一株宽谱裂解性溶藻弧菌噬菌体ФV170的分离鉴定及生物学特性

【背景】溶藻弧菌(Vibrio alginolyticus)是水产养殖中重要的条件致病菌,对海水养殖业造成了极大的危害。传统的抗生素疗法引发的耐药问题已经成为全球面临的严峻挑战之一,而作为可替代抗生素的噬菌体疗法已被证实能够有效治疗弧菌病。【目的】深入研究溶藻弧菌噬菌体ФV170的生物学特性,为该菌株在水产动物病害控制中的应用提供数据支持。【方法】以溶藻弧菌V170为宿主菌,采用斑点法从凡纳滨对虾养殖水体中筛选噬菌体,并以双层平板法对噬菌体进行纯化、生长、效价等方面的研究;利用电镜观察噬菌体形态;通过酶切方法分析噬菌体的基因组大小及其类型。【结果】分离得到一株宽谱裂解性噬菌体ФV170,其噬菌斑边缘整齐且通透,12 h直径达1.5 mm。鉴定结果显示,噬菌体ФV170头部为正廿面体的立体对称结构,直径为60 nm-65 nm,尾部长为65 nm-75 nm,宽14 nm-18 nm,核酸类型为dsDNA,基因组大小约为45 kb,对氯仿不敏感,属于有尾噬菌体目(Caudovirales)肌尾噬菌体科(Myoviridae)。此外,噬菌体ФV170可裂解15株溶藻弧菌中的7株,属于种内宽谱;最佳感染复数为0.01;一步生长曲线显示潜伏期为10 min,裂解量为101.3;对65°C以上温度敏感。【结论】分离得到一株宽谱裂解性溶藻弧菌噬菌体,该噬菌体具有治疗海水养殖过程中溶藻弧菌病的潜力。     

副溶血弧菌噬菌体Vpas_PP24的分离鉴定及生物学特性

【背景】副溶血弧菌是南美白对虾养殖中常见的致病菌,传统的抗生素防治办法不仅低效,而且越来越难以满足食品安全和绿色环保及可持续发展的要求。副溶血弧菌的生物防治是南美白对虾养殖业可持续发展的必由之路。噬菌体是天然安全的活体抗菌剂,因其对特定细菌的专一性感染和高效性裂解而备受关注。【目的】分离一株能高效裂解副溶血弧菌的烈性噬菌体,为探索副溶血弧菌的噬菌体防治方法提供基础研究。【方法】以28株病虾来源的副溶血弧菌为宿主菌,用双层琼脂平板法从海鲜市场污水中分离副溶血弧菌噬菌体;点斑法测定噬菌体的裂解谱,并对筛选到的宽裂解谱噬菌体进行透射电镜(transmission electron microscopy,TEM)观察、生物学特性测定和全基因组序列分析。【结果】分离筛选到一株副溶血弧菌烈性噬菌体,命名为Vpas_PP24。透射电镜观察显示该噬菌体头部为二十面体,有一长尾,头部长约92 nm,宽约46 nm,尾部长约147 nm,属于有尾噬菌体目长尾噬菌体科。其基因组全长83 482 bp,预测有118个开放阅读框(open reading frames,ORFs),具有已知功能的有13个,不含非编码RNA、毒力基因及抗生素抗性基因。基因组一致性对比显示噬菌体Vpas_PP24可能为弧菌噬菌体的一个新种。Vpas_PP24对28株副溶血弧菌的裂解率为54%,对其他种属的116株弧菌的总裂解率为16%;最佳感染复数(multiplicity of infection,MOI)为0.000 1,效价可达3.0×10¹⁰ PFU/mL。一步生长曲线显示Vpas_PP24的潜伏期为10 min,暴发期为150 min,暴发量为30 PFU/cell。该噬菌体在温度<50 ℃、pH 4.0-11.0范围内活性稳定,对糜蛋白酶、木瓜蛋白酶和对虾肝胰腺酶提取液的水解作用不敏感,但蛋白酶K可快速使其失活,紫外辐照也能使Vpas_PP24失活。宿主菌对该噬菌体的不敏感突变频率为2×10^-5。【结论】分离筛选到一株裂解谱较宽、基因型较新、生物学性质较稳定的副溶血弧菌噬菌体,该噬菌体具有进一步开发成为新型副溶血弧菌抗菌剂的潜力。     

