Enhancing bioremoval of textile dyes by eight fungal strains from media supplemented with gelatine wastes and sucrose

Eight Aspergillus strains were found to be successful in removing textile dyes from liquid media. These fungal strains were grown on medium containing: gelatine wastes and sucrose, as sources of nitrogen and carbon to test the possible speed up of the dyes removing while fungus biomass is building up in the media. The growth of fungal strains ranged from 10 to 110 mg biomass dry weight/100 ml medium. This growth induced high decolorization percentages, which ranged 33-95% within eight days. Two textile dyes Direct brown and Polar red were included in the study. The growth of the fungal strains as well as decolorization percentage of the dyes increased after 5, 6, and 8 days from incubation time with most tested strains. With Direct brown dye the strains number 2, 5, 31 and 37 recorded the highest percentage of decolorization (91, 92, 93 and 95 respectively) after incubation for 6 days. Fungal strains Aspergillus 5 and 31 gave the highest mycelium dry weight being 110 mg. Most of fungal strains induced 86 to 95 percentage of decolorization after 6 days incubation with Polar red dye. The possible toxicity of the remaining supernatant media after fungal biomass removal was tested by Ames test to assess the residual mutagenic agents remaining after dye removal, using three strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538). The results showed that the toxicity of the dyes, measured by Ames test could be removed by the dye absorption on the fungal biomass.     

High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray

Here we report the development of an oligonucleotide microarray method that can identify fungal pathogens in a single reaction. Specific oligonucleotide probes targeted to internal transcribed spacer 2 were designed and synthesized. Fungal DNA was amplified by universal primers, and the PCR product was hybridized with the oligonucleotide microarray. A series of specific hybridization profiles corresponding to species were obtained. The 122 strains of fungal pathogens, including standard and clinically isolated strains, used to test the specificity, stability, and sensitivity of the microarray system belonged to 20 species representing 8 genera. We found that the microarray system can successfully discriminate among the fungal pathogens to the species level, with high specificity and stability. The sensitivity was 15 pg/ml of DNA. This oligonucleotide microarray system represents a rapid, simple, and reliable alternative to conventional methods of identifying common clinical fungal isolates.     

Cytological effects of fungal metabolites produced by fungi isolated from Egyptian poultry feedstuffs

The cytogenetic effects of fungal metabolites produced by 113 strains belonging to 36 fungal species and isolated form 5 substrates of commercial poultry feedstuffs were tested for their effect on the growing root meristems of Allium cepa. The fungal metabolites of Paecilomyces canescens, Aspergillus fumigatus, Syncephalastrum racemosum, Aspergillus terreus and Mucor hiemalis strongly suppressed cell division. Metabolites from other strains had less effect on cell division but permitted the appearance of several abnormalities through different mitotic stages. In general, chromosomal aberrations were more obvious with metabolites of Aspergillus species, Mucor circinelloides and Cladosporium cladosporioides. The mutagenic effects produced by these fungal metabolites reflect the risk that might take place through the consumption of these contaminated feedstuffs.     

Activity of hydrolytic enzymes in fungi isolated from diabetic pregnant women: Is there any relationship between fungal alkaline and acid phosphatase activity and glycemic control?

Ability to respond to environmental changes and secretion of hydrolases are considered to be important for Candida virulence. In this study we determined and compared the activities of 19 different hydrolases of the fungal strains isolated from diabetic and non-diabetic pregnant women. We also looked for the presence of a relationship between hydrolase activities and glycemic control, and, furthermore, evaluated the influence of gestational age on the activity of hydrolases. Mycological examinations were performed for 119 diabetic pregnant women: 47 with diabetes mellitus type I (DM), 72 with gestational diabetes (GDM), and for 132 healthy women (CON). Samples were collected from the vagina, rectum and oral cavity and cultured on Sabouraud media. The fungal hydrolase activities were evaluated using the API ZYM test (bioMerieux). For the 19 different fungal hydrolases tested, 13 activities were present in the isolated fungal strains. The activity of alkaline phosphatase (ALP) in vaginal strains (p=0.028) and acid phosphatase (ACP) in strains from the vagina (p=0.006) and rectum (p=0.049) was significantly lower in DM than in GDM and CON women. In conclusion, we describe for the first time that fungi isolated from pregnant diabetic women have lower activity of both phosphatases compared to fungi isolated from healthy women. Furthermore, similar differences of mean ALP and ACP activities were observed in the course of pregnancy in strains from the vagina and rectum of DM and CON women. However, strains from DM had lower activity at each stage of pregnancy. The highest activity of ALP and ACP was detected at the beginning, then declined, and had the lowest values between the 24(th) and 33(rd) week of gestation. After that period the activity of both phosphatases increased.     

