Multilocus sequence identification of Penicillium species in cork bark during plank preparation for the manufacture of stoppers

Despite several studies reporting Penicillium as one of the most frequent fungal genera in cork planks, the isolates were rarely identified to species level. We conducted a detailed study to identify Penicillium species from the field to the factory environment prior to and after boiling the cork planks. A total of 84 samples were analyzed. Of the 486 Penicillium isolates phenotypically identified, 32 representative or unusual strains were selected for identification by multilocus DNA sequence type. Cork proved to be a rich source of Penicillium biodiversity. A total of 30 taxa were recognized from cork including rarely seen species and 6 phylogenetically unique groups. Spores of some species lodged deep in cork can survive the boiling process. P. glabrum, P. glandicola and P. toxicarium, species with high CFU numbers in the field, are still frequently present in cork after boiling. Other species are killed by the boiling treatment and replaced by Penicillium species originating from the factory environment. Species known to contribute to cork taint were isolated at all stages. Good manufacturing practices are necessary at all stages in the preparation of cork planks to minimize the load of Penicillium species that produce cork taint.     

Necrotizing pneumonia caused by Penicillium chrysogenum.

We report a case of necrotizing pneumonia due to Penicillium chrysogenum in a 57-year-old woman operated on for lung cancer. The residual right lower pulmonary lobe was infiltrated by Penicillium chrysogenum. The patient underwent a second pulmonary right lobectomy and was successfully treated with oral itraconazole. To our knowledge, this is the first case of pneumonia due to P. chrysogenum.     

Phylogeny and PCR identification of the human pathogenic fungus Penicillium marneffei.

The phylogenetic position of the human pathogenic fungus Penicillium marneffei was assessed from the nucleotide sequences of the nuclear and mitochondrial ribosomal DNA regions. Phylogenetic analysis determined that P. marneffei is closely related to species of Penicillium subgenus Biverticillium and sexual Talaromyces species with asexual biverticillate Penicillium states. Knowledge of the phylogenetic position of P. marneffei facilitated the design of unique oligonucleotide primers, from the nuclear ribosomal DNA internal transcribed spacer region, for the specific amplification of P. marneffei DNA. These primers were successful at selectively amplifying DNA from six isolates of P. marneffei and excluding the other species tested, which included Penicillium subgenus Biverticillium and Talaromyces species and several well-known fungal pathogens, namely, Aspergillus fumigatus, Coccidioides immitis, Histoplasma capsulatum, and Pneumocystis carinii. The primers that we have developed for the specific amplification of P. marneffei have the potential to be incorporated in a PCR identification system which could be used for the identification of this pathogenic agent from clinical material.     

Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of Penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two-dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS-PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.     

Isolation and characterization of a cDNA clone encoding one IgE-binding fragment of Penicillium brevicompactum

BACKGROUND: The abundance of allergenic Penicillium species has been associated with an increased incidence of childhood asthma and pulmonary bleeding. Penicillium brevicompactum has been identified as the most prevalent indoor species of this genus. However, detailed studies on the allergens of the ubiquitous Penicillium species are still lacking. For the characterization of allergens of prevalent Penicillium species, molecular cloning of the allergen genes of P. brevicompactum was performed in the present study. METHODS: A phage cDNA library of P. brevicompactum was constructed in Uni-ZAP XR vector using mRNA isolated from the organism. The cDNA library of P. brevicompactum was screened using pooled atopic sera. RESULTS: Screening of P. brevicompactum cDNA library resulted in one positive clone encoding an estimated molecular weight of 11 kDa polypeptide, rich in acidic residues (>20%), with a pI of 3.87. This clone was designated as Pen b 26 and found to be reactive only against the atopic sera obtained from individuals sensitive to P. brevicompactum. The amino acid sequence analysis of Pen b 26 revealed that it had strong homology to the 60S acidic ribosomal protein P1 family from different eukaryotic sources, predominantly fungal aero-allergens. Other features of Pen b 26 including having high alpha-helical content (>50%), alanine-rich residues (>20%), and a well-conserved C-terminal epitope region fits well into the common properties of 60S acidic ribosomal proteins. CONCLUSIONS: The results obtained suggest that the allergenic clone, Pen b 26 is a 60S acidic ribosomal protein P1 of P. brevicompactum and shows strong similarity to other P1 family proteins.     

