Genomic characterization of cocirculating Corynebacterium diphtheriae and non-diphtheritic Corynebacterium species among forcibly displaced Myanmar nationals, 2017-2019

Abstract

Respiratory diphtheria is a serious infection caused by toxigenic Corynebacterium diphtheriae, and disease transmission mainly occurs through respiratory droplets. Between 2017 and 2019, a large diphtheria outbreak among forcibly displaced Myanmar nationals densely settled in Bangladesh was investigated. Here we utilized whole-genome sequencing (WGS) to characterize recovered isolates of C. diphtheriae and two co-circulating non-diphtheritic Corynebacterium (NDC) species - C. pseudodiphtheriticum and C. propinquum. C. diphtheriae isolates recovered from all 53 positive cases in this study were identified as toxigenic biovar mitis, exhibiting intermediate resistance to penicillin, and formed four phylogenetic clusters circulating among multiple refugee camps. Additional sequenced isolates collected from two patients showed co-colonization with non-toxigenic C. diphtheriae biovar gravis, one of which exhibited decreased susceptibility to the first-line antibiotics and harboured a novel 23-kb multidrug resistance plasmid. Results of phylogenetic reconstruction and virulence-related gene contents of the recovered NDC isolates indicated they were likely commensal organisms, though 80.4 %(45/56) were not susceptible to erythromycin, and most showed high minimum inhibition concentrations against azithromycin. These results demonstrate the high resolution with which WGS can aid molecular investigation of diphtheria outbreaks, through the quantification of bacterial genetic relatedness, as well as the detection of virulence factors and antibiotic resistance markers among case isolates.

Keywords: Corynebacterium; diphtheria; genomic epidemiology; outbreak; whole-genome sequencing.

Fig. 1.

Phylogenetic reconstruction of Bangladesh C. diphtheriae isolates from 38 024 core variable sites using maximum likelihood. Four detected clusters are shaded in different colours. The corresponding microbiological characteristics of each isolate are indicated next to the tree. Filled circles represent biovar (mitis, yellow), toxigenicity (toxigenic, purple), susceptibility to penicillin (intermediate resistance, blue) or macrolides (resistance, red). Scale bar indicates substitutions per site.

Fig. 2.

Geographical and temporal origins of C. diphtheriae isolates from the Bangladesh outbreak. (a) Geographical locations (camp) of the C. diphtheriae case clusters identified during the outbreak from 9 December 2017 to 7 April 2018. (b) Weekly identified C. diphtheriae clusters in Bangladesh during the same investigation period. All the collection dates have been normalized into week numbers. The 7 day intensive study period was between 19–25 December 2017 (weeks 3 and 4).

Fig. 3.

Map of novel plasmid pSRPD from the C. diphtheriae isolate PC0697. G+C % content is the inner black tilde; GC skew is the green/purple tildes; blue ring represents comparative plasmid analysis against reported plasmid (pLRPD). The outermost ring represents predicted CDSs, which are portrayed by arrows with important ones labelled with either gene name or predicted gene function. The CDSs in red represents the pbp-containing transposable unit (PCU).

Fig. 4.

Phylogeny reconstructions for NDC isolates from 31 946 core variable sites using maximum likelihood. Isolates belonging to C. pseudodiphtheriticum were highlighted in blue while C. propinquum in purple. Grey circles represent collapsed clades of Bangladesh NDC isolates from the two species. Scale bar indicates substitutions per site.