噬菌体PF18的生物学特性及其对肺炎克雷伯菌所致小鼠全身感染疗效的初步研究

目的研究新分离噬菌体PF18的生物学特性及其对肺炎克雷伯菌所致小鼠全身感染的疗效。方法调查PF18的噬菌谱,观察其感染肺炎克雷伯菌临床株F18的噬菌斑形态。电镜下明确其形态分类。提取噬菌体PF18的基因组并进行酶切鉴定。观察PF18尾静脉注射治疗全身感染小鼠的生存状态变化。结果 PF18在F18上可形成直径约为5mm完全透明且周围有晕环的噬菌斑,电镜显示其属于有尾噬菌体目,长尾噬菌体科。PF18感染F18的潜伏期为17min,爆发量约为200PFU/细胞。PF18的基因组可被EcoRI、BamHI两种限制性内切酶降解,但不能被HindⅢ,kpnI降解。PF18治疗组小鼠1d内生存率为100%,1周内生存率可达30%,而无治疗对照组小鼠1d能全部死亡,差异有统计学意义(P0.01)。结论 PF18潜伏期短,爆发量大,且治疗肺炎克雷伯菌所致小鼠的全身感染有一定疗效,有望作为对抗多重耐药肺炎克雷伯菌感染的生物抗菌剂,应对PF18进行深入研究。     

鲍曼不动杆菌噬菌体ZZ2生物学特性研究

目的研究鲍曼不动杆菌裂解性噬菌体ZZ2的生物学特性。方法电镜观察ZZ2形态,并研究其噬菌谱、生长曲线及稳定性等生物学特性。结果ZZ2可在3株鲍曼不动杆菌（AB09V,AB0901,AB0904）菌苔上形成裂解性噬菌体所具有的完全透明的滴斑区,其中AB09V是其最敏感的宿主菌。电镜观察ZZ2具典型的有尾噬菌体目肌尾病毒科病毒的形态特征;一步生长曲线显示ZZ2感染AB09V的潜伏期为12min,裂解量为（191±5）PFU／细胞。稳定性试验显示ZZ2在pH4～9及50℃和60℃环境均具良好稳定性。结论噬菌体ZZ2能杀灭鲍曼不动杆菌,可作为生物抗菌剂使用。     

沙雷菌裂解性噬菌体PS2生物学特性及其全基因组测序

目的明确一株粘质沙雷菌裂解性噬菌体PS2的生物学特性及全基因组序列。方法电镜观察PS2的形态;双层平板法分析PS2噬菌谱、生长曲线及理化稳定性;采用MiniBEST Viral RNA/DNA Extraction Kit提取噬菌体基因组,酶解作用鉴定基因组类型;构建噬菌体PS2测序文库,采用MiSeqTM系统测序,Velvet 1.2.08进行序列拼接。结果 PS2为肌尾型噬菌体。其感染S2菌株的潜伏期为21min,爆发量为70PFU。PS2在pH5到pH10的环境中具有较好的稳定性,在50℃和60℃的环境中活性也无明显变化。PS2基因组为双链DNA,全长167 266bp,平均GC含量为41.7%,共预测到276个ORF。BLASTN初步比对结果提示其与多株T4-like噬菌体基因组核酸序列具有显著相似性。结论分离鉴定了一株粘质沙雷菌属噬菌体,进行了生物学特性、全基因组测序和生物信息学初步分析,为噬菌体治疗多重耐药细菌及噬菌体生物信息学深入分析奠定了基础。     

