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

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.

Opposite nucleotide usage biases in different parts of the Corynebacterium diphtheriae spaC gene.

In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.

Structural and antigenic features of the synthetic SF23 peptide corresponding to the receptor binding fragment of diphtheria toxin.

The SF23 peptide corresponding to the receptor binding fragment of diphtheria toxin (residues 508-530) has been synthesized. This fragment forming a protruding beta hairpin has been chosen because it is the less mutable B-cell epitope. Affine chromatography and ELISA show that antibodies from the sera of persons infected by toxigenic Corynebacterium diphtheriae and those immunized by diphtheria toxoid are able to bind the synthetic SF23 peptide. There are antibodies recognizing the SF23 peptide in the serum of horses hyperimmunized with diphtheria toxoid. Analysis of circular dichroism spectra show formation of beta hairpin by the peptide. Taken together, the results showed that the structure of the less mutable epitope of C. diphtheriae toxin was reproduced by the short SF23 peptide. Since antibodies against that epitope should block its interactions with cellular receptor (heparin-binding epidermal growth factor), the SF23 peptide can be considered as a promising candidate for synthetic vaccine development. Fluorescence quenching studies showed the existence of chloride and phosphate binding sites on the SF23 molecule. Phosphate containing adjuvants (aluminum hydroxyphosphate or aluminum hydroxyphosphate sulfate) are recommended to increase the SF23 immunogenic properties.

Multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis.

BACKGROUND AND OBJECTIVE: Rapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening. MATERIALS AND METHODS: Targets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella parapertussis in clinical samples. A total of 121 human clinical specimens obtained within 2012-2013 were used to evaluate the multiplex real-time PCR assay. Clinical specimens were also tested for culture and conventional PCR. Sensitivity and specificity for culture, conventional PCR, and multiplex real-time PCR were measured in comparison with a clinical standard for B. pertussis infection. RESULTS: The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for IS481, IS1001 and 10 genomic equivalents per reaction for BP0026 target. When the B. pertussis assays were compared with a clinical standard for B. pertussis infection, sensitivity was 5, 59 and 89% the specificity was 100, 100 and 100% for culture, conventional PCR, and multiplex real-time PCR, respectively. CONCLUSIONS: Developed multiplex real-time PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections which is suitable for implementation in a routine laboratory diagnostics.

Identification of Corynebacterium diphtheriae gene involved in adherence to epithelial cells.

Corynebacterium diphtheriae the causative pathogen of human diphtheria infects the nasopharynx or skin. Although diphtheria has been extensively studied, little is known about the two key aspects of C. diphtheriae invasiveness: colonization and invasion. The role of adhesive properties in establishing the infection of C. diphtheriae strains, independent of toxin production, still needs to be clarified. In this study, we describe a novel gene involved in adherence to epithelial cells. Transformation of C. diphtheriae 225, biotype gravis, ribotype St-Petersburg by EZ:TN(KAN-2)Tnp Transposome was undertaken. A C. diphtheriae 225 Tn5 insertion library of 2800 mutants was created. Five hundred and eighty five transformants were qualitatively screened for reduced adherence to HEp-2 cells by an adherence assay. One mutant strain consistently exhibiting 15.2% of the wild-type adherence was isolated. The DNA flanking the transposon was identified by inverse PCR and subsequent sequencing. The disrupted gene was 94% identical to the C. diphtheriae DIP1621 gene that belongs to unclassified genes. In conclusion, the disruption of the C. diphtheriae DIP1621 gene led to decreased adherence to epithelial cells; its exact function remains to be established.

Molecular epidemiology of C. diphtheriae strains during different phases of the diphtheria epidemic in Belarus.

BACKGROUND: The reemergence of epidemic diphtheria in Belarus in 1990s has provided us with important information on the biology of the disease and the diversity of the causative agent Corynebacterium diphtheriae. Molecular investigations were conducted with the aim to analyze the genetic variability of C diphtheriae during the post-epidemic period. METHODS: The biotype and toxigenicity status of 3513 C. diphtheriae strains isolated from all areas in Belarus during a declining period of diphtheria morbidity (1996-2005) was undertaken. Of these, 384 strains were isolated from diphtheria cases, 1968 from tonsillitis patients, 426 from contacts and 735 from healthy carriers. Four hundred and thirty two selected strains were ribotyped. RESULTS: The C diphtheriae gravis biotype, which was prevalent during 1996-2000, was "replaced" by the mitis biotype during 2001-2005. The distribution of toxigenic C. diphtheriae strains also decreased from 47.1% (1996) to 5.8% (2005). Changes in the distribution of the epidemic ribotypes Sankt-Peterburg and Rossija were also observed. During 2001-2005 the proportion of the Sankt-Peterburg ribotype decreased from 24.3% to 2.3%, in contrast to the Rossija ribotype, that increased from 25.1% to 49.1%. The circulation of other toxigenic ribotypes (Otchakov, Lyon, Bangladesh), which were prevalent during the period of high diphtheria incidence, also decreased. But at the same time, the proportion of non-toxigenic strains with the Cluj and Rossija ribotypes dramatically increased and accounted for 49.3% and 30.1%, respectively. CONCLUSION: The decrease in morbidity correlated with the dramatic decrease in the isolation of the gravis biotype and Sankt Peterburg ribotype, and the prevalence of the Rossija ribotype along with other rare ribotypes associated with non-toxigenic strains (Cluj and Rossija, in particular).

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.