New insights into the possible role of bacteriophages in host defense and disease

BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics – resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds β3 integrins (primarily αIIbβ3). Therefore, phages could bind β3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. TESTING THE HYPOTHESIS: Binding of KGD+ phages to β3 integrin+ cells may be detected using standard techniques involving phage – mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage – cell interactions may be assessed by examining the possible biological effects of β3 blockade (e.g., anti-metastatic activity). IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of β3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer

What are the limitations on the wider therapeutic use of phage?

Bacterial resistance to antibiotics poses a serious health threat. Since research into new antibiotics is not progressing at the same rate as the development of bacterial resistance, widespread calls for alternatives to antibiotics have been made. Phage therapy is an ideal alternative candidate to be investigated. However the success of phage therapy may be hampered by a lack of investment support from large pharmaceutical companies, due to their narrow spectrum of activity in antibiotics, very large costs associated with clinical trials of the variety of phages needed, and regulatory requirements remaining unclear. Intellectual property is difficult to secure for therapeutic phage products for a variety of reasons, and patenting procedures vary widely between the US and the EU. Consequently, companies are more likely to invest in phage products for decontamination or veterinary use, rather than clinical use in humans. Some still raise questions as to the safety of phage therapy overall, suggesting the possibility of cytotoxicity and immunogenicity, depending on the phage preparation and route. On the other hand, with patients dying because of infections untreatable with conventional antibiotics, the question arises as to whether it is ethical not to pursue phage therapy more diligently. A paradigm shift about how phage therapy is perceived is required, as well as more rigorous proof of efficacy in the form of clinical trials of existing medicinal phage products. Phage therapy potential may be fulfilled in the meantime by allowing individual preparations to be used on a named-patient basis, with extensive monitoring and multidisciplinary team input. The National Health Service and academia have a role in carrying out clinical phage research, which would be beneficial to public health, but not necessarily financially rewarding

Use of newly isolated phages for the control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilms

Pseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections. that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells

The influence of the mode of administration in the dissemination of three coliphages in chickens

Escherichia coli can cause severe respiratory and systemic infections in chickens, and it is often associated with significant economic losses in the poultry industry. Bacteriophages (phages) have been shown to be potential alternatives to the antibiotics in the treatment of bacterial infections. To accomplish that, phage particles must be able to reach and remain active in the infected organs. The present work aims at evaluating the effect of the route of administration and the dosage in the dissemination of 3 coliphages in the chicken’s organs. In vivo trials were conducted by infecting chickens orally, spray, and i.m. with 106, 107, and 108 plaque-forming units/mL suspensions of 3 lytic phages: phi F78E (Myoviridae), phi F258E (Siphoviridae), and phi F61E (Myoviridae). Birds were killed 3, 10, and 24 h after challenge and the phage titer was measured in lungs and air sacs membranes, liver, duodenum, and spleen. When administered by spray, the 3 phages reached the respiratory tract within 3 h. Oral administration also allowed all phages to be recovered in lungs, but only phi F78E was recovered from the duodenum, the liver, and the spleen. These differences can be explained by the possible replication of phi F78E in commensal E. coli strains present in the chicken gut, thus leading to a higher concentration of this phage in the intestines that resulted in systemic circulation of phage with consequent phage in organs. When phages were administered i.m., they were found in all of the collected organs. Despite this better response, i.m. administration is a nonpracticable way of protecting a large number of birds in a poultry unit. In general, the results suggest that oral administration and spray allowed phages to reach and to remain active in the respiratory tract and can, therefore, be considered promising administration routes to treat respiratory E. coli infections in the poultry industry.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BDE/15508/200

Phage Therapy:What Have We Learned?

In this article we explain how current events in the field of phage therapy may positively influence its future development. We discuss the shift in position of the authorities, academia, media, non-governmental organizations, regulatory agencies, patients, and doctors which could enable further advances in the research and application of the therapy. In addition, we discuss methods to obtain optimal phage preparations and suggest the potential of novel applications of phage therapy extending beyond its anti-bacterial action

