Genomic Characterization of Skin and Soft Tissue Streptococcus pyogenes Isolates from a Low-Income and a High-Income Setting

Abstract

Streptococcus pyogenes is a leading cause of human morbidity and mortality, especially in resource-limited settings. The development of a vaccine against S. pyogenes is a global health priority to reduce the burden of postinfection rheumatic heart disease. To support this, molecular characterization of circulating S. pyogenes isolates is needed. We performed whole-genome analyses of S. pyogenes isolates from skin and soft tissue infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where there is a high burden of such infections. To act as a comparator to these LIC isolates, skin infection isolates from Sheffield, United Kingdom (a high-income country [HIC]), were also sequenced. The LIC isolates from The Gambia were genetically more diverse (46 emm types in 107 isolates) than the HIC isolates from Sheffield (23 emm types in 142 isolates), with only 7 overlapping emm types. Other molecular markers were shared, including a high prevalence of the skin infection-associated emm pattern D and the variable fibronectin-collagen-T antigen (FCT) types FCT-3 and FCT-4. Fewer of the Gambian LIC isolates carried prophage-associated superantigens (64%) and DNases (26%) than did the Sheffield HIC isolates (99% and 95%, respectively). We also identified streptococcin genes unique to 36% of the Gambian LIC isolates and a higher prevalence (48%) of glucuronic acid utilization pathway genes in the Gambian LIC isolates than in the Sheffield HIC isolates (26%). Comparison to a wider collection of HIC and LIC isolate genomes supported our findings of differing emm diversity and prevalence of bacterial factors. Our study provides insight into the genetics of LIC isolates and how they compare to HIC isolates. IMPORTANCE The global burden of rheumatic heart disease (RHD) has triggered a World Health Organization response to drive forward development of a vaccine against the causative human pathogen Streptococcus pyogenes. This burden stems primarily from low- and middle-income settings where there are high levels of S. pyogenes skin and soft tissue infections, which can lead to RHD. Our study provides much needed whole-genome-based molecular characterization of isolates causing skin infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where infection and RHD rates are high. Although we identified a greater level of diversity in these LIC isolates than in isolates from Sheffield, United Kingdom (a high-income country), there were some shared features. There were also some features that differed by geographical region, warranting further investigation into their contribution to infection. Our study has also contributed data essential for the development of a vaccine that would target geographically relevant strains.

Keywords: FCT region; bacterial genomics; emm types; group A Streptococcus; skin infection; vaccine.

FIG 1

Distribution of emm type, pattern, and cluster differs by site. (A) Frequency of emm types found in the wider LIC genome collection and Gambian isolate genomes combined (LIC; orange) compared to the that found in the wider HIC genome collection and Sheffield isolate genomes combined (HIC; black) (B) An emm pattern of A-C, D, or E was assigned to 98/107 Gambian LIC isolates (patterns for the remaining 9 were undetermined) and all 142 Sheffield HIC isolates. The same emm patterns were also assigned to 766/781 (15 were undetermined) LIC isolates from Gambia, Kenya, and Fiji (All LIC) and all 4,820 HIC isolates from Sheffield, BSAC (England/Wales), PHE (United Kingdom), and ABCs (United States) (All HIC). (C) An emm cluster was also assigned to 98/107 Gambian LIC isolates (the remaining 9 were excluded) and all 142 Sheffield HIC isolates, as well as to all LIC and all HIC isolates. Pie charts represent the percentage of isolates associated with each pattern/cluster.

FIG 2

Phylogenetic analysis of the 107 Gambian LIC isolate genomes. A maximum likelihood phylogeny was constructed from the core gene alignment (1,382,412 bp) using RAxML (51) with 100 bootstraps. Isolates clustered by emm type except for those indicated, whereby two lineages were represented by a single emm genotype: star, emm25; filled square, emm65; open square, emm85; open circle, emm89; filled circle, emm209. Also shown is the presence (black)/absence (white) of the superantigen genes speA, speC, speG, speH to speM, speQ, speR, ssa, and smeZ and DNase genes spd1 and spd3; four other DNase genes (sda1, sda2, sdn, and spd4) were tested for but were not found in any isolate. In all cases except one (red dot), speA was located within the prophage-like element Φ10394.2. One isolate had a gene that appeared to be a fusion of 5′ speK and 3′ speM (red triangles). Antimicrobial resistance genes (AMR) tetM, tetL, dfrG, and dfrK were also identified in some isolates (white, absent; black, present). The positivity for serum opacity factor (sof) is also shown, although for one emm55 isolate, this gene would produce a truncated variant of serum opacity factor (gray). The scale bar represents substitutions per site. emm types are colored for easy visualization, and type numbers are also given.

FIG 3

Phylogenetic analysis of the 142 Sheffield HIC isolate genomes. A maximum likelihood phylogenetic tree was generated with the core gene alignment (1,185,813 bp) by using RAxML (51) with 100 bootstraps. All isolates clustered by emm type. The presence (black)/absence (white) of superantigen genes speA, speC, speG, speH to speM, speQ, speR, ssa, and smeZ and DNase genes sda1, sda2, sdn, spd1, spd3, and spd4 is indicated. Antimicrobial resistance genes (AMR) tetM, ermA, and ermB were also identified in some isolates (white, absent; black, present). The positivity for serum opacity factor (sof) is also shown, but in all emm12 types, this gene would produce a truncated variation of serum opacity factor (gray). The scale bar represents substitutions per site. emm types are colored for easy visualization, and type numbers are also given.

FIG 4

Comparison of superantigen and DNase gene carriage in LIC isolates and HIC isolates. The proportions of the Gambian LIC isolates (gray bars) and the Sheffield HIC isolates (white bars) as well as the wider LIC isolates (Kenya, Fiji, Gambia; n = 781) (orange bars) and the wider HIC isolates (BSAC, PHE, ABCs, Sheffield; n = 4,820) (black bars) carrying the respective genes were determined by BLAST analysis and mapping.

FIG 5

Arrangement of genes in the Mga regulon of Gambian LIC and Sheffield HIC isolates. The genes within the mga regulon for each isolate were determined, and Mga regulon type I to VI was assigned. The majority of emm types in both the Sheffield HIC isolates and the Gambian LIC isolates were type I, with the M-like protein genes mrp and enn flanking the M protein gene emm. The previously assigned emm pattern A-C, D, or E (based on the emm type) is also given. The streptococcal inhibitor of complement (sic) gene was identified only in Sheffield HIC emm1 isolates, and the distantly related to sic (drs) gene was found only in Sheffield HIC emm12 isolates. The gene pgs encodes Pgs, a 15.5-kDa protein of unknown function (27).

FIG 6

FCT arrangement patterns identified in Gambian LIC and Sheffield HIC isolates. FCT regions were extracted from de novo assemblies, and the FCT type was assigned based on the predicted function and order of genes within the extracted region. The emm types of isolates with each FCT type are shown for Sheffield HIC and Gambian LIC isolates. A new FCT region was identified (FCT10) as similar to that of FCT5 but with an additional gene encoding for a fibronectin binding protein after the sortase genes. For all emm types, there was at least one isolate with a designated FCT type in a single contiguous region. The only exception to this was emm118 (*), where the FCT was estimated to be FCT4 and the new FCT10 for each of the two isolates as the FCT region was split over two contigs. In FCT1, transposases were found in Sheffield HIC emm6 and emm108, and in FCT9, transposases were found in Sheffield HIC emm75 and Gambian LIC emm4. fbp, fibronectin binding protein; cwp, cell wall protein; ap, ancillary protein; trp, transposase.