Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant Enterococcus faecalis in Kenya
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Cultural Conditions
2.2. Bacteriophage Isolation, Purification, and Propagation
2.3. Phage Stability
2.3.1. Thermal Stability
2.3.2. pH Stability
2.4. Host Range Analysis
2.5. Determination of Optimal Multiplicity of Infection
2.6. Lytic Properties of Phage vB_Efs8_KEN04
2.7. One-Step Growth Curve
2.8. Adsorption Assay
2.9. Isolation of Phage-Resistant Mutants
2.10. Determination of the Nature of Phage Receptor
2.11. Biofilm Formation Assay
2.12. Biofilm Inhibition Assay by Phage
2.13. Biofilm Disruption Assay by Phage
2.14. Genomic DNA Extraction
2.15. Genome Sequencing and Bioinformatic Analysis of Sequencing Data
2.16. Phylogenetic Tree and Comparative Genomics of Phage Genomes
2.17. Phage Host Range Prediction Based on Phage Receptor-Binding Proteins
2.18. Statistical Analysis
3. Results
3.1. Bacteriophage Isolation and Purification
3.2. Host Range Analysis
3.3. Host Range Prediction Based on Receptor-Binding Proteins
3.4. Phage Stability
3.5. Lytic Characteristics of Phage vB_Efs8_KEN04
3.6. Adsorption Efficiency and One-Step Growth Curve
3.7. Phage Mutation Rate and Nature of Phage Receptors
3.8. Biofilm Formation of Enterococcus faecalis
3.9. Biofilm Inhibition and Disruption by Phage vB_Efs8_KEN04
3.10. Genome Characteristics of Enterococcus faecalis Phage vB_Efs8_KEN04
- (i)
- DNA replication, transcription, translation, and nucleotide metabolism: A total of 25 CDSs were predicted to encode for DNA replication, transcription regulation, translation, and metabolism-related proteins, such as HNH homing endonuclease, DNA helicase, exonucleases, transcriptional repressor, DNA helicase, DNA primase, and a transcriptional regulator, RNA polymerase beta subunit, and thymidylate synthase.
- (ii)
- Structural and packaging proteins: 27 CDS were predicted to encode for tail, head, and packaging proteins such as portal proteins, head proteins, tail fiber proteins, head maturation proteases, virion structural proteins, tail proteins, tail assembly chaperones, minor and major head proteins, and terminase large and small subunits.
- (iii)
- Host lysis and adhesion proteins: Two CDS were predicted to encode holin and endolysin proteins. BLASTp analysis of the phage vB_Efs8_KEN04 genome revealed no similarities to the genes encoding integrase or excisionase. The genome of phage vB_Efs8_KEN04 lacks genes encoding toxins, virulence factors, antibiotic resistance genes, and CRISPR. These data indicate that phage vB_Efs8_KEN04 is a strictly lytic phage that can be used to treat E. faecalis infection.
- (iv)
- Sixteen CDS encode for moron, auxiliary metabolic genes, and host takeover.
3.11. Phylogenetic Analysis
4. Discussion
5. Conclusions
6. Limitations
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No. | Bacterial Isolates | Sequence Types (ST) | Origin | Spot Assay | Efficiency of Plating (EOP) |
---|---|---|---|---|---|
1 | E. faecalis EFS8 * | 1904 | Urinary tract infection | ++ | 1 |
2 | E. faecalis EFS1 | 6 | Skin and soft tissue infection | ++ | 0.15 |
3 | E. faecalis EFS4 | 947 | Skin and soft tissue infection | + | <0.001 |
4 | E. faecalis EFS5 | 6 | Skin and soft tissue infection | ++ | 0.076 |
5 | E. faecalis EFS6 | 6 | Skin and soft tissue infection | ++ | 0.05 |
6 | E. faecalis EFS9 | 6 | Urinary tract infection | ++ | 0.01 |
7 | E. faecalis EFS10 | 6 | Urinary tract infection | ++ | 1.1 |
8 | E. faecalis EFS11 | 368 | Urinary tract infection | ++ | 0.0004 |
9 | E. faecalis EFS13 | 59 | Skin and soft tissue infection | ++ | 0.5 |
10 | E. faecalis EFS14 | 6 | Skin and soft tissue infection | ++ | 1.7 |
11 | E. faecalis EFS15 | 16 | Urinary tract infection | + | <0.001 |
12 | E. faecalis EFS17 | 6 | Skin and soft tissue infection | + | <0.001 |
13 | E. faecalis EFS18 | 368 | Urinary tract infection | ++ | 0.0011 |
14 | E. faecalis EFS19 | 1907 | Urinary tract infection | ++ | 0.12 |
15 | E. faecalis EFS21 | 44 | Skin and soft tissue infection | + | <0.001 |
