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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.

It remains unclear how much the accessory genome contributes in the complex processes of establishment and virulence of bacterial infections. P. aeruginosa is one of the most common opportunistic human pathogens and can establish difficult-to-eradicate infections. Genome-integrated viruses, known as prophages, are frequent components of this bacterium’s large accessory genome and can contribute to the virulence of P. aeruginosa. However, systematic interpretations of the contributing role of prophages in the evolution and fitness of the ubiquitous P. aeruginosa in its diverse niches are still in their infancy. This study provides insights into these roles by exploring the activity, abundance and persistence of prophages belonging to P. aeruginosa from the cystic fibrosis (CF) lung. We selected a cohort of 12 CF patients with a high-resolution history of difficult-to-eradicate P. aeruginosa infections. Nanopore technology was used to sequence high-contiguity genomes of one early isolate per patient. Subsequently, we applied a strategy that combined bioinformatics, antibiotic-assisted inductions, lysate sequencing and genomics to identify intact prophages in the host genomes and assess their long-term survival in follow-up isolates. From these data, we observed that CF P. aeruginosa genomes harbour a high abundance of intact prophages which can sometimes self-induce. We identified 29 intact prophages with a wide genomic diversity and some unique prophage genomes with minimum similarity to available genomes. All induced prophages were retraced in follow-up isolates for a tested period of 4 to 9 years with minimal genomic changes. In addition to elucidating the role of prophages in P. aeruginosa, we expect our findings to assist in developing novel diagnostics and phage-based therapies for P. aeruginosa infections.

The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida, an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly efficient genome editing tool for P. putida and P. aeruginosa. This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second Standard European Vector Architecture vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within a week, with an efficiency of >83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas, to complement the existing range of Cas9-based editing methods, and facilitates genomic engineering efforts of this important

It remains unclear how much the accessory genome contributes in the complex processes of establishment and virulence of bacterial infections. P. aeruginosa is one of the most common opportunistic human pathogens and can establish difficult-to-eradicate infections. Genome-integrated viruses, known as prophages, are frequent components of this bacterium’s large accessory genome and can contribute to the virulence of P. aeruginosa. However, systematic interpretations of the contributing role of prophages in the evolution and fitness of the ubiquitous P. aeruginosa in its diverse niches are still in their infancy. This study provides insights into these roles by exploring the activity, abundance and persistence of prophages belonging to P. aeruginosa from the cystic fibrosis (CF) lung. We selected a cohort of 12 CF patients with a high-resolution history of difficult-to-eradicate P. aeruginosa infections. Nanopore technology was used to sequence high-contiguity genomes of one early isolate per patient. Subsequently, we applied a strategy that combined bioinformatics, antibiotic-assisted inductions, lysate sequencing and genomics to identify intact prophages in the host genomes and assess their long-term survival in follow-up isolates. From these data, we observed that CF P. aeruginosa genomes harbour a high abundance of intact prophages which can sometimes self-induce. We identified 29 intact prophages with a wide genomic diversity and some unique prophage genomes with minimum similarity to available genomes. All induced prophages were retraced in follow-up isolates for a tested period of 4 to 9 years with minimal genomic changes. In addition to elucidating the role of prophages in P. aeruginosa, we expect our findings to assist in developing novel diagnostics and phage-based therapies for P. aeruginosa infections.

P3.6-007

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Strain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae, being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae. Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LM

The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus (Nouzillyvirus). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro, this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E