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.     

Lack of a correlation between the vitamin requirement and the ecological position of wood-decay fungi

Forty-eight species of wood-destroying basidiomycetous fungi which are represented by 74 strains were examined for their dependence on exogenous vitamin sources such as thiamine and biotin. In a series of up to 11 consecutive transfers to vitamin-free glucose-salt medium batches in a 4-week rhythm it was shown that (i) the level of vitamin heterotrophy did not depend on whether the fungus was a parasite or a saprophyte, (ii) among both parasites and saprophytes vitamin-independent and highly vitamin-dependent isolates could be detected, (iii) almost 70% of the brown-fungi, but only 30% of the white-rot fungi dependend comparatively little on exogenous vitamin sources, (iv) the degree of vitamin heterotrophy varied in part dramatically among several strains of a given fungal species, (v) under permanent vitamin starvation none of the 74 fungal strains completely ceased growing, but several strains suffered an irreversible physiological collapse whose nature is still unknown. It is concluded that on natural timber resources both vitamin-dependent and vitamin-independent fungal strains can probably develop their normal pathovirulence and kratovirulence properties. Under conditions of vitamin starvation the equilibrium between the vitamin-dependent decay fungus and the vitamin-independent competitor may nevertheless become disturbed.     

Evaluating the resistance to posaconazole by E-test and CLSI broth microdilution methodologies of <Emphasis Type="Italic">Candida</Emphasis> spp. and pathogenic moulds

E-test methodology was compared with Clinical and Laboratory Standards Institute (CLSI) broth microdilution, particularly concerning the detection of resistance to posaconazole among clinical fungal isolates. The susceptibility of a large set of fungal strains (n = 300) was evaluated following 24 and 48 h in two different culture media (RPMI 1640 and Sabouraud agar). Fungal strains were highly susceptible to posaconazole; however, few less susceptible strains were found, mostly regarding Candida albicans, Candida glabrata, Acremonium sp., Cladosporium sp. and Scedosporium apiospermum. Broth microdilution and E-test methods provided similar results for posaconazole-susceptible strains, while the less susceptible fungal strains (10.3% of the strains showed MIC ≥2 μg/mL) resulted in higher discrepancies between the two methodologies, particularly concerning Candida spp. E-test susceptibility values were critically affected by the pH of the culture media. Sabouraud medium provided similar susceptibility results for moulds to those for RPMI, soon after 24 h. Posaconazole resistance was rare in this study, but routine susceptibility methods, such as the E-test, should be able to detect fungal strains with reduced susceptibility. E-test methodology still needs improvements to recognise accurately strains less susceptible to posaconazole.     

急诊监护病房肺部感染的临床研究

目的探讨急诊监护病房（EICU）最常见肺部感染痰菌分布特点及耐药性。方法对125例EICU肺部感染患者痰培养结果及药物敏感试验进行分析。结果共检出致病菌109株,以革兰阴性杆菌（G-菌）为主,共71株（65.1%）,前四位分别为铜绿假单胞菌22株（20.2%）,其中嗜麦芽寡养单胞菌14珠（12.8%）,肺炎克雷伯菌10株（9.2%）,鲍氏不动杆菌9株（8.3%）;革兰阳性菌（G＋菌）28株（25.7%）,其中金黄色葡萄球菌16株（14.7%）;真菌10株（9.2%）,其中白色念珠菌占5.6%。药物敏感试验显示铜绿假单胞菌对抗生素多重耐药率较高,嗜麦芽寡养单胞菌对环丙沙星耐药率较低,肺炎克雷伯菌和鲍氏不动杆菌仍然对碳青酶烯类敏感。结论该组EICU肺部感染以G-杆菌为主;MRSA和真菌感染的比例较高,应根据药敏试验合理使用抗菌药物。     

Identification of Candida albicans clinical isolates by PCR amplification of an EFB1 gene fragment containing an intron-interrupted open reading frame.

