Phage steering of antibiotic-resistance evolution in the bacterial pathogen, Pseudomonas aeruginosa

Abstract

Background and objectives: Antimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in Eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads but also select for phage resistance mechanisms that trade-off with antibiotic resistance-so called 'phage steering'.

Methodology: Recent work has shown that the phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity of the pathogenic bacterium Pseudomonas aeruginosa. We tested the robustness of this approach to three different antibiotics in vitro (tetracycline, erythromycin and ciprofloxacin) and one in vivo (erythromycin).

Results: We show that in vitro OMKO1 can reduce antibiotic resistance of P. aeruginosa (Washington PAO1) even in the presence of antibiotics, an effect still detectable after ca.70 bacterial generations in continuous culture with phage. Our in vivo experiment showed that phage both increased the survival times of wax moth larvae (Galleria mellonella) and increased bacterial sensitivity to erythromycin. This increased antibiotic sensitivity occurred both in lines with and without the antibiotic.

Conclusions and implications: Our study supports a trade-off between antibiotic resistance and phage sensitivity. This trade-off was maintained over co-evolutionary time scales even under combined phage and antibiotic pressure. Similarly, OMKO1 maintained this trade-off in vivo, again under dual phage/antibiotic pressure. Our findings have implications for the future clinical use of steering in phage therapies. Lay Summary: Given the rise of antibiotic-resistant bacterial infection, new approaches to treatment are urgently needed. Bacteriophages (phages) are bacterial viruses. The use of such viruses to treat infections has been in near-continuous use in several countries since the early 1900s. Recent developments have shown that these viruses are not only effective against routine infections but can also target antibiotic resistant bacteria in a novel, unexpected way. Similar to other lytic phages, these so-called 'steering phages' kill the majority of bacteria directly. However, steering phages also leave behind bacterial variants that resist the phages, but are now sensitive to antibiotics. Treatment combinations of these phages and antibiotics can now be used to greater effect than either one independently. We evaluated the impact of steering using phage OMKO1 and a panel of three antibiotics on Pseudomonas aeruginosa, an important pathogen in hospital settings and in people with cystic fibrosis. Our findings indicate that OMKO1, either alone or in combination with antibiotics, maintains antibiotic sensitivity both in vitro and in vivo, giving hope that phage steering will be an effective treatment option against antibiotic-resistant bacteria.

Keywords: antibiotic resistance; bacteriophage; combination therapy; phage steering.

Figure 1.

Percent (± 95% CI) of MIC of phage-resistant bacteria tested for three antibiotics relative to ancestral phage-susceptible bacteria. PAO1 bacteria resistant to OMKO1 were obtained after 24 h of in vitro growth in the presence of the phage. MIC levels were determined for each of three antibiotics; all three were significantly reduced from the initial ancestor level (t-test, Welch two-sample test)

Figure 2.

Levels of antibiotic resistance [MIC ± 95% CI] of bacteria evolved in the presence of an antibiotic only, phage only or both phage and antibiotic combined. (a) Erythromycin, (b) tetracycline and (c) ciprofloxacin. Bacteria were isolated after 10 serial transfers (20 days) of treatment and their MICs determined. Bacteria from antibiotic treatments were only tested against the same antibiotic; bacteria from phage-only treatments were tested against all three antibiotics. Phage OMKO1 either reduced the emergence of resistance or sensitized the bacteria to the antibiotic. Dashed red line is the mean MIC for the ancestor

Figure 3.

Mean (±95% CI) proportion of surviving wax moth larvae under the four different treatments. Larvae were infected with a lethal dose of PAO1 and observed for 48 h. All three treatment lines provided a greater mean survival time. Both phage alone (blue triangles) and phage plus erythromycin (purple diamonds) produced an additive survival rate when compared with erythromycin alone (red squares), representative markers are staggered to the left or right of the control for ease of reading. Mean survival of 24 replicates

Figure 4.

Mean MIC (±95% CI) of recovered PAO1 bacteria from wax moth infection assay. Bacteria were recovered from 8 wax moth larvae per treatment. Both the phage and phage + antibiotic (erythromycin) lines produced clear reductions in the MIC level post-infection. The reason for the control line MIC above the ancestor is unknown, but could be associated with increased expression of the efflux system responsible for observed antibiotic resistance [54]. Dashed red line is the mean MIC for the ancestor