假单胞菌胆固醇酯酶的生产Ⅰ.菌种筛选和发酵条件的研究

通过平板初筛和摇瓶复筛,从371株菌中筛得一株有工业生产价值的胆固醇酯酶产生菌,并对它进行了发酵条件的研究。用所获得的胆固醇酯酶配制的三酶试剂具有良好的稳定性,适用于临床检测。     

甜菜碱促进脱氮假单胞菌合成维生素B12

采用摇瓶发酵并分批补料的培养方式，考察了甜菜碱在起始培养基中的浓度及补加浓度对脱氮假单胞菌合成维生素B12水平及其合成途径中δ-氨基乙酰丙酸合酶酶活力的影响。结果表明，添加适当浓度的甜菜碱可提高δ-氨基乙酰丙酸合酶的酶活力，促进维生素B12的合成。     

铜绿假单胞菌耐亚胺培南机制研究

目的研究铜绿假单胞菌对亚胺培南的耐药机制。方法取临床分离的铜绿假单胞菌35株(对亚胺培南耐药21株,对亚胺培南敏感14株)。采用外膜蛋白图谱,对其外膜蛋白OprD2进行SDS PAGE分析。同时进行超广谱β 内酰胺酶（ESBLs）和AmpC酶检测。结果铜绿假单胞菌耐药组OprD2相对含量明显低于敏感组(P＜0.01),且检出产ESBLs 9株,AmpC酶5株,同时产AmpC酶和ESBLs共2株。结论OprD2减少及产生ESBLs和AmpC酶是铜绿假单胞菌对亚胺培南耐药的主要原因。     

亚胺培南耐药铜绿假单胞菌产碳青霉烯酶耐药机制及其分子流行病学特征研究

目的:检测和分析亚胺培南耐药铜绿假单胞菌(IRPA)碳青霉烯酶耐药机制,研究其分子流行病学特征,为防治院内IRPA感染提供参考。方法:抽取2016—2017年间临床分离出的IRPA菌株80株资料(产/非产碳青霉烯酶IRPA菌株,分别为32株和48株),分析采用改良碳青霉烯灭活试验(mCIM法)和改良Hodge法和聚合酶链反应(PCR)法检测碳青霉烯酶基因,其显示阳性者进行测序比较产/非产碳青霉烯酶IRPA菌株的耐药谱差异性,以及分析对携带不同种类碳青霉烯酶菌株的耐药及流行病学特征。结果:分离出的80株IRPA中,其中32株(40.00%)mCIM表型阳性,15株(18.75%)为改良Hodge试验阳性,PCR检测结果显示VIM-2基因阳性13株(16.25%)、KPC-2基因阳性19株(23.75%),NDM及OXA-48基因均为阴性;产碳青霉烯酶IRPA菌株对头孢他啶、头孢吡肟、哌拉西林-他唑巴坦、头孢哌酮-舒巴坦和阿米卡星的耐药率分别为87.50%、87.50%、68.75%、68.75%和68.75%显著高于非产碳青霉烯酶IRPA菌株分别为37.50%、35.42%、22.92%、22.92%和35.42%(P<0.05);MLST及PFGE分型结果显示IRPA菌株11个基因型,以ST235/A、ST244/D及ST639/K型为主,其中产酶IRPA菌株主要流行在ST235/A型、ST235/G型、ST244/D型、ST639/K型及ST1029/G型中。结论:本地区分离出的IRPA菌株以产VIM-2、KPC-2型MBL为主,其对临床常用的多种抗菌药物具有高度耐药性,应加强对产酶菌株的检测和监控,防止该菌株对抗菌药物的耐药性传播和流行。     