一株副溶血弧菌噬菌体的分离鉴定及生理特性

为了探寻有效的控制副溶血弧菌的方法, 从水产品市场的污水中分离出来一株烈性噬菌体qdvp001。采用双层平板法分离纯化噬菌体qdvp001, 电镜观察其形态特征, 并利用双层平板法测定其生理特性, 包括热稳定性试验、最适pH、最佳感染复数及一步生长曲线, 然后提取基因组进行酶切和序列分析。结果显示该烈性噬菌体qdvp001头部直径大约为79 nm, 尾长大约118 nm, 属于肌尾噬菌体科。它对60 °C以下的温度耐受力较强, 最适pH为7.0?8.0左右, 最佳感染复数是0.000 1, 感染宿主菌的潜伏期约20 min, 裂解期约70 min, 并获得部分DNA片段的序列。将获得的DNA序列在NCBI上进行比对, 结果显示, qdvp001与其他噬菌体的同源性较低。该噬菌体很可能是一种新发现的噬菌体。     

一株产L-天冬氨酸酶大肠埃希氏菌的噬菌体分离和生物学特性

【目的】从大肠埃希氏菌CICC 11021S发酵液中分离一株噬菌体,对其生物学特性进行研究。【方法】采用双层平板法分离噬菌体CICC 80003;利用透射电镜观察噬菌体形态;提取噬菌体基因组,核酸内切酶处理并进行凝胶电泳;分析噬菌体最佳感染复数、一步生长曲线、p H和温度稳定性、宿主谱。考察CICC 80003对CICC 11021S生长和L-天冬氨酸酶活力的影响。【结果】CICC 80003噬菌斑圆形透明,有明显晕环;头部规则,直径约50-60 nm,尾部长约120-130 nm;基因组能被核酸内切酶Bam H I和Mlu I切开;最佳感染复数0.1,潜伏期5 min,裂解期25 min,平均裂解量约86个;最适p H值8.0;90°C温育15 min,噬菌体全部失活;能裂解大肠埃希氏菌和沙门氏菌的部分菌株。发生噬菌体污染时,CICC 11021S无法正常生长,基本检测不到L-天冬氨酸酶活力。【结论】CICC 80003属于长尾噬菌体科ds DNA噬菌体,液体环境中能够彻底裂解大肠埃希氏菌CICC 11021S。     

副溶血弧菌CHY5-M1M噬菌体的分离鉴定及生物学特性分析

副溶血弧菌引起的疾病给水产养殖行业带来巨大损失,而随着抗生素的禁用,人们开始探索治疗水产动物病菌的新型方法,如噬菌体治疗法。从青岛市城阳水产品批发市场采集了15份海产品养殖水及下水道污水样品,以副溶血弧菌作为宿主菌,采用点滴法分离纯化获得12株副溶血弧菌噬菌体,通过双层平板法研究副溶血弧菌噬菌体CHY5-M1M的最佳感染复数、一步生长曲线、温度和pH的稳定性以及对紫外线的敏感度等生物学特性。结果显示,CHY5-M1M噬菌体效价为8.65×10¹³ CFU·mL^-1,且在100感染复数时效价最高;一步生长曲线的潜伏期为20 min,裂解时长100 min,140 min后达到平稳期;噬菌体在温度为40 ℃时效价最高,高于60 ℃时活性开始下降;噬菌体pH适应范围为5~11;噬菌体浓度随紫外照射时间增加明显下降;噬菌体基因组共43 193 bp,包含44个基因,无毒力因子基因和抗生素耐药基因。研究结果表明,噬菌体CHY5-M1M在开发新型抗弧菌药物、预防治疗严重海水动物弧菌病等方面具有良好的应用前景,有望作为未来的抗生素替代产品。     

黄芩素对副溶血弧菌噬菌体裂解宿主的影响研究

论文以副溶血弧菌噬菌体为研究对象,通过研究黄芩素对副溶血弧菌噬菌体裂解宿主的影响来反映黄芩素的抗病毒效果,同时还研究了将黄芩素制备成环糊精包合物后对副溶血弧菌噬菌体裂解其宿主作用的影响。结果表明,黄芩素及其环糊精包合物均抑制了副溶血弧菌噬菌体对其宿主的裂解,且抑制作用均与它们的浓度与作用时间（一定时间范围内）成正相关;将黄芩素制备成环糊精包合物后有利于黄芩素对副溶血弧菌噬菌体裂解抑制作用的增强,在相同浓度下,黄芩素环糊精包合物对副溶血弧菌噬菌体作用60 min就能完全抑制了副溶血弧菌噬菌体对其宿主的裂解作用,而黄芩素则需要120 min。可见,利用副溶血弧菌噬菌体对其宿主的裂解作用来研究一些药效成分的抗病毒作用是一种简单、方便、成本较低的有效方法。     