耐药肺炎克雷伯菌噬菌体的分离及特性研究

目的筛选能特异性裂解耐药肺炎克雷伯菌的噬菌体并对其生物学及全基因组学特性进行分析。方法以耐药肺炎克雷伯菌为宿主菌分离噬菌体,用双层琼脂平板法纯化噬菌体并测定其生物学特性,透射电镜观察噬菌体形态。提取噬菌体DNA,使用illumina平台测序噬菌体基因组并分析其特性。结果从上海交通大学医学院附属瑞金医院污水中筛选纯化得到1株肺炎克雷伯菌属噬菌体,命名为vB_KpnP_1209。透射电镜观察该噬菌体由正二十面体头部和尾部组成,属于有尾噬菌体目、短尾噬菌体科。其宿主菌裂解率为16%。vB_KpnP_1209在pH 3～10及温度4～50℃的环境中具有较高活性。一步生长曲线表明,噬菌体在裂解宿主菌时的潜伏期为10 min,爆发期持续10 min左右,20 min后进入平稳期,裂解量为170 PFU/cell(plaque-forming units per cell)。吸附曲线显示,20 min时其吸附率达到最大值99%。vB_KpnP_1209基因组全长40 222 bp,编码45个开放阅读框(open reading frame, ORF),CG含量50.99%。全部ORF中有11个ORFs编码已知功能蛋白。vB_KpnP_1209与Klebsiella phage K11(EU734173.1)全基因组一致性为95.18%。vB_KpnP_1209属于Studiervirinae亚科Przondovirus属,其基因组中未发现tRNA、耐药基因及毒力因子基因。结论筛选的vB_KpnP_1209基因组容量小、结构规整,有潜力成为研究耐药肺炎克雷伯菌噬菌体的模式噬菌体。其生物学特性稳定,安全性高,有望用于治疗耐药肺炎克雷伯菌所致的难治性感染。     

分支杆菌噬菌体Leo生物学特性及抗结核作用

目的 明确分支杆菌噬菌体Leo的生物学特性及其抗结核作用,为“鸡尾酒疗法”筛选候选噬菌体。方法 采用双层平板法制备噬菌体Leo,观察噬菌斑形态;电镜观察噬菌体颗粒形态;提取噬菌体DNA,限制性内切酶酶切分析确定其核酸类型;以不同MOI扩增噬菌体Leo,确定最佳MOI及最低MOI;通过一步生长曲线,确定潜伏期及裂解量;采用终点滴定反浊法测定耻垢分支杆菌对噬菌体Leo的耐受突变率;检测噬菌体Leo对酒精、温度的耐受性及在不同pH下的裂解能力;通过体外杀结核分枝杆菌实验,明确噬菌体Leo对结核分枝杆菌的杀灭作用。结果 Leo噬菌斑圆形透明,边缘清晰,含对称的多面体头部,直径(70±3.0)nm, 可弯曲的尾部,尾长(211±31.7)nm;其基因组能被限制性内切酶HindⅢ、BglⅠ切开;最佳MOI为0.0 0001,最低MOI为0.0 001,潜伏期为150 min,裂解量74,耐受突变率为10^-7。Leo对温度、酒精敏感,在pH 7.4和pH 5.0时均能裂解宿主菌。Leo作用于结核分支杆菌72 h后,Leo组菌量明显低于空白组(P <0.05)。结论 分支杆菌噬菌体Leo属长尾噬菌体科,双链DNA噬菌体,能够杀灭结核分支杆菌,可作为“鸡尾酒疗法”的候选噬菌体。     

猕猴桃果实内生菌株的鉴定及其发酵液抑菌活性初探

目的从新鲜猕猴桃果实中分离鉴定内生细菌并分析其生物学特性及抑菌谱。方法采用平板对峙法初筛选猕猴桃株发酵液对链格孢菌有明显拮抗作用的内生菌、打孔法复筛猕猴桃菌株发酵液对铜绿假单胞菌的抑菌活性,采用平板倾注法测定不同浓度内生菌对标准菌株和临床菌株的抑菌活性。基于内生菌的形态特征、生化特性、电镜扫描结果及16SrDNA基因序列同源性分析,采用MEGA5.0软件N-J法建立系统发育树,进行遗传进化分析。结果从猕猴桃果实分离获得3株内生细菌。平板对峙生长初筛试验显示其中一株具有抑菌活性,该菌株发酵液对链格孢菌有抑制作用;打孔法复筛实验显示发酵液对铜绿假单胞菌的抑菌活性最明显,其发酵液能抑制标准菌株大肠埃希菌、金黄色葡萄球菌及临床菌株中的革兰阴性菌大肠埃希菌、阴沟肠杆菌、肺炎克雷柏菌生长,不能抑制临床革兰阳性菌金黄色葡萄球菌、溶血性葡萄球菌及肺炎链球菌的生长。该菌株被鉴定为粪肠碱杆菌(16rDNA同源性为99%)。结论获得了一株对部分临床革兰阴性菌有抑制作用的猕猴桃果实内生菌,鉴定为粪肠碱杆菌,为主要抑菌成分有待进一步研究。     