Antibodies against synthetic oligosaccharide antigens reactive with Extracellular polysaccharides produced by moulds

Aspergillus and Penicillium species produce extracellular polysaccharides (EPS) of which (1→5)‐interlinked β‐D‐galactofuranosides are immunodominant. Synthetic tetra‐ and heptamers of (1→5)‐interlinked P‐D‐galactofuranosides conjugated to tetanus toxoid were used to produce antibodies in rabbits. The antibodies obtained with the tetramer conjugate reacted only with EPS of a few Penicillium and Aspergillus species. Antibodies obtained with the heptamer conjugate were reactive with EPS of all the Penicillium and Aspergillus species tested. The reaction with P. digitatum EPS could be inhibited by the synthetic heptamer of β‐(1→5) interlinked galactofuranosides. No reactions were observed with EPS of moulds belonging to any other genus.     

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.

BACKGROUND: Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. OBJECTIVE: The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. METHODS: BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. RESULTS: Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. CONCLUSION: Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.     

Immunoreactivity of a 38-kilodalton Penicillium marneffei antigen with human immunodeficiency virus-positive sera.

Penicillium marneffei produced and secreted a 38-kDa antigen that appeared to be specific for this dimorphic fungus. This component could not be detected in antigenic extracts of Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, and two other species of Penicillium by immunoblot analysis against the sera from patients with culture-confirmed penicilliosis marneffei. Antibody reactive with this antigen was found in a large proportion of human immunodeficiency virus (HIV)-positive patients, indicating a presumptive diagnosis of P. marneffei infection. A small number of asymptomatic HIV-seropositive patients and HIV-seropositive patients with other fungal infections were also found to be positive by this analysis, suggesting that subclinical or mixed fungal infections involving P. marneffei are not uncommon.     

Effect of inoculation with Penicillium albidum, a phosphate-solubilizing fungus, on the growth of Trifolium pratense cropped in a volcanic soil

Volcanic soils in the south of Chile have an elevated quantity of total P, which is scarcely available due to its high P fixation capacity. One strategy for increasing the availability of P for the vegetables that grow there would be to use phosphate-solubilizing microorganisms. In one assay conducted in a greenhouse on a volcanic soil, the effect of inoculation with Penicillium albidum, a phosphate-solubilizing fungus, was studied on the growth of red clover (Trifolium pratense L). Some chemical and biological properties of the soil were also evaluated. There were three treatments: a) active inoculum [In(+)], b) inactive inoculum (autoclaved) [In(-)] and c) without inoculum [In(0)], each one done in three replicates. The In(+) significantly (P < 0.050) increased the growth of the plants, contributing particularly to root development. The P mobilized to the shoot with In(+) was higher than twofold related to In(0) and In(-) treatments; however, the In(+) plants had similar concentration of shoot P. In the soil, available-P was not statistically different (P < 0.050) among the treatments but phosphatase activity in In(+) was higher (P < 0.050) in comparison to In(0). The results suggest that Penicillium albidum contributed to growth and nutrition of the red clover through the induction of root development and enhancing phosphate mobilization from the soil and into the plant. It is concluded that Penicillium albidum, under greenhouse conditions, in soils deficient in available P can increase the inoculation potential for volcanic soils in Chile. Anyway further studies are required, especially in organic farming where the use of soluble P fertilizer is avoided.     

Pulmonary infection with Penicillium citrinum in a patient with multiple myeloma

A 60-year-old male patient undergoing chemotherapy for multiple myeloma Stage II presented to our hospital with complaints of cough, haemoptysis, fever and loose stools. Sputum sample was sent for fungal culture. Fungal culture on Sabouraud dextrose agar yielded bluish-green velvety growth with orange-to-red diffusible pigment on the reverse. The isolate was identified as Penicillium species, probably Penicillium citrinum or Penicillium pinophilus. As the isolate did not exhibit thermal dimorphism, the possibility of the fungal isolate being Penicillium marneffei was ruled out. The isolate was sent for molecular identification and confirmation, which was identified as P. citrinum. His HIV status was negative. In this case, his immunocompromised state due to multiple myeloma and chemotherapy could have predisposed him to this fungal infection, which is an emerging infection and a rare manifestation seen in high-risk patients receiving targeted therapies.     

