井冈山地区龙脑樟来源真菌多样性和抗菌活性研究

摘要：目的 探究井冈山地区龙脑樟来源真菌的物种多样性和抗菌活性,为新型抗菌活性天然产物的研发提供菌种资源。方法 采用组织切块及研磨法分离植物来源真菌;依据形态学特征观察、ITS测序对菌株进行鉴定;菌株发酵产物经乙酸乙酯萃取,粗浸膏经琼脂纸片扩散法进行抗菌活性检测。结果 从龙脑樟树的叶片、树皮、种子及树周苔藓中分离纯化出真菌197株,经ITS测序比对归属于29个属,显示出较好的真菌多样性。对真菌室温静置发酵后获得的代谢粗提物进行抗菌活性筛选,发现其中50株真菌的次级代谢产物具有抗菌活性,从菌株JDWHS-7-081发酵产物中获得1个化合物。结论 井冈山地区龙脑樟来源真菌具有丰富的物种多样性,筛选获得的抗菌活性菌株具有潜在的研究价值。     

D-对羟基苯甘氨酸产生菌的筛选研究

目的采用多种筛选方法以获得D-海因酶及N-氨甲酰水解酶的阳性菌株。方法通过以底物(对羟基苯海因)为唯一氮源的培养基从土样和水样中初步分离,然后利用双层琼脂法和微孔快速筛选法对分离到的菌株以及实验室保藏的菌株进行筛选,得到D-海因酶阳性菌株。用茚三酮显色法及纸色谱法进一步筛选N-氨甲酰水解酶阳性菌株。结果最终获得D-海因酶阳性菌株54株,从这54株菌中筛选到2株产N-氨甲酰水解酶的阳性菌株,从而获得了可同时产生双酶的菌株。结论几种不同筛选方法和测定方法的配合使用提高了阳性菌株的获得机会,为进一步诱变得到高产菌株,建立基因工程菌,获得基因工程药物奠定了基础。     

黄河三角洲植物真菌的分离、活性菌株的筛选及活性产物的鉴定

目的 从黄河三角洲植物中分离真菌,筛选具有抗菌或抗肿瘤活性菌株,分离鉴定活性成分。方法 蔗糖密度梯度法分离菌株,对其发酵物进行抗菌活性和细胞毒活性筛选,采用硅胶柱色谱、凝胶柱色谱对发酵产物进行分离、纯化,运用核磁共振、质谱等手段鉴定化合物的结构。结果 从黄河三角洲的9种植物样品中分离纯化真菌136株,筛选得到具有抑菌活性菌株25株、具有细胞毒活性菌株17株,并从1株枝孢属真菌Cladosporium sp. OUCMDZ-2046的发酵产物中分离鉴定了1个对白色念珠菌和人乳腺癌细胞株MCF-7具有抑制作用的化合物：桔青霉素。结论 黄河三角洲的植物真菌具有开发为药用活性菌株的潜力。     

腈水解酶活性真菌的筛选及其对氰基吡啶类化合物的转化

目的建立简便、快速的筛选方法,获得具有腈水解酶活性的真菌,并用筛选获得的菌株对氰基吡啶类化合物进行转化。方法建立了基于腈水解反应中体系pH变化为指示的微量比色筛选方法,应用筛选获得的睛水解酶活性菌对2-氰基吡啶、3-氰基吡啶、4-氰基吡啶、蓖麻碱4种化合物进行转化。结果应用微量比色筛选法从23株真菌中筛选获得一株具有腈水解酶活性的真菌As-pergillus niger 3.795;该菌株对4-氰基吡啶表现出较高活性,其全细胞转化4-氰基吡啶生成4-吡啶甲酸(异烟酸)转化率可达84%,且没有检测到副产物。     

