The use of mitochondrial mutants in hybridization of industrial yeast strains

Summary The contributions of each of the parental strains to the genomes of the sporulating and non-sporulating hybrids, Saccharomyces diastaticus (S. cerevisiae) × Hansenula capsulata, S. diastaticus × Hansenula wingei, S. diastaticus × Torulopsis glabrata, S. diastaticus × Candida pseudo tropicalis (Kluyveromyces marxianus) S. diastaticus × Saccharomyces rouxii, S. diastaticus × Saccharomyces kluyveri, and Saccharomyces diastaticus × Saccharomyces bayanus, obtained by protoplast fusion, were determined by the methods of whole nuclear DNA-DNA reassociation. Petite mutants of S. diastaticus NCYC625, and respiratory-competent strains of the other species, were used. In all of the hybrids but one, the DNA from the S. diastaticus parent showed 93.3 to 109.3% homology with the DNA from the hybrids, and the other parents, from –7.7% (S. kluyveri) to 20.0% (S. bayanus). Reassociation between the DNA from S. diastaticus and the DNA from the other parental strains ranges from 4.7 to 19.4%. Reassociation between DNAs from S. diastaticus and that of the S. diastaticus × T. glabrata fusion hybrids were 15.2 and 18.9% respectively. Further investigation of this hybrid is desirable. The fusion products were relatively stable as compared to some fusion hybrids selected by use of nuclear markers, and could be maintained on normal media, with little or no selection pressure, but use of an appropriate carbon source. In most of the hybrids, except for S. diastaticus × T. glabrata, the S. diastaticus parent contributed most of the genome, and only a single chromosome, or a fragment of a chromosome, appeared to be transferred to the Saccharomyces nucleus, to form the genome of the fusion product.     

产乳糖酶酵母Kluyveromyces fragilis株培养产酶发酵条件的优化

目的：为进一步提高产乳糖酶酵母Kluyueromyces fragilis(K.fragilis)株的酶产量,对该菌株的培养产酶发酵条件进行优化试验。方法：采用增殖和诱导二步培养法培养酵母K.fragilis株,并采用单因素试验方法分析碳源、氮源对酵母诱导产酶的影响。结果：确定了K.fragilis酵母株诱导培养的较佳配方,当乳糖逍度为120g/L,醋酸铵浓度为5g/L,pH为6.7时,单位酶产量达122.4IU/ml培养液,结论：该菌株生长产酶情况良好,具有较高的应用价值。     

三种方法检测酵母菌对氟康唑药敏试验一致性的研究

目的:对比研究3种酵母菌体外药敏试验在测定酵母菌对氟康唑药物敏感的可靠性及实用性。方法:以美国实验室标准协会(CLSI)酵母菌纸片扩散法药敏试验(M44-A)为参照,采用丹麦ROSCO纸片扩散法、法国梅里埃ATB FUNGUS3药敏试验,检测82株临床分离的酵母菌对氟康唑的药物敏感度。采用WHONET-5.4和SPSS13.0软件对结果进行分析,比较ROSCO纸片扩散法、ATB FUNGUS3与M44-A结果间的一致性。结果:ROSCO纸片扩散法检测82株临床分离酵母菌对氟康唑的药敏结果与CLSI M44-A检测结果间差异无统计学意义(P>0.05),一致率为80.5%;ATBFUNGUS3检测的药敏结果与CLSIM44-A检测结果间差异有统计学意义(P<0.01),一致率为74.4%,其中光滑假丝酵母菌和白假丝酵母菌的检测结果与参照差异较大。结论:在检测酵母菌对氟康唑体外药物敏感度方面,ROSCO纸片扩散法与M44-A的一致性较好;对部分菌株,则需进一步应用微量肉汤稀释法M27-A2检测,以确定其最低抑菌浓度(MIC)。     

武汉城区致病性酵母菌的药物敏感性

目的:研究武汉城区致病性酵母菌对氟康唑、伊曲康唑的体外药物敏感性。方法:酵母菌收集自武汉市3家大型综合性医院住院患者提供的标本。分别采用美国国家临床实验室标准化委员会的M38A方案的纸片扩散法和M27A2方案的微量稀释法对氟康唑和伊曲康唑进行体外药物敏感实验,分别测定抑菌圈直径和最小抑菌浓度(MIC),统计分离菌株的构成比以及不同种菌株对氟康唑、伊曲康唑的敏感率。结果:共分离出186株菌,白色念珠菌占67.2%,其次为热带念株菌15.6%,光滑念珠菌15.1%,克柔念珠菌1.6%,葡萄牙念珠菌0.54%。111株实验菌株对氟康唑的总敏感率为87.3%,其中白色念珠菌为96.1%,光滑念珠菌为52.9%;对伊曲康唑总敏感率为78.4%,其中白色念珠菌为84.6%,光滑念珠菌为41.2%。结论:武汉城区致病性酵母菌以白色念珠菌分离率最高,其对氟康唑、伊曲康唑有较高的敏感性,光滑念珠菌对两种药物的敏感性明显低于白色念珠菌。实验室重视对菌种的鉴定,积极开展体外药物敏感实验有利于指导临床治疗。     

