Biofilm removal mediated by Salmonella phages from chicken-related source

Salmonella and their biofilm formation are the primary bacterial causes of foodborne outbreaks and crosscontamination. The objective of the study was to investigate the potential of Salmonella phages as an alternative technology for biofilm removal. In this work, 21 Salmonella phages were isolated from a chicken farm and slaughter plant and the phage(CW1)with the broadest spectrum was characterized. Complete genome sequence analysis revealed that the genomes of phage CW1 is composed of 41 763 bp with 58 open reading frames(ORFs)and a holin-endolysin system and it does not encode any virulence or lysogeny. A phage cocktail consisted of CW1(with the broadest spectrum of 70.49%)and CW11, M4 and M10(with a high lytic activity of more than 67.11%)was established. Treatment with the cocktail reduced the cells in the developing biofilm and mature biofilm by 0.79 lg(CFU/cm~2)and 0.4 lg(CFU/cm~2), respectively. More dead cells and scattered extracellular polymeric substances(EPS)were observed by confocal laser scanning microscopy and scanning electron microscopy. Raman analysis found that carbohydrates and proteins were the identification receptors for scattered EPS. This finding suggests that this phage cockta il has potential applications for the sterilization of Salmonella biofilm during meat processing.

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.

Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections

The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this paper is to consider the conditions for the safe, long-term use of phage therapy against various infections caused by Pseudomonas aeruginosa. We describe the selection of the most suitable phages, their most effective combinations and some approaches for the rapid recognition of phages unsuitable for use in therapy. The benefi ts and disadvantages of the various different approaches to the preparation of phage mixtures are considered, together with the specifi c conditions that are required for the safe application of phage therapy in general hospitals and the possibilities for the development of personalized phage therapy.

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,

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

Release the iron: does the infection of magnetotactic bacteria by phages play a role in making iron available in aquatic environments?

Magnetotactic bacteria(MTB)are ubiquitous prokaryotes that orient along magnetic field lines due to magnetosomes’biomineralization within the cell.These structures are ferrimagnetic organelles that impart a magnetic moment to the cell.To succeed in producing magnetosomes,MTB accumulate iron in(i)cytoplasm;(ⅱ)magnetosomes;and(ⅲ)nearby the organelle.It has already been estimated that a single MTB has an iron content of 10 to 100-fold higher than Escherichia coli.Phages are the most abundant entity in oceans and are known for controlling nutrient flow such as carbon and nitrogen by viral shunt and pump.The current work addresses the putative role of phages that infect MTB on the iron biogeochemical cycle.Can phage infection in MTB hosts cause a biogenic iron fertilization-like event in localized microenvironments?Are phages critical players in driving magnetosome biomineralization genes(BGs)horizontal transfer?Further investigation of those events,including frequency of occurrence,is necessary to fully comprehend MTB’s effect on iron cycling in aqueous environments.

THE THERAPEUTIC EFFECT OF INTRATUMORAL INJECTION OF GM-CSF GENE-MODIFIED ALLOGENIC MACROPHAGES ON TUMOR-BEARING MICE

Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GMCSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GMCSF genemodified allogenic macrophages on tumorbearing mice observed. The peritoneal macrophages of C57BL/6 mice were transfected with GMCSF gene mediated by recombinant adenovirus and the subcutaneous CT26 colon adenocarcinomabearing BALB/c mice were treated by intratumoral injection of the above macrophages. The survival time of the tumorbearing mice were prolonged significantly and some tumor mass disappeared completely. The necroses of the tumor cells and massive infiltration of inflammatory cells were observed 6 days after treatment. 30 days after treatment, only the leftover of tumor cells and the inflammatory cells remained. The data indicated that introtumoral injection of GMCSF genemodified allogenic macrophages displayed more potent therapeutic effect on the preestablished tumorbearing mice.

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.

Prophages domesticated by bacteria promote the adaptability of bacterial cells

Prophages are temperate phages integrated into the host bacterial genome.They play an important role in the adaptation and the pathogenicity of bacteria,especially pathogenic bacteria.In this review,we described the distribution of prophages in different hosts and different environments,and focused on the significance of prophages.At the singlecell level,prophages can help the host adapt to harsh external environments by directly carrying virulence genes,encoding regulatory factors and activating lysogeny.At the population level,prophages can influence the overall evolutionary direction and ecological function of the host bacterial community.This review will help us understand the important role of prophages as unique organisms in individual bacteria and microbial populations.

