海洋软体动物中具抗菌活性、溶血性及表面活性细菌的分离、系统进化分析及筛选

考察了生长于日本海的海洋软体动物毛蚶（Anadara broughtonii）中分离的可培养的异养菌活性,主要包括对其它微生物的拮抗性、溶血性及低分子量代谢产物的表面活性研究。共分离到149株菌并进行了形态学鉴定,其中27株通过16S rRNA基因测序分析的系统发育研究,大多数菌株与变形菌纲和变形菌门α亚群有关联,而与厚壁菌门、放线菌纲噬纤维黄杆菌则相差较大。具有溶血性的菌株数量上大都为假交替单胞菌属,嗜水单胞菌属和芽孢杆菌属。6株革兰阳性菌属于芽孢杆菌属,类芽孢杆菌属和糖丝菌属。     

罗布泊地区沙生植物根际放线菌多样性及生物活性的研究

目的研究罗布泊地区沙生植物根际放线菌多样性及其生物活性,以期发现新药用微生物资源。方法采用2种分离方法,6种分离培养基,从两种沙生植物24份根际土样中分离放线菌;对其中131株放线菌发酵液及乙酸乙酯提取液采用抗菌、杀线虫模型进行活性筛选,并根据活性测定结果结合菌株形态特征对其中66株进行16S rRNA基因序列分析。结果共分离到放线菌154株,53株在其中一个或两个活性筛选模型中都显示为阳性,总阳性率为40.46%,16S rRNA基因序列分析表明这66株放线菌分布在12个属,除了优势菌链霉菌(Streptomyces)外,还发现了拟诺卡氏菌属(Nocardiopsis),拟无枝酸菌属(Amycolatopsis),疣孢菌属(Verrucosispore),喜冷杆菌属(Cryobacterium),异壁放线菌属(Actinoalloteichus),假诺卡氏菌属(Pseudonocardia),普氏菌属(Prauserella),链单孢菌属(Streptomonospora),贫养杆菌属(Modestobacter),放线产孢菌属(Actinomycetospora),芽生球菌属(Blastococcus)等稀有放线菌属,菌株CA15-2与GenBank数据库中最近种Nocardiopsis nikkonensis YU1183-22T(GenBank登录号:AB491226)的相似性约为94%,可能为潜在的新属或新种。结论罗布泊地区沙生植物根际存在较为丰富的放线菌资源并能产生多种活性次级代谢产物,值得进一步的研究。     

两株红树林根际土壤放线菌的鉴定和抗菌活性研究

目的对分离自广西北仑河口红树林根际土壤的两株放线菌(B200、B205)进行抗菌活性研究及鉴定。方法观察两株放线菌的形态特征;通过PCR扩增、测定并比对16S rRNA基因序列,采用邻接法构建系统发育树并进行分析;通过液体发酵、萃取等方法,分别获得发酵液水相、发酵液酯相和菌丝丙酮浸提液三类样品;样品通过纸片扩散法进行抗菌活性测定。结果从红树林根际土壤分离的两株放线菌,其发酵液具有较强的广谱抗菌活性,菌株的16S rRNA基因序列对比结果显示,菌株B200和B205分别与有效发表菌株Agromyces subbeticus DSM16689~T的相似率为98.01%和Micromonospora rosaria DSM 803~T的相似率为99.73%,菌株B200可能为壤霉菌属潜在新种,B205初步鉴定是小单孢菌Micromonospora rosaria DSM 803~T的变种。结论分离自广西北仑河口红树林根际土壤的两株放线菌其次级代谢产物有较强抗菌活性,具有从中发现新抗生素的潜力。     

4种药用植物内生放线菌的分离与初步研究

目的分离并研究具有抑菌活性的药用植物内生放线菌。方法使用高氏一号培养基定向筛选药用植物内生放线菌,采用滤纸扩散法评价分离菌株的抑菌活性,进而通过形态学、生理生化等特征的分析对其进行鉴定。结果共分离得到63株内生放线菌。对金黄色葡萄菌、表皮葡萄球菌、枯草芽孢杆菌、大肠杆菌、铜绿假单胞菌等表现出选择性抑菌作用的菌株有17株,其中Y6菌株抗菌谱较广、抑菌作用较强。初步鉴定结果表明Y6菌株是链霉菌属的一个成员。结论 Y6对金黄色葡萄球菌的抑菌效果较好。     

