嗜热芽孢杆菌噬菌体的分离及特征研究

目的：从腾冲热海温泉中分离嗜热芽孢杆菌噬菌体,并初步分析其特征。方法：采用双层平板法分离纯化嗜热芽孢杆菌噬菌体,对分离得到的噬菌体进行电镜形态观察,按照感染复数（MOI）分别为0．01、0．1、1．0、10和100加入噬菌体纯培养液和宿主菌,55℃、160r／min培养8h后测定噬菌体滴度,并进行噬菌体的热稳定性和pH稳定性分析。结果：从腾冲热海温泉中分离得到的噬菌体为二十面体型;其感染宿主菌NHH4形成清晰的噬菌斑,最适MOI为1．0,最适感染温度为55℃,最适感染pH值为7．5。将这株噬菌体命名为TBIP1。结论：从腾冲热海温泉中分离得到的噬菌体TBIP1为典型的二十面体型,当MOI为1．0时,TBIP1感染其宿主菌产生的子代噬菌体滴度最高。     

腾冲热海一株嗜酸热硫化叶菌的分离与鉴定

从云南腾冲热海酸性温泉中分离纯化出一株极端嗜酸热菌株K4-1,并对其进行形态观察、生长特征、碳源和能源利用及16S rRNA基因分析.该菌株细胞形态为不规则球形,有单生鞭毛,严格好氧,兼性自养,能利用元素硫作为能源,也能利用酵母膏、精氨酸或核糖作为碳源和能源.其最适生长温度为75℃,最适pH为3.5.通过16S rRNA基因序列相似性对比对该菌株进行鉴定,结果表明该菌株与硫化叶茵属标准菌株的相似性介于86.6%～94.3%之间,与分离自腾冲热海的腾冲硫化叶菌Sulfolobus tengchongensis RT8-4菌株序列相似性最高,达到98.9%,可将菌株K4-1鉴定为硫化叶菌属菌株.菌株K4-1的16S rRNA基因序列号为EU729124.     

腾冲热海七株高温厌氧菌的分离及鉴定

摘要：【目的】了解中国云南腾冲热海环境中厌氧菌的分类学特征及生理生化特性。【方法】利用Hungate厌氧操作技术,从云南腾冲热海80-93℃温泉中分离出7株高温厌氧菌,对其进行形态、生长特征及16S rRNA基因序列分析,确定菌株的系统发育地位。【结果】7株菌株细胞形态均为杆状,不产芽孢,革兰氏阴性,严格厌氧,在70℃生长良好。其中较典型的菌株RH0802能在55-80℃温度范围内生长,最适生长温度为70℃;生长pH值范围为5.5-8.5,最适pH值为7.0,能利用葡萄糖、淀粉、甘露醇、甘露糖、核糖、麦芽糖、纤维二糖、木糖、果糖、半乳糖、木聚糖、甘油,不能利用蔗糖、丙酮酸。16S rRNA基因序列相似性的比较分析表明,其中5株菌与Caldanaerobacter属菌株的最高相似性均在98%以上,而RH0804与RH0806分别为96%和93%。菌株RH0802-RH0808的序列登录号分别为FJ748766、FJ748762、FJ748761、FJ748763、FJ748765、FJ748764和FJ748767,在中国普通微生物菌种保藏中心的保藏号分别为CGMCC1.5134-1.5140。【结论】分离自腾冲热海的7株嗜热厌氧菌与Caldanaerobacter属菌株有较高相似性,可将这7株菌株鉴定为Caldanaerobacter属菌株（Caldanaerobacter. sp）,其中菌株RH0804和RH0806有成为新种的可能。     

