Bacteriophages, revitalized after 100 years in the shadow of antibiotics

The year 2015 marks 100 years since Dr.Frederick Twort discovered the"filterable lytic factor",which was later independently discovered and named "bacteriophage" by Dr.Felix d’Herelle.On this memorable centennial,it is exciting to see a special issue published by Virologica Sinica on Phages and Therapy.In this issue,readers will not only fi nd that bacteriophage research is a

Isolation and Characterization of Bacteriophages from Laban Jameed

Laban jameed is a dried salty dairy product obtained by fermentation of milk using a complex population of lactic acid bacteria. Jameed is considered a traditional food product in most eastern Mediterranean countries and is usually made from sheep or cow milk. The aim of this study was to assess phage contamination of jameed dairy product. Two phages were isolated: one from sheep milk jameed (PPUDV) and the other from cow milk jameed (PPURV). Each of the two bacteriophages was partially characterized. The PPUDV phage was identified as a single stranded DNA virus with an approximately 20 kb genome that was resistant to RNase, whereas PPURV phage possessed a double stranded RNA genome of approximately 20 kb and was resistant to DNase. The phage bacterial strain hosts were identified as Lactobacillus helveticus and Bacillus amyloliquefaciens for PPUDV and PPURV, respectively. One step growth curve using a double layer plaque assay test was carried out to monitor the phage life cycle after host infection. PPUDV showed a latent period of about 36 h, burst period of 70 h and a burst size of about 600 Plaque Forming Units (PFU) per infected cell. PPURV phage showed a latent period of about 24 h, burst period of 47 h and a burst size of about 700 PFU per infected cell. SDS-PAGE analysis of total viral proteins showed at least three major bands (27, 40, and 45 kDa) for PPUDV. This is the first study to report the isolation of both DNA and RNA bacteriophages from laban jameed. This study adds new insights into the complexity of dairy contamination and fermentation microbiology of the jameed revealing the existence of two viral genomes in this highly dried and salty dairy product.

Therapeutic Potential of Staphylococcal Bacteriophages for Nasal Decolonization of <i>Staphylococcus aureus</i>in Mice

Bacteriophages represent a rich and unique resource of anti-infectives to counter the global problem of antibiotic resis- tance. In this work, we assessed the bactericidal activity of two virulent staphylococcal phages, K and 44AHJD, against S. aureus isolates of clinical significance, and tested their efficacy in vivo. The phage cocktail lysed >85% of the clinical isolates tested. Both the phages were purified by ion-exchange column chromatography following propagation in bioreactors. The purity profiles of the ion-exchange purified phages were compared with those of phages purified using cesium chloride density gradient ultracentrifugation, and infectiousness of the purified phages was confirmed by plaque forming assay. The in vivo efficacy of a phage cocktail was evaluated in an experimental murine nasal colonization model, which showed that the phage cocktail was efficacious. To our knowledge, this is the first report of phage use in an in vivo model of nasal carriage.

Determining the Minimum Inhibitory Concentration of Bacteriophages: Potential Advantages

The minimum inhibitory concentration (MIC) is the concentration at which an antibacterial agent experiences the complete inhibition of organism growth. Bacteriophages represent a rich and unique resource of anti-infectives to counter the growing world-wide problem of antibiotic resistance. In this study, we compared the host range of lytic bacteriophages and temperate phagesbelonging to various genera, namely Staphylococcus, E. coli and Salmonella, with a range of clinical isolates using two methods: the classical agar overlay method and a newly developed MIC method. MIC was only observed with isolates that were susceptible to phage infection, which correlated with the agar overlay assay, whereas no MIC was detected with isolates that were resistant to phage infection. The simple MIC method was useful in determining phage adsorption and host range, and detecting possible prophage contamination in phage preparations. Interestingly, this method was also applicable to strain differentiation through phage susceptibility testing using a 96-well, high throughput format that proved to be easy, cost-effective, fast and reliable.

