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.

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.

噬菌体药物开发及质控研究进展

噬菌体治疗作为抗生素治疗细菌感染的替代方法之一,近年来受到越来越多科学家们的关注,该法主要用于针对由耐药菌及多重耐药菌(Multi-drug resistant,MDR)引起的公共卫生安全问题。如今,噬菌体在农业和兽医治疗方面及其在西方国家用于人类治疗方面越来越受到重视,过去几十年西方世界进行了许多临床试验和治疗探索,并且国外某些授权药店正在销售人用噬菌体药物。但噬菌体用于人类治疗时仍然存在一些问题,如噬菌体的高度特异性限制了其应用范围和噬菌体制剂管理法规不完善使得其不能进行规模化生产等。本文结合我国现行药典关于生物制品生产、制备及质量控制要求和欧洲药品管理局(The European Medicines Agency,EMA)近年来以噬菌体治疗药物批准途径的探索为参照,着重对噬菌体库构建与控制、噬菌体制备与质控和临床前测试等进行分析。

OmniLog TM微生物鉴定系统在青海高原野生型鼠疫噬菌体宿主谱检测中的应用研究

目的采用两种微量法检测青海高原野生型鼠疫噬菌体的宿主谱,为今后噬菌体生态学研究和基于宿主范围噬菌体分类研究提供科学依据。方法基于OmniLog TM微生物鉴定系统微量法和微量点滴法,检测3株鼠疫噬菌体对2株鼠疫耶尔森菌(鼠疫疫苗株EV 76、614F)和4株非鼠疫耶尔森菌(假结核耶尔森菌PTB3、PTB5、大肠杆菌V517、小肠结肠炎耶尔森菌52302-2)的裂解情况。结果476号、087号和072204号鼠疫噬菌体均能成功感染鼠疫疫苗株EV 76、614F并依赖其生长繁殖,且基于OmniLog TM系统33℃培养48 h也能够观察到试验孔中四唑类染料颜色随着噬菌体数量的增加和宿主菌数量的减少而变淡。利用微量点滴法检测3株鼠疫噬菌体对4株非鼠疫耶尔森菌敏感性结果显示,28℃和37℃时476号鼠疫噬菌体对假结核耶尔森菌PTB5敏感,而087号和072204号对其不敏感;476号、087号和072204号鼠疫噬菌体对假结核耶尔森菌PTB3、大肠杆菌V517、小肠结肠炎耶尔森菌52302-2这3株非鼠疫耶尔森菌均不敏感,且基于OmniLog TM系统观察到试验孔与对照孔中细菌生长曲线和四唑类染料颜色分别呈现相似对应的结果。然而,以鼠疫疫苗株EV 76、614F为宿主菌,37℃时072204号鼠疫噬菌体微量点滴于固体培养基上不显示噬菌斑。结论基于OmniLog TM检测系统的噬菌体宿主范围测定法可以作为一种替代传统检测宿主谱的测定方法,在噬菌体与宿主菌相互作用研究中具有较好的应用价值。

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。

Defense and anti-defense mechanisms of bacteria and bacteriophages

In the post-antibiotic era,the overuse of antimicrobials has led to a massive increase in antimicrobial resistance,leaving medical doctors few or no treatment options to fight infections caused by superbugs.The use of bacteriophages is a promising alternative to treat infections,supplementing or possibly even replacing antibiotics.Using phages for therapy is possible,since these bacterial viruses can kill bacteria specifically,causing no harm to the normal flora.However,bacteria have developed a multitude of sophisticated and complex ways to resist infection by phages,including abortive infection and the clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR-associated(Cas)system.Phages also can evolve and acquire new anti-defense strategies to continue predation.An in-depth exploration of both defense and anti-defense mechanisms would contribute to optimizing phage therapy,while we would also gain novel insights into the microbial world.In this paper,we summarize recent research on bacterial phage resistance and phage anti-defense mechanisms,as well as collaborative win-win systems involving both virus and host.

固定化c-ω-转氨酶催化合成(4S)-四氢萘酮

以T4噬菌体衣壳为载体,探索了海绵假弧菌-ω-转氨酶(P-ω-TA)的自组装固定化,以将P-ω-TA与T4噬菌体非必需小外壳蛋白(Soc)融合的方式实现了P-ω-TA在T4噬菌体衣壳上的亲和固定及较高的位点结合数。对固定化P-ω-TA的适应性、回收性能、(S)-型异构体选择性拆分及(1S,4S)-去甲基舍曲林的催化能力进行了测试。结果表明,固定化的P-ω-TA保持了完整的活性及适应性,可通过离心操作轻松地回收,并进行重复使用。在5次回收和重复使用过程中,每次酶活回收率均>91%,经5次催化,最终酶活保持率约为84%。固定化P-ω-TA保持了在底物手性中心处的(S)-型异构体选择性拆分以及对(1S,4S)-去甲基舍曲林的催化能力。