广西茅尾海红树林根围淤泥放线菌多样性及抗菌活性

目的 对广西茅尾海红树林保护区植物根部淤泥中的放线菌进行分离鉴别,研究放线菌的多样性和抗菌活性。方法 选用10种分离培养基,经稀释涂布法分离放线菌;通过PCR扩增,测序后获得菌株16S rRNA基因序列;邻接法构建系统发育树并进行同源性分析,探测放线菌的多样性;针对发酵液经乙酸乙酯萃取后的有机相、水相及菌丝丙酮浸泡液共3类样品,采用纸片扩散法开展抗菌活性研究。结果 从8份红树林植物根围淤泥样品中分离纯化得到244株放线菌,分布于5个目13个科29个属,优势菌属为链霉菌属和小单孢菌属;16S rRNA基因序列相似性低于98.65%的放线菌共有4株,可能为潜在新物种;抗菌活性筛选结果表明,83株放线菌的抗菌活性阳性率达71.1%。结论 广西茅尾海红树林保护区植物根围淤泥中抗菌活性放线菌的多样性丰富。     

塔克拉玛干沙漠南麓土壤放线菌资源勘探及抗菌活性筛选

目的探测塔克拉玛干沙漠南麓放线菌多样性及抗菌活性,以期发现新药用微生物资源,为新抗生素发现奠定基础。方法采用10种分离培养基,以稀释涂布法分离放线菌;采用16S rRNA基因序列分析放线菌多样性;放线菌液体发酵,发酵液乙酸乙酯萃取,菌丝体丙酮浸提;对获得的提取浓缩物,采用纸片扩散法进行抗菌活性筛选。结果从17份土壤样品,共纯化到368株放线菌;其中166株经过16S rRNA序列分析的放线菌分布于16个科的24个属,链霉菌属和拟诺卡菌属为优势菌属;菌株SC8A-24的16Sr RNA基因序列与最近有效菌株Nocardioides salaries CL-Z59T(DQ401092)的相似率为96.41%,为潜在的新种;发酵96株放线菌,其中62株在至少一个抗菌活性筛选中显示为阳性,总阳性率为64.58%。结论塔克拉玛干沙漠南麓土壤中存在较为丰富的药用放线菌资源,具有从中发现放线菌新物种及新结构抗生素的潜力。     

三株红树林来源链霉菌的鉴定及抗菌活性研究

目的研究及鉴定广西茅尾海红树林植物根围淤泥中的3株链霉菌(K57M、K51M、K131M)及其抗菌活性。方法在4种培养基上分离放线菌,采用ISP2培养基进行菌株纯化;通过PCR扩增和测序,获得菌株16S rRNA基因序列,并进入数据库比对;采用邻接法构建系统发育树,并进行同源性分析,以此来研究放线菌的多样性和新颖性;牛津杯法开展3株菌株的抗菌活性初筛,采取纸片扩散法对制备的E1-3、A1-3和M1-3共3类样品复筛抗菌活性。结果经16S rRNA基因序列比对分析发现,菌株K57M可能为潜在的新放线菌菌株。抗菌活性筛选结果表明,K57M、K51M、K131M这3株链霉菌对不同的药敏菌株具有较好的抗菌活性。结论来源于广西茅尾海红树林根围淤泥中的链霉菌具有较强的抗菌活性,为我们挖掘天然活性的抗生素类化合物提供了极大的可能性。     

海洋中度嗜盐菌whb36的分离、鉴定和生物活性研究

目的从海洋中度嗜盐细菌中发现具有抗菌和抗肿瘤活性的物质。方法从盐场的底泥和海水中分离嗜盐细菌,采用抗菌模型和细胞毒模型进行活性筛选,对活性较强的菌株whb36进行形态学和生理生化特性研究,测定其16S rRNA序列并通过同源性比对进行系统发育分析。结果菌株whb36为中度嗜盐菌;whb36与Halomonas ventosae在形态和生理生化特征方面最接近,16S rRNA序列相似性为99%。whb36的粗提物具有较强的抗菌和抗肿瘤活性。结论中度嗜盐细菌whb36可以提供新的活性物质。     

