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.

Keywords: Corynebacterium diphtheriae; Diphtheria; Endemic; Epidemic; Non-toxigenic; Sore throat; Toxigenic; Vaccine; Virulence.

Fig. 1

A maximum-likelihood tree derived from concatenated nucleotide sequenced alignment of the core genome. The scale bar represents nucleotide substitutions per nucleotide site. The strain designations of isolates of biovar belfanti, gravis, intermedius and mitis are presented in brown, red, blue and green colour, respectively. The presence of virulence genes is mapped on the tree in black whereas a white box shows the absence of genes

Fig. 2

A distribution of genotypes (STs) and disease states (asymptomatic, diphtheria and sore throat) among C. diphtheriae isolates in difference provinces of Belarus

Fig. 3

A ML tree from the nucleotide sequence alignment of the tox gene. The scale bar represents the number of nucleotide substitutions per site. SNPs separating the clones from the vaccine strain PW8 are mapped on the branches