展示p53蛋白表位噬菌体phage-SD的制备及应用

[目的]利用噬菌体展示技术制备一种可用于检测血清p53抗体的噬菌体.[方法]利用基因工程手段将编码p53蛋白N端20~31位的多肽SD的基因克隆到丝状噬菌体载体fADL-le的pIII蛋白基因中,制备展示该外源肽的噬菌体phage-SD并进行了Western blot鉴定.在此基础上,分别以phage-SD和重组p53蛋白为检测抗原,利用ELISA方法对乳腺癌患者血清p53抗体进行检测.[结果]成功制备出能特异性识别血清p53抗体的噬菌体phage-SD.ELISA检测结果表明,60例乳腺癌患者中phage-SD检测到13例p53抗体阳性患者,检出率为21.67%,与重组p53蛋白检测效率(21.67%)一致.[结论]成功制备一种灵敏度高、特异性强的可用于血清p53抗体检测的噬菌体phage-SD,为新型血清p53抗体检测试剂的开发及临床应用奠定基础.

A review on re-emerging bacteriophage therapy in the era of XDR

In the present medicine world antibiotic resistance is one of the key threats to universal health coverage.Researchers continue to work hard to combat this global health concern.Phage therapy,an age-old practice during the early twentieth century,was outshined by the discovery of antibiotics.With the advent of widespread antibiotic resistance,phage therapy has again redeemed itself as a potential alternative owing to its adeptness to target bacteria precisely.Limited side effects,the ability to migrate to different body organs,a distinct mode of action,and proliferation at the infection site,make phages a profitable candidate to replace conventional antibiotics.The progressive outcome of numerous in vitro studies and case reports has validated the clinical efficacy of phage therapy.The bright perspective of using phages to treat bacterial infections has fueled enormous medical research to exploit their potential as therapeutics.The gaps in the information about phages and the lack of consent for clinical trials is major hurdle for consideration of phage therapy.Crafting phage therapy as a reality in medicine requires a coordinated effort from different fraternities.With this review,we aim to emphasize the importance of phage therapy in modern medicine.This review explains their historical journey,basic phage biology,cross-talk with the host immunity,obstacles with phage therapy,and their possible remedies.Comprehensive data on the various significant clinical trials of phage therapy has been presented.We evaluated the efficacy of antibiotics and phage therapy in part and in combination,along with recent progress and future perspectives of phage therapy.

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus(ASFV)is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies(mAbs)and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein(p72)single-chain antibody and fused with an IgG Fc fragment(scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide(221MTGYKH226)of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

噬菌体裂解酶在畜禽生产中的应用研究进展

畜禽生产中抗生素的长期、大量不规范使用,导致细菌耐药性问题日益严重,给畜禽细菌性疾病的预防和治疗带来极大困难,甚至已经威胁到公共卫生安全。噬菌体裂解酶是噬菌体编码的一种肽聚糖水解酶,能够高效、特异地杀灭细菌且具有抗生物被膜活性,已经成为一种新型抗菌药物,逐渐被纳入细菌尤其是耐药菌感染的治疗策略。畜禽生产中,噬菌体裂解酶已经成功应用于金黄色葡萄球菌、链球菌等革兰阳性菌引起的畜禽细菌性疾病的防控与治疗中,其在大肠杆菌、沙门菌等革兰阴性菌上的应用尚处于体外研究阶段。笔者对噬菌体裂解酶的结构特点、作用机制及其在畜禽生产中的应用进行综述,探究噬菌体裂解酶在畜禽生产中的应用现状及前景,以期为噬菌体裂解酶更进一步地应用于防控和治疗细菌性疾病提供参考。

