phage therapy for multidrug-resistant bacterial infections ......daniel mora, tamu thomas walsh,...
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Phage therapy for multidrug-resistant bacterial infectionsPast history and future prospects
Jason J. Gill, PhD
Department of Animal Science
Center for Phage Technology
Texas A&M University
Discovery of bacterial viruses,1915-17
• Bacteriophages were discovered independently by Frederick Twort (1915) and Felix d’Herelle(1917)
• Felix d’Herelle: self-trained French-Canadian microbiologist at the Pasteur Institute
• Found in filtrate of fecal material from soldiers who survived acute dysentery
• Formed “plaques” on lawns of Shigella dysenteriae bacteria
• Named “bacteriophage”: from Greek phagein: to eatPlaque
Bacteriophages: Therapeutics from the beginning
Summers, Felix d’Herelle and the Origins of Molecular Biology
• d’Herelle pursued phage and promoted them for treatment of bacterial disease
• Phages saw extensive therapeutic use from 1920’s –1940’s
• Clinical success of phage was mixed
• Knowledge base non-existent• No knowledge of virus
morphology, DNA, or mechanism of action
•Abandoned in the West after ~1940
Modern phage therapy
•Modern interest in phage therapy in the West began in the 1980’s and has been studied more intensively since ~2000
•Over 40 recent studies have shown efficacy of phages in animal challenge models against many pathogens
• Several phage products have been approved for human consumption to enhance food safety
•Use in animal production and human medicine still experimental, multiple clinical trials ongoing
Bacteriophages in the modern era
•The top predators of bacteria in nature
•Highly diverse and host-specific
•Tails & tail fibers:• Bind specific surface
feature on bacterial cell (“the receptor”)
• Major specificity determinant
100 nm
Lytic pathway
Lysogenic pathway
Virulent bacteriophage life cycle
Lytic pathway
Lysogenic pathway
Temperate bacteriophage life cycle
Temperate phage
Temperate phages form lysogens,
immune to further infection by the
same phage
Temperate phage
Temperate phages often carry one or
more virulence factors in their
genomes
ToxinsImmune evasionAntimicrobial resistance
Phages are highly diverse
• Tailed phages are ancient and have been co-evolving with bacteria for billions of years
• Phages tend to be adapted to their hosts at the species or strain level
• Two phages that infect the same host may have nodetectable DNA sequence similarity
• Phages also carry large numbers of “hypothetical” genes: predicted genes with no known functionSulcius et al. 2011, Oceanologia
Phages are complex viruses
DNA length (kbp)
Genome of E. coli phage LL5
Piya, Lessor et al., in revision
Genes colored in white: function unknown
Phage advantages
•Phage are generally not toxic• Numerous animal and human trials; multiple phages,
multiple routes, no adverse effects• Phage are found in foods, soils, water and as part of the
gut flora (~1031 phages in biosphere)
•Phage replicate at the site of infection•Phage are specific to the target pathogen, often at
the species or strain level• Less collateral damage to other microbial flora
•Resistance to phage is generally unlinked to antibiotic resistance
Phage limitations
•Phage are specific•Multiple phages will often be needed to cover
most strains of a given pathogen•A phage product may require ongoing monitoring
and revision
•Bacteria can mutate to become resistant to phage•Use two or more phages that target independent
receptors•Phage-resistant mutants may be less fit
Case study: Tom Patterson
•Contracted drug-resistant A. baumannii while in Cairo, Egypt
•Medivac to UCSD hospital in December 2015
•MDR Acinetobacter growing from all drains as well as sputum and peritoneal fluid
•We received strain Feb. 28, 2016
•No phages in the CPT collection infected this strain
March 15, 2016
Schooley et al. AAC, 2017
Phage hunting:Culture-based phage enrichment
Sample slurry in culture medium
Centrifuge & filter sterilize
Add bacterial host culture
Centrifuge & filter sterilize
Plate to desired hosts
Incubate overnight, 37 ºC
Phage sensitivity 1 day before treatment
