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.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

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.

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.