Antigen characterization of major cork moulds in Suberosis (cork worker's pneumonitis) by immunoblotting

BACKGROUND: We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry. METHODS: Serum-specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting. RESULTS: Both HP and asthmatic patients' sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12-13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51-55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi. CONCLUSIONS: Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12-13.5 and 33 kDa proteins of P. glabrum and the 16 and 51-55 kDa proteins of C. sitophila may be major antigens in Suberosis.     

The prevalence of IgE antibody reactivity against the alkaline serine protease major allergen of Penicillium chrysogenum increases with the age of asthmatic patients.

BACKGROUND: Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. OBJECTIVE: The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups. METHODS: Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. RESULTS: Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. CONCLUSIONS: Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.     

cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.     

伏立康唑治疗艾滋病相关马尔尼菲青霉菌感染的临床观察

目的初步了解伏立康唑治疗艾滋病（AIDS）合并播散性马尔尼菲青霉菌病（PSM）的疗效和安全性。方法选择2008年1月至2010年6月期间在广州市第八人民医院感染科住院经血/骨髓培养确证的18例艾滋病合并PSM患者,全部给予伏立康唑规范治疗4周,观察其临床疗效及安全性。结果 18例患者CD4＋T细胞计数均小于50copies/μl,以反复发热、乏力、咳嗽、消瘦、皮疹等为主要临床表现。经用伏立康唑治疗14d后,有效6例（33.3%）,显效7例（38.9%）,无效5例（27.8%）,总有效率72.2%。治疗28d时,有效11例（61.1%）,显效3例（16.7%）,无效4例（22.2%）,总有效率77.8%。治疗后TB疗前-疗后为（12.68±20.74）μmol/L（P=0.02）、ALT疗前-疗后（20.80±35.17）U/L（P=0.02）、AST疗前-疗后（86.01±110.07）U/L（P〈0.01）,三个指标均较治疗前降低,且差异有统计学意义;CR疗前-疗后（0.497μmol/L,P=0.62）、BUN疗前-疗后（0.631mmol/L,P=0.53）、K＋疗前-疗后[（0.15±0.90）mmol/L,P=0.49],三个指标治疗前后差异无统计学意义。结论伏立康唑治疗AIDS合并PSM疗效较确切,肝肾损害等不良反应少,可达预期疗效。     

Putative structure and characteristics of a red water-soluble pigment secreted by Penicillium marneffei.

The dimorphic fungus, Penicillium marneffei, produces and secretes a brick red pigment, during growth at temperatures below 30 degrees C. It generally diffuses into commonly used media like Sabouraud dextrose agar and malt extract agar. The pigment was purified by reverse-phase liquid chromatography and subjected to structural determination by elemental and spectral analysis using atomic absorption (AAS), ultra violet and visible (UV-VIS), fluorescence, infra red (IR), nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS-MS). The pigment showed a buffering ability in aqueous solutions, maintaining an alkaline pH of 8.0. It behaved as a colorimetric pH indicator over a wide acidic and alkaline pH range, with discoloration occurring ostensibly through hydrolysis of key chemical groups at extremely acidic pH ( approximately 2.0). The pigment was found to have some structural resemblance with the copper-colored pigment (herquinone) produced by Penicillium herquei as both pigments contain the phenalene carbon framework. The notable differences between herquinone and the pigment produced by P. marneffei are (i) the latter's apparent dimerization through a sulphur-sulphur (disulfide) bond and (ii) the presence of 1,1,3,3-tetramethyl-2,3-dihydropyrrole moiety in the latter instead of 2,3,3-trimethyl-2,3-dihydrofuran moiety found in the former. The delineation of the structure of the pigment produced by Penicillium marneffei may help in understanding certain aspects of the biology of this pathogenic fungus.     

