Multireceptor phage cocktail against Salmonella enterica to circumvent phage resistance.

Non-Typhoidal Salmonella (NTS) is one of the most common food-borne pathogens worldwide, with poultry products being the major vehicle for pathogenesis in humans. The use of bacteriophage (phage) cocktails has recently emerged as a novel approach to enhancing food safety. Here, a multireceptor Salmonella phage cocktail of five phages was developed and characterized. The cocktail targets four receptors: O-antigen, BtuB, OmpC, and rough Salmonella strains. Structural analysis indicated that all five phages belong to unique families or subfamilies. Genome analysis of four of the phages showed they were devoid of known virulence or antimicrobial resistance factors, indicating enhanced safety. The phage cocktail broad antimicrobial spectrum against Salmonella, significantly inhibiting the growth of all 66 strains from 20 serovars tested in vitro. The average bacteriophage insensitive mutant (BIM) frequency against the cocktail was 6.22 × 10-6 in S. Enteritidis, significantly lower than that of each of the individual phages. The phage cocktail reduced the load of Salmonella in inoculated chicken skin by 3.5 log10 CFU/cm2 after 48 h at 25°C and 15°C, and 2.5 log10 CFU/cm2 at 4°C. A genome-wide transduction assay was used to investigate the transduction efficiency of the selected phage in the cocktail. Only one of the four phages tested could transduce the kanamycin resistance cassette at a low frequency comparable to that of phage P22. Overall, the results support the potential of cocktails of phage that each target different host receptors to achieve complementary infection and reduce the emergence of phage resistance during biocontrol applications.

Degradation of Listeria monocytogenes biofilm by phages belonging to the genus Pecentumvirus.

Listeria monocytogenes is a pathogenic foodborne bacterium that is a significant cause of mortality associated with foodborne illness and causes many food recalls attributed to a bacteriological cause. Their ability to form biofilms contributes to the persistence of Listeria spp. in food processing environments. When growing as biofilms, L. monocytogenes are more resistant to sanitizers used in the food industry, such as benzalkonium chloride (BAC), as well as to physical stresses like desiccation and starvation. Lytic phages of Listeria are antagonistic to a broad range of Listeria spp. and may, therefore, have utility in reducing the occurrence of Listeria-associated food recalls by preventing food contamination. We screened nine closely related Listeria phages, including the commercially available Listex P100, for host range and ability to degrade microtiter plate biofilms of L. monocytogenes ATCC 19111 (serovar 1/2a). One phage, CKA15, was selected and shown to rapidly adsorb to its host under conditions relevant to applying the phage in dairy processing environments. Under simulated dairy processing conditions (SDPC), CKA15 caused a 2-log reduction in Lm19111 biofilm bacteria. This work supports the biosanitation potential of phage CKA15 and provides a basis for further investigation of phage-bacteria interactions in biofilms grown under SDPC. IMPORTANCE: Listeria monocytogenes is a pathogenic bacterium that is especially dangerous for children, the elderly, pregnant women, and immune-compromised people. Because of this, the food industry takes its presence in their plants seriously. Food recalls due to L. monocytogenes are common with a high associated economic cost. In food-processing plants, Listeria spp. typically reside in biofilms, which are structures produced by bacteria that shield them from environmental stressors and are often attached to surfaces. The significance of our work is that we show a bacteriophage-a virus-infecting bacteria-can reduce Listeria counts by two orders of magnitude when the bacterial biofilms were grown under simulated dairy processing conditions. This work provides insights into how phages may be tested and used to develop biosanitizers that are effective but are not harmful to the environment or human health.

Bacteriophage Taxonomy: A Continually Evolving Discipline.

While taxonomy is an often underappreciated branch of science, it serves very important roles. Bacteriophage taxonomy has evolved from a discipline based mainly on morphology, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the sequence-based approach that is taken today. The Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a holistic approach to classifying prokaryote viruses by measuring overall DNA and protein similarity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with the National Center for Biotechnology Information (NCBI) and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

Comparative genomics and proteomics analysis of phages infecting multi-drug resistant Escherichia coli O177 isolated from cattle faeces.

