Phage Biology Phage Therapy Basics Brief History Mario
Phage Biology & Phage Therapy Basics & Brief History Mario Vaneechoutte Faagtherapie als alternatief voor antibioticatherapie? Vesalius University College Ghent March 19 th, 2019
Phage biology Phages are viruses Bacterioviruses (Bacterio)phages Macroscopic life Multicellular eukaryotes Viruses Archaeoviruses Nucleo. Cytoplasmic Large DNA Viruses NCLDVs
Phage biology Genome sizes 5 -70 kbp: 50 000 bp Bacteria: 1 – 5 Mbp: 5 000 bp NCLDVs: 1 000 bp Hatfull GF. 2008. Bacteriophage genomics. Curr Opin Microbiol 11: 447 -453.
Phage biology Genome and virion structure 25% Myoviridae Caudovirales: tailed phages n = 5000: 96% of phages: Caudovirales 61% Siphoviridae 14% Podoviridae
Phage biology Morphology of the virion = viral particle (protein) Escherichia coli phage T 4 (Myoviridae)
Phage biology Virion structure Head Tail Endplate Fibers
Phage biology Infection cycle Adherence and injection of phage genome by E. coli T 4 -bacteriophage
Phage biology Lytic infection cycle: time course Endolysine/holine
Phage biology Infection cycles chronic, lytic, lysogenic Chronic phages (e. g. E. coli phage M 13): Phage particles (virions) are formed immediately after infection. Virions leave the host cell without lysis. Virulent phages (e. g. E. coli phage T 4): Phage particles (virions) are formed immediately after infection. The bacterial cell is lysed and virions are released. only lytic cycle = Therapeutic phages Temperate phages (e. g. E. coli phage Lambda): The phage genome can be incorporated into the bacterial genome as a prophage and duplicated together with the bacterial genome: lysogenic phase Later on, the prophage can be activated and start a lytic cycle: lytic phase
Phage biology Infection cycles Chronically infecting phages: E. coli M 13 Enveloped phage
Phage biology Infection cycles Virulent phages vs Temperate phages Lytic cycle Lysogenic cycle Bacterium + prophage = lysogen Burst size = 6
Phage biology Infection cycle Killing from without Empty: after genome injection Before genome injection Too many phages ‘killing from without’ by severe damage to bacterial cell wall = immediate antibacterial effect: OK, but bacteria are killed without infection no multiplication of phages
Phage biology: Infection cycles Virulent phages clear plaques = all cells lysed Temperate phages opaque plaques = not all cells lysed
Quantification/Titration of lysogenic phage D 3112 Double agar overlay (DAO) method Phage Dilution 10 -6 Dilution 10 -10 Dilution 10 -8 Dilution 10 -9 Calculate phages/ml of original stock: 100 µl of a 10 -10 dilution was spotted 20 plaques are counted = 200 plaques per ml = 2 x 1012 phages/ml of original stock = 2000 x 109 phages (more correct: pfu) Compare to 7. 5 x 109 human world population Hans: q. PCR instead of DAO?
Phage therapy: history M. E. Hankin in 1896: The first evidence for a viral-like agent with antibacterial properties Ganges river in India capable of passing through a porcelain filter reduces titres of the bacterium Vibrio cholerae Hankin suggested that it might help to decrease the incidence of cholera in people using water from the Ganges. Adhya & Merril. 2006. The road to phage therapy. Nature 443: 754 -755.
Phage therapy: history Frederick Twort 1915 Félix d'Hérelle 1917
Phage therapy: history 1917: d'Herelle F. 1917. Sur un microbe invisible antagoniste des bacilles dysentériques. Acad Sci Ser D 165: 373. 1921: First phage therapy report published by Bruynoghe & Maisin… see next slide 1923: George Eliava starts the Microbiology Institute in Tbilisi, Georgia: ������ � Sakar t ve l o 1925: d’Herelle F. Essai de traitement de la peste bubonique par le bactériophage. La Presse Med 33: 1393 -1394. 1934: Eliava learns about phage therapy from d’Herelle. 1936: d'Herelle is invited by (the Georgian) Stalin and Eliava to the (Eliava) Institute. 1937: Eliava is executed (by Beria).
Phage therapy: history First published report: BELGIUM Bruynoghe R & Maisin J. 1921. Essais de thérapeutique au moyen du Bactériophage du Staphyloccoque. J Compt Rend Soc Biol 85: 1120 -1121.
