-salmon conidial masses. They form a monophyletic clade to which Nalanthamala psidii and N. diospyri are related based on analyses of the internal transcribed spacer regions and 5.8S rDNA (ITS rDNA), LSU rDNA, and partial beta-tubulin gene. Few polymorphic sites in the ITS rDNA and beta-tubulin gene indicate that Nalanthamala psidii comprises two lineages, one of which has been detected only in South Africa.
We report a fatal case of Candida auris that was involved in mixed candidemia with Candida tropicalis, isolated from the blood of a neutropenic patient. Identification of both isolates was confirmed by amplification and sequencing of internal transcribed spacer and D1/D2 domain of large subunit in rRNA gene. Antifungal susceptibility test by E-test method revealed that C. auris was resistant to amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole and voriconazole. On the other hand, C. tropicalis was sensitive to all antifungal tested. The use of chromogenic agar as isolation media is vital in detecting mixed candidemia.
A yeast-like organism was isolated from the skin scraping sample of a stasis dermatitis patient in the Mycology Unit Department of Medical Microbiology, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The isolate produced no pigment and was not identifiable using chromogenic agar and API 20C AUX. The fungus was identified as Metschnikowia sp. strain UM 1034, which is close to that of Metschnikowia drosophilae based on ITS- and D1/D2 domain-based phylogenetic analysis. However, the physiology of the strain was not associated to M. drosophilae. This pathogen exhibited low sensitivity to all tested azoles, echinocandins, 5-flucytosine and amphotericin B. This study provided insight into Metschnikowia sp. strain UM 1034 phenotype profiles using a Biolog phenotypic microarray (PM). The isolate utilized 373 nutrients of 760 nutrient sources and could adapt to a broad range of osmotic and pH environments. To our knowledge, this is the first report of the isolation of Metschnikowia non-pulcherrima sp. from skin scraping, revealing this rare yeast species as a potential human pathogen that may be misidentified as Candida sp. using conventional methods. Metschnikowia sp. strain UM 1034 can survive in flexible and diverse environments with a generalist lifestyle.
Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 μg/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.
The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.
Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.
This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture-wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony-forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture-wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR-RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI-5.8S-ITSII region, enabling swift and precise identification of Candida species.
The mycoheterotrophic lifestyle has enabled some plant lineages to obtain carbon from their mycorrhizal symbionts. The mycoheterotrophic genus Epirixanthes (Polygalaceae) consists of six species from tropical Asia. Although it is probably closely related to the chlorophyllous genus Salomonia and linked to arbuscular mycorrhizal fungi, lack of DNA sequence data has thus far prevented these hypotheses from being tested. Therefore, the evolutionary history of Epirixanthes remains largely unknown.
Invasive candidiasis is one of the most common nosocomial fungal infections worldwide. Delayed implementation of effective antifungal treatment caused by inefficient Candida diagnosis contributes to its notoriously high mortality rates. The availability of better Candida diagnostic tools would positively impact patient outcomes. Here, we report on the development of a single-tube, dual channel pentaplex molecular diagnostic assay based on Multiplex Probe Amplification (MPA) technology. It allows simultaneous identification of C. auris, C. glabrata and C. krusei, at species-level as well as of six additional albicans and non-albicans pathogenic Candida at genus level. The assay overcomes the one-channel one-biomarker limitation of qPCR-based assays. Assay specificities are conferred by unique biomarker probe pairs with characteristic melting temperatures; post-amplification melting curve analysis allows simple identification of the infectious agent. Alerting for the presence of C. auris, the well-characterised multi-drug resistant outbreak strain, will facilitate informed therapy decisions and aid antifungal stewardship. The MPA-Candida assay can also be coupled to a pan-Fungal assay when differentiation between fungal and bacterial infections might be desirable. Its multiplexing capacity, detection range, specificity and sensitivity suggest the potential use of this novel MPA-Candida assay in clinical diagnosis and in the control and management of hospital outbreaks.
Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.
Cryptococcus albidus and C. laurentii were the predominant non-neoformans cryptococci isolated during an environmental sampling study for C. gattii at Klang Valley, Malaysia. Cryptococcus gattii was not isolated from any of the environmental samples. Cryptococcus albidus and C. laurentii were isolated mainly from vegetative samples of Eucalyptus trees and bird droppings. Upon testing on canavanine-glycine-bromothymol blue (CGB) agar, all the C. albidus isolates remained unchanged. Interestingly, a total of 29 (76.3%) C. laurentii isolates formed blue colours on the CGB agar. Sequence analysis of ITS1-5.8rDNA-ITS2 gene sequences (468 bp) of four CGB-blue C. laurentii isolates demonstrated the closest match (99%) with that of C. laurentii CBS 7140. This study demonstrated the diverse environmental niche of C. albidus and C. laurentii in Malaysia.