耐亚胺培南铜绿假单胞菌耐药相关基因的研究

目的分析我院2005年1~12月临床分离34株耐亚胺培南铜绿假单胞菌对常用抗生素耐药相关基因存在状况,为临床抗感染治疗提供依据。方法应用PCR方法检测13种β-内酰胺类抗菌药物耐药相关基因,分别为blaTEM、blaSHV、blaCARB、blaOXA-10、blaPER、blaVEB、blaGES、blaDHA、blaIMP、blaVIM、blaGIM、blaSPM、oprD2;3种氨基糖苷类修饰酶基因,分别为:aac(6′)-I、aac(6′)-II和ant(2")-I;整合酶基因I(intI1)。结果耐药相关基因blaTEM、blaCARB、blaDHA、blaIMP、oprD2、aac(6′)-I、aac(6′)-II和ant(2")-I、intI1阳性率分别为:5.9%、32.4%、14.7%、17.6%、97.1%、21.2%、33.3%、48.5%、3.0%;其余耐药基因均未检测到。结论携带多种耐药相关基因是耐亚胺培南的铜绿假单胞菌对多种抗生素耐药的重要原因之一,膜孔蛋白基因oprD2缺失不是本组铜绿假单胞菌对亚胺培南耐药的主要原因。     

小儿铜绿假单胞菌感染对亚胺培南耐药性分析及机制探讨

目的 探讨患儿铜绿假单胞菌感染对亚胺培南的耐药性及可能的作用机制.方法 取临床分离的铜绿假单胞菌亚胺培南耐药11株.采用改良三维试验方法 检测ESBLs和AmpC酶.采用外膜蛋白图谱,紫外吸收法测定OprD2含量.采用Etest试验观察氰氯苯腙对亚胺培南最低抑菌浓度(MIC)的影响,以研究细胞内主动外排泵出机制.结果 11株铜绿假单胞菌耐药株中检出单产ESBLs 4株,单产AmpC酶3株,其中同时产AmpC酶和ESBLs共1株.耐药株OprD2相对含量明显低于敏感组(P＜0.01).有27.3%菌株存在主动外排泵出机制.结论 超广谱β-内酰胺酶(ESBLs)和AmpC酶的产生、OprD2的减少及存在主动外排泵出机制是我院住院患儿铜绿假单胞菌对亚胺培南抗生素耐药的主要原因.     

565株铜绿假单胞菌对碳青酶烯类药物耐药性分析

目的:分析我院近年来铜绿假单胞菌对碳青酶烯类药物的耐药特点及变迁,为指导临床用药提供依据.方法:收集2006年1月至2009年12月间临床各标本中分离的铜绿假单胞菌,使用Vitek-32微生物分析系统进行细菌鉴定(GNI+卡)和药敏试验(GNS448卡),对结果进行回顾性分析.结果:所有临床标本中分离出铜绿假单胞菌565株,对亚胺培南和美洛培南的药敏情况有4种:亚胺培南和美洛培南均敏感,占78.58%;前者敏感、后者耐药,占9.03%;前者耐药、后者敏感,有3株,占0.53%;两者均耐药占11.85%.且两种药物耐药率逐年升高.结论:亚胺培南和美洛培南对铜绿假单胞菌抗菌活性有不同,临床应根据药敏结果用药,同时应注重碳青酶烯类药物耐药性变化.     

舟山地区耐亚胺培南铜绿假单胞菌耐药特征及感染危险因素分析

目的:为临床控制耐亚胺培南铜绿假单胞菌感染提供参考依据。方法:选择2013年2月-2014年2月舟山地区3家三级医院感染耐亚胺培南铜绿假单胞菌的患者114例,从其临床标本中分离出铜绿假单胞菌共计114株,分析菌株耐药特征及产碳青霉烯酶基因多样性;感染危险因素分析以同期住院的对亚胺培南敏感的铜绿假单胞菌感染的101例患者作为对照组,通过单因素及Logistic多因素回归分析,探讨耐亚胺培南铜绿假单胞菌感染的危险因素。结果:114株铜绿假单胞菌均对多黏菌素B敏感,对其他9种抗菌药物均有不同程度耐药;其产碳青霉烯酶基因携带情况以IMP、VIM基因为主。长时间住院、机械通气、之前使用过亚胺培南及早期联合应用抗菌药物是耐亚胺培南铜绿假单胞菌感染危险因素。结论:舟山地区耐亚胺培南铜绿假单胞菌耐药现象较为严重,避免长时间住院、早期联合应用抗菌药物等可降低耐亚胺培南铜绿假单胞菌感染率。     