中国东海及东太平洋海山区来源抗菌活性海洋微生物的筛选研究

摘要：目的 研究中国东海及东太平洋区域来源微生物的抗菌活性,意在筛选具有良好抗菌活性的海洋微生物,并初步研究活性菌株代谢产物的结构,为探求新型抗生素提供基础。方法 以金黄色葡萄球菌、大肠杆菌和白色念珠菌为指示菌株,采用牛津杯法进行抗菌活性微生物初筛和复筛。通过HPLC-DAD结合TLC对活性突出的4株真菌及1株细菌进行活性提取物指纹图谱分析。对广谱抗菌活性菌株WBX-38进行菌种鉴定及活性代谢产物分离纯化,利用核磁共振和质谱等手段对活性化合物进行结构鉴定。采用微量稀释法测定活性化合物的最小抑菌浓度（MIC）。结果与结论 从中国东海及东太平洋来源样品中共筛选90株海洋来源菌株,获得抗菌活性菌株16株,4株为海洋细菌,12株为海洋真菌。通过HPLC-DAD结合TLC分析,发现5株活性菌株均具有独特的色谱行为。海洋真菌WBX-38通过菌种鉴定,确认为海洋来源曲霉（Aspergillus sp.）。从其发酵液提取物中分离获得1个活性化合物5-hydroxymethylfuran-3-carboxylic acid,该化合物对3种指示菌均具有一定生长抑制活性。     

闽浙马尾杉内生真菌分离纯化及产石杉碱甲菌株LC-MS／MS筛选

目的通过组织块分离法从闽浙马尾杉中分离内生真菌,利用LC-MS／MS筛选产石杉碱甲的内生真菌。方法将采集到的新鲜植物闽浙马尾杉进行表面灭菌消毒及将植物的根际土壤稀释,接种于含有3％的链霉素的PDA平板培养基上,挑取尖端菌丝进行分离、纯化和保藏;通过石杉碱甲菌株LC-MS／MS法对分离到的内生真菌进行快速的筛选,筛选出产石杉碱甲的内生真菌。结论通过组织块及稀释平板法从闽浙马尾杉植物中分离到306株内生真菌,其中根（G）8株,茎（J）65株,叶（Y）173株,茎尖（M）60株。通过LC-MS／MS筛选法共筛选到产石杉碱甲菌株15株,其中根（c）o株,茎（J）2株,叶（Y）11株,茎尖（M）2株。     

真菌细胞壁溶解剂的筛选新方法

建立了啤酒酵终琼脂平板方法筛选溶解真菌细菌壁葡聚糖的活性物质，用已知抗真菌抗生素证实了方法的有效性，并已筛选到一些有溶菌活性的菌株。     

黄连内生真菌抑乙酰胆碱酯酶活性菌株的筛选及鉴定

目的 从黄连(Coptis chinensis)植株内分离筛选具有较高乙酰胆碱酯酶抑制活性的内生真菌，并对筛选出的强抑制活性菌株进行菌种分类及鉴定，为阿尔茨海默病新药的研发提供潜在的菌种资源。方法 采用改良Ellman法对黄连植株内分离获得的内生真菌菌株进行乙酰胆碱酯酶抑制活性测定，对筛选的活性较强菌株，根据菌株的形态学特征结合分子生物学方法进行菌种鉴定。结果 在黄连根、茎和叶部共分离获得24株内生真菌，有9株对乙酰胆碱酯酶有抑制活性。活性菌株中SCLH12抑制活性较强，经初步鉴定该菌株为枝孢菌属的Cladosporium tenuissimum。结论 从黄连根部筛选分离出乙酰胆碱酯酶抑制活性菌株，其中菌株SCLH12(Cladosporium tenuissimum)具有较强乙酰胆碱酯酶抑制活性，可为开发新的乙酰胆碱酯酶抑制剂(AChEI)奠定基础。     

丛生形盔珊瑚共附生真菌的分离及其抗菌活性的筛选

目的本试验旨在研究珊瑚共附生真菌抗菌活性。方法采用96孔板培养法结合酶标仪测定。以6种常见的致病或致食品腐败的细菌为指示菌,对其共附生真菌次级代谢产物进行抗菌活性筛选,对抗菌能力及抗菌谱进行研究。结果结果表明:共分离丛生形盔珊瑚共附生真菌19株,均具有不同程度的抗菌活性。高活性菌株占52.63%,其中4-4、4-5两菌株表现出良好的抗菌活性。结论丛生形盔共附生真菌是潜在的抗菌资源,具有较高的应用潜力和综合开发前景。     

