Evolutionary impact on phage-bacteria-host interactions: An opportunity for phage therapy
The dynamic interplay of bacteriophage, bacteria and the mammalian host during phage therapy
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As anticipated from in vitro systems, phage resistance represents one of the greatest challenges to the sustainable development of phage therapy.32,86,87 The application of high titers of therapeutic phage can induce a strong selective pressure for the selection of phage-resistant pathogenic bacteria. The proliferation of phage resistant bacteria can, in turn, lead to therapeutic failure (Figure 3A).25,37,49,88,89 The emergence of bacterial pathogens resistant to virulent phage drastically reduces the ability of phage to clear an infection. Likewise, the emergence of resistance would also inhibit the use of engineered temperate phage to reprogram target bacteria. Finding therapeutic strategies that are robust against phage-resistance is critical for the success of phage therapy.90 Anticipating such evolution of resistance is paramount to understanding the consequences of phage therapy in vivo. However, this requires taking into account how principles of coevolution change when moving from the test tube to animal models and ultimately to clinical applications.
Figure 3. The impact of evolution in phage-bacteria-host interactions
(A) Bacteria can develop phage-resistance which limits phage efficacy to control infections.
(B) To restore the positive therapeutic outcome it may be possible to select combinations of phages so that no individual bacteria can develop resistance to all phages.
(C) Alternatively, tradeoffs can be exploited so that phage resistance by bacteria comes with a fitness cost.
(D) Phage can evolve new mechanisms to infect resistant bacteria. Hence, phage can be ‘trained’ in vitro against the bacteria population to be cleared, and phage sampled at the end of the coevolutionary training may be used to counter resistance mechanisms that will most likely emerge during therapy.
The Future of Bacteriophage Therapy Will Promote Antimicrobial Susceptibility
Efflux pump inhibitor (EPI) mechanisms (A to D) compared to phage steering (E). The figure illustrates (A) substrate competition causing efflux of EPIs (black circles) while retaining antimicrobials (red circles), (B) altered antimicrobial structure by EPIs (red squares) prevents recognition by the efflux system, (C) disruption of pump assembly, (D) disruption of the proton motive force required for efflux, and (E) phage steering using the outer efflux protein as a receptor to block efflux in addition to actively destroying pathogens.
Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa
Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections......
Average cell densities (OD600) of PA01-ΔmexR and PA01-ΔoprM over time in the presence of Tetracycline (10 mg/L) and phage OMKO1 (green and red lines). PAO1 ∆mexR (blue, green) overexpresses mex-OprM and readily grows in TET to high densities alone due to active efflux of TET (blue) but is susceptible to phage infection (green). PAO1 ∆oprM grows poorly in the presence of TET (red) but is resistant to phage OMKO1 (yellow).
Our study showed that phage OMKO1 is a naturally occurring virus that forces a desired genetic trade-off between phage resistance and antibiotic sensitivity, which should benefit phage therapy efforts against MDR bacteria such as P. aeruginosa. Isolation of phage OMKO1 from nature suggested that other phages might have evolved to utilize OprM or other surface-exposed proteins of Mex systems as binding sites. These types of phage could be highly useful for developing therapeutics, because target bacteria are expected to inevitably evolve phage resistance resulting in antibiotic susceptibility. Previous studies similarly demonstrated the evolutionary interplay between phage selection and maintenance of antibiotic resistance in bacterial pathogens. For example, phage binding may rely on surface proteins coded by plasmid genes, causing phage to select against plasmid maintenance in bacterial populations, thereby reducing the prevalence and spread of plasmid-borne antibiotic resistance genes40. Other studies also suggest that combined use of phages and antibiotics is superior to either selection pressure alone, indicating that the dual approach is promising as an antimicrobial strategy41,42. Our study demonstrates that phage OMKO1 is also a promising evolutionary-based phage adjunctive, which can be used to directly exploit a genetic trade-off between efflux mediated antibiotic resistance and phage resistance. Taken together, these examples illustrate the potentially valuable approach by which an evolutionary-based antibiotic adjunctive could greatly improve clinical outcomes and reduce the spread of antibiotic resistant infections. The clinical utility of phages such as OMKO1 is vital because selection using this phage restores usefulness of antibiotics that are no longer considered to be therapeutically valuable.
