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.

A Novel Aeromonas popoffii Phage AerP_220 Proposed to Be a Member of a New Tolavirus Genus in the Autographiviridae Family.

Aeromonas popoffii is one of the environmental Aeromonas species. A number of factors of virulence have been described for this species and it has been reported as a causative agent of urinary tract infection. The first A. popoffii bacteriophage AerP_220 along with its host strain A. popoffii CEMTC 4062 were isolated from river water. The phage has a podovirus morphotype, shows a narrow host range and is lytic against the host strain. The AerP_220 genome comprises 45,207 bp and does not contain genes responsible for antibiotic resistance and toxin production. Fifty-nine co-directional putative ORFs were found in the AerP_220 genome. Thirty-three ORFs encoded proteins with predicted functions; the products of 26 ORFs were hypothetical proteins. AerP_220 genome analysis revealed that this phage can be considered a novel species within the Autographiviridae family. Comparative genomic and proteomic analysis revealed that AerP_220 along with the Aeromonas phage vB_AspA_Tola (OM913599) are members of a new putative Tolavirus genus in the family Autographiviridae. The Gajwadongvirus and proposed Tolavirus genera along with Pantoea phage Nufs112 and phage Reminis could form a new Tolavirinae subfamily within the Autographiviridae family.

Evaluation of the Microbiological Effect of Colloidal Nanosilver Solution for Root Canal Treatment.

BACKGROUND: The goal of endodontic treatment, along with the preparation of the root canal and giving it a shape corresponding to the obturation technique, is the drug treatment of the canal. The aim of this study was to determine the antibacterial effect of a colloidal solution of nanosilver at its various dilutions on root canal microorganism. MATERIALS AND METHODS: A solution of silver nanoparticles at a concentration of 10,000 ppm (1.0%) was diluted in various concentrations (10 solutions from 1% to 0.0025%). Cultures used for research: Str. agalacticae ATCC 3984, E. faecalis ATCC 323, St. aureus ATCC 4785, C. albicans ATCC 10231. After thawing, cultures of microorganisms were introduced into a liquid nutrient medium: cerebral heart broth for bacterial cultures and Sabouraud broth for C. albicans. The cultivation was carried out at a temperature of 37 °C for 24 h. A bacterial suspension for inoculation was prepared from a microbial sediment according to a turbidity standard of 0.5 McFarland in saline. Then, 100 µL of the obtained suspension of microorganisms was inoculated by the "lawn" method using a spatula on the Muller-Hinton medium. Solutions of silver nanoparticles were introduced into wells prepared in agar with a sterile metal punch. Further incubation was carried out for 24 h at 37 °C. RESULTS: colloidal solution of silver nanoparticles at concentrations of 1%, 0.75%, 0.5% inhibited the growth of Str. agalacticae ATCC 3984 with a growth retardation zone of 6-7 mm. The E. faecalis ATCC 29212 strain was sensitive to solutions of silver nanoparticles at concentrations of 1%, 0.75%, 0.5% with a growth inhibition zone of 6-7 mm. Strain St. aureus 4785 demonstrated sensitivity to solutions of silver nanoparticles at concentrations of 1%, 0.75%, 0.5%, 0.1%, 0.05% with a growth retardation zone of 6-8 mm. CONCLUSION: colloidal solutions of silver nanoparticles have antimicrobial action against gram-positive bacteria (Str.agalacticae ATCC 3984, St. aureus ATCC 4785, E. faecalis ATCC 29212) and yeast-like fungi of the genus Candida (C. albicans ATCC 10231, C. albicans 672 and C. albicans D-225M), but this action is strain-specific and depends on the concentration of the solution.

Er,Cr:YSGG Laser Surface Modification Effect on Dentin Bonding to Zirconia: An In Vitro Study.

