Development of a Bacteriophage Cocktail to Constrain the Emergence of Phage-Resistant Pseudomonas aeruginosa.

Yuhui Yang,Qian Chen,Wei Shen,Fuquan Hu,Kun Xiong,Shuai Le,Jing Wang,Xuesong He,Xiaoling Jin,Jonathon L Baker,Qiu Zhong

doi:10.3389/fmicb.2020.00327

Yuhui Yang, Qian Chen + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2020.00327

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Abstract

With the emergence of multidrug-resistant and extensively drug-resistant bacterial pathogens, phage therapy and other alternative or additional therapeutic modalities are receiving resurgent attention. One of the major obstacles in developing effective phage therapies is the evolution of phage resistance in the bacterial host. When Pseudomonas aeruginosa was infected with a phage that uses O-antigen as receptor, phage resistances typically achieved through changing or loss of O-antigen structure. In this study, we showed that dsRNA phage phiYY uses core lipopolysaccharide as receptor and therefore efficiently kills the O-antigen deletion mutants. Furthermore, by phage training, we obtained PaoP5-m1, a derivative of dsDNA phage PaoP5, which is able to infect mutants with truncated O-antigen. We then generated a cocktail by mixing phiYY and PaoP5-m1 with additional three wide host range P. aeruginosa phages. The phage cocktail was effective against a diverse selection of clinical isolates of P. aeruginosa, and in the short-term constrained the appearance of the phage-resistant mutants that had beleaguered the effectiveness of single phage. Resistance to the 5-phage cocktail emerges after several days, and requires mutations in both wzy and migA Thus, this study provides an alternative strategy for designing phage cocktail and phage therapy.

Highlights

Pseudomonas aeruginosa is a common opportunistic pathogen that causes infections of the bloodstream, urinary tract, burn wounds, and is one of the major pathogens infecting the airways of cystic fibrosis patients and P. aeruginosa infections can be life-threatening (Jonckheere et al, 2018; Waters and Grimwood, 2018)
P. aeruginosa is a master of antibiotic resistance, with intrinsic resistance to several drugs via low membrane permeability and expression of efflux pumps, and has a remarkable capacity to mutate and horizontally acquire additional traits leading to multidrug-resistant (MDR) and extensively-resistant (XDR) strains (Lopez-Causape et al, 2018)
As to the phage agents, one of the major barriers to successful phage therapy is the rapid emergence of phage-resistant mutants (Rohde et al, 2018)

Summary

Introduction

Pseudomonas aeruginosa is a common opportunistic pathogen that causes infections of the bloodstream, urinary tract, burn wounds, and is one of the major pathogens infecting the airways of cystic fibrosis patients and P. aeruginosa infections can be life-threatening (Jonckheere et al, 2018; Waters and Grimwood, 2018). The host range and killing efficiency of phage cocktails targeting P. aeruginosa have been studied in vitro and in vivo (Roach et al, 2017; Forti et al, 2018). Phage resistance is a somewhat more difficult problem to address, as bacteria have evolved a number of different mechanisms to defend themselves against bacteriophages, including prevention of phage adsorption and DNA injection, restriction enzymes, and CRISPR/Cas systems (Hyman and Abedon, 2010). These mechanisms are highly effective and emerge rapidly. The emergence of phage resistance has been reaffirmed by experts in the field as a key issue regarding the feasibility of phage therapy (Rohde et al, 2018; Kortright et al, 2019)

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