两株大白鲨鳃部来源青霉生物碱及其生物活性研究r*

目的 探索大白鲨鳃部共附生真菌菌株 Penicillium sp. BP2T2 和 Penicillium sp. TBG2-2 的次生代谢产物。方法 利用柱色谱和高效液相色谱对发酵产物进行分离纯化,综合运用质谱、一维与二维核磁共振谱、比旋光测定和圆二色谱来鉴定化合物结构,采用结晶紫法评价化合物细胞毒活性,采用改良的微孔板法评价卤虫 Artemia salina 致死活性,采用纸片法评价其抗菌活性。结果 从菌株 Penicillium sp. BP2T2 发酵产物中分离鉴定到 1 个生物碱 Meleagrin （1）,从菌株 Penicillium sp. TBG2-2发酵产物中分离鉴定到3个生物碱,分别为fructigenine A （2）、（-）-cyclopenol （3） 和 viridicatol （4）。化合物 1、2 和 4 显示了对人结肠癌 HCT116 细胞株的细胞毒性,其半抑制浓度（IC50）分别为 5.1 μg/mL、40.5 μg/mL 和 59.0 μg/mL,并显示了对金黄色葡萄球菌或大肠埃希氏菌弱的抑菌活性,25 μg/片剂量下抑菌圈直径为7.5~8.0 mm。化合物 2 对卤虫的 24 h 半致死浓度为 125 μg/mL。结论 首次报道从鲨鱼鳃真菌中分离得到化合物 1~3 以及化合物 1、2 和 4 对 HCT116 细胞株的细胞毒性,鲨鱼鳃真菌作为研究较少的真菌资源在产生生物碱等次生代谢产物方面具有一定潜力。     

某院假丝酵母菌感染的临床特点及药物敏感性分析

目的了解医院假丝酵母菌感染特点及药物敏感情况,为临床抗真菌治疗提供依据。方法收集医院2011年1月至2012年12月临床送检标本中分离的假丝酵母菌,采用法国梅里埃公司生产的ATB微生物鉴定仪、ID32C真菌鉴定板、ATBFUNGUS3药敏板进行真菌鉴定和药物敏感性试验。结果在标本分离的351株假丝酵母菌中,以白色假丝酵母菌为主（74．93％）,其次是光滑假丝酵母菌（14．53％）。检出的真菌标本以痰所占比例最高（61．54％）．尿液次之（22．79％）。分离的假丝酵母菌对各类抗真菌药物的敏感性以两性霉素B、5-氟胞嘧啶敏感性最高,对氟康唑、伏立康唑、伊曲康唑有不同程度的耐药。结论医院真菌感染以白色假丝酵母菌为主,在临床医疗中应重视真菌的培养鉴定和药物敏感性试验,以指导合理选用抗菌药物,延缓耐药菌的产生。     

In vitro antimicrobial activity of carbapenem antibiotics against Pseudomonas aeruginosa, measured using a low-concentration Mueller-Hinton Agar culture medium]

Ohya et al. noted that the antibacterial activity of carbapenem-family antibiotics against Pseudomonas aeruginosa was significantly enhanced through lowering the basic amino acid concentration in the culture medium. They reported that there was a marked difference in antimicrobial activity of panipenem (PAPM) against Pseudomonas aeruginosa between the culture medium with Mueller-Hinton Agar (MHA) diluted with distilled water and the non-diluted culture medium. We used 2,312 strains of fresh Pseudomonas aeruginosa, isolated from clinical materials, to examine the antibacterial activity of PAPM in non-diluted and diluted culture media. For testing the susceptibility, we employed the Showa disc method and agar plate dilution method. In the Showa disc method, the inhibition diameters of the tested microbial strains showed a larger distribution for both 16-fold and 40-fold diluted MHA, compared to the non-diluted MHA. The MIC values in the agar plate dilution method were also smaller in distribution for the 16-fold as well as the 40-fold diluted MHA, compared to the non-diluted culture media. Approximately 90% of the strains showed decreased MIC values, 2-8 times in the 16-fold diluted MHA and 2-16 times in the 40-fold diluted MHA. From the above results, we confirmed that the in vitro antibacterial activity of PAPM against Pseudomonas aeruginosa was enhanced through lowering the MHA concentration.     