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection

Hepatitis B virus(HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic(vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspec-tives to the growing field of bacteriophage researches directing towards HBV infection.

Bacteriophages in <i>Escherichia coli</i>antimicrobial resistance

The continuous battle between humans and the multitude of pathogenic microorganisms in the environment has sought relief in the form of antimicrobials. But the counter attack by pathogenic organisms in the form of multidrug resistance, acquired by various mechanisms such as transformation, transposition, conjugation and transduction is a major reason for concern. Bacteriophages have contributed in a significant way to dissemination of genes encoding antimicrobial resistance, heavy metal resistance and virulence factors through the phenomenon of transduction. This review aims at compiling information about the different mechanisms by which bacteriophages aid in transferring genes involved in antimicrobial resistance to Escherischia coli in various environments.

Bacteriophages as antimicrobial agents against major pathogens in swine： a review

In recent years, the development of antibiotic resistant bacteria has become a global concern which has prompted research into the development of alternative disease control strategies for the swine industry. Bacteriophages （viruses that infect bacteria） offer the prospect of a sustainable alternative approach against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes. This paper reviews the use of phages as an antimicrobial strategy for controlling critical pathogens including Salmonella and Eschefich[a coli with an emphasis on the application of phages for improving performance and nutrient digestibility in swine operations as well as in controlling zoonotic human diseases by reducing the bacterial load spread from pork products to humans through the meat,

Disruption of <i>Mycobacterium smegmatis</i>Biofilms Using Bacteriophages Alone or in Combination with Mechanical Stress

Environmental mycobacteria are capable of forming biofilms in low-nutrient environments, and these biofilms may act as reservoirs for opportunistic infections. The purpose of this study was to determine if bacteriophages could disrupt existing biofilms of acid-fast staining Mycobacterium smegmatis. Using the MBEC 96-well plastic peg assay system, M. smegmatis biofilms were created and then tested for their stability in the presence of mycobacteriophages isolated from a Minnesota sphagnum peat bog. All phages tested were lytic and were observed to have weak, intermediate, and strong abilities to disrupt M. smegmatis biofilms. The formation of biofilms was severely impaired in the presence of mycobacteriophages. Phage treatment was also shown to augment?M. smegmatis biofilm disruption by mechanical forces of sonication or water flow. Our study shows that, as with biofilms of Gram-positive and Gram-negative bacteria, mycobacterial biofilms are also susceptible to destruction by bacteriophages.

噬菌体裂解酶研究进展

噬菌体裂解酶是双链DNA噬菌体所特有的细胞壁水解酶。研究表明,所有噬菌体裂解酶在结构上具有相似性,即含有2个结构域:比较保守的N端催化区和差异较大的C端特异性结合区。裂解酶的高亲和性与种属特异的细胞壁糖基有关,而后者常常是细菌存活的必要成分。所以,细菌难以产生对裂解酶的抗性。本文简要综述噬菌体裂解酶的研究进展。

酸奶噬菌体的防治

从上海市光明乳业股份有限公司车间发酵罐中分离出4株酸奶噬菌体,电镜下均呈蝌蚪形。酸奶噬菌体防治实验结果表明:使用来今1207处理液或来今1208处理液,虽然一定浓度的处理液(100mg/kg的来今1207处理液,0.2%的来今1208处理液)可以达到杀灭噬菌体的效果,但是会影响乳酸菌的正常生长,所以处理酸奶发酵生产设备如工厂管道、发酵罐和种子罐时,需要用清水洗去残留液才能投入生产,否则会导致乳酸菌生长和酸奶发酵的不正常。而来今1205处理液的浓度为0.1%时就可以达到杀灭噬菌体的效果,且不影响乳酸菌的生长,适合于作为酸奶噬菌体的杀灭处理液。

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Beyond bacteria:Role of non-bacterial gut microbiota species in inflammatory bowel disease and colorectal cancer progression

