Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis
Abstract
:1. Introduction
2. Results
2.1. Virion and Plaque Morphology
2.2. Host Range
2.3. Phage Genome Analysis
2.4. Phage Protein Analysis
2.5. Sensitivity of Virions to External Conditions
2.6. Bacteriophage Development
2.7. Effects of the Bacteriophage on Bacterial Biofilm
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
4.2. Isolation of the vB_EfaS-271 Bacteriophage from Urban Sewage
4.3. Preparation of the vB_EfaS-271 Lysate
4.4. Determination of the Bacteriophage vB_EfaS-271 Host Range
4.5. Electron Microscopic Examination of Phage vB_EfaS-271 Morphology
4.6. Examination of the Morphology of vB_EfaS-271 Plaques
4.7. The Effects of External Factors on the Stability of vB_EfaS-271 Virions
4.8. vB_EfaS-271 Phage Adsorption Rate Assay
4.9. One-Step Growth Experiment
4.10. Lysis Profile of the Bacterial Hosts after vB_EfaS-271 Infection
4.11. Assessment of vB_EfaS-271 Phage Lytic Activity in Biofilms
4.12. Crystal Violet (CV) Staining for the Quantification of Biofilm Biomass
4.13. Detection of the Metabolic Activity of Biofilm Cells after vB_EfaS-271 Infection
4.14. Extraction, Sequencing and Bioinformatic Analysis of the vB_EfaS-271 Genome
4.15. Phylogenetic Analysis
4.16. Mass Spectrometry Analysis of vB_EfaS-271 Proteins
4.17. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BCA | Bicinchonic acid |
BRIG | BLAST ring image generator |
CFU | Cetyltrimethylammonium bromide |
CTAB | Crystal violet |
CV | Dimethyl sulfoxide |
DNase I | Deoxyribonuclease |
DTT | Dithiothreitol |
EHEC | Enterohemorrhagic Escherichia coli |
EM | Emmision |
EPEC | Enteropathogenic Escherichia coli |
ESP | Eschrichia coli secretion protein |
EXC | Excitation |
FDR | False discovery rate |
FU | Fluorescence units |
HPLC | High-performance liquid chromatography |
LA | Luria-Bertani agar |
LB | Luria-Bertani broth |
LIN | Linezolid |
MS | Mass spectrometry |
M.O.I. | Multiplicity of infection |
NCBI | National center for biotechnology information |
NGS | Next generation sequencing |
OD | Optical density |
ORF | Open reading frame |
PBS | Phosphate-buffered saline |
PFU | Plaque forming units |
RNaseA | Ribonuclease A |
SD | Standard deviation |
Stx | Shiga toxin |
TEM | Transmission electron microscopy |
TFA | Trifluoroacetic acid |
TerL | Large subunit of terminase |
TM | Tris-Magnesium sulfate buffer |
TSA | Tryptic soy agar |
TSB | Tryptic soy broth |
VFDB | Virulence Factors of the Pathogenic Bacteria database |
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Bacterial Strain | Phage Sensitivity a |
---|---|
Enterococcus faecalis 230 | − |
Enterococcus faecalis 271 | + |
Enterococcus faecalis 272 | + |
Enterococcus faecalis 273 | − |
Enterococcus faecalis 274 | − |
Enterococcus faecalis 275 | − |
Enterococcus faecalis M2056 | − |
Enterococcus faecalis OG1RF | − |
Enterococcus faecalis V583 | + |
Detected Protein | Predicted Function | Molecular Mass (kDa) | Number of Peptides | Sequence Coverage (%) | Protein Score |
---|---|---|---|---|---|
