A New High-Throughput Screening Method for Phages: Enabling Crude Isolation and Fast Identification of Diverse Phages with Therapeutic Potential

Olsen, Nikoline S.; Hendriksen, Niels Bohse; Hansen, Lars Hestbjerg; Kot, Witold

doi:10.1089/phage.2020.0016

Cited by 42 publications

(35 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We al-so showed that three out of five phages isolated individually from the same sample virome with plaque assay are ineffective of lysis in liquid media or outcompeted by phages from the same virome. This has to be considered when deciding if the single isolated phages are good potential candidates for treatment in phage therapy or biocontrol [17,29,[68][69][70]. Furthermore, our analysis highlighted how much longread sequencing data improves the assembly of viral metagenomes as we saw an increase of 5.64% in the proportion of complete phage genomes from long-read assembly compared to short-read-only assemblies (p-value < 0.0001, Figure 2).…”

Section: Discussionmentioning

confidence: 84%

“…One of the reasons for the lack of high-quality phage genomes is that the methodology for isolating single phage strains with known hosts is laborious, time-consuming, and often the limiting step [17][18][19]. One of two methods (I) direct plating or (II) enrichment approach is most often used to isolates single phages.…”

Section: Introductionmentioning

confidence: 99%

“…Some phages are not capable of making plaques on plates due to limited diffusion in agar in general or low productivity, and isolation of highly effective single phages might be unsuccessful despite them being present and able to lyse the host [15]. All these restrictions are limiting types and numbers of phages isolated, despite attempts to make it more high-throughput [17].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay

Alanin

Junco

Joergensen

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or the ones with highest ability to plaque on solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in envi-ronmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community's shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media corresponds to the metaviromic data. In this study, we observed a significant shift (p = 0.015) of the 47 identified putative Pseudomonas phages with a minimum 2-fold change above 0 in read abundance when adding a Pseudomonas syringae DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the Pseudomonas strain, was highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlights how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.

show abstract

Section: Discussionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay

Alanin

Junco

Joergensen

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Three out of 16 sequenced samples (~19%, two from host HTCC7211, one from OM43 host H5P1) failed to assemble into single viral contigs, in line with previously reported failure rates of 18-39% for phages of Escherichia coli and Salmonella spp . [47]. For 11 of the remaining 13 samples (12 from SAR11 hosts and one from OM43), each individual sequence assembly was identified as a complete viral genome by VirSorter [15] and 95-100% complete using CheckV [48].…”

Section: Resultsmentioning

confidence: 99%

Efficient Dilution-to-Extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria

Buchholz

Michelsen

Bolaños

et al. 2020

Preprint

View full text Add to dashboard Cite

Microbes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated a new SAR11 and three new members of the methylotrophic bacterial clade OM43, and used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first known phages for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that SAR11 viruses have ecotype-specific host-ranges. Similar to the abundant human-associated phage ϕCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny, irrespective of detectable associated genes, and/or host phenotypic bistability, with lysis putatively maintained within a susceptible subpopulation. Broader representation of virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

show abstract

“…Despite the wide diversity of known E. coli phages [30], nearly all phage isolates reported in these studies belonged to five major groups and were either myoviruses of 1) Tevenvirinae or 2) Vequintavirinae subfamilies and close relatives, 3) large siphoviruses of the Markadamsvirinae subfamily within Demerecviridae, or small siphoviruses of 4) diverse Drexlerviridae subfamilies or 5) the genera Dhillonvirus, Nonagvirus, and Seuratvirus of the Siphoviridae family. Podoviruses of any kind were rarely reported, and if, then were mostly Autographiviridae isolated using enrichment cultures that are known to greatly favor such fast-growing phages [24,31]. This pattern does not seem to be strongly biased by any given strain of E.coli as isolation host because a large, very thorough study using diverse E. coli strains reported essentially the same composition of taxonomic groups [23].…”

Section: Fig 2 Overview Of the Basel Collectionmentioning

confidence: 99%

Systematic exploration ofEscherichia coliphage-host interactions with the BASEL phage collection

Maffei

Shaidullina

Burkolter

et al. 2021

Preprint

View full text Add to dashboard Cite

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 66 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL collection (BActeriophage SElection for your Laboratory). This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside ten well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to eleven defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Our results reveal clear patterns in the distribution of phage phenotypes and genomic features that highlight systematic differences in the potency of different immunity systems and point towards the molecular basis of receptor specificity in several phage groups. Strong trade-offs were detected between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

show abstract

A New High-Throughput Screening Method for Phages: Enabling Crude Isolation and Fast Identification of Diverse Phages with Therapeutic Potential

Cited by 42 publications

References 44 publications

Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay

Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay

Efficient Dilution-to-Extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria

Systematic exploration ofEscherichia coliphage-host interactions with the BASEL phage collection

Contact Info

Product

Resources

About