16 | E. faecalis EFS22 | 1908 | Skin and soft tissue infection | + | <0.001 |
17 | E. faecalis EFS23 | 6 | Urinary tract infection | ++ | 3 |
18 | E. faecalis EFS25 | 6 | Surgical site infection | ++ | 1.5 |
19 | E. faecalis EFS26 | 6 | Skin and soft tissue infection | ++ | 1.2 |
20 | E. faecalis EFS27 | 1903 | Urinary tract infection | ++ | 0.14 |
21 | E. faecalis EFS28 | 28 | Skin and soft tissue infection | ++ | 0.6 |
22 | E. faecalis EFS29 | 6 | Blood infection | ++ | 0.8 |
23 | E. faecalis EFS30 | 28 | Skin and soft tissue infection | ++ | 0.8 |
24 | E. faecalis EFS31 | 6 | Urinary tract infection | ++ | 1.2 |
25 | E. faecalis EFS32 | 1903 | Urinary tract infection | ++ | 0.9 |
26 | E. faecalis EFS33 | 1903 | Skin and soft tissue infection | ++ | 6 |
27 | E. faecium EFM5 | 80 | Urinary tract infection | + | <0.001 |
28 | E. faecium EFM1 | Skin and soft tissue infection | − | N/A | |
29 | E. faecium EFM2 | 80 | Skin and soft tissue infection | − | N/A |
30 | E. faecium EFM3 | Skin and soft tissue infection | − | N/A | |
31 | E. faecium EFM4 | 80 | Skin and soft tissue infection | − | N/A |
32 | E. faecium EFM6 | 612 | Skin and soft tissue infection | − | N/A |
33 | E. faecium EFM7 | 761 | Skin and soft tissue infection | − | N/A |
34 | E. faecium EFM8 | 80 | Urinary tract infection | − | N/A |
35 | E. faecium EFM9 | 80 | Skin and soft tissue infection | − | N/A |
36 | E. faecium EFM10 | 2672 | Urinary tract infection | − | N/A |
37 | E. faecium EFM11 | 761 | Surgical site infection | − | N/A |
Protein Name | Phage Name | % Identity | Protein Accession Number | Protein Length | Phage Hosts |
---|---|---|---|---|---|
Tail fiber protein * | Enterococcus phage vB_Efs8_KEN04 | 100% | WZP34890.1 | 1832 | E. faecalis EFS8 |
Putative tail fiber protein | Enterococcus phage MDA2 | 99.89% | QVW28137.1 | 1825 | E. faecium VREfm (VRE001, VRE004, VRE008, VRE1147, VRE1181) [73] |
Putative tail fiber | Enterococcus phage phiM1EF22 | 99.95% | BBE37304.1 | 1822 | E. faecalis KUEF22 [74] |
Tail fiber protein | Enterococcus phage ECP3 | 99.89% | YP_009147083.1 | 1822 | E. faecalis 10K28 [75] |
Putative tail fiber | Enterococcus phage phiEF17H | 99.89% | BBE37101.1 | 1822 | E. faecalis EF17 [74] |
Tail fiber protein | Enterococcus phage vB_OCPT_Carl | 98.68% | UQT00063.1 | 1825 | E. faecalis strains (DP11, EF07, EF116PII, EF11, EF09PII, Ent6, V587, Yi6-1) [76] |
Tail fiber protein | Enterococcus phage EF24C | 98.47% | YP_001504140.1 | 1825 | E. faecalis EF24 [77] |
Tail fiber protein | Enterococcus phage vB_OCPT_Bob | 98.30% | UQT00475.1 | 1825 | E. faecalis strains (B3286, DP6, EF06, DP11, EF07, EF116PII, EF11, EF09PII, Ent6, V587, Yi6-1) [76] |
Tail fiber protein | Enterococcus phage vB_OCPT_Car | 97.10% | UQT00278.1 | 1825 | E. faecalis strains (DP11, EF07, EF116PII, EF11, EF09PII, Ent6, V587, Yi6-1) [76] |
Putative tail fiber | Enterococcus phage Sw5 | 96.38% | USL84310.1 | 1825 | E. faecalis OG1RF |
Tail fiber protein | Enterococcus phage vB_Efa29212_3e | 96.66% | UYB00790.1 | 1825 | E. faecalis ATCC 29212TM [78] |
Tail fiber protein | Enterococcus phage EFLK1 | 95.99% | YP_009219864.2 | 1822 | E. faecalis V583 and E. faecalis V583 phage-resistant mutant (EFDG1r) [79,80] |
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Soro, O.; Kigen, C.; Nyerere, A.; Gachoya, M.; Georges, M.; Odoyo, E.; Musila, L. Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant Enterococcus faecalis in Kenya. Viruses 2024, 16, 1275. https://doi.org/10.3390/v16081275
Soro O, Kigen C, Nyerere A, Gachoya M, Georges M, Odoyo E, Musila L. Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant Enterococcus faecalis in Kenya. Viruses. 2024; 16(8):1275. https://doi.org/10.3390/v16081275
Chicago/Turabian StyleSoro, Oumarou, Collins Kigen, Andrew Nyerere, Moses Gachoya, Martin Georges, Erick Odoyo, and Lillian Musila. 2024. "Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant Enterococcus faecalis in Kenya" Viruses 16, no. 8: 1275. https://doi.org/10.3390/v16081275