肠道致病性大肠埃希氏菌(EPEC)和产肠毒素大肠埃希氏菌(ETEC)的噬菌体型别调查

<正> 1989年宋元堤等用何晓青分离的5株大肠埃希氏菌属分型噬菌体对从腹泻病人和动物分离的235株大肠埃希氏菌进行分型,可分为81个型。作者在1991年用该分型噬菌体对从腹泻病人和健康人分离的 160株ETEC进行分型,可分为17个噬菌体型。现将收集到的78株EPEC和270株ETEC进行分型的结果报告如下。     

噬菌体Chy5的生物学和基因组学特征

目的 筛选能感染结核分枝杆菌的溶源性噬菌体,了解其生物学及遗传学信息。方法 从土壤中分离纯化噬菌体;电镜观察噬菌体形态;单斑法测定宿主谱;提取噬菌体DNA,鸟枪法随机测序。采用DNAStar软件包分析基因组成分,Glimmer3.0、GeneMark软件预测基因功能,同时做共线性分析。分离溶源菌,通过紫外线诱导试验、超免反应验证噬菌体溶源性。结果 分离到一株新的噬菌体Chy5,其头部直径(61.5±1.4) nm,尾部长度(114.1±2.1) nm,为长尾噬菌体,可感染敏感及耐药结核菌株。Chy5基因组全长51 214 bp,G+C含量63.60%,与噬菌体D29基因组最相似,有88个推定基因,含有编码整合酶和阻遏蛋白的基因以及attP位点。经紫外线诱导Chy5溶源菌可形成噬菌斑,Chy5与其溶源菌共培养会发生超免反应。结论 分离到一株新的能感染敏感及耐药结核菌的溶源性噬菌体Chy5,其基因组与噬菌体D29相似,有望应用于构建荧光报告噬菌体。     

鹤庆新发野鼠鼠疫疫源地中鼠疫噬菌体的分离及其裂解谱测定

目的 查明鹤庆新发野鼠鼠疫疫源地内是否存在鼠疫噬菌体,并对所离分鼠疫噬菌体进行形态鉴定及噬菌谱分析。方法 以鹤庆县马厂村为核心,选择5 km范围内的自然村为采样点,采用鼠铗法进行捕鼠,实验室中取鼠盲肠置入改良PBS中,采用双层平皿对样本进行筛选、纯化得到噬菌体,观察噬菌斑形态,电镜检查噬菌体形态,并在22 ℃、24 ℃、28 ℃及37 ℃对21株鼠疫菌与115株非鼠疫菌进行裂解特性分析。结果 采集的354份标本,分离到2株鼠疫噬菌体;2株噬菌体在高于24 ℃温度下可裂解鼠疫菌,低于24 ℃不能裂解鼠疫菌,而非鼠疫菌在4个温度下均不裂解;电镜形态检测2株噬菌体均属于肌尾噬菌体。结论 鹤庆新发野鼠疫疫源地内存在着鼠疫噬菌体,且所分鼠疫噬菌体只有在高于24 ℃时才裂解鼠疫菌,此特性与鼠疫菌在鹤庆县的长期存在相关,且两株噬菌体存在裂解谱较窄,特异性良好,可用于备用诊断噬菌体的筛选株。     

