产黄青霉菌球状菌株原生质体的分离再生和融合

产黄青霉菌球状菌株原生质体的融合是用两株不同遗传标记的菌株作亲本,用玻璃纸平板培养菌丝体,在高渗透性稳定剂中用纤维素酶裂解菌丝细胞壁,制备高浓度(10~6～10~7/ml)原生质体悬浮液。 将原生质体悬浮液接种到高渗性再生培养基上,再生频率可达40％左右。作渗透处理后,测得菌丝断片含量在0.01～0.1％之间。 两菌株的原生质体以1∶1相混合,用分子量6000,30％聚乙二醇处理后,发生了原生质体间的凝聚。将混合物涂布在高渗性合成培养基上,产生了异核体菌落,频率为0.40％左右。将少量异核体分生孢子分离在菌丝生长培养基上后,产生了数量相仿的两亲本型分离子。将大量异核体产生的分生孢子接种到合成培养基上后,能得到少数原养型杂合二倍体,频率为0.035％左右,杂合二倍体纯化后,再以紫外线诱发,获得了以角变和斑点形式出现的二亲本型分离子。     

青霉素生产菌原生质体诱变选育

以青霉素生产菌丝状产黄青霉ＪＳ９４－１８为出发菌株制备菌丝原生质体·并对原生质体进行了紫外诱变，筛选到变异株ＪＳ９４－１８－５８，该菌株摇瓶发酵效价比生产菌株提高８．６３％，５０吨发酵罐试生产平均发酵效价比生产菌株提高１０．４０％。     

A82846B生产菌株选育及发酵培养基的优化

以荒漠拟孢囊菌SIPI-HT47为出发菌株,发酵合成奥利万星中间体A82846B。应用紫外和NTG诱变,结合自身抗性及氯化盐耐受筛选,获得1株高产突变株,命名为FH-32-322,发酵效价较出发菌株提高172.5%。通过PB试验、爬坡试验和响应面试验设计优化发酵培养基组成,确定最佳摇瓶发酵配方。在优化后的发酵培养基上,突变株FH-32-322摇瓶发酵效价达到1 013 mg/L,较原工艺提高85.9%。     

万古霉素产生菌的选育与发酵培养基优化

万古霉素产生菌东方拟无枝酸菌（Amycolatopsis orientalis）5－13经紫外线（30W，253.7nm，距离30cm，照射50s）诱变后，在含万古霉素1000μg/ml的浓度梯度平板上筛选万古霉素抗性变株，得到一株万古霉素抗性变株A.orientalis 6-21，其摇瓶效价较出发菌株提高23％，对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化，与原发酵培养基相比，万古霉素的摇瓶发酵效价提高了14.3%。     

万古霉素产生菌的选育

以东方拟无枝酸菌（Amycolatopsisoriental）05—12为出发菌株,经紫外线（15W。253．7nm,距离18cm,照射5m）诱变处理,在含庆大霉素1000μg·mL^-1的浓度梯度平板上筛选庆大霉素抗性变株,得到一株万古霉素高产交株（Amyeolatopsisoriental）06-4。其摇瓶效价较出发菌株提高23％．对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化,与原发酵培养基相比,万古霉素的摇瓶发酵效价提高了13．4%。     

原生质体再生法选育耐自身代谢产物的卡那霉素链霉菌高产菌株

以卡那链霉菌株KA01为出发菌株,酶法制备原生质体,在含有高浓度的卡那霉素的培养基再生或经UV诱变处理后再生,从再生菌落中分离突变菌株。从中获得一高产突变株KA02,其摇瓶相对效价比出发株提高18%,大罐生产水平提高8%。     

林可霉素高产菌种的选育

以林可霉素产生菌82－7^#为出发菌株,采用氯化锂、紫外线（uV)、甲基磺酸乙酯（EMS）三重复合诱变处理、筛选耐自身产物的高产菌种,从中获得65－12^#菌株。其摇瓶效价较出发菌株提高25％,发酵培养基优化组合后,其摇瓶效价提高28％。应用于30吨发酵罐生产,其发酵指数较出发菌株提高16．8％。     

