Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.

泸州老窖古酿酒作坊空气微生物分布特征研究

本文主要研究了泸州老窖古酿酒作坊内外环境空气真菌和空气细菌的群落结构和分布特征。结果如下： 作坊内外环境空气微生物浓度差别显著，并随季节变换而变化，春、夏季微生物浓度较高，秋、冬季较低，空气真菌在夏季达到最高，细菌在春季最高。 古作坊内外环境检测到的真菌均为16 属，但优势菌属不同，作坊外的优势菌属为青霉属(Penicillium)、曲霉属(Aspergillus)、无孢菌(non-sporing)、枝孢霉属(Cladosporium)和链格孢属(Alternaria)；而作坊内优势菌属为曲霉属、青霉属、酵母菌(Yeast)、无孢菌，作坊内还含有较高浓度的根霉属（Rhizopus）、毛霉属（Mucor）、短梗霉属（Aureobasidiu），枝孢霉属和链格孢属等，曲霉属、酵母菌、根霉属、毛霉属为古酿酒作坊重要的酿酒真菌，青霉属、链格孢属为酿酒不利菌群。对古作坊内曲霉属进行了初步鉴定，主要是小冠曲霉（A.cristatellus）、米曲霉（A.oryzae）、黑曲霉（A.niger）和白曲霉（A.cadidus）。 空气细菌10 属21 种，作坊内外环境的优势菌属均为芽孢杆菌属（Bacillus）、微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonad)，其中芽孢杆菌属在作坊内占有绝对的优势，浓度比在40℅以上，是古酿酒作坊重要的酿酒细菌，另外还检测到较高浓度的乳酸杆菌（lactobucillus），这类菌容易使酒味发涩发苦，为酿酒不利菌。 作坊内外环境空气微生物表现出明显的交流现象。作坊内，青霉属、枝孢霉属、链格孢属、葡萄球菌属等杂菌占有一定比例；而在作坊外,芽孢杆菌属、曲霉属、根霉属(Rhizopus)、酵母菌等处于相对较高水平，绿化环境较好的营沟头作坊内的短梗霉属，枝孢霉属和链格孢属等杂菌含量低于什字头和新街子作坊。 The community structure and distribution characteristic of airborne microbes was investigated in ancient brewage workshops of luzhoulaojiao. The results are as follows: The concentration of airborne microbes was different in interior and exterior environment of ancient workshops, and also varied by seasons. microbial concentration was higher in spring and summer, and lower in fall and winner. The highest levels of airborne bacteria was in spring, but the fungal’s in summer. The identified genus of fungi were 16 in interior and exterior environment of the ancient workshops. But the dominant genus were different , The advantage genus in the interior were Aspergillus, Yeasts, Penicillum and Nonsporing and in the exterior were Penicillum, Nonsporing, Cladosporium, Aspergillus and Aureobasidiu. Rhizopus ,mucor, Aureobasidiu, Cladosporium, Alternaria and all also were at a higher level. Among these, Aspergillus, Yeasts, Rhizopus ,mucor are important vintage flora . Penicillum, Alternaria do harm to vintage. Aspergillus of ancient workshops was identified , the preponderant aspergillus species were A.cristatellus, A.oryzae, A.niger and A.cadidus in ancient brewage workshops. 10 genus 21 species bacteria were identified, the advantage genuses among the interior and exterior of the three workshops were bacillus, microccus, Staphylococcus Pseudomonas. Bacillus, which account for beyond 40℅ of the total bacteria concentration in all sampling pots, was the most dominant genus. Lactobacillus was identified at a high level in ancient workshops, it makes spirit taste bitter and astringent. So it is not a kind of good bacterium for vintage. The fungus in the interior and exterior atmosphere characterized intercommunion phenomenon. Obviously, the concentration of profitless fungus such as Penicillum, Cladosporium, Alternaria appeared in the interior, and the fungus such as Bacillus, Aspergillus, Rhizopus and Yeasts in the exterior were at a relatively high level. the harmfull fungus in yinggoutou workshops such as Aureobasidiu, Cladosporium, Alternaria and all were lower than shenzitou and xinjiezi workshops.

