Spontaneous Phage Resistance in Avian Pathogenic Escherichia coli

Patricia E Sørensen,Hanne Ingmer,Marc Stegger,Patrick Butaye,Sharmin Baig,An Garmyn

doi:10.3389/fmicb.2021.782757

Abstract

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide. The emergence of multidrug-resistant pathogens has renewed the interest in the therapeutic use of bacteriophages (phages). However, a major concern for the successful implementation of phage therapy is the emergence of phage-resistant mutants. The understanding of the phage-host interactions, as well as underlying mechanisms of resistance, have shown to be essential for the development of a successful phage therapy. Here, we demonstrate that the strictly lytic Escherichia phage vB_EcoM-P10 rapidly selected for resistance in the APEC ST95 O1 strain AM621. Whole-genome sequence analysis of 109 spontaneous phage-resistant mutant strains revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of these, a single SNP was detected while two SNPs were identified in a total of nine strains. In total, 34 unique SNPs were detected. In 42 strains, including 18 strains with SNP(s), gene losses spanning 17 different genes were detected. Affected by genetic changes were genes known to be involved in phage resistance (outer membrane protein A, lipopolysaccharide-, O- antigen-, or cell wall-related genes) as well as genes not previously linked to phage resistance, including two hypothetical genes. In several strains, we did not detect any genetic changes. Infecting phages were not able to overcome the phage resistance in host strains. However, interestingly the initial infection was shown to have a great fitness cost for several mutant strains, with up to ∼65% decrease in overall growth. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in APEC. Although acquired resistance to phages is frequently observed in pathogenic E. coli, it may be associated with loss of fitness, which could be exploited in phage therapy.

Highlights

Bacteriophages are viruses that infect bacteria, and are estimated to be the most abundant organisms on Earth with more than 1031 entities (Güemes et al, 2016)
The avian pathogenic E. coli (APEC) ST95 O1:H7 strain, AM621, is part of the in-house collection that was isolated from clinical material suspected of Avian pathogenic Escherichia coli (APEC) infection from Belgium collected during 2013–2014 by Animal Health Care Flanders (Torhout, Belgium)
For the agar plate (AP) method, the highest number of true resistant mutants were isolated from multiplicity of infection (MOI) 100 suspensions (∼42%) and the lowest from MOI 0.1 (0%)

Summary

Introduction

Bacteriophages (phages) are viruses that infect bacteria, and are estimated to be the most abundant organisms on Earth with more than 1031 entities (Güemes et al, 2016). Bacteria can evolve resistance to phage infection through various mechanisms, such as spontaneous mutations, acquisition of restriction-modification (R-M) systems, and adaptive immunity via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system(s). These mechanisms can be used to target different steps of the phage life cycle, including phage attachment, adsorption, replication, and host cell lysis (Barrangou et al, 2007; Labrie et al, 2010). Maintenance of defense systems such as R-M enzymes and CRISPR-Cas, has its own costs associated with enzyme production and expression (Vasu and Nagaraja, 2013; Vale et al, 2015; Bradde et al, 2019)

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