Ongoing diversification of the global fish pathogen Piscirickettsia salmonis through genetic isolation and transposition bursts.

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Genotype-phenotype correlations within the Geodermatophilaceae.

The integration of genomic information into microbial systematics along with physiological and chemotaxonomic parameters provides for a reliable classification of prokaryotes. In silico analysis of chemotaxonomic traits is now being introduced to replace characteristics traditionally determined in the laboratory with the dual goal of both increasing the speed of the description of taxa and the accuracy and consistency of taxonomic reports. Genomics has already successfully been applied in the taxonomic rearrangement of Geodermatophilaceae (Actinomycetota) but in the light of new genomic data the taxonomy of the family needs to be revisited. In conjunction with the taxonomic characterisation of four strains phylogenetically located within the family, we conducted a phylogenetic analysis of the whole proteomes of the sequenced type strains and established genotype-phenotype correlations for traits related to chemotaxonomy, cell morphology and metabolism. Results indicated that the four isolates under study represent four novel species within the genus Blastococcus. Additionally, the genera Blastococcus, Geodermatophilus and Modestobacter were shown to be paraphyletic. Consequently, the new genera Trujillonella, Pleomorpha and Goekera were proposed within the Geodermatophilaceae and Blastococcus endophyticus was reclassified as Trujillonella endophytica comb. nov., Geodermatophilus daqingensis as Pleomorpha daqingensis comb. nov. and Modestobacter deserti as Goekera deserti comb. nov. Accordingly, we also proposed emended descriptions of Blastococcus aggregatus, Blastococcus jejuensis, Blastococcus saxobsidens and Blastococcus xanthilyniticus. In silico chemotaxonomic results were overall consistent with wet-lab results. Even though in silico discriminatory levels varied depending on the respective chemotaxonomic trait, this approach is promising for effectively replacing and/or complementing chemotaxonomic analyses at taxonomic ranks above the species level. Finally, interesting but previously overlooked insights regarding morphology and ecology were revealed by the presence of a repertoire of genes related to flagellum synthesis, chemotaxis, spore production and pilus assembly in all representatives of the family. A rich carbon metabolism including four different CO2 fixation pathways and a battery of enzymes able to degrade complex carbohydrates were also identified in Blastococcus genomes.

Panoramic Insights into Microevolution and Macroevolution of A Prevotella copri-containing Lineage in Primate Guts.

Prevotella copri and its related taxa are widely detected in mammalian gut microbiomes and have been linked with an enterotype in humans. However, their microevolution and macroevolution among hosts are poorly characterized. In this study, extensively collected marker genes and genomes were analyzed to trace their evolutionary history, host specificity, and biogeographic distribution. Investigations based on marker genes and genomes suggest that a P. copri-containing lineage (PCL) harbors diverse species in higher primates. Firstly, P. copri in the human gut consisted of multiple groups exhibiting high genomic divergence and conspicuous but non-strict biogeographic patterns. Most African strains with high genomic divergence from other strains were phylogenetically located at the root of the species, indicating the co-evolutionary history of P. copri and Homo sapiens. Secondly, although long-term co-evolution between PCL and higher primates was revealed, sporadic signals of co-speciation and extensive host jumping of PCL members were suggested among higher primates. Metagenomic and phylogenetic analyses indicated that P. copri and other PCL species found in domesticated mammals had been recently transmitted from humans. Thirdly, strong evidence was found on the extensively horizontal transfer of genes (e.g., genes encoding carbohydrate-active enzymes) among sympatric P. copri groups and PCL species in the same primate host. Our study provides panoramic insights into the combined effects of vertical and horizontal transmission, as well as potential niche adaptation, on the microevolutionary and macroevolutionary history for an enterotype-representative lineage.

TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes.

Microbial systematics is heavily influenced by genome-based methods and challenged by an ever increasing number of taxon names and associated sequences in public data repositories. This poses a challenge for database systems, particularly since it is obviously advantageous if such data are based on a globally recognized approach to manage names, such as the International Code of Nomenclature of Prokaryotes. The amount of data can only be handled if accurate and reliable high-throughput platforms are available that are able to both comply with this demand and to keep track of all changes in an efficient and flexible way. The List of Prokaryotic names with Standing in Nomenclature (LPSN) is an expert-curated authoritative resource for prokaryotic nomenclature and is available at https://lpsn.dsmz.de. The Type (Strain) Genome Server (TYGS) is a high-throughput platform for accurate genome-based taxonomy and is available at https://tygs.dsmz.de. We here present important updates of these two previously introduced, heavily interconnected platforms for taxonomic nomenclature and classification, including new high-level facilities providing access to bioinformatic algorithms, a considerable expansion of the database content, and new ways to easily access the data.