Genetically Engineered Phages: a Review of Advances over the Last Decade

Summary: Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.D.P.P. acknowledges financial support from the Portuguese Foundation for Science and Technology (FCT) through grant SFRH/BD/76440/2011. This work was funded by The Center for Microbiome Informatics and Therapeutics and NSF Expeditions in Computing Program award #1522074 as part of the Living Computing Project. This work was further supported by grants from the Defense Threat Reduction Agency (grants HDTRA1-14-1-0007 and HDTRA1-15-1-0050), the National Institutes of Health (grants 1DP2OD008435,1P50GM098792,1R01EB017755, and 1R21AI12166901), and the U.S. Army Research Laboratory and U.S. Army Research Office, through the Institute for Soldier Nanotechnologies, under contract number W911NF-13-D-0001.S.S.is an FCT investigator (IF/01413/2013). D.P.P., S.S., and J.A. also acknowledge financial support from FCT under the scope of the strategic funding of the UID/ BIO/04469/2013 unit and COMPETE 2020 (grant POCI-01-0145FEDER-006684). T.K.L. is a founder of Sample6 Inc. and Eligo Biosciences, two companies developing phage-based technologies

Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This work investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4 and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45 % reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium adhered bacteria

Measurement of νˉμ\bar{\nu}_{\mu} and νμ\nu_{\mu} charged current inclusive cross sections and their ratio with the T2K off-axis near detector

We report a measurement of cross section $\sigma(\nu_{\mu}+{\rm nucleus}\rightarrow\mu^{-}+X)$ and the first measurements of the cross section $\sigma(\bar{\nu}_{\mu}+{\rm nucleus}\rightarrow\mu^{+}+X)$ and their ratio $R(\frac{\sigma(\bar \nu)}{\sigma(\nu)})$ at (anti-)neutrino energies below 1.5 GeV. We determine the single momentum bin cross section measurements, averaged over the T2K $\bar{\nu}/\nu$-flux, for the detector target material (mainly Carbon, Oxygen, Hydrogen and Copper) with phase space restricted laboratory frame kinematics of $\theta_{\mu}$500 MeV/c. The results are $\sigma(\bar{\nu})=\left( 0.900\pm0.029{\rm (stat.)}\pm0.088{\rm (syst.)}\right)\times10^{-39}$ and $\sigma(\nu)=\left( 2.41\ \pm0.022{\rm{(stat.)}}\pm0.231{\rm (syst.)}\ \right)\times10^{-39}$in units of cm$^{2}$/nucleon and$R\left(\frac{\sigma(\bar{\nu})}{\sigma(\nu)}\right)= 0.373\pm0.012{\rm (stat.)}\pm0.015{\rm (syst.)}$.Comment: 18 pages, 8 figure

Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Author Summary: Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is a necrotizing disease of the skin, subcutaneous tissue and bone. Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin for 8 weeks. However, in advanced stages of the disease, surgical resection of the destroyed skin is still required. The use of bacterial viruses (bacteriophages) for the control of bacterial infections has been considered as an alternative or a supplement to antibiotic chemotherapy. By using a mouse model of M. ulcerans footpad infection, we show that mice treated with a single subcutaneous injection of the mycobacteriophage D29 present decreased footpad pathology associated with a reduction of the bacterial burden. In addition, D29 treatment induced increased levels of IFN-γ and TNF in M. ulcerans -infected footpads, correlating with a predominance of a mononuclear infiltrate. These findings suggest the potential use of phage therapy in BU, as a novel therapeutic approach against this disease, particularly in advanced stages where bacteria are found primarily in an extracellular location in the subcutaneous tissue, and thus immediately accessible by lytic phages.This work was supported by a grant from the Health Services of Fundacao Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/64032/2009, SFRH/BD/41598/2007, and SFRH/BPD/68547/2010 to GT, TGM, and AGF, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Measurements of neutrino oscillation in appearance and disappearance channels by the T2K experiment with 6.6 x 10(20) protons on target

111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee comments111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee comments111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee commentsWe thank the J-PARC staff for superb accelerator performance and the CERN NA61/SHINE Collaboration for providing valuable particle production data. We acknowledge the support of MEXT, Japan; NSERC, NRC, and CFI, Canada; CEA and CNRS/IN2P3, France; DFG, Germany; INFN, Italy; National Science Centre (NCN), Poland; RSF, RFBR and MES, Russia; MINECO and ERDF funds, Spain; SNSF and SER, Switzerland; STFC, UK; and the U. S. Deparment of Energy, USA. We also thank CERN for the UA1/NOMAD magnet, DESY for the HERA-B magnet mover system, NII for SINET4, the WestGrid and SciNet consortia in Compute Canada, GridPP, UK, and the Emerald High Performance Computing facility in the Centre for Innovation, UK. In addition, participation of individual researchers and institutions has been further supported by funds from ERC (FP7), EU; JSPS, Japan; Royal Society, UK; and DOE Early Career program, USA