The increasing prevalence of antimicrobial-resistant (AMR) pathogens has become a major global health concern. To address this challenge, innovative strategies such as bacteriophage therapy must be optimised. Genomic characterisation is a crucial step in identifying suitable phage candidates for combating AMR pathogens. The aim of this study was to characterise seven phages that infect the Escherichia coli O177 strain using a whole genome sequencing. The analysis of genome sequences revealed that these phages had linear dsDNA, with genome sizes spanning from 136, 483 to 166,791 bp and GC content varying from 35.39 to 43.63%. Taxonomically, the phages were classified under three different subfamilies (Stephanstirmvirinae, Tevenvirinae, and Vequintavirinae) and three genera (Phapecoctavirus, Tequatrovirus, and Vequintavirus) within the class Caudoviricetes. In silico PhageAI analysis predicted that all the phages were virulent, with confidence levels between 96.07 and 97.26%. The phage genomes contained between 66 and 82 ORFs, which encode hypothetical and putative functional proteins. In addition, the phage genomes contained core genes associated with molecular processes such as DNA replication, transcription modulation, nucleotide metabolism, phage structure (capsid and tail), and lysis. None of the genomes carried genes associated with undesirable traits such as integrase, antimicrobial resistance, virulence, and toxins. The study revealed high genome and proteome homology among E. coli O177 phages and other known Escherichia phages. The results suggest that the seven phages are new members of the genera Phapecoctavirus, Tequatrovirus, and Vequintavirus under the subfamilies Stephanstirmvirinae, Tevenvirinae, and Vequintavirinae, respectively.

Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage.

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Broad-host-range lytic Erwinia phage Key with exopolysaccharide degrading activity.

In this study, the genome of the lytic broad-host-range phage Key infecting Erwinia amylovora, Erwinia horticola, and Pantoea agglomerans strains was characterized. Key phage has a 115,651 bp long double-stranded DNA genome with the G + C ratio of 39.03%, encoding 182 proteins and 27 tRNA genes. The majority (69%) of predicted coding sequences (CDSs) encode proteins with unknown functions. The protein products of 57 annotated genes were found to have probable functions in nucleotide metabolism, DNA replication, recombination, repair, and packaging, virion morphogenesis, phage-host interaction and lysis. Furthermore, the product of gene 141 shared amino acid sequence similarity and conserved domain architecture with the exopolysaccharide (EPS) degrading proteins of Erwinia and Pantoea infecting phages as well as bacterial EPS biosynthesis proteins. Due to the genome synteny and similarity to the proteins of T5-related phages, phage Key, together with its closest relative, Pantoea phage AAS21, was suggested to represent a novel genus within the Demerecviridae family, for which we tentatively propose the name "Keyvirus".

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains.

Staphylococcus aureus causes nosocomial and intramammary infections in humans and cattle, respectively. A large number of virulence factors are thought to play important roles in the pathogenesis of this bacterium. Currently, genome-wide and data-analysis studies are being used to better understand its epidemiology. In this study, we conducted a genome wide comparison and phylogenomic analyses of S. aureus to find specific virulence patterns associated with clinical and subclinical mastitis strains in cattle and compare them with those of human origin. The presence/absence of key virulence factors such as adhesin, biofilm, antimicrobial resistance, and toxin genes, as well as the phylogeny and sequence type of the isolates were evaluated. A total of 248 genomes (27 clinical mastitis, 43 subclinical mastitis, 21 milk, 53 skin-related abscesses, 49 skin infections, and 55 pus from cellulitis) isolated from 32 countries were evaluated. We found that the cflA, fnbA, ebpS, spa, sdrC, coa, emp, vWF, atl, sasH, sasA, and sasF adhesion genes, as well as the aur, hglA, hglB, and hglC toxin genes were highly associated in clinical mastitis strains. The strains had diverse genetic origins (72 protein A and 48 sequence types with ST97, ST8 and ST152 being frequent in isolates from clinical mastitis, abscess, and skin infection, respectively). Further, our phylogenomic analyses suggested that zoonotic and/or zooanthroponotic transmission may have occurred. These findings contribute to a better understanding of S. aureus epidemiology and the relationships between adhesion mechanisms, biofilm formation, antimicrobial resistance, and toxins and could aid in the development of improved vaccines and strain genotyping methods.