盐单胞菌Halomonas-GY1的分离鉴定与活性产物的研究

目的:从连云港赣榆附近海泥样品中分离嗜盐菌株并鉴定,对产物的活性做初步研究。方法:以Gibbons培养基富集培养并分离纯化菌株,以《伯杰氏系统细菌手册》第1卷(1984)等为主要依据,对分离得到的菌株进行鉴定,琼脂扩散法测定体外抗菌活性,小鼠迟发型变态反应测定免疫活性。结果:分离得到一株盐单胞菌Halomonas-GY1,Halomonas-GY1符合盐单胞菌属特征,但其生理生化特征与几个典型的盐单胞菌属菌种存在差异。体外抗菌试验表明,Halomonas-GY1培养产物对革兰阳性菌以及部分革兰阴性菌有明显的抗菌作用,其多糖提取物对正常小鼠的迟发型变态反应有增强作用,对环磷酰胺所致免疫低下小鼠迟发型变态反应有恢复作用。结论:Halomonas-GY1为盐单胞菌菌株,其培养产物对革兰阳性菌及部分革兰阴性菌有明显的抗菌作用,多糖提取物具有明显的免疫保护作用。     

锡林郭勒盟高原盐湖放线菌资源勘探及抗菌活性筛选

摘要：目的 探究锡林郭勒盟高原盐湖放线菌的多样性、新颖性及抗菌活性,为新型抗菌活性产物的发掘储备菌种资源。方 法 采用20种分离培养基,以平板涂布法分离放线菌;依据16S rRNA基因的同源性对菌株进行分子鉴定;PCR检测代表菌株的I型聚 酮合酶(PKS I)、II型聚酮合酶(PKS II)和非核糖体多肽合成酶(NRPS)功能基因;菌株的发酵液上清和菌体分别经乙酸乙酯和丙酮提取, 提取样品经纸片扩散法进行抗菌活性检测。目标菌株合成次级代谢产物的能力通过antiSMASH对基因组序列进行分析预测。结果 从 7份盐湖土壤样品中分离获得了365株放线菌,其分布于放线菌纲的8个目15个科28个属,优势菌属为链霉菌属和拟诺卡菌属。分离获 得的链霉菌新颖性突出,13株链霉菌与近缘菌株16S rRNA基因的最高相似度≤98.0%,推测其归属于10个链霉菌属新种。受试放线菌 对革兰阳性菌的抗菌活性显著,并从中筛选出3株抗菌作用强且抗菌谱较广的链霉菌;20株受试放线菌同时含有3种抗生素生物合成基 因。菌株XMNu-295基因组含有24个潜在的次级代谢产物生物合成基因簇,以聚酮类、非核糖体多肽类和萜烯类等为主。结论 锡林 郭勒盟高原盐湖中可培养放线菌多样性较为丰富,新颖性突出,目标菌株值得进一步开展次级代谢产物的化学研究。     

海洋微生物抗菌活性的初步研究

从南海、东海、黄海等海域的海洋底质、生物样品中分离到1003个海洋微生物菌株．包括520株放线菌、111株真菌、222株常温细菌和150株耐热细菌。通过纸片法对其抗菌活性进行筛选．结果发现321株海洋微生物具有抑菌活性，占总分离菌株的32．0％。放线菌、真菌、常温细菌、耐热细菌的活性菌株比例分别为39．6％，32．4％。17．1％，27．3％。具有抑菌活性的菌株比例与样品来源密切相关。生物样品来源的细菌抑菌活性较高的菌株比例较底质样品来源的要高，而海洋放线菌与海洋真菌结果相反。各类海洋微生物菌株的抗菌谱有很大的不同。     