• Strain is sensitive to the 3 new phages C2P24, C2P21 and C1P12, and to AC4 (provided by AmpliPhi)
•Also sensitive to 4 phages isolated by the US Navy MRC
March 14, 2016
Phage sensitivity 12 days post-treatment
•Strain is now insensitive to all phages
•Strain is also resistant to Navy phages but has regained partial sensitivity to minocycline
March 27, 2016
Clinical course
•Treatment course• Feb. 29 – Mar 14: Isolating, producing,
purifying phage • Mar 15: Begin phage treatment • March 28: Patient conscious
•Despite phage resistance, patient continued to improve
•Phage discontinued May 10, 2016
•Patient discharged from hospital Aug. 13, 2016
March 15, 2016
Schooley et al. AAC, 2017
Multiple additional human interventions
Phage-antibiotic synergy
• Increased efficacy in vivo has been observed with combinations of phage and antibiotics
• Phage-resistant mutants may be more sensitive to drugs and/or less virulent due to loss of surface features
Phage-resistant A. baumannii strain TP3 is affected by minocycline and minocycline +
phage (Schooley et al., AAC 2017)
P aeruginosa phage OMKO1 uses the Tet efflux pump OprM as its receptor; phage resistance
leads to drug sensitivity (Chan et al., Sci Rep 2016)
Genomic approaches to phage biology
•Phages are highly diverse, but their genomes are often organized into recognizable modules
•The presence or absence of genes can determine phage characteristics
•Relationships to other known phages can be used to predict function
Genome of B. cenocepacia phage BcepIL02
• Novel phage of B. cenocepacia, few close relatives• 63 kb genome, circularly permuted• Therapeutically effective: ~100-fold reduction in bacterial
loads in mouse lung model• Also a “cryptic temperate” phage, probably not suitable for
therapy in its present form
Gill et al., 2011. J Bacteriol 193:1500-13
K. pneumoniae phage Spivey vs. E. coli phage T5
• Phage T5 is a canonical phage and one of the original seven phages studied in molecular biology
• Binds initially to the LPS O-antigen and then irreversibly to the iron transporter FhuA (TonA)
• Both the LPS-binding tail fiber and FhuA-binding protein are conserved in Spivey
• Spivey-resistant mutants have defects in their FhuA loci
L-shaped tail fiber(O-antigen in T5)
Receptor-binding protein
(FhuA in T5)
T5 (AY543070)
Spivey
Still much to explore in phage genomics
• Phage genomes are under-represented in the databases
• 270,000 bacterial genomes vs. 9,800 phage genomes
• ~30-fold more bacterial genomes
• Because of their size difference, over 1,000 X more bacterial sequence than phage sequence!
Still much to explore in phage genomics
• Phage genomes are under-represented in the databases
• 270,000 bacterial genomes vs. 9,800 phage genomes
• ~30-fold more bacterial genomes
• Because of their size difference, over 1,000 X more bacterial sequence than phage sequence!
270,000
1 Tb
9,800
1 Gb
Organisms
Bases
Still much to explore in phage genomics
•Novel phages can be found almost anywhere
• Sequencing is cheap, annotation tools more available
• Phage genomes are excellent for teaching molecular genetics and genomics
https://cpt.tamu.edu/galaxy-pub
Conclusion
• Phages were discovered in the pre-antibiotic era and were almost immediately developed as antibacterials• Early use of phages was purely empirical and success was
mixed
•Modern revival of phage therapy is spurred by lack of treatment options for MDR bacteria
• Phage diversity and specificity require a thorough understanding of the phage-host interaction using genomic and molecular approaches
• Linkages between phage resistance and virulence or antibiotic sensitivity may be exploitable
Thanks!Center for Phage Technology
Ry Young
Mei Liu
Carlos Gonzalez
Junjie Zhang
Jennifer Herman
Lanying Zeng
Cory Maughmer
Jolene Ramsey
K. pneumoniae project
Lauren Lessor, CPT
Jordyn Michalik, TAMU
Daniel Mora, TAMU
Thomas Walsh, WCMC
Rita Mavridou, WCMC
Vidmantas Petraitis, WCMC
Robert Danner, NIH CC
Karen Frank, NIH CC
Rebecca Weingarten, NIH CC
A. baumannii project
Adriana Hernandez, TAMU
Jacob Lancaster, CPT
Robert Schooley, UCSD
Sharon Reed, UCSD
Jeremy Barr, SDSU
Forrest Rohwer, SDSU
Anca Segall, SDSU
Theron Hamilton, US Navy
Biswas Biswajit, US Navy
Scott Salka, Ampliphi