Isolation, Characterization, and Genome Analysis of a Novel Bacteriophage, Escherichia Phage vB_EcoM-4HA13, Representing a New Phage Genus in the Novel Phage Family Chaseviridae.

Shiga toxin-producing Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction-one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.

Genome sequence and characterization of Streptomyces phage Pablito, representing a new species within the genus Janusvirus.

Streptomycetes are ubiquitous soil bacteria. Here we report the complete and annotated genome sequence and characterization of Streptomyces phage Pablito, isolated from a soil sample in Haarlem, the Netherlands using Streptomyces lividans as host. This phage was able to infect a diverse range of Streptomyces strains, but none belonging to the sister genus Kitasatospora. Phage Pablito has double-stranded DNA with a genome length of 49,581 base pairs encoding 76 putative proteins, of which 26 could be predicted. The presence of a serine integrase protein indicated the lysogenic nature of phage Pablito. The phage remained stable over a wide range of temperatures (25-45 °C) and at pH ≥ 7.0, but lost infectivity at temperatures above 55 °C or when the pH dropped below 6.0. This newly isolated phage is closely related to Streptomyces phage Janus and Hank144 and considered a new species classified in the genus Janusvirus, within the subfamily Arquattrovirinae.

Mining bacterial NGS data vastly expands the complete genomes of temperate phages.

Temperate phages (active prophages induced from bacteria) help control pathogenicity, modulate community structure, and maintain gut homeostasis. Complete phage genome sequences are indispensable for understanding phage biology. Traditional plaque techniques are inapplicable to temperate phages due to their lysogenicity, curbing their identification and characterization. Existing bioinformatics tools for prophage prediction usually fail to detect accurate and complete temperate phage genomes. This study proposes a novel computational temperate phage detection method (TemPhD) mining both the integrated active prophages and their spontaneously induced forms (temperate phages) from next-generation sequencing raw data. Applying the method to the available dataset resulted in 192 326 complete temperate phage genomes with different host species, expanding the existing number of complete temperate phage genomes by more than 100-fold. The wet-lab experiments demonstrated that TemPhD can accurately determine the complete genome sequences of the temperate phages, with exact flanking sites, outperforming other state-of-the-art prophage prediction methods. Our analysis indicates that temperate phages are likely to function in the microbial evolution by (i) cross-infecting different bacterial host species; (ii) transferring antibiotic resistance and virulence genes and (iii) interacting with hosts through restriction-modification and CRISPR/anti-CRISPR systems. This work provides a comprehensively complete temperate phage genome database and relevant information, which can serve as a valuable resource for phage research.

ICTV Virus Taxonomy Profile: Chaseviridae 2022.

Members of the family Chaseviridae are lytic bacterial viruses infecting representatives of the bacterial class Gammaproteobacteria. Chaseviruses have a global distribution. Virions of members of this family have a myovirus morphology (icosahedral head with contractile tail). Genomes are dsDNA of 52-56 kbp with G+C content ranging from 39.3-52.5 %. Chaseviruses, like members of the family Autographiviridae, encode a large single subunit RNA polymerase, but unlike those viruses their promoter sequences have not yet been identified. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Chaseviridae, which is available at ictv.global/report/chaseviridae.

Complete Genome Sequence of a Jumbo Bacteriophage, Escherichia Phage vB_EcoM_EC001.