沙门菌噬菌体YT90的分离鉴定及生物学特性分析

[目的]本文旨在分离针对肠炎沙门菌的烈性噬菌体并研究其生物学特性。[方法]从山东烟台某鸭场采集58份粪便样品,以鸭源肠炎沙门菌sdc-11为宿主分离噬菌体,并对噬菌体分离株进行形态学观察,对其温度和pH值稳定性、感染复数(MOI)、一步生长曲线进行测定和分析,测试其对71株不同血清型沙门菌的裂解谱,以及对沙门菌sdc-11生物被膜形成的抑制和对成熟生物被膜的清除作用,最后进行基因组测序分析。[结果]分离获得1株长尾噬菌体,命名为YT90。该噬菌体在30~60℃和pH值4~10的条件下,均表现出较好的增殖能力,最佳MOI值为10^(-5),潜伏期10 min,裂解期90 min,裂解量为每细胞278 PFU;可裂解66.7%(14/21)肠炎沙门菌、66.7%(28/42)鼠伤寒沙门菌和75.0%(3/4)鸡白痢沙门菌;在96孔细胞板上,YT90具有抑制沙门菌sdc-11生物被膜形成的潜力,并能有效清除成熟生物被膜;全基因组测序发现,YT90的基因组全长121240 bp,不含已知的毒力因子和耐药相关基因,不编码整合酶;基于末端酶大亚基构建的进化树分析表明,YT90与沙门菌噬菌体GRNsp50关系最近,属于根西病毒亚科(Guernseyvirinae)新泽西病毒属(Jerseyvirus)。[结论]从鸭场粪便样品中分离获得1株烈性噬菌体YT90,可裂解多种血清型沙门菌,耐受性强、潜伏期短、裂解量大,在防治沙门菌感染方面有较好的临床应用潜力。

大肠埃希氏菌噬菌体swi2裂解系统的表达及协同抑菌活性测定

为了确定大肠埃希氏菌噬菌体swi2裂解系统的穿孔素(holin)与裂解酶(lysin1、lysin2)在裂解宿主菌过程中的协同作用,根据噬菌体swi2裂解系统的基因序列设计引物,搭桥PCR法分别扩增holin-lysin1、holin-lysin2、lysin1-lysin2基因,以pColdTF为载体,构建重组质粒pCold-holin-lysin1、pCold-holin-lysin2、pCold-lysin1-lysin2,在大肠埃希氏菌BL21中诱导表达,并测定各串联蛋白对大肠埃希氏菌BL21的菌内外裂解活性。结果表明,成功构建了噬菌体swi2裂解系统相关基因两两串联的重组质粒,并在原核系统中实现了holin-lysin1、holin-lysin2、lysin1-lysin2蛋白的可溶性表达。lysin1、lysin2具有天然的菌内和菌外裂解活性,而单独的holin未表现出任何抑菌活性;lysin1与lysin2、holin与lysin1之间无协同作用,而holin与lysin2之间存在协同作用,可以显著增强lysin2的菌内外裂解活性(P<0.05)。研究结果证明了lysin1和lysin2在噬菌体swi2增殖后期替代holin完成裂解宿主菌的过程,两者之间不存在协同作用,这将为进一步阐明噬菌体swi2的裂解机制提供参考。

间充质干细胞对代谢功能障碍相关脂肪性肝炎的作用及机制研究

目的探讨间充质干细胞(Mesenchymal stem cells,MSCs)对代谢功能障碍相关脂肪性肝炎(Metabolic dysfunction-associated steatohepatitis,MASH)的作用及机制。方法(1)体内实验:8周龄C57BL/6小鼠高脂喂养24周后,给予链脲佐菌素一次性注射制作MASH模型。将造模成功小鼠随机分为MASH组(n=6)和MSCs组(n=6),另取6只同期正常饲料喂养小鼠为对照组(n=6)。MSCs组小鼠每周经尾静脉注射1×10^(6)MSCs,连续6周,MASH组和对照组给予同等剂量的磷酸缓冲盐溶液(Phosphate buffer saline,PBS)输注。输注结束后,检测3组小鼠肝功能、血脂;HE染色、油红染色观察肝脏病理改变;qRT-PCR检测小鼠肝脏及血清中炎症因子的表达。(2)体外机制研究:提取6周龄C57BL/6小鼠骨髓巨噬细胞,并分为3组(n=3):对照组、脂多糖(LPS)+干扰素-γ(IFN-γ)组及MSCs组。对照组为未处理的巨噬细胞;LPS+IFN-γ组:给予LPS和IFN-γ诱导为促炎型巨噬细胞;MSCs组:LPS+IFN-γ诱导后再经MSCs共培养24 h。处理结束后,通过流式细胞技术比较3组巨噬细胞表型变化,qRT-PCR比较巨噬细胞促炎因子TNF-α、IL-6和IL-1β的表达。结果(1)MASH小鼠给予MSCs输注后,血清AST、ALT水平低于MASH组[AST:(51.33±5.47)U/L vs.(83.33±4.50)U/L,P<0.05;ALT:(67.67±6.71)U/L vs.(111.33±5.47)U/L,P<0.05]。与MASH组相比,MSCs输注后,总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)呈下降趋势,甘油三酯(TG)下降明显[(0.69±0.24)mmol/L vs.(1.11±0.25)mmol/L,P<0.05],提示脂代谢得到改善。与MASH组相比,MSCs组肝细胞脂肪变减轻,炎细胞灶浸润减少,NAFLD活动度积分(NAFLD activity score,NAS)评分明显降低[(3.10±0.13)vs.(5.66±0.52),P<0.05]。MSCs组肝脏油红染色面积较MASH组明显减少[(36.30%±6.06%)vs.(53.10%±3.06%),P<0.05]。MSCs组炎症因子TNF-α、IL-1β及IL-6表达较MASH组明显下调(P<0.05),提示小鼠MASH明显减轻。(2)体外,LPS+IFN-γ诱导的促炎型巨噬细胞与MSCs共培养后,促炎表型CD11c的表达从79.01%±6.08%下降到39.67%±6.11%(P<0.05),抑炎表型CD206从22.67%±4.04%上升至44.67%±4.16%(P<0.05),炎症因子TNF-α、IL-6和IL-1β表达明显下降(P<0.05),提示巨噬细胞的促炎能力被明显抑制。结论间充质干细胞可以减轻代谢功能障碍相关脂肪性肝炎,可能与抑制促炎型巨噬细胞的促炎能力有关。