产β-内酰胺酶大肠埃希菌临床检出率及耐药性分析

目的了解产β-内酰胺酶大肠埃希菌临床检出率、分布和耐药性,为临床治疗产β-内酰胺酶菌感染提供依据。方法收集临床分离的202株大肠埃希菌,统计菌株来源、分布。菌株分离及培养按常规方法进行;细菌鉴定采用VITET-2Compact全自动微生物分析仪;采用试纸法检测β-内酰胺酶;采用微量肉汤稀释法测定产β-内酰胺酶大肠埃希菌对10种抗生素的MIC值,分析其耐药性。结果 202株大肠埃希菌中检出167株产β-内酰胺酶,检出率为82.67%。产β-内酰胺酶大肠埃希菌的标本来源以尿液为主,其次为口痰、消化道分泌物和伤口分泌物等。科室以普外科、肾内科、泌尿外科和和呼吸内科为主。产β-内酰胺酶菌株对多种抗生素均表现出较强耐药性,其中对氨苄西林的耐药率为100%,对亚胺培南不耐药。结论临床分离大肠埃希菌产β-内酰胺酶检出率高,耐药率较高。治疗产β-内酰胺酶菌感染应根据体外药敏试验结果选用敏感抗生素。     

Isolation and Characterization of Lytic Bacteriophages Active against Clinical Strains of E. coli and Development of a Phage Antimicrobial Cocktail

Pathogenic E. coli cause urinary tract, soft tissue and central nervous system infections, sepsis, etc. Lytic bacteriophages can be used to combat such infections. We investigated six lytic E. coli bacteriophages isolated from wastewater. Transmission electron microscopy and whole genome sequencing showed that the isolated bacteriophages are tailed phages of the Caudoviricetes class. One-step growth curves revealed that their latent period of reproduction is 20–30 min, and the average value of the burst size is 117–155. During co-cultivation with various E. coli strains, the phages completely suppressed bacterial host culture growth within the first 4 h at MOIs 10⁻⁷ to 10⁻³. The host range lysed by each bacteriophage varied from six to two bacterial strains out of nine used in the study. The cocktail formed from the isolated bacteriophages possessed the ability to completely suppress the growth of all the E. coli strains used in the study within 6 h and maintain its lytic activity for 8 months of storage. All the isolated bacteriophages may be useful in fighting pathogenic E. coli strains and in the development of phage cocktails with a long storage period and high efficiency in the treatment of bacterial infections.     

Efficacy Assessment of Phage Therapy in Treating Staphylococcus aureus-Induced Mastitis in Mice

The primary aim of this study was to evaluate the efficacy of phage against mastitis induced by drug-resistant S. aureus in a mouse model. In this study, five S. aureus phages—4086-1, 4086-2, 4086-3, 4086-4, and 4086-6—were isolated from milk samples secreted by mastitis cows. Transmission electron microscopy showed that all the five phages had icosahedral heads and short non-contractile tails, which are typical characteristics of the family Podoviridae. All these phages were species-specific against S. aureus. The one-step growth curve showed a short latency period (10–20 min) and high burst size (up to 400 PFU/infected cell). To evaluate the effectiveness of the phage 4086-1 in the treatment against mastitis, a mouse model of mastitis was challenged with drug-resistant S. aureus. The results showed the proliferation of S. aureus in the mammary glands was significantly inhibited after treating by phage 4086-1. The concentrations of TNF-α and IL-6 decreased significantly, which demonstrated the phages could effectively alleviate the inflammatory responses. Furthermore, the histopathological analysis showed that inflammatory infiltration in the mammary glands was significantly reduced. These results demonstrate that phage may be a promising alternative therapy against mastitis caused by drug-resistant S. aureus.     

T4-like Bacteriophages Isolated from Pig Stools Infect Yersinia pseudotuberculosis and Yersinia pestis Using LPS and OmpF as Receptors

The Yersinia bacteriophages fPS-2, fPS-65, and fPS-90, isolated from pig stools, have long contractile tails and elongated heads, and they belong to genus Tequatroviruses in the order Caudovirales. The phages exhibited relatively wide host ranges among Yersinia pseudotuberculosis and related species. One-step growth curve experiments revealed that the phages have latent periods of 50–80 min with burst sizes of 44–65 virions per infected cell. The phage genomes consist of circularly permuted dsDNA of 169,060, 167,058, and 167,132 bp in size, respectively, with a G + C content 35.3%. The number of predicted genes range from 267 to 271. The phage genomes are 84–92% identical to each other and ca 85% identical to phage T4. The phage receptors were identified by whole genome sequencing of spontaneous phage-resistant mutants. The phage-resistant strains had mutations in the ompF, galU, hldD, or hldE genes. OmpF is a porin, and the other genes encode lipopolysaccharide (LPS) biosynthetic enzymes. The ompF, galU, and hldE mutants were successfully complemented in trans with respective wild-type genes. The host recognition was assigned to long tail fiber tip protein Gp38, analogous to that of T-even phages such as Salmonella phage S16, specifically to the distal β-helices connecting loops.     