他克莫司高产菌株选育及发酵配方优化

目的 选育他克莫司高产菌株,优化培养基配方,提高发酵产量。方法 采用原生质体融合技术对2株筑波链霉菌进行基因组重排,并经响应面设计对发酵培养基配方进行优化。结果 以紫外灭活作为遗传标记经过4轮基因组重排育种后,摇瓶筛选获得3株高产菌株,其中菌株FK22-71菌丝球松散、椭圆状,发酵浸泡液颗粒状。该菌株稳定高产,采用响应面设计优化后的配方在50 L罐发酵产量平均达1684 mg/L。结论 基因组重排技术应用于他克莫司高产菌株选育,可大幅提高发酵产量,为其工业化发酵生产奠定了基础。     

复合诱变筛选avermectins高产菌株

由Streptomyces avermitilis初始菌株经过自然分离可以得到三种不同类型的菌株，即灰色粉末型、白色和光秃型。其中灰色粉末型产素水平最高，摇瓶发酵效价达到6835．1μg／ml，B1组分达到56．3％。经过紫外线和亚硝基胍对初始菌株的孢子的诱变及对其原生质体的诱变再生。并且经过异亮氨酸和链霉素的定向筛选，得到的高产菌株摇瓶发酵效价达到8542．9μg／ml，B。组分达到64．9％；同时通过对高产菌株进行传代培养检验其传代稳定性，结果表明，传代3代后，菌株开始退化，并且其发酵效价有比较显著的下降。     

氮离子注入林可霉素产生菌诱变高产菌株

林可霉素产生菌 6 5 - 1株经 1.5× 10 1 5ions/ cm2 氮离子束诱变处理得到 75 - 8菌株。其摇瓶相对效价较出发菌株 6 5 - 1提高了 31% ,发酵培养基优化后 ,摇瓶相对效价提高了 35 %。应用于 10 0吨发酵罐生产 ,其发酵指数较出发菌株提高了 2 5 .6 %。     

螺旋霉素产生菌螺旋霉素链霉菌遗传育种

本文是利用抗生素抗性与产量之间的相关性来进行螺旋霉素产生菌螺旋霉素链霉菌的遗传育种,旨在获得高效价菌株,从而提高螺旋霉素的生产水平。首先用正交试验法确定出发菌株最佳发酵培养基,并在此基础上通过紫外线诱变,和在螺旋霉素浓度梯度平板上筛选螺旋霉素抗性菌株。按上述方法理性化筛选得到的菌株多为正突变株。从本实验得到一株螺旋霉素抗性菌株SPM~r-248,其抗性较出发菌株提高300％,生产能力较出发菌株提高81.3％。菌株SPM~r-248的高效价生产性能遗传特性稳定。在7m~3罐做发酵放大,与出发菌株相比,发酵效价提高31.4％,发酵指数提高18.8％,具有一定的学术意义和经济价值。     

New milbemycins from Streptomyces hygroscopicus subsp. aureolacrimosus: fermantation, isolation and strucutre elucidation

Twelve new milbemycins have been isolated and characterized from some strains derived from Streptomyces hygroscopicus subsp. aureolacrimosus SANK 60286 and SANK 60526. The metabolites 1 approximately 4 and 9 approximately 11 were produced by strain RM28D-688 SANK 60797 as minor products. The metabolites 5 approximaetly 8 were obtained from a broth of strain 57-338 SANK 61796. Strain MK-1391 SANK 62896 was used for the production of metabolite 12. The new metabolites, eight alpha-class and four beta-class compounds, have new structural features. For example, milbemycins alpha26 and alpha27, have the 26-hydroxy moiety, and other derivatives (milbemycins alpha20 approximately 23) have different side chains at the C-26 position from those of milbemycins alpha11 and alpha14. In addition, 5-hydroxylmilbemycin beta7 (beta12), involved in the major biosynthetic pathway of 25-methyl and 25-ethyl milbemycins, was discovered.     

