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, 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

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.

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.

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 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,

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.

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.

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.

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.

Phages and their potential to modulate the microbiome and immunity

Bacteriophages(hence termed phages)are viruses that target bacteria and have long been considered as potential future treatments against antibiotic-resistant bacterial infection.However,the molecular nature of phage interactions with bacteria and the human host has remained elusive for decades,limiting their therapeutic application.While many phages and their functional repertoires remain unknown,the advent of next-generation sequencing has increasingly enabled researchers to decode new lytic and lysogenic mechanisms by which they attack and destroy bacteria.Furthermore,the last decade has witnessed a renewed interest in the utilization of phages as therapeutic vectors and as a means of targeting pathogenic or commensal bacteria or inducing immunomodulation.Importantly,the narrow host range,immense antibacterial repertoire,and ease of manipulating phages may potentially allow for their use as targeted modulators of pathogenic,commensal and pathobiont members of the microbiome,thereby impacting mammalian physiology and immunity along mucosal surfaces in health and in microbiomeassociated diseases.In this review,we aim to highlight recent advances in phage biology and how a mechanistic understanding of phage-bacteria-host interactions may facilitate the development of novel phage-based therapeutics.We provide an overview of the challenges of the therapeutic use of phages and how these could be addressed for future use of phages as specific modulators of the human microbiome in a variety of infectious and noncommunicable human diseases.

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.

Temperate Stutzerimonas Phage Encoding Toxin-Antitoxin System Genes Represents a Novel Genus

Stutzerimonas have been extensively studied due to their remarkable metabolic and physiological diversity.However,research on its phages is currently limited.In this study,we isolated a novel double-stranded DNA(dsDNA)phage,vB_SstM-PG1,from the marine environment that infects Stutzerimonas stutzeri G1.Its dsDNA genome is 37204 bp long with a G/C content of 64.14%and encodes 54 open reading frames.The phage possesses a tail packaging structure that is different from known Stutzerimonas stutzeri phages and exhibits structural protein characteristics similar to those of temperate phages.In addition,two genes of toxin-antitoxin system,including YdaS_antitoxin and HEPN_SAV_6107,were found in the vB_SstM-PG1 genome and play important roles in regulating host growth and metabolism.With phylogenetic tree and comparative genomic analysis,it has been determined that vB_SstM-PG1 is not closely related to any phages previously identified in the GenBank database.Instead,it has a connection with enigmatic,uncultured viruses.Specifically,the vB_SstM-PG1 virus exhibits an average nucleotide identity of over 70%with six uncultivated viruses identified in the IMG/VR v4 database.This significant finding has resulted in the identification of a novel viral genus known as Metabovirus.

Recombination of T4-like Phages and Its Activity against Pathogenic Escherichia coli in Planktonic and Biofilm Forms

The increasing emergence of multi-drug resistant Escherichia coli(E.coli)has become a global concern,primarily due to the limitation of antimicrobial treatment options.Phage therapy has been considered as a promising alternative for treating infections caused by multi-drug resistant E.coli.However,the application of phages as a promising antimicrobial agent is limited by their narrow host range and specificity.In this research,a recombinant T4-like phage,named WGqlae,has been obtained by changing the receptor specificity determinant region of gene 37,using a homologous recombination platform of T4-like phages established by our laboratory previously.The engineered phage WGqlae can lyse four additional hosts,comparing to its parental phages WG01 and QL01.WGqlae showed similar characteristics,including thermo and pH stability,optimal multiplicity of infection and one-step growth curve,to the donor phage QL01.In addition,sequencing results showed that gene 37 of recombinant phage WGqlae had genetically stable even after 20 generations.In planktonic test,phage WGqlae had significant antimicrobial effects on E.coli DE192 and DE205 B.The optical density at 600 nm(OD600)of E.coli in phage WGqlae treating group was significantly lower than that of the control group(P\0.01).Besides,phage WGqlae demonstrated an obvious inhibitory effect on the biofilm formation and the clearance of mature biofilms.Our study suggested that engineered phages may be promising candidates for future phage therapy applications against pathogenic E.coli in planktonic and biofilm forms.