Phage therapy: history 1934: Félix D’Herelle (1873 – 1949) & George Eliava (1892 – 1937)
Phage therapy: history Phages: discovered during WW I Antibiotics: applied during WW II The interbellum: first golden age for phage therapy in the West Commercialization of phages in France and USA in 1930’s L’Oréal: Bacté-intesti-phage, Bacté-pyo-phage, Bacté-staphylo-phage Eli Lilly: Colo-lysate, Entero-lysate, Staphylo-lysate Phage therapy was abandoned in the West, because of lack of understanding of the high specificity and of mode of action of phages exaggerated claims of effectiveness: urticaria, herpes, eczema the rise of broad-spectrum antibiotics: penicillin in WWII but phage therapy research continued in Georgia, Russia and Poland
Phage therapy: Strengths Current problems in infectious diseases Antibiotic resistance is increasing (see/listen Eva) Limited number of antibiotics in the pipeline Chronic, recurrent infections are due to biofilm: intrinsic poor efficacy of antibiotics due to poor penetration through biofilm matrix lowered metabolism: dormant bacteria Chronic biofilm-related infections: infection of airways in CF-patients: Pseudomonas aeruginosa chronic otitis media: Haemophilus influenzae recurrent urinary tract infection: uropathogenic Escherichia coli bacterial vaginosis: Gardnerella, Atopobium vaginae burn wounds: Pseudomonas aeruginosa, Staphylococcus aureus foreign object infections: catheters, protheses: Staphylococcus spp. , Propionibacterium acnes !!! chronic infection: time to select the best phages.
Phage therapy: Strengths Narrow spectrum No effect on commensal microflora No cross-resistance effects Treatment can be customized/personalized Different kinetics from inert substances (e. g. antibiotics) Phages multiply by factor 10 -50 (burst size): in theory one single dose can be sufficient to treat an infection Less dependent on blood stream: phages pass also BBB (1) Phages are cleared when they have cleared their host No relation to antibiotic resistance: MDR bacteria can be treated. e. g. , ISP: Intravenous Staphylococcal Phage: active against MRSA 1. Dabrowska et al. 2005. Bacteriophage penetration in vertebrates. J Appl Microbiol 98: 7 -13.
Phage therapy: Strengths Some phages can escape resistance: ISP, active against 95% of S. aureus strains including MRSA is used since decades, with almost no resistance development In case of bacterial resistance, phages can evolve: Phages can be ‘trained’ against resistant bacteria One can ‘fish’ for new phages
Phage therapy: Strengths Phages are safe: Theoretical considerations 1 Phages are safe by definition: viruses which infect only bacteria 1. Bacteriophages infect specifically bacteria since they need to recognize bacterial cell wall structures: peptidoglycane, LPS. Even among bacteria: very narrow spectrum of bacterial strains that can be infected. 2. Bacteriophages that were manipulated genetically to infect mammalian cells were not able to multiply inside the mammalian cells after infection. Di Giovine et al. 2001. Binding properties, cell delivery, and gene transfer of adenoviral penton based displaying bacteriophage. Virology 282: 102 -112. 3. No bacteriophage genes can be found in the human genome retro-viruses have left hundreds of genes integrated into the human genome. In summary, bacteriophages have no tropism towards mammalian (eukaryote) cells and cannot multiply in them.
Phage therapy: Strengths Phages are safe: Theoretical considerations 2 4. Bacteriophages are numerous and ubiquitous: but no phage infections have been reported Numerous Estimate of total number of tailed phage particles on Earth: 4 -6 x 1031 = 10 -fold of number of bacterial cells. Compare: Number of stars in Universe: 1020 - 1024 Bergh. 1989. Nature 340: 467 -468 Whitamn et al. 1998. PNAS 95: 6578 -6583 Ubiquitous Up to 109 phages per ml of surface waters In animal sera, in vaccines, in food E. coli phages in 11% of faeces of healthy persons B. fragilis phages in 68% of faeces of healthy persons "We live in a sea of phages" still no infections with phages have been reported
Phage therapy: Strengths Phages are safe: Practical evidence: Humans 1 During the long history of using bacteriophages as therapeutic agents bacteriophages have been administered to thousands/millons of humans (1) (i) orally, in tablet or liquid formulations (105 to 1011 bacteriophages/dose) (ii) rectally (iii) locally: skin, eye, ear, nasal mucosa, burn wounds, rinses and creams (iv) as aerosols or intrapleural injections (v) intravenously: ISP Only one group, from the Hirszfeld Insitute, Wroclaw, Poland (2), renown for its clinical application of bacteriophages reported a few minor side effects (e. g. nausea, fever). Possibly due to the liberation of endotoxins from lysed Gram-negative bacteria, also observed with antibiotic treatment probably not specifically bacteriophage-related. 1. Sulakvelidze et al. 2001. Bacteriophage therapy. Antimicrob Agents Chemother 45: 649 -659. 2. Weber-Dabrowska et al. 2000. Bacteriophage therapy of bacterial infections: an update of our institute’s experience. Arch Immunol Therap Experiment 48: 547 -551.