Isolation and Characterization of Lytic Pseudomonas aeruginosa Bacteriophages Isolated from Sewage Samples from Tunisia

Bacteriophages could be a useful adjunct to antibiotics for the treatment of multidrug-resistant Pseudomonas aeruginosa infections. In this study, lytic P. aeruginosa myoviruses PsCh, PsIn, Ps25, and Ps12on-D were isolated from Tunisian sewage samples. Phage Ps12on-D displayed an adsorption time of ~10 min, a short latency period (~10 min), and a large burst size (~115 PFU per infected cell) under standard growth conditions. All phages were active at broad temperature (4 °C to 50 °C) and pH (3.0 to 11.0) ranges and were able to lyse a wide variety of P. aeruginosa strains isolated from clinical and environmental samples worldwide. Illumina sequencing revealed double-stranded DNA genomes ranging from 87,887 and 92,710 bp with high sequence identity to Pseudomonas phage PAK_P1. All four phages based on sequence analysis were assigned to the Pakpunavirus genus. The presented characterization and preclinical assessment are part of an effort to establish phage therapy treatment as an alternative strategy for the management of multidrug-resistant P. aeruginosa infections in Tunisia.     

DNA from recombinogenic lambda bacteriophages generated by arl mutant of Escherichia coli is cleaved by single-strand-specific endonuclease S1.

When propagated on arl strains (a subclass of Escherichia coli hyper-rec mutants), lambda "Red-" duplication phages accumulated an enhanced potential for recombination. The physical properties of the recombinogenic phages thus obtained ("Arl-" phages) were similar to those of phages grown on arl+ bacteria. However, Arl- phage DNA was cleaved by endonuclease S1 under conditions such that the nuclease is specific for single-stranded DNA;DNA from control phages was S1-resistant. The number of S1 sites (defined by the apparent decrease in single-strand molecular weight) reached a maximum (seven to nine sites per strand of lambda DNA) after five or six rounds of growth on arl bacteria. Similarly, the recombinogenicity of Arl- phages reached a limiting value (recombination frequency, 15%) that was 5 times that of Arl+ phages. Recombinogenicity and S1 susceptibility were accumulated concomitantly during growth on arl+ bacteria. If all increased recombination occurred at the S1 sites, then these regions (about 40 bases each) were about 300 times as recombinogenic as normal DNA regions of the same size, and 1.5 times as recombinogenic as UV-induced lesions. Chromosomal DNA and plasmid DNA (pBR322) from arl cells were more susceptible to nuclease S1 than was DNA from arl+ bacteria. Analysis of the cleavage products suggests that the S1 sites on Arl- lambda phage DNA are located randomly.     

Two Newly Isolated Enterobacter-Specific Bacteriophages: Biological Properties and Stability Studies

In an era of antibiotic therapy crisis caused by spreading antimicrobial resistance, and when recurrent urinary tract infections constitute a serious social and medical problem, the isolation and complex characterization of phages with a potential therapeutic application represents a promising solution. It is an inevitable, and even a necessary direction in the development of current phage research. In this paper, we present two newly isolated myoviruses that show lytic activity against multidrug-resistant clinical isolates of Enterobacter spp. (E. cloacae, E. hormaechei, and E. kobei), the genomes of which belong to a poorly represented phage group. Both phages were classified as part of the Tevenvirinae subfamily (Entb_43 was recognized as Karamvirus and Entb_45 as Kanagawavirus). Phage lytic spectra ranging from 40 to 60% were obtained. The most effective phage-to-bacteria ratios (MOI = 0.01 and MOI = 0.001) for both the phage amplification and their lytic activity against planktonic bacteria were also estimated. Complete adsorption to host cells were obtained after about 20 min for Entb_43 and 10 min for Entb_45. The phage lysates retained their initial titers even during six months of storage at both −70 °C and 4 °C, whereas storage at 37 °C caused a complete loss in their activity. We showed that phages retained their activity after incubation with solutions of silver and copper nanoparticles, which may indicate possible synergistic antibacterial activity. Moreover, a significant reduction in phage titers was observed after incubation with a disinfectant containing octenidinum dihydrochloridum and phenoxyethanol, as well as with 70% ethanol. The observed maintenance of phage activity during incubation in a urine sample, along with other described properties, may suggest a therapeutic potential of phages at the infection site after intravesical administration.     