Bacteriophages in water pollution control:Advantages and limitations

Wastewater is a breeding ground for many pathogens,which may pose a threat to human health through various water transmission pathways.Therefore,a simple and effective method is urgently required to monitor and treat wastewater.As bacterial viruses,bacteriophages(phages)are the most widely distributed and abundant organisms in the biosphere.Owing to their capacity to specifically infect bacterial hosts,they have recently been used as novel tools in water pollution control.The purpose of this review is to summarize and evaluate the roles of phages in monitoring pathogens,tracking pollution sources,treating pathogenic bacteria,infecting bloom-forming cyanobacteria,and controlling bulking sludge and biofilm pollution in wastewater treatment systems.We also discuss the limitations of phage usage in water pollution control,including phage-mediated horizontal gene transfer,the evolution of bacterial resistance,and phage concentration decrease.This review provides an integrated outlook on the use of phages in water pollution control.

Phage engineering： how advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophages

Background： The therapeutic potential of bacteriophages has been debated since their first isolation and characterisation in the early 20th century. However, a lack of consistency in application and observed efficacy during their early use meant that upon the discovery of antibiotic compounds research in the field of phage therapy quickly slowed. The rise of antibiotic resistance in bacteria and improvements in our abilities to modify and manipulate DNA, especially in the context of small viral genomes, has led to a recent resurgence of interest in utilising phage as antimicrobial therapeutics. Results： In this article a number of results from the literature that have aimed to address key issues regarding the utility and efficacy of phage as antimicrobial therapeutics utilising molecular biology and synthetic biology approaches will be introduced and discussed, giving a general view of the recent progress in the field. Conclusions： Advances in molecular biology and synthetic biology have enabled rapid progress in the field of phage engineering, with this article highlighting a number of promising strategies developed to optimise phages for the treatment of bacterial disease. Whilst many of the same issues that have historically limited the use of phages as therapeutics still exist, these modifications, or combinations thereof, may form a basis upon which future advances can be built. A focus on rigorous in vivo testing and investment in clinical trials for promising candidate phages may be required for the field to truly mature, but there is renewed hope that the potential benefits of phage therapy may finally be realised.

Transient carriage and low-level colonization of orally administrated lytic and temperate phages in the gut of mice

Many studies have shown the efficacy of phage therapy in reducing gastrointestinal pathogens.However,it is unclear whether phages can successfully colonize the gut when administered in an adequate amount for a long time.About 1×10^(8)PFU/mL of purified lytic phage PA13076 or temperate phage BP96115 were fed daily to mice via drinking water over 31 days,to elucidate the distribution of phages in the gastrointestinal tract.At day 16 and 31,six different segments of the gastrointestinal tract with their contents,including stomach,duodenum,jejunum,ileum,cecum,colon,and fresh feces,were aseptically collected.The phage titers were determined using the double-layered plate method with S.Enteritidis ATCC 13076 or S.Pullorum SPu-109 used as host cells.The results indicated that a small portion of administered phages survived exposure to gastric acid and entered the intestinal tract.The prevalence of phages in the gastrointestinal tract was lower than 1%of the primary phage count.Highest phage titers were detected in the cecum with 10^(4)~10^(5)PFU/g,and most of the phages were eliminated from the body via feces with 10^(6)PFU/g.On day 16 and day 31,the same level of phage titers in different segments of the gastrointestinal tract indicated that the colonization of phages had reached saturation at day 16.These results demonstrate transient phage carriage and low-level colonization of orally administrated lytic and temperate gut phages in mice.

Morphological diversity of cultured cold-active lytic bacteriophages isolated from the Napahai plateau wetland in China

Dear Editor,Viruses are the most abundant,diverse,and ubiquitous entities(approximately 1031)on Earth.They play major roles in horizontal gene transfer,the regulation of bacterial community structures,as well as nutrient and energy cycles of marine ecosystems(Danovaro et al.,2008).In particular,lytic bacteriophages(phages)can infect and kill bacteria without harming human or