鼠疫耶尔森菌低钙应答V抗原人源单克隆抗体筛选与鉴定

目的筛选抗低钙应答V抗原(LcrV)的人源单克隆抗体,分析抗体基因特点,检测抗体与抗原结合的特异性、亲和力及功能。方法使用PCR方法从鼠疫疫苗临床试验志愿者外周血细胞cDNA扩增抗体轻、重链可变区基因片段,构建ScFv噬菌体抗体文库。用LcrV对文库进行富集筛选,将获得的抗体基因表达成人IgG1后,测定抗体与LcrV抗原结合的特异性、亲和力、免疫调节功能以及保护能力。结果成功构建了鼠疫耶尔森菌人源ScFv抗体文库,库容量为7.54×10^(8)。抗体文库经LcrV筛选后获得了3株抗LcrV抗体,命名为RV-B4、RV-D1和RV-E8。3株抗体重链为VH1-46和VH3-30,轻链分别为VL1-51、VK3-20和VK1-39。3株抗体经ELISA及Western blot验证均与LcrV特异性结合,其与V抗原结合的解离常数(KD)分别为2.1 nmol/L、1.24 nmol/L和42 nmol/L。RV-D1体外降低人THP-1分泌TNF-α,动物攻毒试验中未发现抗LcrV抗体的保护效果。结论从鼠疫疫苗免疫人群获得了靶向低钙应答V抗原的人源抗体,以结合抗体为主,不能有效阻断病菌感染。所获得的单抗为鼠疫免疫基础研究、鼠疫诊断应用提供了候选材料。

高产乙醇肺炎克雷伯菌的危害及感染应对策略

高产乙醇肺炎克雷伯菌(HiAlc Kpn)能够在患者肠道内产生大量内源性乙醇,是非酒精性脂肪肝(NAFLD)的新病因,严重危害着人类健康。抗生素治疗是应对细菌感染的主要方式,然而肺炎克雷伯菌的耐药问题日益严重,且抗生素往往破坏人体肠道菌群的平衡。噬菌体疗法有望在不破坏健康肠道菌群的基础上,通过靶向杀伤HiAlc Kpn治疗NAFLD。同时,应积极寻找影响HiAlc Kpn产乙醇能力的目标靶点,为开发“抗毒力药物”提供理论基础。本文主要阐述HiAlc Kpn的危害性并提出治疗HiAlc Kpn感染的一些应对策略。

噬菌体抗细菌生物膜机制及应用策略的研究进展

细菌生物膜(bacterial biofilms,BF)是细菌在生物或非生物表面所形成的复杂微生物群落,其形成显著增强了细菌毒力和耐药性,与高比例的慢性细菌感染相关,对人类健康造成严重威胁。传统抗生素和常用消毒剂在清除生物膜方面的能力有限,迫切需要一种有效的新策略治疗细菌生物膜。噬菌体(bacteriophage,phage)作为一类能感染并裂解细菌的病毒,具有较高的安全性和特异度,被认为是治疗细菌生物膜有前景的替代方法。该文综述了噬菌体抗细菌生物膜的作用机制、基于噬菌体及其衍生物在防控细菌生物膜形成的应用策略,为开发高效的噬菌体抗细菌生物膜方法提供新思路。