Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa

Abstract

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.

Figure 1. Selection for phage resistance causes a trade-off resulting in significantly reduced Minimum Inhibitory Concentrations (MIC) to four drugs drawn from different antibiotic classes.

LEFT: Average MIC ± SD of four antibiotics for phage sensitive MDR bacteria (left column) and for spontaneous mutants of these bacteria resistant to phage OMKO1 (right column). RIGHT: Fold improvement of MIC for isolated strains resistant to OMKO1 (*p < 0.05, **p < 0.01). For comparison, data for fold-increased sensitivity of transposon knockout PAO1-∆oprM (phage resistant) is displayed as a vertical black line.

Figure 2. Phage OMKO1 selects against the expression of OprM and, consequently, the function of the mexAB/XY-OprM efflux systems.

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).