细菌NDM-1质粒耐药表型与其表达的相关性研究

目的 探讨NDM-1质粒携带菌株耐药表型与其基因表达的相关性，为临床抗菌药物选择提供理论参考。方法 携带NDM-1的质粒电转化入大肠埃希菌TOP10，采用琼脂平板倍比稀释法测定碳青霉烯类药物对野生株和电转化子的最低抑菌浓度(minimal inhibitory concentration, MIC)，TaqMan RT-PCR检测碳青霉烯类耐药菌株的blaNDM-1基因表达及亚-MIC亚胺培南诱导对blaNDM-1基因表达的影响。结果 12株产NDM-1菌株中共有8株电转化成功，电转化子对碳青霉烯类药物的耐药性与野生菌株基本一致。细菌对碳青霉烯类的耐药性与blaNDM-1基因表达成正比。亚-MIC亚胺培南诱导后菌株对碳青霉烯类药物的MIC值和blaNDM-1基因表达均显著高于野生株。结论 碳青霉烯类耐药菌株耐药表型与blaNDM-1基因表达具有正相关性，TaqMan RT-PCR检测可快速准确地为临床治疗提供依据。     

细菌NDM-1质粒耐药表型与其表达的相关性研究

目的 探讨NDM-1质粒携带菌株耐药表型与其基因表达的相关性,为临床抗菌药物选择提供理论参考。方法 携带NDM-1的质粒电转化入大肠埃希菌TOP10,采用琼脂平板倍比稀释法测定碳青霉烯类药物对野生株和电转化子的最低抑菌浓度(minimal inhibitory concentration, MIC),TaqMan RT-PCR检测碳青霉烯类耐药菌株的blaNDM-1基因表达及亚-MIC亚胺培南诱导对blaNDM-1基因表达的影响。结果 12株产NDM-1菌株中共有8株电转化成功,电转化子对碳青霉烯类药物的耐药性与野生菌株基本一致。细菌对碳青霉烯类的耐药性与blaNDM-1基因表达成正比。亚-MIC亚胺培南诱导后菌株对碳青霉烯类药物的MIC值和blaNDM-1基因表达均显著高于野生株。结论 碳青霉烯类耐药菌株耐药表型与blaNDM-1基因表达具有正相关性,TaqMan     

Hydration of nail plate: A novel screening model for transungual drug permeation enhancers

Drug delivery by topical route for the treatment of onychomycosis, a nail fungal infection, is challenging due to the unique barrier properties of the nail plate which imparts high resistance to the passage of antifungal drugs. Permeation enhancers are used in transungual formulations to improve the drug flux across the nail plate. Selection of the effective permeation enhancer among the available large pool of permeation enhancers is a difficult task. Screening the large number of permeation enhancers using conventional Franz diffusion cells is laborious and expensive. The objective of present study was to evolve a simple, accurate and rapid method for screening of transungual drug permeation enhancers based on the principle of hydration of nail plate. The permeation enhancer which affects the structural or physicochemical properties of nail plate would also affect their hydration capacity. Two screening procedures namely primary and secondary screenings were evolved wherein hydration and uptake of ciclopirox olamine by nail plates were measured. Hydration enhancement factor, HEF(24) and drug uptake enhancement factor, UEF(24) were determined for screening of 23 typical permeation enhancers. The Pearson's correlation coefficient between HEF(24) and UEF(24) was determined. A good agreement between the HEF(24) and UEF(24) data proved the validity of the proposed nail plate hydration model as a screening technique for permeation enhancers.     

