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