The use of a single pair of primers, deduced from the intron and exon nucleotide sequences of the Candida albicans EFB1 gene, in polymerase chain reaction (PCR) assays performed with whole cells of both laboratory strains and clinical isolates of Candida species, resulted in the species-specific amplification of a 785 bp DNA fragment in C. albicans strains. Clinical C. albicans isolates were tested, and 85 out of 86 generated the expected PCR-amplified product; other Candida species, both laboratory strains and clinical isolates, as well as laboratory strains belonging to other fungal genera, including medically relevant taxa, failed to amplify any DNA fragment. In addition, unusual C. albicans isolates (glucosamine- and N-acetylglucosamine-negative) from Africa also yielded the expected PCR-generated DNA fragment. These results indicate that genes containing intron sequences may be useful to design species-specific primers for the identification of fungal strains by PCR.     

α-L-rhamnosidase and β-D-glucosidase activities in fungal strains isolated from alkaline soils and their potential in naringin hydrolysis

α-L-Rhamnosidases (EC 3.2.1.40) and β-D-glucosidases (EC 3.2.1.21) obtained from several microbial sources are potential catalysts in food, beverage, and pharmaceutical industries. However, the enzyme preparations currently used have limitations related to the stability and activity of the enzyme as well to their reuse. A microtiter screening was carried out in 55 fungal strains isolated from alkaline soils, to obtain active α-L-rhamnosidases and β-D-glucosidases at pH 9.0. While α-L-rhamnosidase activity was detected in 45% of the strains tested, β-D-glucosidase activity was found only in 27%. Based on the fungal ability to produce α -L-rhamnosidase activity, cultures were supplemented with naringin to study the activities of the enzymes and the potential of the fungal strains on naringin hydrolysis. About 70% of the fungal strains tested increased the activities of both enzymes in the naringin-supplemented cultures as compared to non-supplemented ones. This effect was higher in Acrostalagmus luteo-albus LPSC 427 (15.3 fold) for α-L-rhamnosidase activity and Metarrhizium anisopliae LPSC 996 (51.1 fold) for β-D-glucosidase activity. All the enzyme preparations tested hydrolyzed naringin at pH 9.0, being that obtained from Acremonium murorun LPSC 927 cultures the one which showed highest hydrolysis. Here, different fungal species are reported for the first time for their ability to produce α-L-rhamnosidase and β-D-glucosidase activity at alkaline pH.     

Mineralization of mono-nitrophenols by Bjerkandera adusta and Lentinus squarrosulus and their extracellular ligninolytic enzymes

Nitroaromatic compounds constitute a major class of widely distributed environmental contaminants. Fifty fungal strains were screened for their potential to tolerance with 2-nitrophenol, 3-nitrophenol and 4-nitrophenol on solid medium supplemented with 2% malt extract (MEA). Growth rate (mm/day) was determined at three concentrations (0.25, 0.5 and 1 mM) of all the three nitrophenols. From the fifty fungal strains only Bjerkandera adusta and Lentinus squarrosulus were able to tolerate all the three nitrophenols (NPs). These white-rot fungi (WRF) were chosen for liquid medium studies for the mineralization of mono-nitrophenols and ligninolytic enzyme activity at 0.25 mM concentration. Both varieties completely removed 2-NP and 3-NP while 4-NP was hard to mineralize. AAO (Aryl Alcohol Oxidase) is the main oxidase enzyme in B. adusta while laccase plays important role in L. squarrosulus. MnP (Manganese peroxidase) is the main peroxidase enzyme in both varieties. These fungal strains were capable to degrade nitrophenols and could be used for bioremediation applications on large scale.     

Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.     

儿童真菌感染的病原学研究

目的探讨广州地区儿童真菌感染的病原分布特点及其耐药状况,为防治儿童真菌感染提供实验室依据。方法对患儿感染部位的真菌进行分离培养和鉴定：以ATB^TM FUNGUS3酵母样真菌药敏试验条进行常用抗真菌药物的敏感性分析。结果从患儿标本中分离出558株真菌,主要来自呼吸道有299株,占53．58％;其次是消化道、伤口（创口）、泌尿系统和血液等,分别占28．14％、6．27％、4．66％、3．76％。其中白色假丝酵母菌367株,占65．77％;其次为热带假丝酵母菌、光滑假丝酵母菌、近平滑假丝酵母菌、克柔假丝酵母菌、季也蒙假丝酵母菌等,分别占15．28％、5．02％、4．48％、3．41％、2．69％。从骨髓中检出5株马尔尼菲青霉,从脑脊液中检出3株新型隐球菌。真菌对两性霉素B、5-氟胞嘧啶、氟康唑、伊曲康唑、伏立康唑等总耐药率分别为8．78％、4．84％、10．54％、1．36％、0．85％。结论引起儿童真菌感染的主要病原菌是白色假丝酵母菌。对真菌感染应该有针对性地使用高效的抗真菌药物进行早期治疗。     

