In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli

Abstract

Worldwide, poultry industry suffers from infections caused by avian pathogenic Escherichia coli. Therapeutic failure due to resistant bacteria is of increasing concern and poses a threat to human and animal health. This causes a high demand to find alternatives to fight bacterial infections in animal farming. Bacteriophages are being especially considered for the control of multi-drug resistant bacteria due to their high specificity and lack of serious side effects. Therefore, the study aimed on characterizing phages and composing a phage cocktail suitable for the prevention of infections with E. coli. Six phages were isolated or selected from our collections and characterized individually and in combination with regard to host range, stability, reproduction, and efficacy in vitro. The cocktail consisting of six phages was able to inhibit formation of biofilms by some E. coli strains but not by all. Phage-resistant variants arose when bacterial cells were challenged with a single phage but not when challenged by a combination of four or six phages. Resistant variants arising showed changes in carbon metabolism and/or motility. Genomic comparison of wild type and phage-resistant mutant E28.G28R3 revealed a deletion of several genes putatively involved in phage adsorption and infection.

Keywords: APEC; E. coli; bacteriophages; biofilm; colibacillosis; multidrug-resistant bacteria; phage cocktail; phage-resistant variants.

Figure 1

Morphology of phages. Transmission electron micrographs of negatively-stained phages EW2 (A), AB27 (B), TB49 (C), TriM (D), KRA2 (E), G28 (F) infecting E. coli strain E28 (Scale bars represent 100 nm).

Figure 2

Host range analysis of phages. Serial dilutions of phage lysates were spotted on a lawn of the host bacteria. After overnight incubation plates were examined for plaques. Blue fields indicate the ability of phages to form plaques on the corresponding bacterial strains, gray fields represent no lysis. Experiments were performed at least twice.

Figure 3

Efficacy of bacteriophages and phage combinations in inhibiting the growth of E. coli E28. Optical density of E. coli E28 cultures infected with (A) 4 × 10⁶ PFU of single phages AB27, EW2, G28, KRA2, TB49, and TriM and (B) 4 × 10⁶ PFU or hundredfold dilutions of phage combinations 4-phage (4 φ) (AB27, EW2, G28, an TB49) or 6-phage (6 φ) (4-phage preparation+ KRA2, TriM). Each experiment was performed twice with triplicate determinations. Mean values are presented without SD to maintain clarity; the average SD is given in Table S8.

Figure 4

Effect of the 6-phage preparation on the formation of E. coli biofilms. Bacterial cultures were infected with 1 × 10⁶ PFU/mL of the phage preparation at the beginning of the experiment (t0) and after 24 h (t24). Bacterial counts were determined after 48 h. Data are presented as mean with SEM of triplicate determinations (n = 4). Statistical analysis was performed using two-way ANOVA and Bonferroni corrected post-hoc tests for detection of significant differences. * p < 0.05 and *** p < 0.001.

Figure 5

Comparison of adsorption of G28 to wild type E28 and phage-resistant mutant E28.G28R3. FESEM of E28 wild type (A) and mutant E28.G28R3 (B) 20 min after addition of phage G28. (C) Adsorption of G28 on E28 wild type and mutant E28.G28R3. Mean and SD of three experiments with duplicate determinations are shown. Statistical analysis was performed using two-way ANOVA and bonferroni corrected post-hoc tests for detection of significant differences. Stars indicate comparison of “G28” and “G28 + E28”, crosses signify the comparison of “G28 + E28” and “G28 + E28.G28R3” (⁺ p < 0.05, ^++/** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 6

Deleted region in the genome of phage-resistant E. coli E28.G28R3 harboring genes putatively necessary for phage adsorption. The location of the deleted region is marked in red in the genome of wild type E28. Genes marked in green are linked to putative transposase activity and regulation. A detailed map of the deletion was visualized with EasyFig.