Corrigendum: Genotype-phenotype correlations within the Geodermatophilaceae.

[This corrects the article DOI: 10.3389/fmicb.2022.975365.].

Prevotella illustrans sp. nov., derived from human oropharyngeal abscess puncture fluid.

An obligately anaerobic strain, designated as A2931T, was isolated from oropharyngeal abscess puncture fluid of a patient sampled during routine care at a hospital and further characterized both phenotypically, biochemically and genotypically. This Gram-negative rod-shaped bacterium was moderately saccharolytic and proteolytic. Phylogenetic analyses of full-length 16S rRNA gene and whole-genome sequences revealed it to be best placed in the genus Prevotella, but to be only comparatively distantly related to recognized species, with the closest relationship to Prevotella baroniae (average nucleotide identity and digital DNA-DNA hybridization values both well below the generally accepted thresholds). Strain A2931T had a genomic DNA G+C content of 47.7 mol%. Its most abundant cellular long-chain fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. Taken together, this polyphasic data suggests strain A2931T to represent a novel species within the genus Prevotella, for which the name Prevotella illustrans sp. nov. is proposed. The type strain is A2931T (=DSM 108028T=CCOS 1232T=CCUG 72806T). Interestingly, we found strain A2931T to correspond to the oral taxon Prevotella HMT-820 in the Human Oral Microbiome Database, as supported by overall genome relatedness index analyses >99 %. Thus, our work not only closes one of the gaps of knowledge about hitherto unnamed species isolated from humans, but also will facilitate identification of this taxon both in the clinical microbiology context and in research alike.

Sphingomonas aliaeris sp. nov., a new species isolated from pork steak packed under modified atmosphere.

Species belonging to the genus Sphingomonas have been isolated from environments such as soil, water and plant tissues. Many strains are known for their capability of degrading aromatic molecules and producing extracellular polymers. A Gram-stain-negative, strictly aerobic, motile, red-pigmented, oxidase-negative, catalase-positive, rod-shaped strain, designated DH-S5T, has been isolated from pork steak packed under CO2-enriched modified atmosphere. Cell diameters were 1.5×0.9 µm. Growth optima were at 30 °C and at pH 6.0. Phylogenetic analyses based on both complete 16S rRNA gene sequence and whole-genome sequence data revealed that strain DH-S5T belongs to the genus Sphingomonas, being closely related to Sphingomonas alpina DSM 22537T (97.4 % gene sequence similarity), followed by Sphingomonas qilianensis X1T (97.4 %) and Sphingomonas hylomeconis GZJT-2T (97.3 %). The DNA G+C content was 64.4 mol%. The digital DNA-DNA hybridization value between the isolate strain and S. alpina DSM 22537T was 21.0 % with an average nucleotide identity value of 77.03 %. Strain DH-S5T contained Q-10 as the ubiquinone and major fatty acids were C18 : 1 cis 11 (39.3 %) and C16 : 1 cis 9 (12.5 %), as well as C16 : 0 (12.1 %) and C14 : 0 2-OH (11.4 %). As for polar lipids, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, dimethylphosphatidylethanolamine and sphingoglycolipid could be detected, alongside traces of monomethylphosphatidylethanolamine. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain DH-S5T (=DSM 110829T=LMG 31606T) is classified as a representative of the genus Sphingomonas, for which the name Sphingomonas aliaeris sp. nov. is proposed.

A single-cell genomics pipeline for environmental microbial eukaryotes.

Single-cell sequencing of environmental microorganisms is an essential component of the microbial ecology toolkit. However, large-scale targeted single-cell sequencing for the whole-genome recovery of uncultivated eukaryotes is lagging. The key challenges are low abundance in environmental communities, large complex genomes, and cell walls that are difficult to break. We describe a pipeline composed of state-of-the art single-cell genomics tools and protocols optimized for poorly studied and uncultivated eukaryotic microorganisms that are found at low abundance. This pipeline consists of seven distinct steps, beginning with sample collection and ending with genome annotation, each equipped with quality review steps to ensure high genome quality at low cost. We tested and evaluated each step on environmental samples and cultures of early-diverging lineages of fungi and Chromista/SAR. We show that genomes produced using this pipeline are almost as good as complete reference genomes for functional and comparative genomics for environmental microbial eukaryotes.

Reclassification of Haloactinobacterium glacieicola as Occultella glacieicola gen. nov., comb. nov., of Haloactinobacterium album as Ruania alba comb. nov, with an emended description of the genus Ruania, recognition that the genus names Haloactinobacterium and Ruania are heterotypic synonyms and description of Occultella aeris sp. nov., a halotolerant isolate from surface soil sampled at an ancient copper smelter.