栖热菌噬菌体TSP4 dCTP脱氨酶基因的克隆表达

将编码栖热菌噬菌体TSP4菌株的dCTP脱氨酶tspdCD基因亚克隆到表达载体pET-32a中,并将重组质粒pET-32a-tspdCD转化至大肠杆菌Rosetta(DE3)中进行诱导表达.SDS-PAGE分析结果显示,目的蛋白经IPTG诱导后在大肠杆菌Rosetta(DE3)中以可溶性形式高效表达.通过Ni-NTA agarose亲和层析柱纯化表达的tspdCD,并对其活性,最适作用温度、pH、底物特异性以及金属离子和有机溶剂对其活性的影响进行测定,检测结果表明重组蛋白活性达到4.12U/mg,它的最适作用温度和pH分别为60℃和7.5,最佳反应底物是dCTP,2mmol/L的Ca2+和Mg2+对其活性具有明显的促进作用,而同浓度的Ni2+和Cu2+对其活性产生了明显的抑制作用,10％ (V/V)的乙酸乙酯和异丙醇对其活性有很明显的促进作用,而同体积比的丙酮对其活性产生明显的抑制作用.实现了tspdCD在大肠杆菌系统中的功能性表达,为进一步研究高温噬菌体tspdCD的功能奠定了基础.     

一株假单胞菌属低温噬菌体的分离及其特性研究

目的从纳帕海湿地土样中分离和培养低温菌及其噬菌体,并对其特性进行研究。方法分离纯化低温宿主菌,采用"双层平板法"分离获得噬菌体,通过16S rRNA基因分析对宿主菌进行初步鉴定,对噬菌体进行电镜形态观察,并进行噬菌体基因组酶切片段长度多态性分析及噬菌体生理特性研究。结果从纳帕海土样中分离获得一株裂解性低温噬菌体,命名为SV-12,其宿主菌S-12鉴定为恶臭假单胞菌(Pseudomonas putida)。噬菌体SV-12有囊膜,头部为典型的正六边形,立体对称结构,直径约68.7 nm;尾管长约122.5 nm,直径约25 nm;在4℃时具有侵染活性,4～25℃均能产生边缘清晰、透明的噬菌斑。最适感染温度约为10℃,pH耐受范围较广,在pH=10时具有最多出斑数。结论 SV-12属于肌尾噬菌体科,为裂解性噬菌体,对氯仿极度敏感。基因组为dsDNA,大小约为74 kb。     

从嗜热栖热菌中提取超氧物歧化酶的研究

嗜热栖热菌(Thermus thermophilus)ATCC 27634是一种适于75—80℃高温中生长的微生物。该菌超氧物歧化酶为一胞内酶,分子量为80000,由181个氨基酸组成。本试验对其发酵、破菌、Rnasel酶解、硫酸铵盐析、滤除小分子物质、上DE-32Ⅰ、DE-32Ⅱ柱分离等条件进行了一系列研究,提取到了热稳定性较商的Mn²⁺-SOD,比活力为3000u/mg,回收率达78.0%。     

一株沙门氏菌裂解性噬菌体的分离鉴定及生物学特性

【目的】从贝类样品中分离到一株沙门氏菌裂解性噬菌体SLMP1,对其进行鉴定及生物学特性分析。【方法】采用双层平板法从贝类样品中分离沙门氏菌噬菌体SLMP1,观察噬菌斑特征,分析SLMP1的宿主范围;利用聚乙二醇8000沉淀浓缩SLMP1颗粒,用氯化铯等密度梯度离心纯化;采用透射电子显微镜观察纯化的SLMP1颗粒;采用酚-氯仿法提取SLMP1核酸,通过核酸酶处理分析核酸类型;分析SLMP1的热稳定性、pH稳定性、最佳感染复数、一步生长曲线及裂菌效果。【结果】SLMP1噬菌斑直径约2–3 mm,圆形透明、边缘清晰;SLMP1能裂解肠沙门氏菌肠亚种和鼠伤寒沙门氏菌;SLMP1头部呈二十面体,直径约62 nm,含非收缩性尾部,尾长约110 nm,属于长尾病毒科;SLMP1核酸为双链DNA;SLMP1在30–60 °C稳定,在pH 4.0–11.0稳定,最佳感染复数为0.001,感染宿主菌潜伏期为10 min、裂解期为120 min、裂解量为51;SLMP1在液体环境中具有良好的裂菌效果。【结论】SLMP1属dsDNA长尾科裂解性噬菌体,具有沙门氏菌生物抑菌剂的应用潜力。     

一株可裂解动物源性多重耐药志贺氏菌的噬菌体分离鉴定及其生物学特性研究

从鸡的肠道内分离鉴定多重耐药志贺氏菌(Shigelle)，纯化获得可侵袭、裂解该志贺氏菌的噬菌体，并研究其生物学特性。以养殖场病鸡肠道中分离的志贺氏病原菌为宿主，筛选鉴定噬菌体，聚乙二醇沉淀浓缩噬菌体，通过透射电镜观察噬菌体形态，双层平板法测定噬菌体宿主谱、最佳感染复数、一步生长曲线、酸碱耐受性和热稳定性，测定噬菌体对多重耐药致病性志贺氏菌的裂解效果。通过上述方法，共筛选到26株志贺氏菌分离株，分别命名为BS1~BS26。以志贺氏菌BS26为宿主菌分离得到一株裂解性噬菌体并命名为噬菌体PSF26，鉴定为有尾噬菌体目，长尾噬菌体科，噬菌体对福氏志贺氏菌（Shigella flexneri）和宋内志贺氏菌(Shigella sonnei)分离株均有裂解作用。噬菌体PSF26的最佳感染复数为0.1，感染周期为90 min，包括75 min的潜伏期和15 min的爆发期，裂解量为(58±17) pfu/cell。噬菌体PSF26在pH 5~8，温度0~40 ℃条件下能保持较高活性，对宿主菌的抑菌圈直径为(11.33±1.53) mm。结果表明，噬菌体PSF26爆发期短，裂解量高，有较好的酸碱、温度耐受性，对多重耐药性福氏和宋内志贺氏菌有良好的作用效果，为利用噬菌体防控饲养动物中食源性致病菌感染和饲用抗生素替代技术研发提供参考。     

一株嗜麦芽窄食单胞菌裂解性噬菌体的分离及生物学特性

【背景】嗜麦芽窄食单胞菌是一种广泛存在于医院和自然环境中的条件致病菌,其分离率与耐药率逐年增加。噬菌体是一类能特异性感染并杀灭细菌的病毒。【目的】分离一株新型嗜麦芽窄食单胞菌噬菌体,为临床嗜麦芽窄食单胞菌感染及防控提供补充手段。【方法】以临床分离的嗜麦芽窄食单胞菌为宿主菌,用点板法从医院污水中分离鉴定噬菌体;用双层平板法测定噬菌体效价及一步生长曲线等生物学特性;用透射电镜观察噬菌体形态;提取噬菌体基因组DNA进行全基因组测序,拼接噬菌体基因组并进行注释。【结果】分离到一株嗜麦芽窄食单胞菌裂解性噬菌体,命名为v B＿Sma S＿P11。该噬菌体感染宿主菌的潜伏期小于5 min,快速增殖60 min后达到平稳期,暴发量为100 PFU/cell。透射电镜观察该噬菌体为长尾噬菌体,具有典型的二十面体头和不可收缩的尾部。基因组测序结果表明,该噬菌体基因组全长44 600 bp,GC含量为63.7%,无抗生素耐受基因、毒力基因和t RNA,与NCBI数据库中所有已知嗜麦芽窄食单胞菌噬菌体相比同源性很低。基因组注释显示该噬菌体含有66个开放阅读框(open reading frame,ORF),其...     

一株产异淀粉酶栖热菌的生长和产酶初步研究

异淀粉酶是一类在工业中有重要应用价值的淀粉酶,从云南梁河温泉水样中分离筛选到1株产异淀粉酶的菌株,初步鉴定为栖热菌(Thermus)。该菌在8~19 h为对数生长期,17 h生长最快,20 h后进入稳定期,产酶伴随菌体生长同时发生,进入稳定期后产酶迅速提高,39 h产酶量达最高,初始酶活达4.14 u/mL。     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

Complete genome sequence of a newly isolated lytic bacteriophage,EFAP‐1 of Enterococcus faecalis,and antibacterial activity of its endolysin EFAL‐1

Aims: In this work, we aimed to identify an effective treatment of infections caused by Enterococcus spp. strains resistant to conventional antibiotics. Methods and Results: We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP‐1, that is capable of lysing Enterococcus faecalis bacteria that exhibit resistance to multiple antibiotics. EFAP‐1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP‐1 belongs to the Siphoviridae family. A putative lytic protein of EFAP‐1, endolysin EFAL‐1, is encoded in ORF 2 and was expressed in Escherichia coli. Recombinant EFAL‐1 had broad‐spectrum lytic activity against several Gram‐positive pathogens, including Ent. faecalis and Enterococcus faecium. Conclusions: The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram‐positive pathogens. Significance and Impact of the Study: Bacteriophage EFAP‐1 and its lytic protein, EFAL‐1, can be utilized as potent antimicrobial agents against Enterococcus spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease‐causing Enterococcus spp. in dairy farms.     

Isolation and characterization of a virulent bacteriophage from Staphylococcus carnosus

Abstract A virulent bacteriophage, øSK311, was isolated from Staphylococcus carnosus , an organism used as a starter culture for the production of dry sausage. Electron microscopic studies revealed that this bacteriophage showed some morphological similarities with the Escherichia coli phages T4 and λ. The host range of øSK311 extends over 9 different staphylococcal species. A phage-resistant mutant of S. carnosus could be isolated. Cells of this mutant exhibited a capsule-like structure. The DNA of øSK311 possesses a G + C content of 31.4 mol% and appears to be highly modified.     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.     

明永冰川融水中一株裂解性低温噬菌体的分离及特征

【目的】高山冰川是一类独特的生态系统,本研究探索从明永冰川地区分离和培养低温菌噬菌体,并对其特征进行研究。【方法】利用已分离的低温菌为宿主,采用"双层平板法"从明永冰川融水中分离纯化低温菌噬菌体;对噬菌体及其宿主进行电镜形态观察,并进行噬菌体基因组限制性酶切片段长度多态性分析、衣壳蛋白组成分析及噬菌体生理特征研究。【结果】从明永冰川融水中分离获得一株裂解性低温噬菌体,命名为MYSP03(Mingyong Flavobacterium Siphoviridae Bacteriophage),其宿主菌MYB03鉴定为Flavobacterium菌株。噬菌体MYSP03为长尾型,无囊膜,头部具典型的正多面体立体对称结构,直径约72 nm;尾管长约240 nm,直径约10 nm;4℃时具侵染活性,在4℃-20℃范围内均可产生边缘清晰、透明的噬菌斑,最适感染温度约10℃,pH耐受范围较广,最适感染pH约9.4,对氯仿不敏感,基因组为双链DNA,大小约66 kb。     

两株海洋蛭弧菌的分离及生物学性质

[目的]从深圳湾海泥中分离鉴定蛭弧菌,并对其生物学性质进行初步研究.[方法]通过稀释营养肉汤(dilute nutrient broth,DNB)双层平板法分离蛭弧菌,对所分离的菌株进行电镜形态观测,并进行16S rDNA测序分析,之后结合1994年版伯杰氏鉴定细菌学手册对菌株进行鉴定,最后通过生理试验对其生物学性质进行研究.[结果]从深圳湾海泥中分离出2株蛭弧菌,分别命名为5#-12和5#-sh06,它们可在20℃～35℃范围内生长,最适温度分别是25℃和30℃;生长pH范围6.1～8.6,最适pH均为7.2;2株蛭弧菌可分别裂解46和48株试验菌,各占总试验菌株数(58)的79.3%和82.8%;联合2株蛭弧菌,可裂解56株试验菌,占总试验菌株数的96.6%;同时,它们一起能将所有试验弧菌裂解.[结论]研究结果揭示了蛭弧菌作为一种生物净化因子具有极大的潜在应用价值.     

Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt

AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.