耐亚胺培南的铜绿假单胞菌整合子基因研究

目的通过分析耐亚胺培南的铜绿假单胞菌的整合子所携带的耐药基因的种类及特点，探讨其耐药机制，为指导临床用药，预防院内感染和流行病学调查、新药的研发提供参考。方法根据美国国家临床标准化研究所（CLSI／NCCLS）2004年标准，应用纸片扩散法（KB）进行药敏试验，采用多重PCR方法对临床分离的23株耐亚胺培南的铜绿假单胞菌进行整合酶（IntI）检测，并分析整合酶阳性菌株的耐药基因盒。结果药敏试验显示23株耐亚胺培南的铜绿假单胞菌全部为泛耐株，5株为Ⅰ类整合酶阳性，其中3株为缺失型，无耐药基因盒，其余2株耐药基因为blaVIM4和blaPSE-1；14株为Ⅱ类整合酶阳性，其耐药基因盒均为dfr1-sat1-aadA1；3株Ⅰ、Ⅱ类整合酶均为阳性；7株不含Ⅰ、Ⅱ类整合酶。结论耐亚胺培南的铜绿假单胞菌大多都具备Ⅰ、Ⅱ类整合酶基因和基因盒，这是造成它们成为泛耐株的重要耐药机制。     

耐亚胺培南铜绿假单胞菌及其产金属酶的相关基因blaVIM-2研究

目的了解临床分离耐亚胺培南铜绿假单胞菌(Imipenem-resistant Pseudomonas aeruginosa,IPMRPa)及其耐药特征,探讨该菌产金属酶和相关基因blaVIM-2对抗菌药物耐药性的影响。方法收集3年临床分离铜绿假单胞菌,采用MIC法测定抗生素耐药性;用复合纸片法筛查IPMRPa产金属β-内酰胺酶的菌株;聚合酶链反应(PCR)法对金属酶相关基因(blaVIM-2)检测并测序确定。结果 1426株铜绿假单胞菌中IPMRPa为910株(63.8%),均为多重耐药;非IPMRPa的516株(36.2%)中仅有71株多重耐药,两者的多重耐药率比较有显著性差异(P<0.05)。在IPMRPa与非IPMRPa的耐药性比较中发现:除四环素外的其他测试抗生素耐药率均有显著差异(P<0.05)。910株IPMRPa中经复合纸片法检出产金属β-内酰胺酶168株,占18.6%(168/910);168株采用PCR扩增出135株(80.4%,135/168)携带有blaVIM-2型基因,扩增产物经序列分析均为VIM-2型。结论 IPMRPa多重耐药现象严重,VIM-2型金属酶基因型在产金属β-内酰胺酶的IPMRPa菌株中检出率高,但该基因并不是产金属酶的铜绿假单胞菌对本研究中12种测试抗菌药物耐药的主要原因。     

d-Amino Acid-Based Protein Arginine Deiminase Inhibitors: Synthesis,Pharmacokinetics, and in Cellulo Efficacy

d-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of d-amino acid analogues of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.     

Isolation and identification of antifungal N-butylbenzenesulphonamide produced by Pseudomonas sp. AB2

An antifungal bacterial strain, isolated from a greenhouse soil sample, inhibits growth of microflora nearby. It was selected for further studies of bacterial antifungal properties. This isolate was identified as a Pseudomonas sp. based on carbohydrate utilization, and other biochemical and physiological tests. Petri plate assay revealed that the Pseudomonas sp. exhibited antifungal activity against the plant pathogens, Pythium ultimum, Rhizoctonia solani, Phytophthora capsici, Botrytis cinerea and Fusarium oxysporum. Using direct inhibition bioassay on TLC plates after ethyl acetate extraction of the culture filtrate, we correlated antifungal activity with production of antifungal compounds. An antifungal antibiotic was isolated from the culture filtrate and was identified as N-butylbenzenesulphonamide. ED50, values of the N-butylbenzenesulphonamide against P. ultimum, P. capsici, R. solani, and B. cinerea were 73, 41, 33 and 102 ppm, respectively.     