In the course of screening the surface soils of ancient copper mines and smelters (East Harz, Germany) an aerobic, non-motile and halotolerant actinobacterium forming small rods or cocci was isolated. The strain designated F300T developed creamy to yellow colonies on tryptone soy agar and grew optimally at 28 °C, pH 7-8 and with 0.5-2 % (m/v) NaCl. Its peptidoglycan was of type A4α l-Lys-l-Glu (A11.54). The menaquinone profile was dominated by MK-8(II, III-H4) and contained minor amounts of MK-8(H2), MK-8(H6) and MK-9(H4). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, mono and diacylated phosphatidylinositol dimannosides, and components that were not fully characterized, including two phospholipids, two glycolipids and an uncharacterized lipid. Major whole-cell sugars were rhamnose and ribose. The fatty acid profile contained mainly iso and anteiso branched fatty acids (anteiso-C15 : 0, iso-C14 : 0) and aldehydes/dimethylacetals (i.e. not fatty acids). Sequence analysis of its genomic DNA and subsequent analysis of the data placed the isolate in the group currently defined by members of the genera Ruania and Haloactinobacterium (family Ruaniaceae, order Micrococcales) as a sister taxon to the previously described species Haloactinobacterium glacieicola, sharing an average nucleotide identity and average amino acid identity values of 85.3 and 85.7 %, respectively. Genotypic and chemotaxonomic analyses support the view that strain F300T (=DSM 108350T=CIP 111667T) is the type strain of a new genus and new species for which the name Occultella aeris gen. nov., sp. nov. is proposed. Based on revised chemotaxonomic and additional genome based data, it is necessary to discuss and evaluate the results in the light of the classification and nomenclature of members of the family Ruaniaceae, i.e. the genera Haloactinobacterium and Ruania. Consequently, the reclassification of Haloactinobacterium glacieicola as Occultella glacieicola comb. nov. and Haloactinobacterium album as Ruania alba comb. nov., with an emended description of the genus Ruania are proposed.

List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ.

The List of Prokaryotic names with Standing in Nomenclature (LPSN) was acquired in November 2019 by the DSMZ and was relaunched using an entirely new production system in February 2020. This article describes in detail the structure of the new site, navigation, page layout, search facilities and new features.

A publicly accessible database for Clostridioides difficile genome sequences supports tracing of transmission chains and epidemics.

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile.

Analysis of 1,000+ Type-Strain Genomes Substantially Improves Taxonomic Classification of Alphaproteobacteria.

The class Alphaproteobacteria is comprised of a diverse assemblage of Gram-negative bacteria that includes organisms of varying morphologies, physiologies and habitat preferences many of which are of clinical and ecological importance. Alphaproteobacteria classification has proved to be difficult, not least when taxonomic decisions rested heavily on a limited number of phenotypic features and interpretation of poorly resolved 16S rRNA gene trees. Despite progress in recent years regarding the classification of bacteria assigned to the class, there remains a need to further clarify taxonomic relationships. Here, draft genome sequences of a collection of genomes of more than 1000 Alphaproteobacteria and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa recognized as problematic long ago but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera and of a variety of genera to other families. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which are confirmed as valuable taxonomic markers. Similarly, analysis of the gene content was shown to provide valuable taxonomic insights in the class. Significant incongruities between 16S rRNA gene and whole genome trees were not found in the class. The incongruities that became obvious when comparing the results of the present study with existing classifications appeared to be caused mainly by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. Another probable cause of misclassifications in the past is the partially low overall fit of phenotypic characters to the sequence-based tree. Even though a significant degree of phylogenetic conservation was detected in all characters investigated, the overall fit to the tree varied considerably.

Comparative Genomics Suggests Mechanisms of Genetic Adaptation toward the Catabolism of the Phenylurea Herbicide Linuron in Variovorax.

Biodegradation of the phenylurea herbicide linuron appears a specialization within a specific clade of the Variovorax genus. The linuron catabolic ability is likely acquired by horizontal gene transfer but the mechanisms involved are not known. The full-genome sequences of six linuron-degrading Variovorax strains isolated from geographically distant locations were analyzed to acquire insight into the mechanisms of genetic adaptation toward linuron metabolism. Whole-genome sequence analysis confirmed the phylogenetic position of the linuron degraders in a separate clade within Variovorax and indicated that they unlikely originate from a common ancestral linuron degrader. The linuron degraders differentiated from Variovorax strains that do not degrade linuron by the presence of multiple plasmids of 20-839 kb, including plasmids of unknown plasmid groups. The linuron catabolic gene clusters showed 1) high conservation and synteny and 2) strain-dependent distribution among the different plasmids. Most of them were bordered by IS1071 elements forming composite transposon structures, often in a multimeric array configuration, appointing IS1071 as a key element in the recruitment of linuron catabolic genes in Variovorax. Most of the strains carried at least one (catabolic) broad host range plasmid that might have been a second instrument for catabolic gene acquisition. We conclude that clade 1 Variovorax strains, despite their different geographical origin, made use of a limited genetic repertoire regarding both catabolic functions and vehicles to acquire linuron biodegradation.