丙型肝炎病毒NS3基因酵母表达载体构建及表达

目的　为探讨丙型肝炎病毒 (HCV)非结构蛋白NS3的功能 ,在真核生物酵母细胞中表达HCVNS3基因。方法　用聚合酶链反应 (PCR)的方法以HCV全长质粒pBRTM/HCV 1为模板扩增HCVNS3基因 ,克隆到 pGEM T载体中 ,双酶切后回收连接到酵母表达质粒 pGBKT7中表达。提取酵母蛋白质 ,进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析。结果　成功构建HCVNS3基因酵母表达载体 ,Western免疫印迹显示了HCVNS3在酵母细胞中表达。表达产物在胞内存在 ,相对分子质量为 70 0 0 0。结论　HCVNS3蛋白在酵母中表达成功。     

难辨梭状芽孢杆菌相关性腹泻研究进展

难辨梭状芽孢杆菌是一种革兰阳性肠道病原体。难辨梭状芽孢杆菌相关性腹泻（CDAD）及其引起的假膜性肠炎为消化道多发病。难辨梭状芽孢杆菌可产生毒素A和毒素B，侵入肠黏膜后引起细胞病变，导致一系列感染相关临床表现。免疫学机制在难辨梭状芽孢杆菌感染相关疾病的发病中起重要作用。本文就CDAD的发病机制、临床表现及其检测和治疗作一综述。     

泌尿系感染假丝酵母菌的临床特点及耐药性分析

目的 了解泌尿系感染假丝酵母菌菌株分布及耐药现状,为临床合理选择抗菌药物提供依据.方法 回顾性调查2011年10月～ 2013年12月本院住院患者泌尿系发生假丝酵母菌感染的相关资料及药敏结果.结果 从送检的1688份泌尿系感染患者的尿培养标本中共分离268株假丝酵母菌,检出率为15.87％,菌种以热带假丝酵母菌为主,占52.6％,其次为白色假丝酵母菌61株,占22.76％,克柔假丝酵母菌26株,占9.70％,光滑假丝酵母菌为19株,占7.09％.所有菌株体外药敏实验对两性曲霉B100％敏感,对5-氟胞嘧啶、氟康唑、伊曲康唑、酮康唑的耐药率分别是38.43％、76.49％、73.88％、74.25％.结论 假丝酵母菌是泌尿系发生感染最常见的真菌,耐药性有增长趋势,应对分离菌株进行药敏试验,根据实验室的药敏试验结果指导临床合理.     

In vitro and in vivo activity of a possible novel antifungal small molecule against Candida albicans

Nosocomial infections by fungi are important causes of morbidity and mortality, and the adhesion capacity of yeast on abiotic and biotic surfaces has been considered an important step in this process. Als3 proteins are widely studied for their ability to allow Candida albicans to bind to various surfaces. The objective of the present study was to verify, with more details, the action of F2768-0318 in relation to its antifungal activity as well as its ability to act on C. albicans virulence factors related to adhesion and biofilm formation in vitro and in vivo by inhibiting the Als3 protein. F2768-0318 was assessed in tests of biofilm formation and adhesion on abiotic surfaces (polystyrene plates) and adherence on biotic surfaces, including human endocervical (HeLa) cells, human umbilical vein endothelial cells (HUVECs), and fresh buccal epithelial cells (BEC). Our results showed F2768-0318 was useful in reducing the adhesion and biofilm formation of C. albicans on abiotic surfaces, indicating the possibility of treating hospital materials and preventing biofilm formation on these types of equipment. Further studies are still needed, including optimization of the molecule to allow this molecule to be effective on other types of surfaces, such as human cells.     

Isavuconazole is highly active in vitro against Candida species isolates but shows trailing effect

ObjectiveIsavuconazole is a triazole previously shown to have potent in vitro activity against Aspergillus spp., Mucorales, and Candida spp. Unlike for other azoles, it is unclear if isavuconazole may induce a trailing effect. We studied isavuconazole MICs for a large collection of Candida isolates from blood samples and determined the extent of the trailing effect when using the EUCAST Edef 7.3.1 method.Methods761 molecularly identified Candida isolates from blood samples of 742 patients admitted to the hospital (January 2007 to September 2017) were evaluated and further tested for in vitro susceptibility to isavuconazole following the EUCAST E.Def 7.3.1 test method.ResultsC. albicans showed the highest susceptibility, followed by C. parapsilosis and C. tropicalis (geometric mean MIC 0.003 vs 0.005/0.006, respectively; P < 0.001). In contrast, C. glabrata, and C. krusei had significantly higher MIC values (geometric mean MIC 0.094 vs 0.093, respectively). Isavuconazole MIC distributions were not truncated at the lowest concentration tested, except for C. albicans. Overall, the mean percentage of trailing was 12.9% but differences among species were observed: C. glabrata, C. albicans, and C. tropicalis exhibited higher trailing in comparison to C. parapsilosis and non-Candida yeasts (P < 0.001). The percentage of non-wild-type C. albicans (considering the heavy trailer isolates as wild-type), C. parapsilosis and C. glabrata isolates were 0.56% (2/355), 1.5% (3/200), and 4.65% (4/86), respectively.ConclusionsIsavuconazole showed high in vitro activity against Candida spp., particularly against C. albicans. Trailing effect is commonly observed with isavuconazole, particularly with C. glabrata.