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。

鸭疫里默氏菌噬菌体宿主谱的拓宽

为建立一种拓宽鸭疫里默氏菌噬菌体噬菌谱的方法,将6株噬菌体混合液分别与6株宿主菌、18株多重耐药鸭疫里默氏菌(不能被6株噬菌体中的任何1株裂解)培养,收获培养液中的噬菌体,混合后同样进行30次传代。噬斑分析法测定30代混合液及混合液中单一噬菌体的噬菌谱。结果表明,30代噬菌体混合液可裂解79.2%(19/24)的鸭疫里默氏菌试验菌株;30代噬菌体混合液中分离出的单一噬菌体能裂解的试验菌株数量为2~9株;裂解数最多的5株单一噬菌体,以各自宿主菌传代10次,噬菌谱没有发生变化;将裂解谱互补的单一噬菌体混合,4株噬菌体即可裂解19株试验菌株,与30代噬菌体混合液相同。说明建立的方法可以拓宽噬菌体混合液、单一噬菌体的噬菌谱,为噬菌体鸡尾酒制剂的毒株选择奠定了基础。

噬菌体疗法在动物细菌感染中的应用研究进展

近年来,随着畜禽养殖业的飞快发展,畜禽养殖过程中抗生素的不合理使用情况越发频繁,细菌耐药性问题也越发严重,“禁抗、减抗、限抗”已成畜牧业发展的必然趋势。因噬菌体治疗动物细菌性疾病具有专一性强、不易产生抗性等优点,噬菌体已经作为抗生素替代品应用于动物疾病治疗领域。笔者对噬菌体疗法的主要应用方式和其在家禽、猪、犬、猫和牛细菌性传染病中的应用研究进行总结,以期为噬菌体在动物疫病防控方面的应用提供理论参考。

微生物组在乳腺肿瘤中的研究进展

乳腺癌是女性最常见的恶性肿瘤,探索其发生发展的机制和治疗策略是目前研究的难点。该文重点综述了微生物群在乳腺癌发展及治疗中的研究进展。研究显示,乳腺肿瘤内微生物群对于促进肿瘤转移有着不可忽视的作用;同时,肠道微生物的变化不仅与乳腺癌的发生密切相关,也对治疗效果有显著影响,尤其体现在药物作用机制及免疫治疗的响应性方面。此外,噬菌体展示技术通过筛选出肿瘤特异性蛋白质,为靶向治疗提供了新途径。微生物群和噬菌体作为潜在的诊断工具,已经在癌症风险评估方面展现出新的研究潜力。未来应深入研究微生物群对乳腺癌的调控机制及乳腺癌与肠道微生物的交互作用,以期为乳腺癌的进一步诊断和治疗提供更多新思路。

口腔噬菌体的研究进展

噬菌体是专门感染细菌的病毒,是人体口腔微生态的重要组成部分。口腔中有多种噬菌体,其在口腔微生物群落中与细菌相互作用、共同进化,在维持口腔微生态系统的稳定中具有重要作用,发掘噬菌体与细菌之间的作用关系有利于了解口腔微生物群落的变化因素,而且可以利用噬菌体调控口腔微生态,保持口腔健康。随着抗生素耐药菌株的不断出现,传统的抗生素疗法遇到了重大挑战,由于噬菌体具有特异性、无毒性、自我增殖性等优势,噬菌体疗法已成为目前控制微生物感染的研究热点,为龋病、牙周病和口腔念珠菌病等口腔感染性疾病提供了新的研究方向。本文综述了噬菌体的生物学特征、主要的口腔噬菌体的研究现状、噬菌体在口腔微生态中的作用,以及噬菌体疗法在控制口腔致病菌方面的潜在应用,为噬菌体的研究提供参考。

大肠埃希菌裂解性噬菌体vB_EcoP-R1的特性及其抗细菌生物膜活性分析

目的从医院污水中分离大肠埃希菌特异性噬菌体,分析其生物学特性,并检测其对细菌生物膜的裂解作用。方法通过双层琼脂平板法分离纯化噬菌体,透射电镜观察其形态,并测定其一步生长曲线、体外裂解曲线、温度及酸碱稳定性。全基因测序分析噬菌体基因组组成及特性,结晶紫染色法评估噬菌体对宿主菌生物膜的清除效果。结果从医院污水中新分离1株大肠埃希菌特异性噬菌体,命名为vB_EcoP-R1;透射电镜下显示形态与尾病毒目足病毒科噬菌体成员相似,在-20~50℃的温度范围内以及在pH值5.0~11.0酸碱条件下能稳定存活。vB_EcoP-R1基因组全长为40875 bp,GC含量为49.94%,不含毒力基因和耐药基因。vB_EcoP-R1能够裂解临床分离的大肠埃希菌,且显示高度种属特异性。噬菌体作用于宿主菌预成形生物膜4 h后,对生物膜的清除率达到80.16%。结论vB_EcoP-R1是1株新的大肠埃希菌特异性噬菌体,裂解性强且种属特异性高,可裂解宿主菌已形成的生物膜,有望用于大肠埃希菌感染的替代治疗。