vB_EfaS-271_08 | portal protein | 43.0 | 18 | 83.03 | 144.10 |
vB_EfaS-271_09 | prohead protease | 21.1 | 3 | 63.64 | 10.64 |
vB_EfaS-271_10 | major capsid protein | 45.3 | 38 | 91.34 | 1412.60 |
vB_EfaS-271_11 | major tail protein | 7.0 | 5 | 93.94 | 79.72 |
vB_EfaS-271_14 | head-tail joining protein | 15.3 | 2 | 54.81 | 1.94 |
vB_EfaS-271_15 | head-tail joining protein | 14.2 | 3 | 44.63 | 8.62 |
vB_EfaS-271_16 | major tail protein | 19.9 | 7 | 65.96 | 4.38 |
vB_EfaS-271_18 | tail tape measure protein | 156.0 | 44 | 78.64 | 131.99 |
vB_EfaS-271_19 | tail protein | 78.9 | 34 | 77.13 | 168.36 |
vB_EfaS-271_20 | minor tail protein | 88.4 | 44 | 88.65 | 161.67 |
vB_EfaS-271_21 | tail fiber protein | 9.2 | 2 | 97.53 | 1.61 |
vB_EfaS-271_23 | lysin | 39.9 | 8 | 45.21 | 4.04 |
vB_EfaS-271_25 | DNA polymerase B-like protein | 87.5 | 11 | 73.00 | 11.70 |
vB_EfaS-271_28 | unknown protein | 26.5 | 8 | 70.21 | 13.99 |
vB_EfaS-271_49 | DNA primase | 60.7 | 31 | 89.58 | 149.50 |
External Factors (Time and Conditions of Incubation) a | Percentage of Viability of vB_EfaS-271 under Certain Conditions ± SD |
---|---|
−80 °C (16 h) | 100.0 ± 0.0 |
−20 °C (16 h) | 100.0 ± 1.9 |
20 °C (24 h) | 100.0 ± 0.0 |
30 °C (24 h) | 100.0 ± 0.5 |
37 °C (24 h) | 100.0 ± 0.0 |
40 °C (40 min) | 100.0 ± 7.9 |
42 °C (40 min) | 42.6 ± 11.3 |
62 °C (40 min) | 1.4 ± 0.5 |
95 °C (5 min) | 0.0 ± 0.0 |
pH 2 (1 h; 37 °C) | 0.0 ± 0.0 |
pH 4 (1 h; 37 °C) | 71.4 ± 2.8 |
pH 10 (1 h; 37 °C) | 100.0 ± 0.0 |
pH 12 (1 h; 37 °C) | 90.0 ± 12.3 |
Osmotic shock (15 min; RT) | 78.8 ± 10.5 |
0.09% SDS (20 min; 45 °C) | 10.3 ± 2.6 |
0.1% CTAB (1 min; RT) | 0.0 ± 0.0 |
0.1% Sarkosyl (10 min; RT) | 100 ± 9.8 |
63% Ethanol (1 h; RT) | 0.2 ± 0.0 |
90% Acetone (1 h; RT) | 0.9 ± 0.4 |
50% DMSO (1 h; RT) | 82.4 ± 12.5 |
Chloroform (1.5 h; 4 °C) | 55.6 ± 9.6 |
10% dish soap (5 min; RT) | 93.0 ± 6.4 |
10% soap (2 min; RT) | 77.8 ± 11.1 |
Line-Antibacterial 70 (5 min; RT) | 81.5 ± 6.4 |
0.5% Virkon (30 min; RT) | 0.0 ± 0.0 |
5% Viruton Pulver (30 min; 30 °C) | 0.0 ± 0.0 |
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Topka-Bielecka, G.; Bloch, S.; Nejman-Faleńczyk, B.; Grabski, M.; Jurczak-Kurek, A.; Górniak, M.; Dydecka, A.; Necel, A.; Węgrzyn, G.; Węgrzyn, A. Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis. Int. J. Mol. Sci. 2020, 21, 6345. https://doi.org/10.3390/ijms21176345
Topka-Bielecka G, Bloch S, Nejman-Faleńczyk B, Grabski M, Jurczak-Kurek A, Górniak M, Dydecka A, Necel A, Węgrzyn G, Węgrzyn A. Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis. International Journal of Molecular Sciences. 2020; 21(17):6345. https://doi.org/10.3390/ijms21176345
Chicago/Turabian StyleTopka-Bielecka, Gracja, Sylwia Bloch, Bożena Nejman-Faleńczyk, Michał Grabski, Agata Jurczak-Kurek, Marcin Górniak, Aleksandra Dydecka, Agnieszka Necel, Grzegorz Węgrzyn, and Alicja Węgrzyn. 2020. "Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis" International Journal of Molecular Sciences 21, no. 17: 6345. https://doi.org/10.3390/ijms21176345