Phage therapy: Strengths Phages are safe: Practical evidence: Humans 2 "Vaccination" study in Tbilisi, Georgia (1965) 17. 044 children ingested bacteriophages against Shigella dysenteriae No adverse effects were reported. Babalova et al. 1968. Preventive value of dried dysentery bacteriophage. Zh. Mikrobiol. Epidemiol. Immunobiol. 2: 143 -145.
Phage therapy: Strengths Phages are safe: Practical evidence: Humans 3 E. coli bacteriophage phi. X 174 is IV injected since decades to test the influence on the immune response of different medicines. E. g. IV injection of 109 phi. X 174 bacteriophages/kg body weight, twice, in 18 patients with chronic renal failure. Bearden et al. 2005. Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phi. X 174. Am. J. Transplant. 5: 50 -57. This research group uses this approach since the early 70 s without reporting any adverse effects. Ochs et al. 1971. Immunologic responses to bacteriophage phi. X 174 in immunodeficiency diseases. J. Clin. Investigation 50: 2550 -2558. Wedgwood et al. 1975. The recognition and classification of immunodeficiency diseases with bacteriophage phi. Chi 174. Birth Defects Orig. Artic. Ser. 11: 331 -338.
Phage therapy: Strengths Phages are efficient: Practical evidence: Animals 1 Diarrhoea causing E. coli in mice, calves, lambs and piglets, Treatment with bacteriophages reduces the number of bacteria from 107 to 102 in 2 hours, and stops the associated fluid loss. survival of all treated animals, compared to the placebo group Institute for Animal Disease Research in Houghton, Cambridgeshire, UK Smith WH and Huggins MB. 1983. Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs. J. Gen. Microbiol. 129: 2659 -2675. Smith, et al. 1987. The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages. J. Gen. Microbiol. 133: 1111 -1126.
Phage therapy: Strengths Phages are efficient: Practical evidence: Animals 2 Intramuscular injection (single) in one leg with bacteriophage MW to treat intramuscular E. coli infection in the other leg in mice is more effective than multiple IM administration of antibiotics + phages can ‘migrate/travel’ through the body to other sites of infection Smith WH and Huggins MB. 1982. Succesful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol. 128: 307 -318.
Phage therapy: Strengths Phages are efficient: Practical evidence: Humans 1 "Vaccination" study in Tbilisi, Georgia (1965) 30. 769 children aging 6 months to 7 years old 17. 044 children ingested bacteriophages against Shigella dysenteriae 13. 725 children, living at the opposite side of the streets, served as a control group. Dysentery incidence in control group is 2. 6 fold higher than phage treated group. Babalova et al. 1968. Preventive value of dried dysentery bacteriophage. Zh Mikrobiol Epidemiol Immunobiol 2: 143 -145.
Phage therapy: Strengths Phages are efficient: Practical evidence: Humans 2 Chanishvili N. 2012. Phage therapy--history from Twort and d'Herelle through Soviet experience to current approaches. Adv Virus Res 83: 3 -40. Weber-Dabrowska B. et al. 2000. Bacteriophage therapy of bacterial infections: an update of our institute’s experience. Arch Immunol Therap Experiment 48: 547 -551. 1307 patients with suppurative bacterial infections caused by multidrug-resistant bacteria of different species were treated with specific bacteriophages (BP). full recovery in 1123 cases (85. 9%) transient improvement in 134 cases (10. 9%) ineffective only in 50 cases (3. 8%) The results confirm the high effectiveness of phage therapy in combating bacterial infections which do not respond to treatment with the available antibiotics.