紫外诱变复合重金属离子抗性突变选育霉酚酸高产菌株

以霉酚酸产生菌1321#为出发菌株,采用紫外线诱变并复合重金属Cr6 、Cu2 抗性株筛选,获得一株遗传性状稳定且摇瓶发酵水平提高86.6%的164#高产菌株。经发酵配方优化,该菌株的摇瓶单位再度提升了5.5%,显示出了高产潜力。     

基于深孔板培养高通量筛选rakicidin B1高产菌的研究

目的 获得rakicidin B1的高产突变菌株。方法 建立深孔板发酵rakicidin B1的方法,结合常压室温等离子体技术(ARTP)及核糖体工程对rakicidin B1的产生菌进行高通量诱变选育。结果 获得一株高产突变菌株R36-27,其摇瓶发酵产量达到490mg/L左右,较出发菌株提高了237.9%;经遗传稳定性考察验证了该菌株稳定性较好。结论 采用ARTP为诱变源,以庆大霉素抗性为选择压力,结合深孔板高通量筛选,可以快速有效地获得高产菌株。     

Effect of tumor necrosis factor on human tumor cell lines sensitive and resistant to cytotoxic drugs, and its interaction with chemotherapeutic agents

We investigated the cytotoxic effects of recombinant tumor necrosis factor (TNF) alone and in combination with interferon-gamma (IFN-gamma) and/or cytotoxic drugs on a variety of human tumor cell lines (U937, IGROV-1, HT29, LoVo, MCF7 and U20S), including cell lines with in vitro acquired resistance (LoVo/DX and MCF7/DX selected for resistance to doxorubicin (DX) and characterized by pleiotropic drug resistance; U20S selected for resistance to cisplatin (CDDP], using MTT assay. U937 and MCF7 were sensitive to the cytotoxic effect of TNF, whereas all the other cells were insensitive up to 1000 U/ml (the maximum tested dose). Surprisingly, TNF was cytotoxic (30-40% cytotoxicity) against two resistant lines (LoVo/DX and U20S/Pt) but not against the parent sensitive lines. Treatment with increasing doses of TNF after 6 h incubation with a subtoxic concentration of IFN-gamma produced a synergistic effect in four cell lines (U937, HT29, LoVo/DX and MCF7), whereas in the other five the cell killing of the combination was comparable with that achieved by TNF alone. The combination of subtoxic doses of TNF and increasing doses of drugs targeted at DNA topoisomerase II (i.e. DX, actinomycin D and VP16) produced an additive cytotoxic effect in all cell lines. The same results were obtained combining TNF and CDDP, except in U20S/Pt cells in which TNF synergistically increased CDDP cytotoxicity. The combination of TNF and IFN-gamma enhanced cytotoxicity about 20-fold for DX and 6-fold for CDDP, evaluated in terms of the modification index, against LoVo/DX and U20S/Pt cells respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

新生儿重症监护室多重耐药菌感染分析

目的：了解我院新生儿重症监护室（NICU）多重耐药菌的菌种和耐药情况。方法对本院NICU2011年4月-2012年3月发生多重耐药菌感染的44例新生儿细菌监测结果进行分析。结果共检出多重耐药菌48株,革兰阴性菌38株,占79.17%,以鲍曼/溶血不动杆菌最多（17株）,产超广谱β内酰胺酶（ESBL）阳性14株,占29.17%;鲍曼/溶血不动杆菌对亚胺培南耐药率为76.47%,仅对阿米卡星、妥布霉素敏感,其它革兰阴性菌对氨苄青霉素、氨曲南、除头孢西丁外的头孢类抗生素、哌拉西林耐药率高。革兰阳性菌10株,占20.83%,对阿莫西林/克拉维酸、头孢唑啉、亚胺培南、氨苄青霉素、苯唑青霉素、青霉素耐药率为100%,对利奈唑烷、利福平、万古霉素、奎奴普丁/达福普丁敏感。48株多重耐药菌对3-10类抗生素耐药,平均对6.44＋1.66类抗生素耐药。治愈34例,好转3例,死亡2例,自动出院后死亡5例,病死率为15.91%（7/44）。结论本院NICU多重耐药菌感染病原菌以革兰阴性菌为主,对大部分抗生素耐药。     

