Genomic analyses reveal two distinct lineages of Corynebacterium ulcerans strains

Corynebacterium ulcerans is an important zoonotic pathogen which is causing diphtheria-like disease in humans, globally. In this study, the genomes of three recently isolated C. ulcerans strains, 4940, 2590 and BRAD-2649, respectively from an asymptomatic carrier, a patient with pharyngitis and a canine host were sequenced to investigate their virulence potential. A comparative analysis was performed including the published genome sequences of 16 other C. ulcerans isolates. C. ulcerans strains belong to two lineages; 13 strains grouped together in Lineage-1 and 6 strains in Lineage-2. Consistent with the zoonotic nature of C. ulcerans infections, isolates from both, the human and canine hosts clustered in both the lineages. Most of the strains possessed spaDEF and spaBC gene clusters along with the virulence genes cpp, pld, cwlH, nanH, rpfI, tspA and vsp1. The gene encoding Shiga-like toxin was only present in one strain and 11 strains carried the tox gene encoding the diphtheria-like toxin. However, none of strains 4940, 2590 and BRAD-2649 carried any toxin genes. These strains varied in the number of prophages in their genomes, which suggests these are playing an important role in introducing diversity in C. ulcerans. The pan-genomic analyses revealed a variation in the number of membrane-associated and secreted proteins that may contribute to the variation in the pathogenicity between different strains

ANALYSIS OF THE CLINICAL EFFICIENCY OF RESTORATIVE FILLING MATERIALS

Aim. To conduct a clinical assessment of short- and long-term surface sealing in patients having a different hygienic status of the oral cavity.Materials and methods. For clinical trials, a group of 250 male and female patients aged 25–45 years old and diagnosed with Black’s class III and V carious lesions was selected. All patients were divided into three groups depending on the state of oral hygiene and the applied methods of aesthetic restoration: control group — defect restoration by Filtek Z-250 microhybrid composite (3M ESPE); comparison group — Filtek Z-250 restoration (3M ESPE) + Easy Glaze (Voco) surface sealing on the day of restoration; and the main group with three subgroups: 2α subgroup — Restavrin restoration + Easy Glaze sealant on the day of treatment; 2β subgroup — Restavrin restoration + Easy Glaze sealant on the day of treatment and re-sealing with a frequency of 1 time per year; 2γ subgroup — Restavrin restoration + Easy Glaze sealant on the day of treatment and re-sealing with a frequency of 1 time per 6 months. The quality of caries cavities was assessed by USPHS criteria, including anatomical shape (AS), marginal pigmentation (MP), marginal adaptation (MA), the presence of secondary caries (SC) and sensitivity (S). The evaluation was conducted on the day of the visit and following 6, 12 and 24 months.Results. A comparison of the clinical evaluation of photocomposite restorations by the Filtek Z-250 (3M ESPE) microhybrid composite in the control group of patients, in comparison group and in the main Restavrin (Technodent) group showed that the Easy Glaze (Voco) surface sealing of the Restavrin composite restorations carried out in the 2α subgroup of the main group onе time on the day of restoration, in the 2β subgroup with re-sealing after 12 months, and in the 2γ subgroup with sealing every 6 months had allowed the maximum number of restorations to be corresponded to the “satisfactory” value according to the AS, MP, MA, SC, S evaluation criteria throughout the entire observation period.Conclusion. The Restavrin (Technodent, Russia) polymeric nanohybrid fi lling material is shown to produce the most effective and durable restorations compared to other materials under study. The applicatiyon of Restavrin followed by the Easy Glaze (Voco) sealing protective system with a frequency dependent on the hygienic status of the oral cavity allows therapeutic methods for dentin caries treatment to be optimized

A method for estimation of immunogenic determinants mutability: case studies of HIV1 gp120 and diphtheria toxin

There is a need for the method which helps to choose the less mutable immunogenic determinant for the design of recombinant or synthetic vaccines and ELISA test-systems. With the help of our method based on the directional mutational pressure theory one is able to estimate direction of symmetric and asymmetric mutational pressure in a gene coding for a protein of interest, and to find out which of its immunogenic determinants are less prone to missense mutations and so, to immune escaping. Three original computer algorithms (“VVK Sliding Window”, “VVK VarInvar” and “VVK Protective Buffer” available via www.barkovsky.hotmail.ru) have been created to perform all the necessary calculations and tests. “VVK Sliding Window” calculates nucleotide usage in fourfold and twofold degenerated sites, as well as usage of missense, nonsense and synonymous sites for each kind of nucleotide mutation along the length of a coding region, while “VVK Protective Buffer” calculates those indexes in a set of sequences. “VVK VarInvar” calculates percentage of variable sites in a set of aligned sequences, as well as nucleotide usage in invariable sites. We tested our method on HIV1 gp120 protein and on diphtheria toxin. The less mutable epitopes have been found for both proteins. Finally, we showed that antibodies recognizing the less mutable epitope of gp120 can be found in 80,22% of HIV1-infected persons

Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s

Abstract Background Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 – 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains. Results C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10−7 substitutions per strain per year) than ST5 (5.6 × 10−7 substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains. Conclusions Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones

Genotypic and Phenotypic Characteristics of Corynebacterium diphtheriae Strains Isolated from Patients in Belarus during an Epidemic Period

One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with BstEII and PvuII revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C. diphtheriae. This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production

Additional file 1: Table S1. of Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s

Details of C. diphtheriae strains analysed in this study. (XLSX 21 kb

Additional file 3: Figure S1. of Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s

A ML tree from core genomic alignment of ST5 strains; Figure S2. Gubbins analysis of recombination in the core genome of C. diphtheriae. Predicted recombination events on internal branches are shown in red and those occurred at terminal branches are shown in blue; Figure S3. A plot of root-to-tip divergence (Y-axis) against the sampling dates (X-axis); A. within ST5 and B. within ST8; Figure S4. A. CDS BLAST map of ST5 using strain ISS 4060 as the reference. B. CDS BLAST map of ST8 using strain NCTC 13129 as the reference; Figure S5. A ML tree from the binary data of the presence or absence of genes in the accessory genome. The scale bar with a distance of 0.1 represents the difference of 341.7 genes; Figure S6. A plot showing the average global vaccine coverage, reported vaccination in Belarus and the reported number of diphtheria cases between 1992 and 2015. (PDF 5948 kb

Additional file 2: Table S2. of Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s

Number and toxigenicity of isolates collected between 1996 and 2014 in Belarus and those selected for genomic analyses in this study; Table S3. Distribution of isolates from epidemic (≤ year 2000) and postepidemic period (≥ year 2001) in major groups. All minor groups are pooled together for statistical analysis; Table S4. Distribution of isolates from asymptomatic carriage, diphtheria and sore throat patients in major groups. All minor groups are pooled together for statistical analysis; Table S5. Allelic variation in the direct repeat and spacer sequences among the CRISPR loci of ST8 and ST5 isolates. (PDF 396 kb