This letter emphasizes the need to expand discussions on gut microbiome’s role in inflammatory bowel disease(IBD)and colorectal cancer(CRC)by including the often-overlooked non-bacterial components of the human gut flora.It highlights how viral,fungal and archaeal inhabitants of the gut respond towards gut dysbiosis and contribute to disease progression.Viruses such as bacteriophages target certain bacterial species and modulate the immune system.Other viruses found associated include Epstein-Barr virus,human papillomavirus,John Cunningham virus,cytomegalovirus,and human herpes simplex virus type 6.Fungi such as Candida albicans and Malassezia contribute by forming tissue-invasive filaments and producing inflammatory cytokines,respectively.Archaea,mainly methanogens are also found altering the microbial fermentation pathways.This correspondence,thus underscores the significance of considering the pathological and physiological mechanisms of the entire spectrum of the gut microbiota to develop effective therapeutic interventions for both IBD and CRC.

噬菌体生物扩增法与改良罗氏比例法在结核杆菌药敏检测中的比较

目的 采用噬菌体生物扩增法 (PhaB法 )与传统比例法对结核分枝杆菌临床株进行利福平、异烟肼药物敏感试验 ,建立一种快速、敏感、特异、价廉的结核杆菌药敏试验方法。方法 分别采用噬菌体生物扩增法和改良罗氏比例法检测 5 2株结核分枝杆菌临床株对利福平、异烟肼的敏感性 ,将二者结果进行比较。结果 比例法检测结果为利福平耐药株的 34株中 ,噬菌体生物扩增法检测结果为 32株耐药 ,2株敏感 ;1 8株敏感株中噬菌体方法检测结果为 1 7株敏感 ,1株耐药。噬菌体方法检测结核菌对利福平的灵敏度为 94 .1 %(32 / 34) ,特异度为 94 .4 %(1 7/ 1 8) ,符合率为 94 .2 %(49/5 2 )。比例法检测为异烟肼耐药的 38株菌中 ,噬菌体方法检测 35株耐药 ,3株敏感 ;1 4株异烟肼敏感株中 ,噬菌体方法检测 1 1株敏感 ,3株耐药。噬菌体方法检测异烟肼敏感性的灵敏度为 92 .1 %(35 /38) ,特异度为 78.6 %(1 1 / 1 4 ) ,符合率为 88.5 %(46 / 5 2 )。结论 用噬菌体生物扩增法可快速、准确地检测结核分枝杆菌对利福平的敏感性 ,灵敏度高 ,与WHO推荐使用的比例法符合率高 ,可在 4 8h得到结果。异烟肼耐药性检测用噬菌体方法的特异度不如利福平 ,有待进一步探讨其原因。

一株海洋弧菌噬菌体——VP9的分离和特性研究

1997年夏季,从皱纹盘鲍脓疱病发病区采集海水样品58份,以河流弧菌Ⅱ(Vibriofluvialis Ⅱ)为指示菌,从这些样品中分离出多株噬菌体,对其中VP9进行了电子显微镜观察分析,并对其生物学特性进行了初步的研究.结果表明:VP9具有典型的廿面体头部及具有尾鞘的尾部,并具有尾椎.VP9对热敏感,热稳定性较差,在65℃,5min内全部失活.在pH6～12之间有较高的稳定性.

参量阵差分Pattern时延差编码冰下水声通信方法

为提高差分Pattern时延差编码水声通信方法的通信速率以及抗多途能力,使其能有效适用于冰下水声环境,提出了基于参量阵的差分Pattern时延差编码水声通信方法,推导了Pattern码的参量发射原理,分析了参量阵发射对该方法性能的影响,利用参量阵发射产生低频宽带窄指向性声柬,减少了声线触碰上下边界的次数,提高了系统抗多途的能力。冰下水域外场试验结果表明:本方法可有效抑制多途效应,同时低频宽带特性提高了系统通信速率。

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production.This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum.Eleven bacteriophages isolated from soil and water samples collected in Wuhan,China,were used to infect P.carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county,Kenya.The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P.carotovorum strain.The phages could lyse 20 strains of P.carotovorum,but not Pseudomonas fluorescens control strains.Among the 11 phages,Pectobacterium phage Wc5r,interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P.carotovorum strains.Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control.Phage cocktail applied at a concentration of 1×10^9 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices,resulted in>90%reduction of soft rot symptoms.This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.

