Resistance Evolution against Phage Combinations Depends on the Timing and Order of Exposure

Abstract

Phage therapy is a promising alternative to chemotherapeutic antibiotics for the treatment of bacterial infections. However, despite recent clinical uses of combinations of phages to treat multidrug-resistant infections, a mechanistic understanding of how bacteria evolve resistance against multiple phages is lacking, limiting our ability to deploy phage combinations optimally. Here, we show, usingPseudomonas aeruginosaand pairs of phages targeting shared or distinct surface receptors, that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance. Whereas sequential exposure allowed bacteria to acquire multiple resistance mutations effective against both phages, this evolutionary trajectory was prevented by simultaneous exposure, resulting in quantitatively weaker resistance. The order of phage exposure determined the fitness costs of sequential resistance, such that certain sequential orders imposed much higher fitness costs than the same phage pair in the reverse order. Together, these data suggest that phage combinations can be optimized to limit the strength of evolved resistances while maximizing their associated fitness costs to promote the long-term efficacy of phage therapy.IMPORTANCEGlobally rising rates of antibiotic resistance have renewed interest in phage therapy where combinations of phages have been successfully used to treat multidrug-resistant infections. To optimize phage therapy, we first need to understand how bacteria evolve resistance against combinations of multiple phages. Here, we use simple laboratory experiments and genome sequencing to show that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance evolution in the opportunistic pathogenPseudomonas aeruginosa. These findings suggest that phage combinations can be optimized to limit the emergence and persistence of resistance, thereby promoting the long-term usefulness of phage therapy.

Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence

“…It appears that the evolution pressure in Pspo is exerted at the level of the phage receptor. Similar results were shown in P. aeruginosa when infected with LPS-recognizing phages [37]. Extrapolating the fraction of naturally phage-resistant Pspo mutants in our tests, we can hypothesize that in naturally infected seeds, only a Pspo minority is resistant against the cocktail of the two phages.…”

High-throughput mapping of the phage resistance landscape inE. coli

“…In addition, these methods have the capability to identify fitness costs associated with broadly seen phage resistance phenotypes in a competitive natural environment, and thus improve our understanding of microbial ecology in general [13,114,206,207,[210][211][212]. Such systems-level insights will be valuable both in uncovering new mechanisms in host-phage interaction and perhaps in developing different design strategies for targeted microbial community interventions, engineering highly virulent or extended host-range phages and rationally formulated phage-cocktails for therapeutic applications [89,204,208,[213][214][215][216][217][218][219][220][221][222][223][224][225][226]. Alternatively, identifying phage resistance determinants may also enable engineering of bacterial strains with phage defense systems crucial in a number of bioprocesses such as in the dairy industry [227,228], biocontainment strategies for bioproduction industry [229,230] or to facilitate bacterial vaccine discovery and development [231][232][233].…”

Application of Campylobacter jejuni Phages: Challenges and Perspectives

“…Such a fitness cost can be maximized by simultaneous pressure from multiphage applications. In response, the pressure exerted on bacteria from combinations of phages would have to be met with multiple mutations in a given bacterial cell, which means that resistant bacteria would possess less cellular fitness and could eventually be outgrown by a non-resistant phenotype [74]. This was exactly the case with resistant C. jejuni phenotypes in some studies discussed earlier.…”

“…Today it is widely known that resistance of bacterial strains to bacteriophages can be a major drawback in the development of therapeutic phage applications. There is also some concern that development of phage resistant bacterial strains could lead to limited long-term sustainability of phage applications, as it is the case now with antibiotic drugs [74]. Resistance of C. jejuni to their bacteriophages is making the wider use of the latter challenging.…”