Biosynthesis and characterization of Au-nanostructures by metal tolerant fungi

Microorganisms being eco-friendly and encompassing special features are widely employed in the biosynthesis of different metal nanoparticles (NPs). In the present study, the potential of different fungal strains has been explored for the synthesis of various morphologies of gold nanostructures. Preliminary confirmation of nanosized particles formation has been done with visual observation as the synthesized gold nanoparticles exhibited a variety of colours covering whole visible spectra. Crystallographic and morphological characterization of synthesized nano-materials associated with different fungal strains have been done using X-ray diffraction (XRD) and transmission electron microscope (TEM). Nanoparticles morphology dependence on different fungal strains has been studied and described in detail. A wide variation in shape and size of gold nanoparticles with different strains has been reported as a function of strain characteristics.     

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.     

A rapid method for ploidy determination in fungal cells

Summary Haploid and diploid strains of Schizosaccharomyces pombe were used to show that a method of determining ploidy developed for plant cells and based on the integration of DAPI nuclear fluorescence could be applied to a fungal system. The fluorescence of nuclei stained with DAPI was quantified using a computer controlled imaging system and found to correlate precisely with ploidy. The method has potentially wide applications for ploidy determination in less well characterised fungal species involved in plant infection and industrial processes.     

Tumour necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.

Tumour necrosis factor (TNF) was detected in serum from mice challenged with Paracoccidioides brasiliensis. The serum TNF levels of mice challenged with an avirulent strain were significantly higher than those of mice challenged with a virulent strain, and the same was observed for the TNF levels of mice challenged with a cell wall fraction (F1) from the two fungal strains. Fraction F1 consisted of chitin and beta-glucan; but although the chitin contents were similar for the two strains, the avirulent strain allowed a greater content of beta-glucan. The beta-glucan, purified from both strains, increased serum TNF levels in an identical dose-dependent manner, whereas purified chitin did not induce serum TNF levels. P. brasiliensis, the F1 fractions and beta-glucan induced macrophages to secrete TNF in vitro. The differences in TNF levels, induced by the different fungal strains, were correlated with the beta-glucan concentrations in the cell walls of both the avirulent and virulent strains of P. brasiliensis. These findings support a role for TNF in the pathogenicity of P. brasiliensis.     

Development and validation of a quantitative PCR assay for diagnosis of scedosporiosis

Scedosporium apiospermum and Scedosporium prolificans are fungal pathogens that can cause severe human infections, including disseminated mycosis in immunocompromised patients. Two real-time PCR (RT-PCR) assays for the diagnosis of these species were developed and validated for the classification of clinical strains and for the detection of DNA in clinical samples by use of a murine model of invasive infection. A total of 14 clinical strains and 141 samples, including blood, serum, and lung samples from infected CD1 mice, were analyzed. Each RT-PCR methodology used a species-specific molecular beacon probe targeting a highly conserved region of the fungal ribosomal DNA gene. Results showed 100% specificity and a detection limit of 10 fg of DNA for both assays. The sensitivities for the S. prolificans-specific PCR assay were 100% for cultured clinical strains, 95.5% for lung tissues, 85% for serum, and 83.3% for blood. For S. apiospermum, the sensitivities were 100% for clinical strains and 97.2%, 81.8%, and 54.5% for lung tissues, serum, and blood, respectively. Both techniques can be useful for clinical diagnosis, and further studies are warranted.     

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory.     

Sporothrix nivea n. sp.--a methanol- and hydrocarbon-metabolizing fungus

Two fungal strains of the genus Sporothrix, isolated from industrial waste water, were characterized with respect to some morphological and physiological properties. After comparison with other species of the genus Sporothrix, the fungal strains are described as Sporothrix nivea, n. sp. Among 28 carbon sources tested, such as sugars, polysaccharides, alcohols, organic acids, and hydrocarbons, 23 are utilized. The most remarkable property of Sporothrix nivea is the utilization of both n-alkanes and methanol.