细菌生物被膜及其防控策略研究进展

生物被膜是细菌在压力环境下形成的一种嵌在胞外聚合物基质中的多细胞三维聚集物,它的形成依赖于环境因素的变化。生物被膜结构的发展包括可逆附着、不可逆附着与早期结构发展、生物被膜成熟、细胞分散等4个主要阶段。生物被膜导致常规抗生素难以渗透,使细菌对抗生素不再敏感。生物被膜的耐药机制有外排泵、群体感应等。了解生物被膜形成机制对成功防控生物被膜至关重要,目前的预防策略侧重于阻断生物被膜的形成过程,包括阻止细菌附着在底物上,阻止生物被膜基质的合成阶段,抑制群体感应信号级联以及中断细菌之间的通信等。治疗策略主要是控制和消除形成的生物被膜,方式包括噬菌体、抗菌肽、天然药物、纳米复合材料、光动力疗法、联合用药等。生物被膜的防控策略包括但不限于以上方法,未来的研究可能集中在提高这些方法的效率、纳米材料的临床应用可行性、拓宽噬菌体宿主谱、提高抗生物被膜制剂的稳定性、联合使用多种策略、新型的防控策略等多个方面。同时,进一步深入研究生物被膜的形成机制与调控机制,将为开发新的防控策略提供理论基础。

从噬菌体展示随机肽库中筛选豌豆凝集素的结合肽

目的:从噬菌体展示随机肽库中筛选能与豌豆凝集素(PSA)特异结合的短肽。方法:①用豌豆凝集素(PSA)作为靶蛋白,对噬菌体展示的随机六肽库进行亲和筛选;②α-甲基-D-甘露糖苷对筛选出的噬菌体和PSA结合的影响实验(点印迹法);③选择性地合成了3条6肽ARMWSF、RYDYSY、LRLRQL,用不同浓度的6肽对PSA、ConA与HRP的结合进行竞争抑制实验。结果:经过三轮筛选后,这些噬菌体展示肽有明显的富集,从第三轮挑选的22个克隆的插入氨基酸序列可分为三大类;点印迹结果表明,这些噬菌体展示肽能与PSA特异结合,而α-甲基-D-甘露糖苷不同程度地抑制这种结合;LRLRQL不溶于水,ARMWSF和RYDYSY对PSA和HRP的结合有抑制,而对ConA和HRP的结合没有明显抑制。结论:人工合成的2条6肽和PSA的结合部位与α-甲基-D-甘露糖苷和PSA结合的部位并非相同。

4株酸奶噬菌体的分离

从上海光明乳业股份有限公司车间发酵罐中分离出4株酸奶噬菌体。1号噬菌体头部呈六角形,平均直径为95.1nm,头部剖面平均面积为8184.8nm2,尾部的平均直径为18.3nm,平均长度为403.4nm;2号噬菌体头部呈六角形,平均直径为85.4nm,头部剖面平均面积为8046.2nm2,尾部的平均直径为23.3nm,平均长度为549.4nm;3号噬菌体头部呈六角形,平均直径为95.5nm,头部剖面平均面积为9792.3nm2,尾部的平均直径为19.8nm,平均长度为450.5nm;4号噬菌体头部呈六角形,平均直径为119.3nm,头部剖面平均面积为12586.1nm2,尾部的平均直径为23.6nm,平均长度为520.5nm。

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients.Several A.baumannii strains are multidrug resistant and infect wounds,bones,and the respiratory tract.Current studies are focused on finding new effective agents against A.baumannii.Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A.baumannii are currently being conducted.As shown in animal models,phages against multidrug-resistant A.baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.