三角叶黄连内生真菌的多样性及其抑菌活性的筛选

目的分离和鉴定三角叶黄连Coptis deltoidea根状茎、叶柄及叶中的内生真菌,并检测其抑菌活性。方法用平板法分离菌株,对照真菌鉴定手册,根据菌株的菌落形态、大小、颜色、生长速率、质地、生长培养基的颜色变化以及菌丝体和孢子的形态特征进行分类鉴定;选择大肠杆菌、金黄色葡萄球菌作为指示菌,采用杯碟法对内生真菌的发酵上清液进行体外抑菌试验。结果从三角叶黄连中分离获得的297株内生真菌,分属2目、3科、32属;抑菌实验表明:22株内生真菌的发酵液对指示菌均有不同程度的抑制作用,其中6号内生真菌同时对两种致病菌的抑菌效果明显。结论三角叶黄连内生真菌在种类、不同部位呈多样性分布,且部分内生真菌的代谢产物具有较强的抑菌作用,可作为筛选抑菌活性物质的新资源。     

Synthesis and Antimicrobial Evaluation of Dibenzo[b,e]oxepin-11(6H)-one O-Benzoyloxime Derivatives

A series of dibenzo[b,e]ox(thi)epin-11(6H)-one O-benzoyloximes has been synthesized and structurally elucidated by means of IR, (1)H-NMR, (13)C-NMR, MS, and elemental analysis. The newly developed compounds were screened at concentrations of 200-25 μg/mL for their antibacterial activity against Gram+ve organisms such as Methicillin-Resistant Staphylococcus Aureus (MRSA), Gram-ve organisms such as Escherichia coli (E. coli), and at the same concentration range for their antifungal activity against fungal strain Aspergillus niger (A. niger) by the cup plate method. Ofloxacin and ketoconazole (10 μg/mL) were used as reference standards for antibacterial and antifungal activity, respectively. The dibenzo[b,e]oxepines 6a-c and 6e-h showed low antimicrobial activity (MIC 125-200 μg/mL) compared to the reference substances, whereas a major improvement (MIC 50-75 μg/mL) was achieved with the synthesis of the corresponding bromomethyl derivative 6d. Moreover, replacement of oxygen by its bioisosteric sulfur led to isomeric dibenzo[b,e]thi-epine derivatives 6g,h which significantly exhibited higher antimicrobial activity (MIC 25-50 μg/mL) against all tested culture strains used in the present study, demonstrating that a change of chemical class from dibenzo[b,e]oxepine to dibenzo[b,e]thiepine significantly improves the antimicrobial activity. Further variation, such as the oxidation of the thiepine sulfur to the corresponding isomeric dibenzo[b,e]thiepine 5,5-dioxide derivative 9, comparatively failed to exhibit high activity (MIC 200 μg/mL) against S. aureus, E. coli or A. niger.     

Synthesis and antimicrobial activities of highly functionalised novel β-lactam and thiazolidine-grafted tetrahydrobenzothiophenes

A series of biologically active N-(3-chloro-2-oxo-4-phenylazetidin-1-yl)/3-N-(4-oxo-2-arylthiazolidin-3-yl)-2-(methylsulfonamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamides derivatives have been synthesised in good yield. The structures of compounds have been established on the basis of IR, ¹H, ¹³C NMR, and elemental analysis. The structure–activity relationships have been studied by screening of antimicrobial activity over a representative panel of bacterial and fungal strains using two-fold serial dilution method. All these synthesised compounds exhibited significant activities against all bacterial and fungal strains.     

The combination effect of ampicillin and dicloxacillin on the strains of Bacteriodes fragilis isolated from clinical specimens (author's transl)]

The antimicrobial activities of ampicillin (ABPC) and dicloxacillin (MDIPC) alone were tested in 100 strains of B. fragilis isolated from clinical specimens, and combined activity of ABPC and MDIPC was investigated employing a chequer board titration method (Box method) with agar plates and liquid media. The following results were obtained. 1. Of the 100 strains tested, approximately 33% were shown to have MICs of ABPC greater than 100 mcg/ml, whereas 92% of strains were greater than 100 mcg/ml to MDIPC. 2. In the combination, antimicrobial activities were demonstrated additive or synergistic action in almost strains tested. 3. A combined effect of these drugs was proved by agar dilution method for the strains, for which the same effect was shown by the tube dilution method. 4. For ABPC-resistant strains, no increase in antibacterial activity of these drugs by combination was demonstrated.