假单胞菌产壳聚糖酶突变菌株的筛选

以假单胞菌Y1为出发菌株 ,分别通过亚硝基胍 (NTG) ,Co6 0 ,UV(紫外 )诱变 ,采用透明圈法筛选 ,获得了产壳聚糖酶较好的突变菌株假单胞菌Y8,其所产酶活力达到 3.0U·ml- 1,酶活力提高近 6倍 ,并具有较好的遗传稳定性。 3种诱变方法中 ,UV诱变效果最好。     

Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Pseudomonas sp. A92.

Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), was isolated from a culture broth of Pseudomonas sp. A92 by Diaion HP-20 column chromatography, solvent extraction and reverse phase HPLC. Spectroscopic analyses of the compound yielded 3-(1-hydroxyoctyl)-5-methyl-2(5H)-furanone as the proposed structure. In the presence of oxidized low density lipoprotein, acaterin inhibited the synthesis of cholesteryl ester in macrophage J774 by 50% at a concentration of 45 microM. Acaterin also inhibited ACAT activity in the rat liver microsomes by 50% at a concentration of 120 microM. Kinetic studies suggested that inhibition of ACAT by acaterin was noncompetitive with respect to oleoyl-CoA.     

Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a pufferfish

Bacteria isolated from the skin of the pufferfish Fugu poecilonotus were screened for tetrodotoxin production. Tetrodotoxin and its derivatives were detected by HPLC analyses in broth cultures of a Pseudomonas sp. Upon alkali treatment the compounds yielded, as do tetrodotoxin and its derivatives, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline, the identity of which was confirmed, after methylation or trimethylsilylation, by HPLC, mass spectrometry and gas chromatography - mass spectrometry.     

Lysis of methicillin-resistant Staphylococcus aureus by 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga

Previously 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas sp. AMSN isolated from a marine alga, had demonstrated a high level of anti-methicillin-resistant Staphylococcus aureus (MRSA) comparable with that of vancomycin. In this study, this substance had bacteriolytic activity against MRSA at 1 microg/ml as well as similar activity against Vibrio parahaemolyticus at 24 microg/ml that suggests a novel antibacterial mode of action by this substance. Heat and pH stability tests showed it to be stable at temperatures ranging from 35 to 70 degrees C and at pHs ranging from 2-7. It was not acutely toxic to mice at levels up to 100 mg/kg.     

挤出滚圆法制备中药复方当归补血微丸及其性质考察*

目的：应用挤出滚圆法制备中药复方当归补血微丸，研究微丸制备的最佳工艺和处方。方法：用新型的挤出滚圆造粒机制备当归补血微丸；采用单因素考察和正交设计筛选最优处方和工艺条件；考察了微丸的粉体学性质、收率和体外溶出效果。结果：用挤出滚圆技术制备的当归补血微丸圆整度好，大小均匀，收率高，体外溶出迅速。结论：挤出滚圆法可以制备中药微丸。该工艺简便易行，制得的微丸质量好。     

高效液相色谱法测定板蓝根中精氨酸含量

目的:建立板蓝根中精氨酸的含量测定方法.方法:采用高效液相色谱法(HPLC法)测定.结果:精氨酸的浓度在48～240μg/mL范围内与峰面积线性关系良好,r=0.999 5;平均回收率为99.55%,RSD=0.73%.结论:HPLC法操作简便、准确,可作为板蓝根中精氨酸的含量测定和质量控制方法.