100-year-old enigma solved: identification, genomic characterization and biogeography of the yet uncultured Planctomyces bekefii.

The first representative of the phylum Planctomycetes, Planctomyces bekefii, was described nearly one century ago. This morphologically conspicuous freshwater bacterium is a rare example of as-yet-uncultivated prokaryotes with validly published names and unknown identity. We report the results of molecular identification of this elusive bacterium, which was detected in a eutrophic boreal lake in Northern Russia. By using high-performance cell sorting, P. bekefii-like cell rosettes were selectively enriched from lake water. The retrieved 16S rRNA gene sequence was nearly identical to those in dozens of metagenomes assembled from freshwater lakes during cyanobacterial blooms and was phylogenetically placed within a large group of environmental sequences originating from various freshwater habitats worldwide. In contrast, 16S rRNA gene sequence similarity to all currently described members of the order Planctomycetales was only 83%-92%. The metagenome assembled for P. bekefii reached 43% genome coverage and showed the potential for degradation of peptides, pectins, and sulfated polysaccharides. Tracing the seasonal dynamics of P. bekefii by Illumina paired-end sequencing of 16S rRNA gene fragments and by fluorescence in situ hybridization revealed that these bacteria only transiently surpass the detection limit, with a characteristic population peak of up to 104 cells ml-1 following cyanobacterial blooms.

Analysis of 1,000 Type-Strain Genomes Improves Taxonomic Classification of Bacteroidetes.

Although considerable progress has been made in recent years regarding the classification of bacteria assigned to the phylum Bacteroidetes, there remains a need to further clarify taxonomic relationships within a diverse assemblage that includes organisms of clinical, piscicultural, and ecological importance. Bacteroidetes classification has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees and a limited number of phenotypic features. Here, draft genome sequences of a greatly enlarged collection of genomes of more than 1,000 Bacteroidetes and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa proposed long ago such as Bacteroides, Cytophaga, and Flavobacterium but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which can be considered valuable taxonomic markers. We detected many incongruities when comparing the results of the present study with existing classifications, which appear to be caused by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. The few significant incongruities found between 16S rRNA gene and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences and the impediment in using ordinary bootstrapping in phylogenomic studies, particularly when combined with too narrow gene selections. While a significant degree of phylogenetic conservation was detected in all phenotypic characters investigated, the overall fit to the tree varied considerably, which is one of the probable causes of misclassifications in the past, much like the use of plesiomorphic character states as diagnostic features.

Whole-Genome Sequencing Redefines Shewanella Taxonomy.

The genus Shewanella encompasses a diverse group of Gram negative, primarily aquatic bacteria with a remarkable ecological relevance, an outstanding set of metabolic features and an emergent clinical importance. The rapid expansion of the genus over the 2000 s has prompted questions on the real taxonomic position of some isolates and species. Recent work by us and others identified inconsistencies in the existing species classification. In this study we aimed to clarify such issues across the entire genus, making use of the genomic information publicly available worldwide. Phylogenomic analyses, including comparisons based on genome-wide identity indexes (digital DNA-DNA hybridization and Average Nucleotide Identity) combined with core and accessory genome content evaluation suggested that the taxonomic position of 64 of the 131 analyzed strains should be revisited. Based on the genomic information currently available, emended descriptions for some Shewanella species are proposed. Our study establishes for the first time a whole-genome based phylogeny for Shewanella spp. including a classification at the subspecific level.

TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy.

Microbial taxonomy is increasingly influenced by genome-based computational methods. Yet such analyses can be complex and require expert knowledge. Here we introduce TYGS, the Type (Strain) Genome Server, a user-friendly high-throughput web server for genome-based prokaryote taxonomy, connected to a large, continuously growing database of genomic, taxonomic and nomenclatural information. It infers genome-scale phylogenies and state-of-the-art estimates for species and subspecies boundaries from user-defined and automatically determined closest type genome sequences. TYGS also provides comprehensive access to nomenclature, synonymy and associated taxonomic literature. Clinically important examples demonstrate how TYGS can yield new insights into microbial classification, such as evidence for a species-level separation of previously proposed subspecies of Salmonella enterica. TYGS is an integrated approach for the classification of microbes that unlocks novel scientific approaches to microbiologists worldwide and is particularly helpful for the rapidly expanding field of genome-based taxonomic descriptions of new genera, species or subspecies.

Still Something to Discover: Novel Insights intoEscherichia coli Phage Diversity and Taxonomy.

The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.