Phage therapy: Weaknesses 1. Narrow spectrum Disadvantage: species and clone need to be identified before application not sufficient time in case of acute (life-threatening) infection Advantage: commensal microflora not affected “Elk nadeel heb sijn foordeel” (Johan Cruyff) Solutions: 1. Use of phage mixtures (cocktails) 2. Application in chronic infections: time to select appropriate phages 3. Broad spectrum phages (e. g. ISP, most S. aureus) exist. 4. Add phages to antibiotics broader spectrum less chance of development of antibiotic resistance less chance of development of phage resistance ethically easier to apply phages on top of standard care
Phage therapy: Weaknesses 2. Bacterial resistance Major strategies of bacteria for developing phage resistance: 1. Mutation of cell wall receptors which are used by phages as adherence ligand 2. DNA restriction/modification systems: nonmodified (phage) DNA is restricted. 3. CRISPR/Cas: adaptive immunity in bacteria! CONSIDERATIONS & SOLUTIONS: Mutant bacteria can become susceptible for other phages. Mutant bacteria can loose virulence. E. coli K 1 -phages induce phage-resistant E. coli but these are K 1 negative: reduced virulence (1). Phages can co-evolve (in fact they do so since 3. 4 billion years). Phages can be propagated in vivo/in vitro to adapt to resistant hosts: training. New phages can be found: fishing for phages. Use phages in combination with antibiotics to prevent development of resistance against antibiotics and phages 1. Smith WH and Huggins MB. 1982. Succesful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol. 128: 307 -318.
Phage therapy: Weaknesses 3. Temperate phages may cause horizontal gene transfer: transduction Transfer of resistance/virulence between bacteria Many prophages are essential for bacterial virulence: Bacterial species Toxin genes encoded by prophages Clostridium botulinum Botulins C, D, E Corynebacterium diphtheriae Diphtheria toxin Escherichia coli Shiga-like toxin Staphylococcus aureus Enterotoxins A, D, E, staphylokinase, TSST-1 Streptococcus pyogenes (GAS) Erythrogenic toxin: scarlet fever Use virulent phages: (almost) no lateral gene transfer and they are more efficient in eradicating bacteria …
Phage therapy: Strengths Phages can penetrate into biofilm matrix 1 Hanlon et al. 2001. Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 67: 2746 -2753. Sillankorva et al. 2004. Pseudomonas fluorescens infection by bacteriophage Phi. S 1: the influence of temperature, host growth phase and media. FEMS Microbiol. Lett. 241: 13 -20. 85% biomass reduction in planktonic as well as biofilm growth. Hughes et al. 1998. Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiology 144: 3039 -3047.
Phage therapy: Strengths Phages can penetrate into biofilm matrix 2 Polysaccharide depolymerase
Phage therapy: Strengths Phages can penetrate into biofilm matrix 3 Plaque: killed bacteria Halo: diffusing depolymerase Dubrovin et al. 2012. Atomic force microscopy analysis of the Acinetobacter baumannii bacteriophage AP 22 lytic cycle. PLo. S ONE 7: e 47348.
Phage therapy: Strengths Phages can penetrate into biofilm matrix 4 Inactive Active polysaccharide depolymerases Pan Y-J et al. 2017. Klebsiella phage ΦK 64 -1 encodes multiple depolymerases for multiple host capsular types. J Virol 91: e 02457 -16.
Phage therapy: Strengths Phages can penetrate into biofilm matrix 5 Yves Briers: also research into endolysins
Phage therapy: history & Belgium First published report: BELGIUM Bruynoghe R & Maisin J. 1921. Essais de thérapeutique au moyen du Bactériophage du Staphyloccoque. J Compt Rend Soc Biol 85: 1120 -1121.
Phage therapy: history & Belgium Vandamme Erick. 2000. Phage therapy Viruses against bacteria. Antibiotics Today: 38 -41. Fiers W. et al. 1976. Complete nucleotide sequence of bacteriophage MS 2 RNA: primary and secondary structure of the replicase gene. Nature 260: 500 -507.
19 May 2008 Maya goes fishing for phages from the Ghent University Hospital sewage University College Ho. Gent
Phage therapy: history 1934: phages from France to Georgia Félix D’Herelle & George Eliava
Phage therapy: history 2005: phages from Sakartvelo to Belgium Jean-Paul Pirnay & Maia Merabishvili 12 October 2009
Phage therapy: regulatory framework Crossing the Rubicon In Belgium, since last year, magistral phages can be used to treat patients Gilbert Verbeken, Daniel De Vos & Jean-Paul Pirnay
Phage therapy: regulatory framework Magistral phages can be used to treat patients in Belgium
The magic (surrealistic? ) Belgian solution: The magistral phage preparation
Phage biology & phage therapy Summary Phages are everywhere: The world is a phage. We live in a sea of phages. Phages are safe Phages are efficient for many patients/cases also against antibiotic resistant bacteria (and against bacteria in biofilm: chronic infection) Phages are not always efficient! Phages can be applied as magistral preparations: (only) in Belgium (in EC)! Phage ISP is highly active against MRSA!