多重耐药铜绿假单胞菌的耐药性分析

目的分析2007年—2009年我院铜绿假单胞菌多重耐药(multidrug resistant,MDR)菌株对多种抗菌药物的耐药性变迁。方法收集2007年1月—2009年12月我院细菌室分离的铜绿假单胞菌,采用琼脂扩散法作敏感试验。结果 3年来共分离铜绿假单胞菌422株,其中MDR菌株100株,占23.70%。分离出的MDR菌株分布在呼吸科普通病房(13.00%)、呼吸重症监护病房(RICU)(29.00%)、外科病房(9.00%)、外科监护病房(SICU)(20.00%)、急诊科(5.00%)和其他内科病房(14.00%)。各科室中,分离出的MDR菌株数占所有标本分离的该菌株数的比例最高的是RICU(42.39%,39/92),其次为SICU(37.04%,20/54)。痰标本分离的MDR菌株占76.00%(76/100),其次为尿液(5.00%,5/100)。铜绿假单胞菌MDR菌株的分离率在3年期间无显著性差异。3年期间铜绿假单胞菌MDR菌株对头孢哌酮/舒巴坦耐药率最低(分别为25.00%、37.93%、40.48%);对所有检测的抗菌药物耐药率各年间无显著性差异。RICU与SICU分离的MDR菌株对亚胺培南、美罗培南耐药率显著高于其他普通病房,对其余检测的抗菌药物耐药率与其他病房无显著性差异。结论我院铜绿假单胞菌MDR菌株的耐药情况近年来十分严峻,在RICU与SICU,MDR菌株对碳青霉烯类耐药尤其严重,因此需严密监测细菌耐药性,指导合理使用抗菌药物。     

Studies on Clostridium difficile and antimicrobial associated diarrhea or colitis

Clostridium difficile has been implicated as the major cause of antimicrobial associated colitis or diarrhea. C. difficile was found in stools from 21 of 120 healthy subjects. C. difficile was found in stools from 8 of 9 patients with antimicrobial associated pseudomembranous colitis and in stools from 16 of 96 patients with antimicrobial associated diarrhea. The cytopathic toxin to HeLa cell neutralized by antitoxin to C. difficile was found in stools from all patients with antimicrobial associated colitis or diarrhea. Of 21 strains isolated in the stools from healthy subjects, 19 strains were toxigenic. Some of them were highly toxigenic strains. Such highly toxigenic strains, however, did not give rise to any clinical symptoms probably because of the limited number of this organisms in the normal flora of human intestines; the number of C. difficile ranged between less than 10 and 10(2) level per gram of stool when the organism was found. Of the 24 isolates from antimicrobial associated colitis or diarrheal patients, 4 were highly toxigenic. The susceptibilities of 65 isolates to various antimicrobial agents were determined by the agar dilution technique. All of the 65 strains were inhibited by low concentration of rifampicin, metronidazole, vancomycin, ampicillin and amoxicillin. The isolates were highly resistant to gentamicin, cefoxitin, cefotaxime and cefmenoxime. Wide variations in susceptibility of C. difficile strains to erythromycin, clindamycin and lincomycin were found. The studies have shown that subinhibitory concentrations of clindamycin, cephalothin and amoxicillin cause an increase in cytotoxin levels of C. difficile during broth culture. The increase in supernatant toxin levels occurs concomitant with a decrease in sonicated cell extract toxin levels. The data suggest that a number of factor can cause a release of toxin from C. difficile into the surrounding medium or the intestine.     

Antimicrobial activity of prenylflavanones

Among two flavanones [YS01, YS02] and eight prenylflavanones [YS03-YS10], preliminary screening with fifteen test bacterial strains showed that YS06 was the most active agent. YS06 exhibited highly significant antimicrobial action when tested against 228 bacterial strains comprising two Gram-positive and six Gram-negative genera. The in vitro susceptibility test was carried out by determining the minimum inhibitory concentration (MIC) of YS06 by agar dilution technique. Twenty-two out of fifty strains of Staphylococcus aureus were inhibited at 25 to 50 micrograms/mL of the agent. YS06 also inhibited strains of Salmonella, Shigella and a few strains of Escherichia coli strains were also highly sensitive to YS06, while Kleibsiella spp. and Pseudomonas aeruginosa were much less sensitive. In in vivo study, YS06 offered significant protection (p < 0.001 according to chi-square test) to Swiss albino mice (challenged with 50 minimum lethal dose (MLD, virulent bacterium) at concentrations of 160 and 80 micrograms/mouse.     