Sialic Acid-Dependent Recognition of Laminin by Penicillium marneffei Conidia

Immunofluorescence microscopy demonstrated that laminin bound to the surface of Penicillium marneffei conidia. Attachment of P. marneffei conidia in an adherence assay was inhibited by soluble laminin and anti-laminin antibody. N-Acetylneuraminic acid abolished adherence, indicating an interaction mediated by a sialic acid-specific lectin.     

Distinct immunologic properties of Penicillium marneffei yeasts obtained from different in vitro growth conditions

A dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis, a life-threatening disseminated disease in immunocompromised hosts predominantly found in southeast Asia and southern China. P. marneffei is the only known Penicillium that possesses a dimorphic characteristic. Since it is difficult to produce large amount of P. marneffei yeasts in vivo for experimentation purpose, yeast cells were produced in different in vitro conditions as alternatives. We interested in investigating the immunologic properties of yeast cells from different culture preparations. It was found that yeast cells obtained from brain heart infusion broth and Sabouraud dextrose broth did not resemble those resided in clinical specimens. A solution of 1% peptone, on the other hand, could induce a direct conidial transition into fission yeasts. Ability of yeast cells in each preparation to activate macrophages was determined by analyzing surface expression of CD40 and CD86 co-stimulatory molecules after two days of co-cultivation. Every P. marneffei yeast cell preparation demonstrated such ability. However, the ones from Sabouraud dextrose broth seemed to induce less phagocytosis. Additionally, although distinct antigenic profiles and lack of conformity in antigenic expression were observed among yeast cells from different culture conditions, most major immunogenic bands were present when Western analysis was performed using polyclonal antisera from penicilliosis patients. The results of the study raise attention on immunological and biochemical characteristics of P. marneffei yeasts if such preparations are to be used in future laboratory investigations.     

A case of disseminated mycosis in a German shepherd dog due to Penicillium purpurogenum.

The genus Penicillium is among the most common contaminant fungi in the environment. Around 15 species are known to cause opportunistic human mycoses, in immunocompromised patients. Until now, Penicillium purpurogenum has been involved in only three human cases of pulmonary diseases but no infections in animals have been reported. Most disseminated mycoses in dogs are caused by members of the genus Aspergillus, with the predisposing factors in these cases being difficult to define. The case reported here involved a 4-year-old female German shepherd dog (GSD) with forelimb instability and back pain. Clinical examination showed hyperthermia, generalized lymphadenomegaly and kyphosis. Radiological findings of the spine revealed areas of discospondilitis involving thoracic and lumbar vertebrae. Microscopic observations of fine needle aspiration biopsies (FNAB) of lymph-nodes showed regular, septate, branching fungal hyphae. Itraconazole therapy was started but the subject died six days later. Disseminated necrotic areas were detected in enlarged lymph-nodes, liver and spleen. Vertebral granulomas within lytic areas in T10-T11 and L2-L3, were observed. Cultures inoculated with samples obtained from lymph-node FNAB and bioptic material from necropsied organs revealed the presence of pure cultures of Penicillium, subsequently identified as P. purpurogenum. Apart from female GSD's suspected predisposition to disseminated mycoses described in literature, no other predisposing factors were ascertained in this case.     

Production of antigenic extracellular polysaccharides by penicillium aurantiogriseum and penicillium digitatum

Antigenic extracellular polysaccharides (EPS) were produced by Penicillium digitatum and P. aurantiogriseum under various conditions, including different carbon and nitrogen sources, glucose concentrations and incubation periods. With lactate as carbon source no antigenic EPS was produced. EPS consisted mainly of glucose, mannose and galactose. Differences were seen in the monosaccharide composition. The amount of glucose in the medium (1–30 g/l) influenced growth and the production of antigenic EPS moderately. The antigenicity of P. aurantiogriseum EPS, but not that of P. digitatum, decreased with prolonged incubation. P. aurantiogriseum, but not P. digitatum, grew well with nitrate as the nitrogen source.