Isolation and Characterization of Novel Lytic Phages Infecting Multidrug-Resistant Escherichia coli

Abstract

Urinary tract infections (UTIs) are the second most frequent bacterial infections worldwide, with Escherichia coli being the main causative agent. The increase of antibiotic-resistance determinants among isolates from clinical samples, including UTIs, makes the development of novel therapeutic strategies a necessity. In this context, the use of bacteriophages as a therapeutic alternative has been proposed, due to their ability to efficiently kill bacteria. In this work, we isolated and characterized three novel bacteriophages, microbes laboratory phage 1 (MLP1), MLP2, and MLP3, belonging to the Chaseviridae, Myoviridae, and Podoviridae families, respectively. These phages efficiently infect and kill laboratory reference strains and multidrug-resistant clinical E. coli isolates from patients with diagnosed UTIs. Interestingly, these phages are also able to infect intestinal pathogenic Escherichia coli strains, such as enteroaggregative E. coli and diffusely adherent E. coli. Our data show that the MLP phages recognize different regions of the lipopolysaccharide (LPS) molecule, an important virulence factor in bacteria that is also highly variable among different E. coli strains. Altogether, our results suggest that these phages may represent an interesting alternative for the treatment of antibiotic-resistant E. coli. IMPORTANCE Urinary tract infections affect approximately 150 million people annually. The current antibiotic resistance crisis demands the development of novel therapeutic alternatives. Our results show that three novel phages, MLP1, MLP2, and MLP3 are able to infect both laboratory and multidrug-resistant clinical isolates of Escherichia coli. Since these phages (i) efficiently kill antibiotic-resistant clinical isolates of uropathogenic Escherichia coli (UPEC), (ii) recognize different portions of the LPS molecule, and (iii) are able to efficiently infect intestinal pathogenic Escherichia coli hosts, we believe that these novel phages are good candidates to be used as a therapeutic alternative to treat antibiotic-resistant E. coli strains generating urinary tract and/or intestinal infections.

Keywords: Escherichia coli; LPS; antibiotic resistance; antibiotic-resistant pathogens; bacteriophages; lipopolysaccharide; multidrug-resistant clinical isolates; phage predation; phage-host interactions; urinary tract infections.

FIG 1

Genomic and phylogenetic characterization of novel phages. (A) Characterization of MLP phage genomes. (B) Proteome-based phylogenetic tree of phages constructed by comparison of 311 related taxa using VipTree. Inner and outer rings indicate phage and host taxa at the family level, respectively. (C to E) Phylogenetic analysis of MLP1 (C), MLP2 (D), and MLP3 (E). Bootstrap support (>60) is shown for each node.

FIG 2

Morphologic and host range analyses of different pathogens. (A) Transmission electron micrographs of MLP phages. The size bars correspond to 100 nm. (B) Host ranges of MLP1, MLP2 and MLP3 among pathogenic E. coli strains were evaluated based on the presence (black) or absence (white) of lysis plaques. (C) Maximum-likelihood whole-genome phylogenetic analysis of all strains used in this study. Bootstrap support is shown for each node.

FIG 3

Efficiency of plating (EOP) and killing dynamics of UPEC strains exposed to MLP phages. (A) EOP was determined in UPEC cultures challenged with MLP phages (MOI of >1). EOP was calculated as the ratio between the titer of each phage on a test strain and the titer on CFT073 wild type (n = 3). Dotted lines represent the limits of detection of the assay. Statistical significance was determined using the two-tailed Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (B) Growth measurements for bacterial cultures treated with MLP1, MLP2, MLP3, and the untreated control. Curves represent the mean values from 4 independent experiments.

FIG 4

Efficiency of plating (EOP) and killing dynamics of InPEC strains exposed to phages. (A) EOP was determined in cultures exposed to MLP phages (MOI of >1) (n = 3). Dotted lines represent the limits of detection of the assay. Statistical significance was determined using the two-tailed Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (B) Growth measurements in the presence of MLP1, MLP2, MLP3, and the untreated control. Curves represent the mean values from 4 independent experiments.

FIG 5

Characterization of phage-resistant E. coli mutants. The efficiencies of plating (EOPs) of MLP phages (A), morphology of plaques (B), and LPS profiles of variants (C) were determined on isolated resistant mutants. O-Ag, O antigen.

FIG 6

LPS profiles of pathogens used in this study. LPS profiles of UPEC (A) and InPEC strains (B) were analyzed by Tricine-SDS-PAGE. O-Ag, O antigen.