Objective: This study investigated the effect of dentin surface treatment with the erbium, chromium-doped yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser on the bond strength of zirconia to dentin. Background data: Although it is well-known that resin cement (RC) provides adequate bond strength of zirconia restorations to the tooth structure, many clinicians were not convinced in bonding reliability to zirconia materials. So, they preferred cementing their crowns with glass-ionomer cement (GIC). Pretreating the dentin surface is recommended to improve the adhesion of crowns cemented with GIC or RC. Recently, the Er,Cr:YSGG laser has been widely used for a cavity preparation, conditioning the tooth structure. However, there is not enough research on the bond strength of zirconia crowns cemented on a Er,Cr:YSGG laser-treated dentin. Methods: Forty-eight molars were cut horizontally at the crest of curvature. The flat dentin surface of 24 molars was left untreated, whereas the dentin surface of the other half was treated using an Er,Cr:YSGG laser 2.78 µm (4.5 W, 8.18 J/cm2, 90 mJ, 60 µs, 50 Hz, 60% air and 80% water). In addition, 48 plates of zirconia were prepared to be cemented with GIC or RC on the dentin surface. So, the molars were divided into four groups as follows: GIC+untreated dentin; GIC+treated dentin; RC+untreated dentin; and RC+treated dentin. All the specimens were subjected to shear bond strength test. The mode of failure was determined and additional samples were prepared to evaluate the cement-dentin interface using a scanning electron microscope. Data were analyzed with two-way analysis of variance accompanied by univariate analyses. Results: The bond strength of zirconia cemented with GIC or bonded with RC significantly increased on a Er,Cr:YSGG laser-treated dentin surface (p < 0.05). Conclusions: Dentin preconditioning with an Er,Cr:YSGG laser significantly improved the bond strength of zirconia plates to dentin as compared with untreated dentin.

Evaluation the Relationship between Mandibular Molar Root Apices and Mandibular Canal among Residents of the Moscow Population using Cone-Beam Computed Tomography Technique.

Background: The relationship between the inferior alveolar nerve and the root apices of the mandibular molars and premolars is of clinical importance. The aim of this study was to determine the relationship between the mandibular canal (MC) and the mandibular molar root apices using cone-beam computed tomography (CBCT) scanning among residents of the Moscow population. Materials and Methods: Three hundred CBCT scans for patients aged 20-70 years were analyzed. Patients were divided into three age groups: young group (20-44 years), middle-age group (45-59 years), and elderly group (60-70). The distance from the MC and the mandibular molar root apices was measured in each group in the coronal view of CBCT scans. Statistical analysis was set on P < 0.05. Results: The mean distance from the mesial root apices of the first, second, and third molars to the MC was 4.92, 2.85, and 2.24 mm, respectively. The distal root showed to be the closest root to the MC in multirooted teeth. The young age group showed smaller distances to MC than other age groups (P < 0.05). Females showed smaller distances to MC in mandibular molars than males (P < 0.05). Conclusions: The distance between the root apices and the MC has to be taken into consideration when performing surgical or endodontic procedures.

The Application of Nano Silver Argitos as a Final Root Canal Irrigation for the Treatment of Pulpitis and Apical Periodontitis. In Vitro Study.

BACKGROUND: Endodontic treatment of various forms of pulpitis with variations of root canal system anatomy should be performed with high quality. The use of various antibacterial agents is aimed at maintaining the success of endodontic treatment. The aim of this study was to evaluate the penetration and fixation of the nano-silver solution on the dentinal surface during endodontic treatment. MATERIALS AND METHODS: the study was carried out on 70 extracted single-rooted teeth, randomly divided into two groups. In the teeth of the first group, the smear layer was removed after canal preparation with 17% EDTA solution; in the second group, the smear layer was not removed. In both groups, for the final treatment of the canal, a colloidal 1% solution of нанo серебра nanosilver was used. Samples were cut and prepared for analysis using micro-CT, scanning electron microscopy (SEM), X-ray microanalysis and energy dispersive spectrometry (elemental mapping). RESULTS: in 100% of cases in groups of teeth with a preserved smear layer, the ability of a 1% colloidal solution of nanosilver with particles of 1-2 nm to be fixed on dentin with a removed and preserved smear layer and to leave a film on the dentinal surface was established. In the samples with removed smear layer, silver was found in 73.5% of cases. CONCLUSION: The nano-silver solution with a particle size of 1-2 nm proved its ability to penetrate the dentinal surfaces and create a final film covering the dentinal surface of the root canal before applying the sealer.