溶藻弧菌噬菌体的分离

从海产品中检出29株溶藻弧菌噬菌体,从中选出4株进行鉴定。据噬菌斑特征可分为两类：一类是透明,一类是不透明。但大小各有差异,直径均在0．5—3.0mm。电镜可见形态也可分成两种类型：即头部呈长轴六角形,尾部细长,结构简单;另一类头部呈等轴六角形,但棱角不甚明显,尾部很短。这些噬菌体的增殖效价均可达108-9pfu／ml,对溶藻弧菌的综合裂解率为72.22％,单株平均裂解率在9.72—44．4％。4株噬苗体的特异性高,原液与612株属外常见菌作交叉裂解试验,均未出现交叉裂解现象;与697株属内弧菌I昊I试,也仅对副溶血弧菌有39.0％的交叉裂解,但将原液稀释至10RTD时,大部分交叉现象消失,表明两菌间有明显的亲缘关系。     

多重耐药肺炎克雷伯菌噬菌体KP002的生物学特性及基因组学研究

以上海某些医院临床分离到的多重耐药肺炎克雷伯菌为宿主菌,从不同环境的污水中分离获得1株肺炎克雷伯菌噬菌体KP002。电子显微镜显示其为有尾噬菌体,头部直径约70nm,尾长约80nm,尾宽约20nm。对其生物学特性进行研究,结果显示此株噬菌体在pH 3~9及4~50℃的环境中具有较高活性;6min吸附率达95%以上;潜伏期为10min,爆发期为50min;裂解量为172pfu/cell。结果表明,该噬菌体对pH值和温度适应范围较宽。对其全基因组进行测序分析,结果显示其基因组为环状双链DNA,全长47 173bp,GC含量为48%。本研究筛选获得1株对pH值和温度适应范围较宽的耐药肺炎克雷伯菌烈性噬菌体KP002,为建立耐药肺炎克雷伯菌的噬菌体库以用于治疗临床多重耐药菌感染提供了新的思路。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

一株新型旱獭源性宽谱大肠杆菌噬菌体的分离鉴定及基因组分析

[目的]分离喜马拉雅旱獭肠内容物样本中的噬菌体,并研究其生物学特性和基因组特征。[方法]以大肠杆菌为宿主菌,利用双层琼脂平板法从喜马拉雅旱獭肠内容物样本中分离噬菌体;用透射电镜观察形态特征;测定其最佳感染复数、一步生长曲线、酸碱耐受度及宿主裂解谱等生物学特性,并进行全基因组测序。[结果]从喜马拉雅旱獭肠内容物样本中分离得到一株裂解性大肠杆菌噬菌体,命名为vB_EcoM_TH18,其噬菌斑呈无晕环的透亮圆形,透射电镜观察发现该噬菌体头部直径为(90±5) nm,尾部长度为(115±5) nm;最佳感染复数为1;一步生长曲线显示其潜伏期为10 min,110 min后进入平台期,平均裂解量为15 PFU/mL;在pH 4.5-9.5的范围内具有稳定活性;可裂解多种致病型和血清型大肠杆菌和宋内志贺氏菌,无法裂解沙门氏菌、屎肠球菌、金黄色葡萄球菌、肺炎克雷伯杆菌及鲍曼不动杆菌;基因组测序结果表明,其基因组长度为133 882 bp,GC含量为39.95%。基因组共注释到210个编码序列(CDS)和13个tRNAs,不含毒力基因及耐药基因。BLASTn比对结果表明该基因组与Avunavirus属噬菌体Av-05同源性为95.17%。基于噬菌体全基因组、主要衣壳蛋白和终止酶大亚基分别构建系统进化树,结果表明vB_EcoM_TH18是一株肌尾噬菌体科(Myoviridae) Avunavirus属的新型噬菌体。[结论]从喜马拉雅旱獭肠内容物中成功分离并鉴定了一株新型宽谱大肠杆菌噬菌体vB_EcoM_TH18,可裂解多种致病型和血清型的大肠杆菌及宋内志贺菌。     

粘质沙雷菌裂解性噬菌体ФSM9-3Y的分离和鉴定

目的从医院污水中分离粘质沙雷菌噬菌体,并分析其生物学特性,为进一步研究针对耐药性粘质沙雷菌的噬菌体制剂提供依据。方法采用双层琼脂平板法分离纯化针对粘质沙雷菌的裂解性噬菌体,观察噬菌体对宿主菌的裂解特异性,通过负染法电镜观察噬菌体的形态结构,提取噬菌体核酸进行酶切电泳,测定噬菌体的最佳感染复数和一步生长曲线,SDS-PAGE电泳初步分析噬菌体的结构蛋白和非结构蛋白。结果从医院污水分离出7株可裂解粘质沙雷菌的噬菌体,对其中一株噬菌体(命名为ФSM9-3Y)的生物学特征进行了初步研究。电镜显示噬菌体呈蝌蚪状,头部为20面体立体对称、直径约70 nm;尾部长约50 nm。ФSM9-3Y的最佳感染复数为1。一步生长曲线表明;ФSM9-3Y的潜伏期约30 min,暴发时间70 min,暴发量为629 PFU/cell。凝胶电泳显示噬菌体基因组为双链DNA、大小约54 kb。SDS-PAGE呈现至少包括13种蛋白,相对分子质量范围在25～130 kD,其中主要蛋白的相对分子质量约为48 kD。结论此次分离的噬菌体ФSM9-3Y为裂解性噬菌体,根据形态和结构特征,粘质沙雷菌噬菌体ФSM9-3Y属于有尾病毒目,肌尾噬菌体科。     

