In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. Phage Isolation, Purification, and Propagation
2.3. Morphology of Phages and Analysis of Phage Bacteria Interactions
2.4. Host Range and Efficiency of Plating (EOP) Analysis
2.5. One-Step Growth Assays
2.6. Stability of Phages
2.7. Identification of a Suitable E. coli Strain Serving as a Basis for In Vivo Experiments
2.8. Lytic Efficacy of Phages and Phage Preparations
2.9. In Vitro Biofilm Model
2.10. Isolation of Phage-Resistant Variants
2.11. Analysis of the Carbon Consumption of Phage-Resistant Variants Using the Biolog GN2 MicroPlateTM Assay
2.12. Growth and Swimming Motility of Phage-Resistant Variants
2.13. DNA Isolation from Phages and Bacteria
2.14. Library Preparation and Whole Genome Sequencing
2.15. Bioinformatic Analyses
3. Results
3.1. Identification and Characterization of Bacteriophages Active against E. coli Field Isolates of Human and Animal Origin
3.1.1. Six Phages were Isolated That Represent Different Morphological Subgroups of Myoviruses and Show Overlapping Host Ranges and Different Growth Characteristics
3.1.2. All Bacteriophages Showed Lytic Activity under Conditions Relevant for In Vivo Investigation
3.2. Effectivity of the Phages against Model Strain E28
3.2.1. E. coli Strain E28 was Shown to Allow Selective Quantification and Distinction from Other E. coli Strains, thus Being a Suitable Model Strain for In Vivo Experiments
3.2.2. Phage Combinations Show Dose Dependent Lysis of E. coli Model Strain E28
3.2.3. The Six-Phage Preparation Effectively Reduces Biofilm Formation by E28 and DH5α but Not by ECOR10
3.3. Genomic Analysis of G28
3.4. Phenotypic and Genotypic Characterization of Phage-Resistant Variants of E. coli E28
3.4.1. Some Phage-Resistant Strain E28 Variants Show Decreased Growth, Reduced Metabolic Ability, and No Motility
3.4.2. E28.G28R3 Shows Altered Adsorption Behavior and Changes in the Genome
4. Discussion
4.1. Selection of an E. coli Model Strain and Cocktail Composition for a Subsequent Animal Experiment Testing Phage Application against Intestinal Colonization of APEC
4.2. Cocktail Composition Based on Diversity of Phages
4.3. Phages Show Sufficient In Vitro Stability for Testing In Vivo
4.4. In Vitro Determination of an Efficient Cocktail against E28
4.5. Phages Were Composed to Prevent the Development of Resistance
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strain | E28 (WT) | E28.AB27R2 | E28.EW2R3 | E28.G28R3 | E28.KRA2R3 | E28.TB49R2c | E28.TriMR3 | |
---|---|---|---|---|---|---|---|---|
sensitivity against phages | ||||||||
G28 | + | + | + | − | + | (−) | + | |
EW2 | + | + | − | (−) | − | (−) | + | |
KRA2 | + | + | + | + | − | + | + | |
AB27 | + | (−) | + | + | + | + | + | |
TriM | + | + | + | − | + | (−) | − | |
TB49 | + | + | + | (−) | + | − | + | |
metabolic activity | ||||||||
negative | 36 | 38 | 36 | 48 | 36 | 48 | 36 | |
positive | 51 | 47 | 50 | 6 | 51 | 3 | 51 | |
reduced | 2 | 0 | 25 | 0 | 28 | 0 | ||
increased | 0 | 1 | 8 | 0 | 8 | 0 | ||
growth behavior | ||||||||
swimming motility diameter [mm] | 26.3 ± 4.5 | 24.0 ± 7.7 | 19.0 ± 9.0 | 1.0 ± 0.0 | 20.0 ± 5.4 | 1.0 ± 0.0 | 33.3 ± 4.9 | |
growth (OD600nm, 24 h at 37 °C) | 1.33 ± 0.04 | 1.03 ± 0.01 | 1.33 ± 0.05 | 0.53 ± 0.02 | 1.34 ± 0.05 | 0.59 ± 0.03 | 1.34 ± 0.06 |
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Korf, I.H.E.; Kittler, S.; Bierbrodt, A.; Mengden, R.; Rohde, C.; Rohde, M.; Kroj, A.; Lehnherr, T.; Fruth, A.; Flieger, A.; et al. In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli. Viruses 2020, 12, 1470. https://doi.org/10.3390/v12121470
Korf IHE, Kittler S, Bierbrodt A, Mengden R, Rohde C, Rohde M, Kroj A, Lehnherr T, Fruth A, Flieger A, et al. In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli. Viruses. 2020; 12(12):1470. https://doi.org/10.3390/v12121470
Chicago/Turabian StyleKorf, Imke H. E., Sophie Kittler, Anna Bierbrodt, Ruth Mengden, Christine Rohde, Manfred Rohde, Andrea Kroj, Tatiana Lehnherr, Angelika Fruth, Antje Flieger, and et al. 2020. "In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli" Viruses 12, no. 12: 1470. https://doi.org/10.3390/v12121470