基因组重排构建柔红霉素高产菌株

目的构建柔红霉素的高产菌株,提高柔红霉素发酵水平。方法用基因组重排法构建菌株,用摇瓶发酵测定效价。结果使柔红霉素发酵效价提高50%。结论对于柔红霉素发酵效价的提高,基因组重排是一个可行的方法。     

Synthesis and structure-activity relationships of 5-substituted 6,8-difluoroquinolones, including sparfloxacin, a new quinolone antibacterial agent with improved potency

A series of 5,7-disubstituted 1-cyclopropyl-6,8-difluoro-4(1H)-oxoquinoline-3-carboxylic acids (10-36) were prepared; the C-5 substituent in these compounds comprised halo, hydroxy, mercapto, and amino groups and the C-7 functional group included variously substituted piperazines. In vitro antibacterial screening results indicated that the amino group was optimal among the C-5 substituents. A combination of the C-5 amino group and the C-7 3,5-dimethylpiperazinyl appendage in this series conferred the best overall antibacterial property with lack of adverse drug interactions. Compound 36k [named sparfloxacin, originally AT-4140, 5-amino-1-cyclopropyl-6,8-difluoro-7-(cis-3,5-dimethyl-1-piperazinyl)- 4(1H)-oxoquinoline-3-carboxylic acid] was superior to ciprofloxacin in both in vitro and in vivo potency and hence was selected as a promising candidate for an improved therapeutic agent.     

某院综合ICU耐碳青霉烯革兰阴性杆菌的环境流行调查

摘要：目的 分析某院重症监护病房(ICU)环境物表和洗手池耐碳青霉烯革兰阴性杆菌(carbapenem-resistant organism, CRO)的污染现状及其耐药情况,从而为ICU病房CRO防控提供支持依据。方法 采用mSuper CARBA显色培养基对某院综合 ICU水池和患者周围高频接触物进行采样和培养,采用YZB/国1402-2008细菌鉴定及药敏测试仪进行菌株鉴定和药敏试验。 结果 某院综合ICU病房22个洗手池和11例CRKP定植/感染患者周围物表共设置110个采集位点,其中22个水池的池壁、池孔 44个采集位点,患者周围高频接触物表66个采样位点。CRO的总体分离率较高(51.82%,57/110),其中患者周围高频接触物 表的分离率为15.15%(10/66),10个采样位点共分离出14株CRO,检出菌以CRKP(4/14)、CRAB(5/14)、CRPA(2/14)为主;洗 手池的分离率为97.73%(43/44),43个采样位点共分离93株菌株,检出菌以CRKP(13/93,13.98%)、CRPA(29/93,31.18%)、 CRCF(19/95,20.43%)为主。CRKP、CRPA和CRCF的耐药率均较高。17株CRKP对16种常用抗菌药物的耐药率均＞35.29%,对 美罗培南耐药率为100%;29株CRPA对黏菌素的耐药率低(6.45%);19株CRCF对阿米卡星、左氧氟沙星和米诺环素耐药率相对 低(5.26%~21.05%),对其余药物耐药率为52.63%~100.00%。结论 该院综合ICU洗手池CRO定植率较高,而患者周围高频接触 物表CRO定植率相对偏低,应加强落实洗手池的消毒,采取减少通过洗手池交叉传播的预防控制措施,同时加强对患者周围环 境物表的环境清洁与消毒措施,预防和控制CRO医院感染的发生。