This paper studied associations between ocular symptoms, rhinitis, throat and dermal symptoms, headache and fatigue in students by ethnicity and in relation to exposure to chemical microbial markers and fungal DNA in vacuumed dust in schools in Malaysia. A total of 462 students from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated (96% response rate). Dust was vacuumed from 32 classrooms and analysed for levels of five types of endotoxin as 3-hydroxy fatty acids (C10, C12, C14, C16 and C18 3-OH), muramic acid, ergosterol and five sequences of fungal DNA. Multiple logistic regression was applied. Totally 11.9% reported weekly ocular symptoms, 18.8% rhinitis, 15.6% throat and 11.1% dermal symptoms, 20.6% headache and 22.1% tiredness. Totally 21.1% reported pollen or furry pet allergy (atopy) and 22.0% parental asthma or allergy. Chinese students had less headache than Malay and Indian had less rhinitis and less tiredness than Malay. Parental asthma/allergy was a risk factor for ocular (odds ratio=3.79) and rhinitis symptoms (OR=3.48). Atopy was a risk factor for throat symptoms (OR=2.66), headache (OR=2.13) and tiredness (OR=2.02). There were positive associations between amount of fine dust in the dust samples and ocular symptoms (p<0.001) and rhinitis (p=0.006). There were positive associations between C14 3-OH and rhinitis (p<0.001) and between C18 3-OH and dermal symptoms (p=0.007). There were negative (protective) associations between levels of total endotoxin (LPS) (p=0.004) and levels of ergosterol (p=0.03) and rhinitis and between C12 3-OH and throat symptoms (p=0.004). In conclusion, the amount of fine dust in the classroom was associated with rhinitis and other SBS symptoms and improved cleaning of the schools is important. Endotoxin in the school dust seems to be mainly protective for rhinitis and throat symptoms but different types of endotoxin could have different effects. The ethnic differences in symptoms among the students deserve further attention.
Leaf spot diseases are mainly caused by fungi including Fusarium. In the present study several species of Fusarium were isolated from the leaf spot lesion of mango (Mangifera indica L.) Based on morphological characteristics, TEF-1α sequences and phylogenetic analysis, five species were identified as F. proliferatum, F. semitectum, F. mangiferae, F. solani and F. chlamydosporum. Pathogenicity test indicated that representative isolates of F. proliferatum, F. semitectum and F. chlamydosporum were pathogenic on mango leaves causing leaf spot with low to moderate virulence. Nevertheless, abundance of spots on the leaf can disrupt photosynthesis which in turn reduced growth, and lead to susceptibility to infection by opportunistic pathogens due to weakening of the plant. Fusarium solani and F. mangiferae were non-pathogenic and it is possible that both species are saprophyte which associated with nutrient availability on the surface of the leaf through decaying leave tissues. The occurrence of Fusarium spp. on the leaf spot lesion and the effect from the disease needs to be considered when developing disease management method of mango cultivation as numerous spot on the leaves could effect the photosynthesis process and finally giving low yield and less quality of mango.
The genetic heterogeneity and antifungal susceptibility patterns of Candida parapsilosis isolated from blood cultures of patients were investigated in this study. Randomly amplified polymorphic DNA (RAPD) analysis generated 5 unique profiles from 42 isolates. Based on the major DNA fragments of the RAPD profiles, the isolates were identified as RAPD type P1 (29 isolates), P2 (6 isolates), P3 (4 isolates), P4 (2 isolates) and P5 (1 isolate). Sequence analysis of the internal transcribed spacer (ITS) gene of the isolates identified RAPD type P1 as C. parapsilosis, P2 and P3 as Candida orthopsilosis, P4 as Candida metapsilosis, and P5 as Lodderomyces elongisporus. Nucleotide variations in ITS gene sequences of C. orthopsilosis and C. metapsilosis were detected. Antifungal susceptibility testing using Etests showed that all isolates tested in this study were susceptible to amphotericin B, fluconazole, ketoconazole, itraconazole and voriconazole. C. parapsilosis isolates exhibited higher MIC(50) values than those of C. orthopsilosis for all of the drugs tested in this study; however, no significant difference in the MICs for these two Candida species was observed. The fact that C. orthopsilosis and C. metapsilosis were responsible for 23.8 and 4.8 % of the cases attributed to C. parapsilosis bloodstream infections, respectively, indicates the clinical relevance of these newly described yeasts. Further investigations of the ecological niche, mode of transmission and virulence of these species are thus essential.
Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.
The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.