Antibiotic Resistance and Pathogenomics of Staphylococci Circulating in Novosibirsk, Russia.

A total of 394 strains of staphylococci found in humans and pets in Novosibirsk, Siberian Russia, were characterized in terms of antibiotic resistance and corresponding genes. Two coagulase-positive and 17 coagulase-negative species were identified. The majority of isolates, with the exception of S. haemolyticus and hospital S. epidermidis isolates, were sensitive to most of the tested antibiotics, and isolates from pets displayed the lowest level of resistance. Nevertheless, methicillin-resistant (MRS) and/or multidrug-resistant (MDR) isolates were found in all prevailed species, including coagulase-negative. A set of genes corresponding to the detected resistance was identified: mecA (beta-lactam resistance), aac(6')-Ie-aph(2″)-Ia, aph(3')-IIIa, ant(4')-Ia (aminoglycoside-modifying enzymes), ermA/ermC, and msrA (macrolide resistance). Complete genome analysis for ten MDR S. epidermidis and five MDR S. haemolyticus isolates revealed additional antibiotic resistance genes mphC, qacA/qacB, norA, dfrC/dfrG, lnuA, BseSR, and fosB. NorA, dfrC, and fosB were present in all S. epidermidis genomes, whereas mphC and msrA were identified in all S. haemolyticus ones. All investigated MDR S. epidermidis and four of five S. haemolyticus strains were moderate or strong biofilm producers, whereas multiple genes responsible for this function and for virulence and pathogenicity were identified mostly in S. epidermidis, but were less frequently represented in S. haemolyticus.

A New Enterobacter cloacae Bacteriophage EC151 Encodes the Deazaguanine DNA Modification Pathway and Represents a New Genus within the Siphoviridae Family.

A novel Enterobacter cloacae phage, EC151, was isolated and characterized. Electron microscopy revealed that EC151 has a siphovirus-like virion morphology. The EC151 nucleotide sequence shows limited similarity to other phage genomes deposited in the NCBI GenBank database. The size of the EC151 genome is 60,753 bp and contains 58 putative genes. Thirty-nine of them encode proteins of predicted function, 18 are defined as hypothetical proteins, and one ORF identifies as the tRNA-Ser-GCT-encoding gene. Six ORFs were predicted to be members of the deazaguanine DNA modification pathway, including the preQ0 transporter. Comparative proteomic phylogenetic analysis revealed that phage EC151 represents a distinct branch within a group of sequences containing clades formed by members of the Seuratvirus, Nonagvirus, and Vidquintavirus genera. In addition, the EC151 genome showed gene synteny typical of the Seuratvirus, Nonagvirus, and Nipunavirus phages. The average genetic distances of EC151/Seuratvirus, EC151/Nonagvirus, and EC151/Vidquintavirus are approximately equal to those between the Seuratvirus, Nonagvirus, and Vidquintavirus genera (~0.7 substitutions per site). Therefore, EC151 may represent a novel genus within the Siphoviridae family. The origin of the deazaguanine DNA modification pathway in the EC151 genome can be traced to Escherichia phages from the Seuratvirus genus.

Influence of Caudovirales Phages on Humoral Immunity in Mice.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

An In Vitro Evaluation Study of the Geometric Changes of Root Canal Preparation and the Quality of Endodontic Treatment.

