Designing phage cocktails to combat the emergence of bacteriophage-resistant mutants in multidrug-resistant
Klebsiella pneumoniae
Authors: Yoo Seongjun , Lee Kang-Mu , Kim Nayoung , Vu Thao Nguyen , Abadie Ricardo , Yong Dongeun 
Published: November 2023

ABSTRACT
Phage therapy, an emerging strategy in the fight against recalcitrant bacterial infections, encounters a significant hurdle posed by the development of phage-resistant bacterial strains. To address this concern, we suggest a novel phage cocktail. By subjecting bacterial strains to a gradual selection process, we isolated and identified three novel phages augmenting one previously isolated phage. This diverse phage set formed the basis of a phage cocktail, displaying enhanced efficacy in curtailing the emergence of phage resistance, particularly evident in the notable stability achieved by three- and four-phage combinations. Our investigation further illuminated distinctive attributes exhibited by phage-resistant mutants: a concomitant reduction in growth rate and virulence offset by a noteworthy escalation in biofilm formation. The therapeutic modality underpinning our approach encompasses both the direct lethality of phages toward bacteria and the intricate landscape of bacterial genetic adaptations. Of intrigue was our observation that bacterial strains previously impervious to a given phage could regain susceptibility following resistance development against an unrelated phage. This phenomenon demonstrates the potential of phage cocktails as an efficacious countermeasure. Consequently, we suggest an innovative approach involving the design of phage cocktails that selectively encompass phages retaining susceptibility to bacteria that have developed resistance to other phages within the cocktail. Furthermore, we advocate for the comprehensive profiling of phage host ranges targeting both resistant strains and wild-type counterparts. The compilation of such intricate host-range data holds the promise of establishing a repository of diverse phages, meticulously tailored for refined and personalized phage therapy interventions.

IMPORTANCE
In this study, we aimed to design a novel and effective bacteriophage cocktail that can target both wild-type bacteria and phage-resistant mutants. To achieve this goal, we isolated four phages (U2874, phi_KPN_H2, phi_KPN_S3, and phi_KPN_HS3) that recognized different bacterial surface molecules using phage-resistant bacteria. We constructed three phage cocktails and tested their phage resistance-suppressing ability against multidrug-resistant Klebsiella pneumoniae. We argue that the phage cocktail that induces resensitization of phage susceptibility exhibited superior phage resistance-suppressing ability. Moreover, we observed trade-off effects that manifested progressively in phage-resistant bacteria. We hypothesize that such trade-off effects can augment therapeutic efficacy. We also recommend collating phage host range data against phage-resistant mutants in addition to wild-type bacteria when establishing phage banks to improve the efficiency of phage therapy. Our study underscores the importance of phage host range data in constructing effective phage cocktails for clinical use.