A Genomic Analysis of the Bacillus Bacteriophage Kirovirus kirovense Kirov and Its Ability to Preserve Milk.

Bacteriophages are widely recognized as alternatives to traditional antibiotics commonly used in the treatment of bacterial infection diseases and in the food industry, as phages offer a potential solution in combating multidrug-resistant bacterial pathogens. In this study, we describe a novel bacteriophage, Kirovirus kirovense Kirov, which infects members of the Bacillus cereus group. Kirovirus kirovense Kirov is a broad-host-range phage belonging to the Caudoviricetes class. Its chromosome is a linear 165,667 bp double-stranded DNA molecule that contains two short, direct terminal repeats, each 284 bp long. According to bioinformatics predictions, the genomic DNA contains 275 protein-coding genes and 5 tRNA genes. A comparative genomic analysis suggests that Kirovirus kirovense Kirov is a novel species within the Kirovirus genus, belonging to the Andregratiavirinae subfamily. Kirovirus kirovense Kirov demonstrates the ability to preserve and decontaminate B. cereus from cow milk when present in milk at a concentration of 104 PFU/mL. After 4 h of incubation with the phage, the bacterial titer drops from 105 to less than 102 CFU/mL.

Bacteriolytic Potential of Enterococcus Phage iF6 Isolated from "Sextaphag®" Therapeutic Phage Cocktail and Properties of Its Endolysins, Gp82 and Gp84.

The number of infections caused by antibiotic-resistant strains of bacteria is growing by the year. The pathogenic bacterial species Enterococcus faecalis and Enterococcus faecium are among the high priority candidate targets for the development of new therapeutic antibacterial agents. One of the most promising antibacterial agents are bacteriophages. According to the WHO, two phage-based therapeutic cocktails and two medical drugs based on phage endolysins are currently undergoing clinical trials. In this paper, we describe the virulent bacteriophage iF6 and the properties of two of its endolysins. The chromosome of the iF6 phage is 156,592 bp long and contains two direct terminal repeats, each 2108 bp long. Phylogenetically, iF6 belongs to the Schiekvirus genus, whose representatives are described as phages with a high therapeutic potential. The phage demonstrated a high adsorption rate; about 90% of iF6 virions attached to the host cells within one minute after the phage was added. Two iF6 endolysins were able to lyse enterococci cultures in both logarithmic and stationary growth phases. Especially promising is the HU-Gp84 endolysin; it was active against 77% of enterococci strains tested and remained active even after 1 h incubation at 60 °C. Thus, iF6-like enterococci phages appear to be a promising platform for the selection and development of new candidates for phage therapy.

Isolation and Characterization of Two Novel Siphoviruses Novomoskovsk and Bolokhovo, Encoding Polysaccharide Depolymerases Active against Bacillus pumilus.

This study describes two novel bacteriophages infecting members of the Bacillus pumilus group. Even though members of the group are not recognized as pathogenic, several strains belonging to the group have been reported to cause infectious diseases in plants, animals and humans. Bacillus pumilus group species are highly resistant to ultraviolet radiation and capable of forming biofilms, which complicates their eradication. Bacteriophages Novomoskovsk and Bolokhovo were isolated from soil samples. Genome sequencing and phylogenetic analysis revealed that the phages represent two new species of the genus Andromedavirus (class Caudoviricetes). The phages remained stable in a wide range of temperatures and pH values. A host range test showed that the phages specifically infect various strains of B. pumilus. The phages form clear plaques surrounded by halos. Both phages Novomoskovsk and Bolokhovo encode proteins with pectin lyase domains-Putative depolymerases. Obtained in a purified recombinant form, the proteins produced lysis zones on the lawn of a B. pumilus strain. This suggests that Novomoskovsk and Bolokhovo may be effective for the eradication of B. pumilus biofilms.

Novel Bacillus-Infecting Bacteriophage B13-The Founding Member of the Proposed New Genus Bunatrivirus.

In this work, we describe a novel temperate bacteriophage, Bacillus phage B13. Bacillus-infecting phages are widespread and abundant, though often overlooked including because of their temperate lifestyle. B13 was isolated from its bacterial host via mitomycin C induction. Its host range was determined, and its pH and thermal stability were evaluated. The whole genome of B13 was sequenced and annotated. The genome is 36,864 bp long and contains 53 genes. The tail genes of B13 suggest that the phage has a siphovirus morphotype. It was found both in vitro and in silico that the phage uses the 3'-cos DNA packaging strategy, and the phage genome termini were located. Comparative analyses revealed that B13 has no close relatives and should therefore be assigned to a new viral genus, for which we propose the name Bunatrivirus.

vB_BcM_Sam46 and vB_BcM_Sam112, members of a new bacteriophage genus with unusual small terminase structure.