Many, many thanks to Maya Merabishvili Nina Chanishvili Christine Rohde Gilbert Verbeken Daniel De Vos Jean-Paul Pirnay Hans Duyvejonck Leen Van Simaey, Jonas Van Belleghem Els Van Mechelen, Stefan Vermeulen Eva Van Braeckel, Stefanie Vermeersch Petra Schelstraete Pharmacists Lab. MCT/LBR/Eliava DSMZ, Braunschweig Lab. MCT LBR/Ho. Gent LBR Ho. Gent UZ Gent Pharmacy
The Belgian solution: The magistral preparation But this is not yet the end of Belgian surrealism … Magistral phages patients can be treated BUT: Magistral phages cannot be used for a comparative clinical trial For a comparative trial you need: GMP phages = cost x 10 -50 As a consequence: 1. Treat 30 patients with phages: Feel free to use magistral phages 2. Treat 30 patients with phages … and also with saline (placebo) and compare the outcome This is a comparative trial GMP phages must be used Cost of treating with saline = x 10 -50
Phage therapy: Strengths Phages are safe: Practical evidence: Humans 3 105 E. coli T 4 -bacteriophages/ml raw preparations (incl. 2 µg endotoxin/ml) were administered in drinking water to 15 healthy adult volunteers. One day after a single dose exposure, bacteriophages could be recovered in the faeces of the volunteers. No adverse effects were observed No Ig. A, Ig. M or Ig. G antibodies against T 4 -bacteriophage up to one month after … although other studies (with higher doses and long term administration) find antibody development Bruttin A, Brüssow H. 2005. Human volunteers receiving Escherichia coli phage T 4 orally: a safety test of phage therapy. Antimicrob Agents Chemother 49: 2874 -2878.
Phage therapy: Strengths Phages are efficient: Practical evidence: Animals 1 Biswas et al. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun 70: 204 -210. Cheng et al. 2005. Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Antimicrob. Agents Chemother. 49: 111 -117. Wagenaar et al. 2005. Phage therapy reduces Campylobacter jejuni colonization in broiler chickens. Vet. Microbiol. 19: 275 -283.
Phage therapy: Weaknesses 3. Temperate phages may cause horizontal gene transfer: transduction Transfer of resistance/virulence between bacteria
Phage therapy: Weaknesses 3. Temperate phages may cause horizontal gene transfer: transduction Transfer of resistance/virulence between bacteria 1. Prophage DNA accounts for half of the 1. 3 Mb of additional DNA found in the food pathogen E. coli O 157, but absent in the reference strain K 12. Ohnishi et al. 2001. Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Trends Microbiol. 10: 481 -485. 2. Many prophages are essential for bacterial virulence: Bacterial species Toxin genes encoded by prophages Clostridium botulinum Botulins C, D, E Corynebacterium diphtheriae Diphtheria toxin Escherichia coli Shiga-like toxin Staphylococcus aureus Enterotoxins A, D, E, staphylokinase, TSST-1 Streptococcus pyogenes (GAS) Erythrogenic toxin: scarlet fever
Phage therapy: Weaknesses 3. Temperate phages may cause horizontal gene transfer: transduction Transfer of resistance/virulence between bacteria 1. Bacteriophages and bacteria interact naturally in our bodies (gut!) gene exchange (also by plasmids and by transformation) is a natural, frequent and unavoidable phenomenon. 2. Increased horizontal/lateral gene transfer: also due to antibiotics Treatment with aminoglycoside and quinolone antibiotics induces hyper-transformability of Streptococcus pneumoniae, leading to increased uptake of foreign DNA by the bacterial cells which might include incorporation of virulence and antibiotic resistance genes: Prudhomme et al. 2006. Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae. Science 313: 89 -92. 3. Use virulent phages: (almost) no lateral gene transfer (and they are more efficient in eradicating bacteria …)
Phage therapy: regulatory framework The final step towards application is too steep Verbeken G, Huys I, Pirnay JP, Jennes S, Chanishvili N, Scheres J, Górski A, De Vos D, Ceulemans C. 2014. Taking bacteriophage therapy seriously: a moral argument. Biomed Res Int 2014: 621316.
Phage therapy: regulatory framework So, fortunately for phage therapy: Belgium is also the country of surrealism phage magistral phage
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