尿路感染病原菌中产超广谱β-内酰胺酶菌的药敏分析

目的研究尿路感染病原菌中产超广谱β-内酰胺酶菌的耐药状况,为临床合理用药提供参考。方法收集本院2008年1月～2008年12月尿液标本分离出的病原菌326株,用Kirby-Bauer纸片扩散法进行抗菌药物的敏感性实验,ESBLs纸片法筛选和确证试验。操作按NCCLS-2006年标准进行。结果326株病原菌中分离出革兰阴性杆菌223株,其中产ESBLs的有99株,占44．40％,分别为大肠埃希菌73株,肺炎克雷伯菌26株。产ESBLs大肠埃希菌对20种抗菌药的敏感性排在前3位的是美罗培南（100％）、亚胺培南-西司他丁（100％）、阿米卡星（84．9％）。产ESBLs肺炎克雷伯菌对20种抗菌药物的敏感性排在前3位的是芙罗培南（100％）、亚胺培南-西司他丁（100％）、哌拉西林-他唑巴坦（73．1％）。结论本院尿路感染病原菌中分离出的产超广谱β-内酰胺酶菌多重耐药率高,临床应参考药敏结果,合理选用抗菌药物。     

1999-2003年广东和辽宁地区大肠埃希菌和肺炎克雷伯菌临床分离株耐药性比较分析

目的调查1999-2003年国家细菌耐药性监测网广东和辽宁地区大肠埃希菌和肺炎克雷伯菌临床分离株的耐药性,比较不同来源菌株之间耐药率的差别。方法药物敏感性试验采用纸片扩散法,用WHONET5软件进行结果分析。结果 5年间共收集大肠埃希菌6 816株和肺炎克雷伯菌4 411株。尿液、痰和血液等是大肠埃希菌和肺炎克雷伯菌临床分离株的主要标本来源。1999-2003年广东和辽宁地区大肠埃希菌临床分离株对亚胺培南最为敏感,其次为阿米卡星和头孢他啶。大肠埃希菌临床分离株,广东地区的菌株对头孢噻肟的耐药率最高,2003 年增加到32．0％,两地区分离的大肠埃希菌对环丙沙星的耐药率高于60%。肺炎克雷伯菌临床分离株对亚胺培南最为敏感,其次为头孢吡肟、头孢他啶、阿米卡星和环丙沙星。在1999-2003年间,广东和辽宁地区的肺炎克雷伯菌临床分离株对头孢噻肟和头孢他啶的耐药率逐年增高,环丙沙星的耐药率也有一定程度的增高。广东和辽宁地区产超广谱β内酰胺酶(ESBLs)大肠埃希菌和肺炎克雷伯菌的检出率逐年增高,2003年广东地区产ESBL 大肠埃希菌和肺炎克雷伯菌的检出率分别为29.6％和30.1％,辽宁地区的检出率分别为18．6％和26．6％。广东和辽宁地区的大肠埃希菌和肺炎克雷伯菌临床分离株头孢噻肟耐药、头孢他啶敏感率明显高于头孢噻肟敏感、头孢他啶耐药率。结论通过不同地区间临床分离株细菌耐药性的比较,了解耐药率间的差别,对于临床合理使用抗菌药物、有效控制耐药菌株的传播具有重要意义。     

Evaluation of synergistic enhancement of antimicrobial activities of piperacillin and amikacin against Pseudomonas aeruginosa using FIC index and disc diffusion method

