A Novel Method to Create Efficient Phage Cocktails via Use of Phage-Resistant Bacteria.

The rapid antiphage mutation of pathogens is a big challenge often encountered in the application of phages in aquaculture, animal husbandry, and human disease prevention. A cocktail composed of phages with different infection strategies can better suppress the antiphage resistance of pathogens. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. Here, we verified that using a resistant pathogen quickly evolved under single phage infection, as the new host can easily obtain phages with different infection strategies. We randomly isolated two lytic phages (i.e., Va1 and Va2) that infect the opportunistic pathogen Vibrio alginolyticus. Whether they were used alone or in combination, the pathogen easily gained resistance. Using a mutated pathogen resistant to Va1 as a new host, a third lytic phage Va3 was isolated. These three phages have a similar infection cycle and lytic ability but quite different morphologies and genome information. Notably, phage Va3 is a jumbo phage containing a larger and more complex genome (240 kb) than Va1 and Va2. Furthermore, the 34 tRNAs and multiple genes encoding receptor binding proteins and NAD+ synthesis proteins in the Va3 genome implicated its quite different infection strategy from Va1 and Va2. Although the wild-type pathogen could still readily evolve resistance under single phage infection by Va3, when Va3 was used in combination with Va1 and Va2, pathogen resistance was strongly suppressed. This study provides a novel approach for rapid isolation of phages with different infection strategies, which will be highly beneficial when designing effective phage cocktails. IMPORTANCE The rapid antiphage mutation of pathogens is a big challenge often encountered in phage therapy. Using a cocktail composed of phages with different infection strategies can better overcome this problem. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. To address this problem, we developed a method to efficiently obtain phages with disparate infection strategies. The trick is to use the characteristics of the pathogenic bacteria that are prone to develop resistance to single phage infection to rapidly obtain the antiphage variant of the pathogen. Using this antiphage variant as the host results in other phages with different infection strategies being efficiently isolated. We also verified the reliability of this method by demonstrating the ideal phage control effects on two pathogens and thus revealed its potential importance in the development of phage therapies.