Overlapping Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis household transmission and mobile genetic element exchange

Abstract

Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

Fig. 1. Maximum-likelihood phylogeny of 294 Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolates from 47,432 parsimony-informative sites.

Sequences were aligned against SDSE reference genome GGS_124 (NC_012891.1) with mobile genetic element regions masked. Distinct genomic sequence clusters determined by PopPUNK as previously defined by a global SDSE dataset, are denoted by alternating blue and grey highlights from internal nodes. Site of isolation is coloured by blue (throat) and red (skin) tips. The inner ring denotes the Lancefield group carbohydrate and the outer ring the community of isolation. Bootstrap supports are shown as branch colour gradients and were calculated using the ultrafast bootstrap approximation demonstrating some uncertainty in deep branches of the phylogeny. Scale bar represents substitutions per site.

Fig. 2. Alluvial plot of the relationship between the largest 7/18 Streptococcus dysgalactiae subspecies equisimilis (SDSE) genomic sequence clusters (representing 237/294 isolates) as determined by PopPUNK against emm type, emm subtype, multilocus sequence type (MLST) and transmission clusters determined using single linkage clustering at a SNP threshold of 99% shared gene content.

The number of isolates in each category is denoted within brackets. From 10 emm types (14 emm subtypes) and 9 MLSTs shown, 60 transmission clusters were determined. Traditional markers such as emm subtype in some cases over-split SDSE clusters as demonstrated by stC839.0 and stC839.2 which differ by only one SNP within their hypervariable emm region and otherwise fall within the same transmission cluster.

Fig. 3. Transmission links between households across consecutive community visits.

Transmission links for Community 1 (a) and Community 3 (b). Households are represented by nodes proportional in size to the number of participants enrolled at each household and coloured by the proportion of Streptococcus pyogenes (red) and Streptococcus dysgalactiae subsp. equisimilis (SDSE, blue) isolates detected in the household across the entire study period. Transmission links are represented by undirected and unweighted edges between households and coloured by species with shared edges highlighted in green. Loops correspond to predicted transmission edges between unique individuals within the same household. Only community visits where transmission edges were predicted are shown. Source data are provided as a Source Data file.

Fig. 4. Co-occurrence of Streptococcus dysgalactiae subsp equisimilis (SDSE) and Streptococcus pyogenes in households in communities 1 and 3 at each community visit (light blue highlights) during the study period.

Detection of SDSE (blue) and S. pyogenes (red) are denoted by points with the size of each point proportional to the number of isolates. Community visits where a household was not sampled are denoted by crosses. SDSE and S. pyogenes co-occurred on 100/486 (21%) of household-visits. Source data are provided as a Source Data file.

Fig. 5. Shared mobile genetic elements (MGE) across Streptococcus dysgalactiae subsp. equisimilis (SDSE) and Streptococcus pyogenes isolates.

a Maximum likelihood trees of SDSE and S. pyogenes with isolates carrying three near-identical (>99% nucleotide identity) MGEs highlighted by tree tip points. Genomic sequence clusters without the three MGEs of interest are collapsed and denoted by blue (SDSE) and red (S. pyogenes) triangles at tree tips. Flows link corresponding shared MGEs across the species but do not imply directionality of transfer. The emm sequence type of genome sequence clusters sharing MGEs are labelled for each species, respectively. Bootstrap supports are shown as branch colour gradients and were calculated using ultrafast bootstrap approximation. Scale bars represent substitutions per site. b A 54 kbp prophage, ϕ1207.3, carrying macrolide efflux resistance genes mef(A) and msr(D) was present with >99.9% nucleotide identity across SDSE and S. pyogenes. A representative SDSE element from isolate NS4595 was aligned against a representative S. pyogenes sequence (NS3871) with percentage nucleotide identity calculated using Hamming distance and plotted in 100 bp sliding windows. The element was present in a cross-species conserved insertion region with flanking core genes highlighted in green. c An 18 kbp integrative conjugative element (ICE)-like MGE carrying the tetracycline resistance gene, tet(M), was present with >99.9% nucleotide identity across species. The element was present at three different genomic insertion regions and thus flanking core genes are not shown. In the example shown, a 12 bp in-frame deletion was present at the 5’ end of tet(M) in the SDSE element which was distant from the active ribosomal binding domain. d A 41 kbp prophage ϕMGAS5005.3 carrying the streptodornase gene sda1, was shared across species with >99.9% nucleotide identity at a cross-species conserved insertion region as has been described previously. Source data are provided as a Source Data file.