Bacteriophage therapy and application of phages for biocontrol necessitate acquisition of suitable phages. The exclusivity of phage–host relations and the risk of phage resistance instigate a need to rapidly isolate and characterize novel phages and continually build sizeable phage libraries. Current methods for phage isolation are both laborious and time consuming, suitable for the isolation of a limited number of phages. The high-throughput screening method for phages upscales and organizes enrichment of phages for fast isolation and identification of potentially hundreds of distinct phages against single hosts. This enables screening of hundreds of samples, in multiple simultaneous setups with varying parameters, increasing the likelihood of isolating multiple distinct phages specific for the given conditions. The efficiency of the method is emphasized by our screening of 200 environmental samples, resulting in the identification of an abundance of unique phage species virulent to Escherichia coli, Salmonella enterica, Enterococcus faecalis, and Pseudomonas aeruginosa.
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References
1.
Gordillo AltamiranoFL, BarrJJ. Phage therapy in the postantibiotic era. Clin Microbiol Rev. 2019; 32:e00066-18.
2.
LeeJYH, MonkIR, Gonçalves da SilvaA, et al.Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis. Nat Microbiol. 2018; 3:1175–1185.
3.
MagiorakosA-P, SrinivasanA, CareyRB, et al.Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–281.
4.
LinDM, KoskellaB, LinHC. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther. 2017; 8:162.
5.
CaflischKM, SuhGA, PatelR. Biological challenges of phage therapy and proposed solutions: A literature review. Expert Rev Anti Infect Ther. 2019; 17:1011–1041.
6.
DedrickRM, Guerrero-BustamanteCA, GarlenaRA, et al.Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. 2019; 25:730–733.
7.
SchooleyRT, BiswasB, GillJJ, et al.Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 2017; 61(10):e00954-17.
8.
AslamS, CourtwrightAM, KovalC, et al.Early clinical experience of bacteriophage therapy in 3 lung transplant recipients. Am J Transplant. 2019; 19:2631–2639.
9.
MonkAB, ReesCD, BarrowP, et al.Bacteriophage applications: Where are we now?. Lett Appl Microbiol. 2010; 51:363–369.
10.
CarstensAB, DjurhuusAM, KotW, et al.A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions. FEMS Microbiol Lett, 2019; 366:(9):fnz101.
11.
SvircevA, RoachD, CastleA. Framing the future with bacteriophages in agriculture. Viruses. 2018; 10:218.
12.
BrüssowH. Hurdles for phage therapy to become a reality—An editorial comment. Viruses. 2019; 11:557.
13.
GratiaA. Des relations numeriques entre bacteries lysogenes et particules de bacteriophage [The numerical relation between lysogenic bacteria and the phage particles which they carry]. Ann Inst Pasteur. 1936; 57:652–676.
14.
KauffmanKM, PolzMF. Streamlining standard bacteriophage methods for higher throughput. MethodsX. 2018; 5:159–172.
15.
HenryM, BiswasB, VincentL, et al.Development of a high throughput assay for indirectly measuring phage growth using the OmniLog TM system. Bacteriophage. 2012; 2:159–167.
16.
HatfullGF. Mycobacteriophages: Windows into tuberculosis. PLoS Pathog. 2014; 10:e1003953.
17.
KotW, VogensenFK, SørensenSJ, et al.DPS—A rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque. J Virol Methods. 2014; 196:152–156.
18.
WoodE. Molecular cloning. A laboratory manual. Biochem Educ. 2003; 11:82.
19.
BankevichA, NurkS, AntipovD, et al.SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19:455–477.
20.
BrettinT, DavisJJ, DiszT, et al.RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015; 5:8365.
21.
BesemerJ, BorodovskyM. GeneMark: Web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 2005; 33:W451–W454.
22.
AltschulSF, GishW, MillerW, et al.Basic local alignment search tool. J Mol Biol. 1990; 215:403–410.
23.
SodingJ, BiegertA, LupasAN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005; 33:W244–W248.
24.
FinnRD, CoggillP, EberhardtRY, et al.The Pfam protein families database: Towards a more sustainable future. Nucleic Acids Res. 2016; 44:D279–D285.
25.
González-TortueroE, SuttonTDS, VelayudhanV, et al.VIGA: A sensitive, precise and automatic de novo VIral Genome Annotator. bioRxiv. 2018; 277509.
26.
ZankariE, HasmanH, CosentinoS, et al.Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012; 67:2640–2644.
27.
KleinheinzKA, JoensenKG, LarsenMV. Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E. coli virulence genes in bacteriophage and prophage nucleotide sequences. Bacteriophage. 2014; 4:e27943.
28.
JoensenKG, ScheutzF, LundO, et al.Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol. 2014; 52:1501–1510.
29.
AdriaenssensE, BristerJR. How to name and classify your phage: An informal guide. Viruses. 9. Epub ahead of print, 2017; 9(4):70.
30.
ÅgrenJ, SundströmA, HåfströmT, et al.Gegenees: Fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups. PLoS One. 2012; 7:e39107.
31.
KumarS, StecherG, TamuraK. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870–1874.
32.
LopesA, TavaresP, PetitM-A, et al.Automated classification of tailed bacteriophages according to their neck organization. BMC Genomics. 2014; 15:1027.
33.
MercantiDJ, RousseauGM, CapraML, et al.Genomic diversity of phages infecting probiotic strains of Lactobacillus paracasei. Appl Environ Microbiol. 2016; 82:95–105.
34.
EdgarRC. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792–1797.
35.
TamuraK, NeiM. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993; 10:512–526.
36.
HsiehTC, MaKH, ChaoA. iNEXT: An R package for rarefaction and extrapolation of species diversity (Hill numbers). Methods Ecol Evol. 2016; 7:1451–1456.
37.
ChaoA, GotelliNJ, HsiehTC, et al.Rarefaction and extrapolation with Hill numbers: A framework for sampling and estimation in species diversity studies. Ecol Monogr. 2014; 84:45–67.
38.
R Studio Team. RStudio: Integrated Development for R. 2016. Available from: http://www.rstudio.com Last accessed July6, 2020.
39.
OlsenNS, KotW, JuncoLMF, et al.Exploring the remarkable diversity of Escherichia coli Phages in the Danish Wastewater Environment, Including 91 Novel Phage Species. bioRxiv. 2020;2020.01.19.911818.
40.
AdriaenssensEM, EdwardsR, NashJHE, et al.Integration of genomic and proteomic analyses in the classification of the Siphoviridae family. Virology. 2015; 477:144–154.
41.
HuaY, AnX, PeiG, et al.Characterization of the morphology and genome of an Escherichia coli podovirus. Arch Virol. 2014; 159:3249–3256.
42.
d'HumièresC, TouchonM, DionS, et al.A simple, reproducible and cost-effective procedure to analyse gut phageome: From phage isolation to bioinformatic approach. Sci Rep. 2019; 9:1–13.
BrussowH, CanchayaC, HardtW-D. Phages and the evolution of bacterial pathogens: From genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev. 2004; 68:560–602.
45.
ReindelR, FioreCR. Phage therapy: Considerations and challenges for development. Clin Infect Dis. 2017; 64:1589–1590.
46.
Calero-CáceresW, YeM, BalcázarJL. Bacteriophages as environmental reservoirs of antibiotic resistance. Trends Microbiol. 2019; 27(7):P570–P577.
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