Bacteriophage vB_SepP_134 and Endolysin LysSte_134_1 as Potential Staphylococcus-Biofilm-Removing Biological Agents.

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus warneri. LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis.

A Novel Podophage StenR_269 Suggests a New Family in the Class Caudoviricetes.

Stenotrophomonas rhizophila was first discovered in soil; it is associated with the rhizosphere and capable of both protecting roots and stimulating plant growth. Therefore, it has a great potential to be used in biocontrol. The study of S. rhizophila phages is important for a further evaluation of their effect on the fitness and properties of host bacteria. A novel phage StenR_269 and its bacterial host S. rhizophila were isolated from a soil sample in the remediation area of a coal mine. Electron microscopy revealed a large capsid (~Ø80 nm) connected with a short tail, which corresponds to the podovirus morphotype. The length of the genomic sequence of the StenR_269 was 66,322 bp and it contained 103 putative genes; 40 of them encoded proteins with predicted functions, 3 corresponded to tRNAs, and the remaining 60 were identified as hypothetical ones. Comparative analysis indicated that the StenR_269 phage had a similar genome organization to that of the unclassified Xanthomonas phage DES1, despite their low protein similarity. In addition, the signature proteins of StenR_269 and DES1 had low similarity and these proteins clustered far from the corresponding proteins of classified phages. Thus, the StenR_269 genome is orphan and the analyzed data suggest a new family in the class Caudoviricetes.

StM171, a Stenotrophomonas maltophilia Bacteriophage That Affects Sensitivity to Antibiotics in Host Bacteria and Their Biofilm Formation.

Stenotrophomonas maltophilia mainly causes respiratory infections that are associated with a high mortality rate among immunocompromised patients. S. maltophilia exhibits a high level of antibiotic resistance and can form biofilms, which complicates the treatment of patients infected with this bacterium. Phages combined with antibiotics could be a promising treatment option. Currently, ~60 S. maltophilia phages are known, and their effects on biofilm formation and antibiotic sensitivity require further examination. Bacteriophage StM171, which was isolated from hospital wastewater, showed a medium host range, low burst size, and low lytic activity. StM171 has a 44kbp dsDNA genome that encodes 59 open-reading frames. A comparative genomic analysis indicated that StM171, along with the Stenotrophomonas phage Suso (MZ326866) and Xanthomonas phage HXX_Dennis (ON711490), are members of a new putative Nordvirus genus. S. maltophilia strains that developed resistance to StM171 (bacterial-insensitive mutants) showed a changed sensitivity to antibiotics compared to the originally susceptible strains. Some bacterial-insensitive mutants restored sensitivity to cephalosporin and penicillin-like antibiotics and became resistant to erythromycin. StM171 shows strain- and antibiotic-dependent effects on the biofilm formation of S. maltophilia strains.

Isolation, Characterization and Genomic Analysis of a Novel Jumbo Phage, AerS_266, That Infects Aeromonas salmonicida.

Aeromonas salmonicida is the causative agent of septicemia in fish, and it is associated with significant economic losses in the aquaculture industry. While piscine Aeromonas infections are mainly treated with antibiotics, the emergence of resistance in bacterial populations requires the development of alternative methods of treatment. The use of phages can be one of them. A novel A. salmonicida jumbo phage, AerS_266, was isolated and characterized. This phage infects only mesophilic A. salmonicida strains and demonstrates a slow lytic life cycle. Its genome contains 243,674 bp and 253 putative genes: 84 encode proteins with predicted functions, and 3 correspond to tRNAs. Genes encoding two multisubunit RNA polymerases, chimallin and PhuZ, were identified, and AerS_266 was thus defined as a phiKZ-like phage. While similar phages with genomes >200 kb specific to Aeromonas hydrophila and Aeromonas veronii have been previously described, AerS_266 is the first phiKZ-like phage found to infect A. salmonicida.

Identification, Characterization, and Genome Analysis of Two Novel Temperate Pseudomonas protegens Phages PseuP_222 and PseuP_224.

Two novel P. protegens bacteriophages PseuP_222 and Pseu_224 and their host P. protegens CEMTC 4060 were isolated from the same sample (Inya river, Siberia). Both phages have siphovirus morphology and belong to lambdoid phages. Comparative genome analysis revealed a low nucleotide and amino acid sequence similarity of PseuP_222 and PseuP_224 between themselves, and between them and other lambdoid phages. Bioinformatics analysis indicated that PseuP_222 and PseuP_224 are members of a genetically diverse group of phages of environmental Pseudomonas spp.; this group is distant from a large group of P. aeruginosa phages. In phylogenetic trees, the positioning of the terminase large subunits, major capsid proteins, tail tape measure proteins, and CI-like repressors of PseuP_222 and PseuP_224 were remote and changed relative to those of the Escherichia lambda phage and lambdoid phages of Pseudomonas spp. However, the nucleoid-associated protein NdpA/YejK and P5-like structural protein from both phages showed high similarity and were not found in lambda phage and other lambdoid phages of Pseudomonas spp. Substantial divergences of the PseuP_222 and PseuP_224 genomes and proteomes indicated that the evolutionary history of these phages was mostly independent and they probably began to use one host only recently.

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

Emerging OP354-Like P[8] Rotaviruses Have Rapidly Dispersed from Asia to Other Continents.

The majority of human group A rotaviruses possess the P[8] VP4 genotype. Recently, a genetically distinct subtype of the P[8] genotype, also known as OP354-like P[8] or lineage P[8]-4, emerged in several countries. However, it is unclear for how long the OP354-like P[8] gene has been circulating in humans and how it has spread. In a global collaborative effort 98 (near-)complete OP354-like P[8] VP4 sequences were obtained and used for phylogeographic analysis to determine the viral migration patterns. During the sampling period, 1988-2012, we found that South and East Asia acted as a source from which strains with the OP354-like P[8] gene were seeded to Africa, Europe, and North America. The time to the most recent common ancestor (TMRCA) of all OP354-like P[8] genes was estimated at 1987. However, most OP354-like P[8] strains were found in three main clusters with TMRCAs estimated between 1996 and 2001. The VP7 gene segment of OP354-like P[8] strains showed evidence of frequent reassortment, even in localized epidemics, suggesting that OP354-like P[8] genes behave in a similar manner on the evolutionary level as other P[8] subtypes. The results of this study suggest that OP354-like P[8] strains have been able to disperse globally in a relatively short time period. This, in combination with a relatively large genetic distance to other P[8] subtypes, might result in a lower vaccine effectiveness, underscoring the need for a continued surveillance of OP354-like P[8] strains, especially in countries where rotavirus vaccination programs are in place.

Evolutionary time-scale of primate bocaviruses.

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1-HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60-300years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9×10(-4)substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60-80years ago, while HBoV4 separated from great apes bocaviruses about 200-300years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed.