液氮超低温冻结保藏细菌十年效果的检测

采用液氮超低温冻结保藏法保藏３９属、１２８种、１０６０株细菌。储藏１０年（部分７年）后,检测存活率为９７．８３％。     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

液氮超低温冻结保藏酵母菌十年的检测报告

采用液氮超低温冻结保藏酵母菌48属129种1630株,保藏十年后检测其存活率为96．5％。     

全球专利微生物菌种近20年的保藏与发放情况分析

本文基于世界知识产权组织(WIPO)公开的"国际保藏单位2001–2019年专利微生物保藏与发放"数据进行统计分析。结果表明:全球专利微生物保藏量呈现稳步增长态势,中国专利菌种保藏量位列全球第一,保藏全球近四成专利菌种(31386株,占比39.86%);2018年后全球菌种发放量陡增,其中美国是专利菌种发放量最多的国家(24153株,占全球发放量的96.63%),中国专利菌种的发放率处于较低水平(1034株,发放率3.29%)。中国专利菌种"重保藏、轻发放"的现象严重,需要适当提升产业化开发利用水平。     

液氮超低温冻保藏酵母菌十年的检测报告

采用液氮超代温冻结保藏酵母菌４８属１２９种１６３０株,保藏十年后检测其存活率为９６．５％。     

专利微生物菌种的保藏与发放统计分析

对WIPO公开的"国际保藏机构2001～2010年专利微生物保藏与发放"数据进行了统计分析。结果表明:全世界的专利菌种保藏量与发放量处于快速增长中;美国是专利菌种保藏与发放量最多的国家;近年中国的专利菌种年保藏量实现三连冠,但专利菌种的发放率处于极低水平。     

石蜡油保藏苏云金芽孢杆菌13年的活性检验

石蜡油法保藏苏云金芽孢杆菌 ,一般可以保藏 1～ 5a,保藏研究发现石蜡油法保存 1 3a的苏云金芽孢杆菌经复壮后菌株仍存活 ,且营养特征、生理特征正常。     

沼气高产菌群保藏条件的比较

目的：利用不同方法对沼气高产菌群进行保藏,比较各方法不同时间的保藏效果。方法：应用液体低温冷藏法、液体石蜡封存法、液氮冷冻保藏法和低温冷冻干燥保藏法保藏的菌种,分别在保藏后1个月、3个月、6个月及1年后复苏菌种,测定其产气速度、产气量。结果：以上几种方法均能够保证所保藏菌群在1个月内得到复苏并产气,低温冷冻干燥法及液氮冷冻法保藏沼气产生菌菌群可达1年以上,产气速度、产气量较为理想,优于液体低温冷藏法及液体石蜡法。     

8种不同方法保藏病原菌效果的对比观察

采用８种不同的菌种保藏方法,对１８种常见病原菌的保藏时间及生物学特性的影响进行了对比观察。结果表明：冷冻干燥法保藏菌种时间最长（本实验室已保存１５年）;肉浸汤琼脂平板法保藏菌种时间最短（２～３月）。保藏时间由长到短依次为：冷冻干燥法＞半固体冷冻法≥半固体斜面加液体石蜡覆盖法＞半固体斜面法＞肉浸汤加液体石蜡覆盖法＞血琼脂平板法＞肉浸汤法＞肉浸汤琼脂平板法。且相同方法对不同菌种保藏时间不同。保藏时间在１年以内的菌种,其生物学特性无明显变异;而经冷冻干燥法保藏时间较长的白喉棒状杆菌、金黄色葡萄球菌、甲型副伤寒沙     

放线菌和霉菌菌种保藏研究报告

用砂管法室温保藏放线菌244株29年。其存活率(%)为:6年96;9年91;11年88;14年78;15年76;17年74;20年70;22年59;25年47;27年40;29年37。保藏29年尚存活的放线菌的抗曾活性与保藏前基本相同。用麸曲法室温保藏木霉、曲霉和青霉等12属共216株,存活率因种而异。保藏12年,不同种属霉菌的存活率为0—100%;保藏至16年,全部失活。     