INTRODUCTION: The geometry of root canals differs in different parts, especially in the apical region, and it is affected by different preparation techniques. The aim of this study was to evaluate the geometric changes of root canal preparation by general dentists regardless of the endodontic instrumentation systems and to study the quality of endodontic treatment by evaluating the untouched areas after mechanical preparation and the smear layer removal. MATERIALS AND METHODS: 100 extracted maxillary canines were collected for the in vitro study from 10 dentists, and the dentists were asked to treat the teeth endodontically. The teeth then were separated and examined under an optical microscope to evaluate the root canal final diameter and the untouched areas. Then, the teeth were examined under a scanned electronic microscope to evaluate the smear layer in coronal, middle, and third parts of the canal. Statistical significance was set as P < 0.05. RESULTS: The mean diameter of the root canal after instrumentation in the coronal and middle thirds was 2.50 ± 1.12 and 1.75 ± 1.24 mm, respectively, and the untouched area percentage observed in the apical thirds was 71%. For smear layer removal, it was better in the coronal and middle thirds than in the apical (P < 0.05). CONCLUSION: The changes in the diameter of the root canal, the percentage of untouched areas after mechanical preparation, and the percentage of smear layer were observed in a higher percent in the apical third than in the coronal and middle thirds, and this raises the question of changing the technique of processing the root canal, especially in the apical third.

Isolation and Characterization of a Novel Klebsiella pneumoniae N4-like Bacteriophage KP8.

Klebsiella pneumoniae is a common pathogen, associated with a wide spectrum of infections, and clinical isolates of K. pneumoniae often possess multiple antibiotic resistances. Here, we describe a novel lytic N4-like bacteriophage KP8, specific to K. pneumoniae, including its genome, partial structural proteome, biological properties, and proposed taxonomy. Electron microscopy revealed that KP8 belongs to the Podoviridae family. The size of the KP8 genome was 73,679 bp, and it comprised 97 putative open reading frames. Comparative genome analysis revealed that the KP8 genome possessed the highest similarity to the genomes of Enquatrovirus and Gamaleyavirus phages, which are N4-like podoviruses. In addition, the KP8 genome showed gene synteny typical of the N4-like podoviruses and contained the gene encoding a large virion-encapsulated RNA polymerase. Phylogenetic analysis of the KP8 genome revealed that the KP8 genome formed a distinct branch within the clade, which included the members of Enquatrovirus and Gamaleyavirus genera besides KP8. The average evolutionary divergences KP8/Enquatrovirus and KP8/Gamaleyavirus were 0.466 and 0.447 substitutions per site (substitutes/site), respectively, similar to that between Enquatrovirus and Gamaleyavirus genera (0.468 substitutes/site). The obtained data suggested that Klebsiella phage KP8 differs from other similar phages and may represent a new genus within the N4-like phages.

Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762.

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-ß-D-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester: [See figure in text]. Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.

Cell wall teichoic acids of streptomycetes of the phenetic cluster 'Streptomyces fulvissimus'.

Structures of the anionic polymers of streptomycetes Streptomyces fulvissimus VKM Ac-994(T), Streptomyces longispororuber VKM Ac-1735(T), Streptomyces aureoveticillatus VKM Ac-48(T) and Streptomyces spectabilis INA 00606 belonging to the phenetic cluster 'S. fulvissimus' were investigated by chemical and NMR spectroscopic methods. A teichoic acid from the cell wall of S. spectabilis INA 00606 was studied in more detail, and this was shown to represent 1,3-poly(glycerol phosphate) substituted with glucosamine (alpha-D-GlcNAc) and L-glutamic acid (non-stoichiometric substitution). For the first time, glutamic acid is identified as an acyl substituent in teichoic acids of streptomycetes. The polymer chain is built of the following fragments: Cell walls of other streptomycetes of the phenocluster under study contain 1,3-poly(glycerol phosphates) with glucosamine as a glycosyl substituent at O-2 of the glycerol phosphate units and L-glutamic acid and lysine as O-2 acyl substituents. Not all amino sugar residues in the polymers of these strains are N-acetylated, and the content of the glucosamine and lysine residues in the polymers of different strains is not the same. Despite certain quantitative differences in the structures of the polymers, one may consider streptomycetes of the phenocluster 'S. fulvissimus' as closely related microorganisms, the details of the structures serving as additional criteria for the determination of the species status of a strain under study.