Here, we report the genome sequence of a jumbo Escherichia phage vB_EcoM_EC001, a myovirus isolated from primary sludge using enterohemorrhagic Escherichia coli O157:H7. The genome is 240,200 bp long and has 270 predicted coding sequences, including a tryptophanyl tRNA gene. It belongs to genus Seoulvirus.

Guidance for creating individual and batch latinized binomial virus species names.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3.

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.

Genomic comparisons and phylogenetic analysis of mastitis-related staphylococci with a focus on adhesion, biofilm, and related regulatory genes.

Mastitis is a common and costly disease on dairy farms, commonly caused by Staphylococcus spp. though the various species are associated with different clinical outcomes. In the current study, we performed genomic analyses to determine the prevalence of adhesion, biofilm, and related regulatory genes in 478 staphylococcal species isolated from clinical and subclinical mastitis cases deposited in public databases. The most prevalent adhesin genes (ebpS, atl, pls, sasH and sasF) were found in both clinical and subclinical isolates. However, the ebpS gene was absent in subclinical isolates of Staphylococcus arlettae, S. succinus, S. sciuri, S. equorun, S. galinarum, and S. saprophyticus. In contrast, the coa, eap, emp, efb, and vWbp genes were present more frequently in clinical (vs. subclincal) mastitis isolates and were highly correlated with the presence of the biofim operon (icaABCD) and its transcriptional regulator, icaR. Co-phylogenetic analyses suggested that many of these adhesins, biofilm, and associated regulatory genes could have been horizontally disseminated between clinical and subclinical isolates. Our results further suggest that several adhesins, biofilm, and related regulatory genes, which have been overlooked in previous studies, may be of use for virulence profiling of mastitis-related Staphylococcus strains or as potential targets for vaccine development.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

A Roadmap for Genome-Based Phage Taxonomy.

Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy.

Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation.

All sequencing projects of bacteriophages (phages) should seek to report an accurate and comprehensive annotation of their genomes. This article defines 14 questions for those new to phage genomics that should be addressed before submitting a genome sequence to the International Nucleotide Sequence Database Collaboration or writing a publication.

VIRIDIC-A Novel Tool to Calculate the Intergenomic Similarities of Prokaryote-Infecting Viruses.

Nucleotide-based intergenomic similarities are useful to understand how viruses are related with each other and to classify them. Here we have developed VIRIDIC, which implements the traditional algorithm used by the International Committee on Taxonomy of Viruses (ICTV), Bacterial and Archaeal Viruses Subcommittee, to calculate virus intergenomic similarities. When compared with other software, VIRIDIC gave the best agreement with the traditional algorithm, which is based on the percent identity between two genomes determined by BLASTN. Furthermore, VIRIDIC proved best at estimating the relatedness between more distantly-related phages, relatedness that other tools can significantly overestimate. In addition to the intergenomic similarities, VIRIDIC also calculates three indicators of the alignment ability to capture the relatedness between viruses: the aligned fractions for each genome in a pair and the length ratio between the two genomes. The main output of VIRIDIC is a heatmap integrating the intergenomic similarity values with information regarding the genome lengths and the aligned genome fraction. Additionally, VIRIDIC can group viruses into clusters, based on user-defined intergenomic similarity thresholds. The sensitivity of VIRIDIC is given by the BLASTN. Thus, it is able to capture relationships between viruses having in common even short genomic regions, with as low as 65% similarity. Below this similarity level, protein-based analyses should be used, as they are the best suited to capture distant relationships. VIRIDIC is available at viridic.icbm.de, both as a web-service and a stand-alone tool. It allows fast analysis of large phage genome datasets, especially in the stand-alone version, which can be run on the user's own servers and can be integrated in bioinformatics pipelines. VIRIDIC was developed having viruses of Bacteria and Archaea in mind; however, it could potentially be used for eukaryotic viruses as well, as long as they are monopartite.