一株奥奈达希瓦氏菌烈性噬菌体的分离、纯化及鉴定

【目的】从环境中分离获得希瓦氏菌烈性噬菌体,并对其性质进行研究。【方法】以4株希瓦氏菌为宿主菌,采用双层平板法从污水样品中分离得到奥奈达希瓦氏菌MR-1烈性噬菌体M1;观察噬菌斑特征;利用超速离心法浓缩M1颗粒,进一步用氯化铯密度梯度离心纯化;采用透射电子显微镜观察纯化的M1颗粒;提取M1核酸,通过核酸酶处理分析其核酸类型及结构;绘制一步生长曲线。【结果】噬菌体M1在双层平板上形成圆形的噬菌斑,清晰透明,边缘光滑,直径为2.3 mm-2.5 mm;经电镜观察,噬菌体M1头部呈二十面体,直径约为55 nm,尾长约为170 nm,尾部可收缩,属于肌尾噬菌体科(Myoviridae);通过酶切分析表明噬菌体M1核酸为线形双链DNA;一步生长曲线显示该噬菌体感染后完成一个复制循环所需要的时间约为15-20 min。【结论】噬菌体M1属肌尾噬菌体科,研究结果为后续研究病毒在地球微生物成岩过程中所起的作用提供了实验材料。     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定

【目的】自然界中噬菌体种类繁多,其裂菌功能在针对细菌耐药方面具有潜在应用价值。不同噬菌体也呈现出显著的基因多样性及宿主特异性。从上海某猪场仔猪肠内容物样品中分离、纯化大肠杆菌的裂解性噬菌体,分析其生物学特性和病毒学特征,为探索应用噬菌体治疗细菌性感染提供研究材料。【方法】采用双层琼脂平板法分离、纯化噬菌体,观察噬菌斑特征,通过电镜观察噬菌体形态特征,测定其裂菌谱、最佳感染复数、一步生长曲线和生物学特性,进行噬菌体全基因组测序和遗传进化分析。【结果】分离、纯化获得一株能高效裂解大肠杆菌K-12菌株的噬菌体,命名为v B_Eco S_SH2(SH2),噬菌斑呈圆形、大而透明、边缘整齐。电镜观察SH2的头部呈二十面体立体对称,尾部较长。噬菌体的潜伏期为10 min,暴发期为60 min,裂解量高达121 PFU/感染细胞,其最佳感染复数为0.1。基因组测序和比对结果表明,SH2的核酸类型为ds DNA,基因组全长为49 088 bp,G+C%含量为45%,Gen Bank登录号为KY985004,结合电镜观察及BLASTp分析,确定其属于有尾噬菌体目长尾噬菌体科成员。同源性及进化分析表明,该噬菌体为大肠杆菌T1样噬菌体的新成员。【结论】分离鉴定了一株裂解效率极高的大肠杆菌T1样噬菌体,并确认其为T1样噬菌体新成员,为研究大肠杆菌噬菌体及其抗菌应用提供了新的实验材料。     

Two generalized transducing phages in Vibrio parahaemolyticus and Vibrio alginolyticus.

Two bacteriophages named phi VP253 and phi VP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2 x 10(-7) to 7.5 x 10(-5) per infected cell at the m.o.i. of approximately 1.0. The phage phi VP143, but not phi VP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.     

Studies on halophilic vibrios causing a food poisoning outbreak in the city of Vladivostok

V. parahaemolyticus and V. alginolyticus strains isolated from patients during an outbreak of an acute enteric disease in Vladivostok in 1997 were studied. All strains were found to possess typical taxonomic signs. V. parahaemolyticus isolated from humans had direct heat stable haemolysin exotoxin. The overwhelming majority of these strains belonged to serovar O3K6. Among the cultures under study 7 phage types were determined: phage types 1, 2, 7, 10 in 8 V. parahaemolyticus strains and phage types 2, 4. 5. 7 in 5 V. alginolyticus strains. The diagnostic halophilic phage lyzed vibrios in 30.2% of strains. The cultures under study were found to be highly sensitive to chloramphenicol, cefotaxime, nalidixic acid and cyprofloxacin. The study proved that the outbreak of alimentary toxicoinfection was caused by vibrios of serogroup O3:K6.     

Ten new temperate bacteriophages of<Emphasis Type="Italic">Citrobacter youngae</Emphasis>

In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.