One of the serious public health concerns is food contaminated with pathogens and their vital activity products such as toxins. Bacillus cereus group of bacteria includes well-known pathogenic species such as B. anthracis, B. cereus sensu stricto (ss), B. cytotoxicus and B. thuringiensis. In this report, we describe the Bacillus phages vB_BcM_Sam46 and vB_BcM_Sam112 infecting species of this group. Electron microscopic analyses indicated that phages Sam46 and Sam112 have the myovirus morphotype. The genomes of Sam46 and Sam112 comprise double-stranded DNA of 45,419 bp and 45,037 bp in length, respectively, and have the same GC-content. The genome identity of Sam46 and Sam112 is 96.0%, indicating that they belong to the same phage species. According to the phylogenetic analysis, these phages form a distinct clade and may be members of a new phage genus, for which we propose the name 'Samaravirus'. In addition, an interesting feature of the Sam46 and Sam112 phages is the unusual structure of their small terminase subunit containing N-terminal FtsK_gamma domain.

Complete Genome Sequence of Bacillus cereus Sensu Stricto VKM B-370, Isolated from the Silkworm Bombyx mori.

Bacillus cereus is a Gram-positive rod-shaped spore-forming bacterium widespread in different environmental niches. Here, we report the complete genome sequence of Bacillus cereus VKM B-370 from the All-Russian Collection of Microorganisms, the host strain for bacteriophages vB_BtS_B83, vB_BcM_Sam46, vB_BcM_Sam112, and Izhevsk.

Putative plasmid prophages of Bacillus cereus sensu lato may hold the key to undiscovered phage diversity.

Bacteriophages are bacterial viruses and the most abundant biological entities on Earth. Temperate bacteriophages can form prophages stably maintained in the host population: they either integrate into the host genome or replicate as plasmids in the host cytoplasm. As shown, tailed temperate bacteriophages may form circular plasmid prophages in many bacterial species of the taxa Firmicutes, Gammaproteobacteria and Spirochaetes. The actual number of such prophages is thought to be underestimated for two main reasons: first, in bacterial whole genome-sequencing assemblies, they are difficult to distinguish from actual plasmids; second, there is an absence of experimental studies which are vital to confirm their existence. In Firmicutes, such prophages appear to be especially numerous. In the present study, we identified 23 genomes from species of the Bacillus cereus group that were deposited in GenBank as plasmids and may belong to plasmid prophages with little or no homology to known viruses. We consider these putative prophages worth experimental assays since it will broaden our knowledge of phage diversity and suggest that more attention be paid to such molecules in all bacterial sequencing projects as this will help in identifying previously unknown phages.

Bacillus-infecting bacteriophage Izhevsk harbors thermostable endolysin with broad range specificity.

Several bacterial species belonging to the Bacillus cereus group are known to be causative agents of food poisoning and severe human diseases. Bacteriophages and their lytic enzymes called endolysins have been widely shown to provide for a supplemental or primary means of treating bacterial infections. In this work we present a new broad-host-range phage Izhevsk, which infects the members of the Bacillus cereus group. Transmission electron microscopy, genome sequencing and comparative analyses revealed that Izhevsk is a temperate phage with Siphoviridae morphology and belongs to the same genus as the previously described but taxonomically unclassified bacteriophages Tsamsa and Diildio. The Ply57 endolysin of Izhevsk phage has broad-spectrum activity against B. cereus sensu lato. The thermolability of Ply57 is higher than that of the PlyG of Wß phage. This work contributes to our current understanding of phage biodiversity and may be useful for further development of efficient antimicrobials aimed at diagnosing and treating infectious diseases and food contaminations caused by the Bacillus cereus group of bacteria.

Complete Genome Sequence of Enterococcus faecium FS86, Used for Propagation of Bacteriophages with Therapeutic Potential.

The Enterococcus faecium FS86 genome consists of a 2,685,395-bp chromosome and a 9,751-bp plasmid. The plasmid harbors mobilization-related genes. The pathogenicity factor genotype is cylA negative, aggA negative, gelE negative, sprE negative, esp negative, eep positive, and efaA positive. E. faecium FS86 belongs to multilocus sequence type 296, together with the probiotic strain E. faecium SF68.

Bacillus Phage vB_BtS_B83 Previously Designated as a Plasmid May Represent a New Siphoviridae Genus.

The Bacillus cereus group of bacteria includes, inter alia, the species known to be associated with human diseases and food poisoning. Here, we describe the Bacillus phage vB_BtS_B83 (abbreviated as B83) infecting the species of this group. Transmission electron microscopy (TEM) micrographs indicate that B83 belongs to the Siphoviridae family. B83 is a temperate phage using an arbitrium system for the regulation of the lysis-lysogeny switch, and is probably capable of forming a circular plasmid prophage. Comparative analysis shows that it has been previously sequenced, but was mistaken for a plasmid. B83 shares common genome organization and >46% of proteins with other the Bacillus phage, BMBtp14. Phylograms constructed using large terminase subunits and a pan-genome presence-absence matrix show that these phages form a clade distinct from the closest viruses. Based on the above, we propose the creation of a new genus named Bembunaquatrovirus that includes B83 and BMBtp14.