丙型肝炎病毒NS3蛋白人源基因工程单链抗体的表达

目的在大肠杆菌XL1-Blue中表达可溶性的抗HCV非结构蛋白NS3的人源单链可变区抗体（single-chainvariablefragmentantibody,ScFv）。方法以重组的HCVNS3为抗原,利用噬菌体抗体库技术筛选含有抗-HCVNS3ScFv基因的噬菌体克隆。从噬菌体抗体阳性克隆中提取质粒,经SfiI/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行DNA序列测定;异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside,IPTG）诱导表达HCVNS3可溶性单链可变区抗体。ELISA和斑点吸印杂交检测其与不同来源的抗原的结合活性。结果筛选到的HCVNS3的单链抗体基因,经限制性内切酶酶切和序列分析表明,该抗体基因由750bp组成,ELISA和斑点吸印杂交结果表明,在大肠杆菌XL1-Blue中表达的HCVNS3的单链抗体,可与不同来源的NS3抗原结合。结论大肠杆菌XL1-Blue表达的NS3-ScFv具有结合不同来源的HCVNS3的活性和特异性。     

Phage Biocontrol of Pseudomonas aeruginosa in Water

Bacteriophage control of harmful or pathogenic bacteria has aroused growing interest, largely due to the rise of antibiotic resistance. The objective of this study was to test phages as potential agents for the biocontrol of an opportunistic pathogen Pseudomonas aeruginosa in water. Two P. aeruginosa bacteriophages (vB_PaeM_V523 and vB_PaeM_V524) were isolated from wastewater and characterized physically and functionally. Genomic and morphological characterization showed that both were myoviruses within the Pbunavirus genus. Both had a similar latent period (50–55 min) and burst size (124–134 PFU/infected cell), whereas there was variation in the host range. In addition to these environmental phages, a commercial Pseudomonas phage, JG003 (DSM 19870), was also used in the biocontrol experiments. The biocontrol potential of the three phages in water was tested separately and together as a cocktail against two P. aeruginosa strains; PAO1 and the environmental strain 17V1507. With PAO1, all phages initially reduced the numbers of the bacterial host, with phage V523 being the most efficient (>2.4 log₁₀ reduction). For the environmental P. aeruginosa strain (17V1507), only the phage JG003 caused a reduction (1.2 log₁₀) compared to the control. The cocktail of three phages showed a slightly higher decrease in the level of the hosts compared to the use of individual phages. Although no synergistic effect was observed in the host reduction with the use of the phage cocktail, the cocktail-treated hosts did not appear to acquire resistance as rapidly as hosts treated with a single phage. The results of this study provide a significant step in the development of bacteriophage preparations for the control of pathogens and harmful microbes in water environments.     

Isolation of a Specialized Transducing Bacteriophage Lambda Carrying the PolC Locus of Escherichia coli

We have isolated a specialized transducing phage carrying the polC locus of E. coli K-12. A strain of E. coli lacking the lambda attachment site was infected with bacteriophage lambda. Lysogens carrying lambda at the tonA locus were isolated by selecting lambda-immune, T5-resistant strains. Transducing phages of dapC, dapD and polC, which map within 0.2 min of tonA, were obtained in lysates prepared from two of the lysogens. The isolated phage, lambdadpolC, is defective but can transduce four different polC temperature-sensitive mutants. After lysogenation with the transducing phage, DNA polymerase III activity is restored to normal levels in extracts of a polC strain lacking polymerase III activity. However, attempts to obtain increased levels of DNA polymerase III in extracts of induced lysogens carrying lambdadpolC have been unsuccessful.