A preliminary study was carried out to see whether or not the synergistic enhancement of antimicrobial activity of piperacillin (PIPC) and amikacin (AMK) in combinations against Pseudomonas aeruginosa can be evaluated using combination discs of these antibiotics. A synergistic antimicrobial effect was observed between these antibiotics, when evaluated using FIC index, against piperacillin-sensitive strains with MIC less than or equal to 12.5 micrograms/ml under the conditions of the present investigation using the agar dilution method. However, no synergy was found between these antibiotics against piperacillin-resistant strains with MIC greater than or equal to 25 micrograms/ml, even when a higher concentrations of the drugs were used. Using strains of P. aeruginosa against which the synergistic effect between these antibiotics was observed, it was found that inhibitory diameters obtained with combination discs of PIPC/AMK at concentrations of 30 micrograms/30 micrograms or 30 micrograms/15 micrograms were more than 3 mm larger than those obtained with discs containing either PIPC or AMK alone. Using strains against which no synergistic effects were observed, however, the above mentioned enlargement of inhibitory zone diameters with combination discs were not clearly found. Further studies are deemed necessary to determine whether or not the combination discs of PIPC and AMK are useful in routine clinical work.     

Antimicrobial susceptibility of Listeria monocytogenes isolated from meningoencephalitis in sheep

The antimicrobial susceptibility to different antimicrobial agents of 41 Listeria monocytogenes strains isolated from sheep with meningoencephalitis and from feedstuff was tested by both microdilution and disk diffusion methods. Both sets of isolates of L. monocytogenes were susceptible to penicillin G, amoxicillin, cephalothin, erythromycin, vancomycin, rifampicin, gentamicin, kanamycin, trimethoprim, sulfisoxazole, chloramphenicol and ciprofloxacin, but resistant to tetracycline and doxycycline (7.3 and 4.9%, respectively). Tetracycline was the most frequent resistance trait in L. monocytogenes strains of animal origin. Four strains (9.8%) also exhibited reduced susceptibility (MIC 4 mg/l) to doxycycline suggesting the need of surveillance studies to monitor the antimicrobial resistance of Listeria strains of animal origin.     

Novel functionalized pyrido[2,3-g]quinoxalinones as antibacterial, antifungal and anticancer agents

A series of twelve novel pyrido[2,3-g]quinoxalinones (3-14), variously substituted at the C-3 position, was synthesized, structurally determined and submitted to a preliminary in vitro evaluation for antibacterial, anticandida and anticancer activities. Results of the antimicrobial screening showed that all compounds, with the exception of 6, 11 and 12, exhibited interesting activity against all strains tested; while compound 10 was found to have encouraging in vitro anticancer activity at a concentration of l0(-4) M.     

2015-2017年荆州市中心医院临床分离菌分布及耐药性分析

深入调查2015-2017年荆州市中心医院临床分离菌的分布及对临床常用抗菌药物的耐药性,为临床合理用药提供依据。菌株抗菌药物敏感性试验采用自动化仪器法或纸片扩散法。回顾性分析我院连续3年临床菌株药敏数据,结果判读根据2017年CLSI。3年我院临床分离株共11049株,其中革兰阳性菌和革兰阴性菌分别为30.2%和69.8%。耐甲氧西林株金黄色葡萄球菌(MRSA)和凝固酶阴性葡萄球菌(MRCNS)的平均检出率分别为33.4%和75.6%。MRSA对复方磺胺甲噁唑敏感率92%~95.9%;MRCNS对利福平敏感率90.7%~90.8%,耐甲氧西林株(MRSA和MRCNS)对其他抗菌药的耐药率均高。未发现万古霉素耐药株。肠球菌属中屎肠球菌较粪肠球菌对多数测试抗菌药物(除四环素外)的耐药率均高,两者中均未检出万古霉素耐药株。肺炎链球菌非脑膜炎株儿童株中青霉素敏感株逐年下降,中介株的检出率有所上升。大肠埃希菌对碳青霉烯类抗生素仍高度敏感。肺炎克雷伯菌对亚胺培南耐药率从1.1%上升到4.5%,对美罗培南耐药率从1%上升到3.8%,耐药率稳步上升。鲍曼不动杆菌对亚胺培南的耐药率从65.5%升高到80.3%,对美罗培南的耐药率从65.4%升高到80.3%。铜绿假单胞菌对亚胺培南耐药率从13.1%升高到21.2% ,对美罗培耐药率从8.3%升高到20.1%。临床分离的耐药菌呈增长趋势,其中碳青霉烯类耐药肺炎克雷伯菌增长趋势明显,应持续进行细菌耐药监测,加强与临床和院感沟通,共同促进院感控制和规范合理用药。