Vitek—AMS系统检测超广谱β—内酰胺酶的评价

目的：评价Vitek-AMS系统检测超广谱β-内酰胺酶（ESBLs)方法的敏感性和特性。方法：用Vitek－32仪器法、双纸片协同试验法检测45株ESBLs阳性和45株ESBLs阴性菌株,并将结果进行比较。结果：Vitek－32仪器法检出45株ESBLs阳性株中的43株,而双纸片协同试验法仅检出38株,45株ESBLs阴性株用两法检测均为阴性。Vitek－AMS仪器法检测ESBLs的方法敏感性为95．5％,特异性为100％,而双纸片协同试验法分别为84．4％和100％。结论：Vitek－AMS系统检测ESBLs的方法敏感性和特异性均优于双纸片同试验法,且操作简便,有条件的实验室可将其作为初筛ESBLs的首选方法。     

ICU病房产超广谱β-内酰胺酶肺炎克雷伯菌的监测和耐药性分析

了解产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌的耐药性,为临床合理使用抗菌药物提供依据。采用自动化细菌鉴定仪(法国生物梅里埃公司Vitek-2和BD Phonenix 100)进行细菌鉴定和药敏试验,应用WHO-NET5.4软件进行耐药性分析。ICU病房共分离出100株肺炎克雷伯菌,其中产ESBLs株48株,占48%,药敏结果显示产ESBLs菌株的耐药率普遍高于不产ESBLs株,产ESBLs株对氨苄西林、头孢呋辛、头孢唑啉等全部耐药,对头孢曲松、头孢噻肟的耐药率也大于80%,对氨基糖苷类的庆大霉素、四环素,对喹诺酮类的环丙沙星、左氧氟沙星以及磺胺类中的复方新诺明耐药率较高(均大于55%),对哌拉西林/他唑巴坦、阿米卡星、头孢西丁等耐药率较低,对亚胺培南、美洛培南高度敏感。产ESBLs肺炎克雷伯菌耐药现象严重,及时监测产ESBLs菌的发生率及其耐药趋势,为临床合理应用抗菌药物,延缓细菌耐药性的产生,控制医院感染具有重要意义。     

社区与医院感染中肺炎克雷伯杆菌和大肠埃希菌耐药性分析

目的探讨社区和医院感染中肺炎克雷伯杆菌和大肠埃希菌产ESBLs的情况及耐药特性。方法采用体外扩散确证试验检测ESBLs,同时用Micro scan wat RA way-40系统全自动细菌鉴定/药敏分析仪及K-B琼脂扩散法进行细菌鉴定和体外药敏试验。结果社区感染标本中分离出肺炎克雷伯杆菌79株,产ESBLs20株,阳性率为25.3%,大肠埃希菌177株,产ESBLs27株,阳性率为15.3%;医院感染标本中分离出肺炎克雷伯杆菌82株,产ES-BLs33株,阳性率为40.2%,大肠埃希菌135株,产ESBLs42株,阳性率为31.1%,社区与医院感染菌株产ESBLs比较差异均有统计学意义(P均<0.05);ESBLs阳性菌株对多种抗生素耐药,其耐药性明显高于ESBLs阴性菌株。结论肺炎克雷伯杆菌和大肠埃希菌产ESBLs菌株在临床分离率较高,医院感染标本要显著高于社区感染标本,并且对多种抗生素具有高度耐药性,产ESBLs菌株耐药性显著高于不产ESBLs菌株,临床上应加强对ESBLs的控制,以防感染流行。     

Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.     

Mycotoxin production from fungi isolated from grapes

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.     

小儿呼吸道感染肺炎克雷伯杆菌耐药性和基因型研究

目的探讨临海地区小儿呼吸道感染肺炎克雷伯杆菌产超β-内酰胺酶（ESBLs和AmpC酶）的耐药性及耐药基因型分布情况。方法采用VITEK-60型全自动细菌鉴定仪鉴定细菌,按CLSI推荐的确证试验检测ESBLs和K-B纸片法测定药敏结果;采用头孢西丁纸片扩散法筛选产AmpC酶阳性菌株,采用PCR检测AmpC酶基因,并对产物进行测序分析基因型。结果113株肺炎克雷伯杆菌ESBLs和AmpC酶总检测率分别为29．20％和18．58％,其中单产ESBLs、单产AmpC酶和同产AmpC酶＋ESBLs检出率分别为23．01％、12．39％和6．19％;AmpC酶阳性菌株的耐药基因型：16株为DHA-1型,5株为ACT-1型。药敏试验：所分离的肺炎克雷伯杆菌对亚胺培南全部敏感,对喹诺酮类耐药率很低,对大多β-内酰胺类抗生素耐药率较高,并且产酶株的耐药性明显高于非产酶株,耐药现象在同产ES-BLS和AmpC酶菌株中更为严重。结论临海地区小儿呼吸道分离的肺炎克雷伯杆菌产ESBLs和AmpC酶检出率较高;AmpC酶以DHA-1基因型流行为主,产酶株呈现出高度多重耐药性。     

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. Methods and Results: Phage–host interactions between eight EPS‐producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (φYsca, φ3, φ5, φ6, φ8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular‐producing strains were more sensitive to phage attacks than the noncapsular‐producing strains. Streptococcus thermophilus CRL1190 (capsular‐producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular‐negative, EPS low‐producing mutant of strain CRL1190 was reduced, especially for φYcsa and φ8. Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. Significance and Impact of the Study: Capsular‐producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.     