Floral Organogenesis and Development of two Taxa in Tribe Hyoscyameae (Solanaceae)-Przewalskia tanguitca and Hyoscyamus niger

Floral organogenesis and development of Przeivalskia langutica Maxim, endemic to China and Hyoscyamus niger L. , which belong to the tribe Hyoscyameae (Solanaceae), were studied using scanning electron microscope. They have three common characters of floral organ initiation and development: 1) initia-tion of the floral organs in the two species follows Hofmeister's rule; 2) the mode of corolla tube development belongs to the "late sympetaly" type; 3) primordia of the floral appendages initiated in a pentameroua pattern and acropetal order. But initiation of the calyx-lobe primordia showed different modes in these two species. The calyx-lobe primordia of H, niger have simultaneously whorled initiation, while those of P, tangulica have helical initiation, but the five calyx-lobe primordia form a ring after all five calyx-lobe primordia occur. The systematic significance of the present results in the genera Hyoscyamus and Przeivalskia is discussed in this paper.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium.

Background
The identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.

Methods
Cystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24 h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.

Results
Treatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.

Conclusion
Our data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not.

Lovastatin production by Aspergillus terreus using lignocellulose biomass in large scale packed bed reactor

The effect of superficial air velocity on lovastatin production by Aspergillus terreus PL10 using wheat bran and wheat straw was investigated in a 7 l and a 1200 l packed bed reactor. Mass transfer and reaction limitations on bioconversion in the 1200 l reactor was studied based on a central composite design of experiments constructed using the superficial air velocity and solid substrate composition as variables and lovastatin production as response.
The surface response prediction showed a maximum lovastatin production of 1.86 mg g-1 dry substrate on day 5 of the bioconversion process when the reactor was operated using 0.19 vvm airflow rate (23.37 cm min-1 superficial air velocity) and 54% substrate composition (wC). Lovastatin production did not increase significantly with superficial air velocity in the 7 l reactor. Variation in temperature and exit CO2 composition was recorded, and the Damköhler number was calculated for lovastatin production at these two scales. The results showed that in larger reactors mass transfer limitation controlled bioconversion while in smaller reactors bioconversion was controlled by reaction rate limitations. In addition, mass transfer limitations in larger reactors reduced the rate of metabolic heat removal, resulting in hot spots within the substrate bed.

A decrease in bulk water and mannitol and accumulation of trehalose and trehalose-based oligosaccharides define a two-stage maturation process towards extreme stress resistance in ascospores of Neosartorya fischeri (Aspergillus fischeri)

Aspergillus infection in pulmonary cavitating lesions with right atrial myxoma

Cardiac myxomas are rare primary tumors with varied clinical presentations that may pose a diagnostic challenge. Here, we describe the case of a 21-year-old man with multiple cavitating lung lesions with aspergillosis and underlying right atrial myxoma, who presented with hemoptysis and weight loss. He was successfully treated with right atrial myxoma resection and antifungal agents, with no recurrence or complications after one year of follow-up.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228–259, 2015a, doi:10.​1016/​j.​copbio.​2015.​02.​010; Ball and Hallsworth in Phys Chem Chem Phys 17:8297–8305, 2015, doi:10.​1039/​C4CP04564E; Cray et al. in Environ Microbiol 15:287–296, 2013a, doi:10.​1111/​1462-2920.​12018). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight−1. Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0–85.7 kJ kg−1). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as ‘halophile’ and ‘polyextremophile’; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species

Fungi of the genus Aspergillus are widespread in the environment. Some Aspergillus species, most commonly Aspergillus fumigatus, may lead to a variety of allergic reactions and life-threatening systemic infections in humans. Invasive aspergillosis occurs primarily in patients with severe immunodeficiency, and has dramatically increased in recent years. There are several factors at play that contribute to aspergillosis, including both fungus and host-related factors such as strain virulence and host pulmonary structure/immune status, respectively. The environmental tenacity of Aspergilllus, its dominance in diverse microbial communities/habitats, and its ability to navigate the ecophysiological and biophysical challenges of host infection are attributable, in large part, to a robust stress-tolerance biology and exceptional capacity to generate cell-available energy. Aspects of its stress metabolism, ecology, interactions with diverse animal hosts, clinical presentations and treatment regimens have been well-studied over the past years. Here, we synthesize these findings in relation to the way in which some Aspergillus species have become successful opportunistic pathogens of human- and other animal hosts. We focus on the biophysical capabilities of Aspergillus pathogens, key aspects of their ecophysiology and the flexibility to undergo a sexual cycle or form cryptic species. Additionally, recent advances in diagnosis of the disease are discussed as well as implications in relation to questions that have yet to be resolved.