新疆尉犁县黑湖嗜盐嗜碱菌的分离及系统发育学分析

【目的】从新疆尉犁县黑湖中筛选分离获得嗜盐嗜碱微生物,并对筛选获得的微生物进行种属鉴定。【方法】采用传统分离鉴定技术,进行形态和生理生化特性研究和基于16S r RNA基因的序列分析。【结果】从样品中分离获得可培养嗜盐嗜碱菌25株,对其进行鉴定。根据生理生化特征、16S r RNA基因序列测定和系统发育分析表明,25株菌分布在古菌Halorubrum、Haloarcula、Natrialba、Halohasta和Halopiger等5个属。其中优势菌群为Halorubrum,次优势菌群为Natrialba。其中DH-66(KU663028)属于Halopiger属,16S r RNA基因序列同源性与该属的模式菌株Halopiger aswanensis 56T同源性最高,为95.75%,预示为潜在的新种(新种鉴定将另行报道)。25株嗜盐嗜碱菌生长条件实验表明,这些菌适应Na Cl的浓度范围为15%-30%、最适浓度为20%-25%,生长的p H范围为7.0-13.0、最适p H为9.0-10.0。各种水解酶类的分析表明,在分离的25株菌中产淀粉酶的菌有5株占20%、产蛋白酶的菌有4株占16%、产酯酶可水解吐温20的菌有15株占60%、可水解吐温40的有7株占28%、可水解吐温80的有4株占16%、产过氧化氢酶的菌有14株占56%。9株菌同时能产4种酶,2株菌同时能产3种酶。表明了嗜盐嗜碱菌产酶的多样性。19株菌硝酸盐还原为阳性。【结论】揭示了新疆尉犁县黑湖嗜盐嗜碱菌生理生化特性的多样性和系统发育多样性,而且蕴藏着较丰富的新的微生物类群,亟待系统研究和进一步开发利用。     

Selective procedure for isolation of microorganisms producing pullulanase and isoamylase

Selective isolation of microorganisms producing pullulanase and isoamylase was accomplished using a two plate detection assay which distinguished both activities and excluded microorganisms producing other extracellular amylases. Over 115,000 colonies tested, 190 strains producing pullulanase and 57 strains producing isoamylase were isolated; extracellular activities of isolated strains were 0.2 to 4.2 and 0.5 to 2.1 nkat/ml of culture, respectively.This paper is dedicated to Prof. Dr. Raúl E. Trucco, in occasion of his 75th anniversary.     

摩根摩根菌β-内酰胺酶检测及耐药性分析

目的了解摩根摩根菌临床分离株产超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC）、金属酶（MBLs）、碳青霉烯酶（KPC）情况,并分析其对17种常见抗菌药物的耐药性。方法 ESBLs和AmpC及MBLs采用三维试验检测,碳青霉烯酶采用改良Hodge试验进行检测,并以K-B法测定17种常见抗菌药物的耐药性。结果 102株摩根摩根菌单产ESBLs 15株,检出率为14.71%;单产AmpC 8株,检出率为7.84%;单产金属β-内酰胺酶（MBLs）3株,检出率为2.94%;所有菌株中未检出碳青霉烯酶（KPC）;同产ESBLs及AmpC 6株,检出率为5.88%;未发现其他双产酶菌株。摩根摩根菌非产酶分离株对17种抗生素的耐药率均低于50.0%;摩根摩根菌产酶分离株对亚胺培南、美罗培南培南的耐药率低于15.0%,与非产酶菌株相比,差异无统计学意义（P〉0.05）;对其余抗生素的耐药率均明显高于非产酶菌株（P〈0.05）。同产ESBLs＋AmpC与耐亚胺培南摩根摩根菌分离株呈多重耐药。结论我院摩根摩根菌分离株产生多种β-内酰胺酶,且对常用抗生素耐药性比较严重,建议临床医师合理使用抗生素,以免耐药菌株的产生。