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Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance


A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 16). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage ΦHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, ΦHKU.vir and a new prophage, ΦHKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever–associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.

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Figure 1: Phylogenetic analysis of emm12 GAS genotypes from Hong Kong isolates.

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We thank the core sequencing and pathogen informatics teams at the Sanger Institute for their assistance. This work was supported by National Health and Medical Research Council of Australia (NHMRC) program grant 565526, Australian Research Council grant DP140102881, a Research Fund for the Control of Infectious Diseases Commissioned Grant from the Hong Kong government and Wellcome Trust grant 098051. M.R.D. is supported by an NHMRC postdoctoral training fellowship (635250). M.J.W. is supported by an NHMRC principal research fellowship (631386).

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Authors and Affiliations



M.R.D., K.-Y.Y., G.D. and M.J.W. were involved in the study design. K.-Y.Y., H.T. and J.H.K.C. provided the assessment of clinical cases and the bacterial samples. M.R.D., M.T.H., P.C., J.H.K.C., C.V., T.C.B. and N.L.B.Z. performed the analyses. M.R.D., P.C. and M.J.W. wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Mark J Walker.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Annual incidence of scarlet fever.

Reported cases of scarlet fever in Hong Kong (a) and mainland China (b) as reported by the Centre for Health Protection, Department of Health, Government of the Hong Kong Special Administrative Region and by the Ministry of Health, the National Health and Family Planning Commission of the People’s Republic of China.

Supplementary Figure 2 Identification of recombined genomic regions on the basis of GAS emm12 whole-genome alignment.

The maximum-likelihood phylogeny excluding recombined regions is represented on the left of the figure, and the four major clades are indicated. The HKU16 reference genome is represented above the position of recombined ‘blocks’ for each taxon in the tree. Recombination events common to multiple related taxa (through common descent) are indicated by a red block or by a blue block, if unique to a single terminal taxon. Recombination is largely limited to the locations of mobile genetic elements, implying high stability within the core emm12 genome. SNPs located within the boundaries of the mobile elements were excluded.

Supplementary Figure 3 Bayesian phylogenetic tree of emm12 GAS strains.

Supplementary Figure 4 Immunoblot detection of the SpeC and SSA gene products from the culture supernatants of representative GAS strains.

Supplementary Figure 5 Characterization and temporal distribution of the novel ssa-carrying prophage elements ΦHKU.ssa, ΦHKU165.4 and ΦHKU.vir within the emm12 population.

Coding sequences referring to the prophage-associated virulence genes speC (purple), spd1 (yellow), spd3 (dark brown) and ssa (pink) are indicated. (a) Representation of ΦHKU.ssa (light brown; 42.9 kb) from strain HKU360 and ΦHKU165.4 (black; 46.0 kb) from strain HKU165. The scale bar is shown in kilobase pairs. (b) BLASTN comparison of ΦHKU.ssa with the prophage sequences ΦMGAS9429.1 and ΦMGAS10750.3. ΦHKU.ssa is a chimeric element of both of these elements and is located at the ΦMGAS10750.3 att site. Red blocks indicate homologous sections of genome sequence as defined by BLASTN. (c) BLASTN comparison of ΦHKU165.4 with the prophage sequences ΦHKU.vir and ΦNZ131. All three prophage elements share the same attachment site but carry different virulence determinants. Red blocks indicate homologous sections of genome sequence as defined by BLASTN. (d) Bayesian phylogeny of the emm12 population with the distribution of ΦHKU.ssa (light brown), ΦHKU165.4 (black) and ΦHKU.vir (blue) indicated on the right. Color-coded branches indicate clones possessing ΦHKU.ssa, ΦHKU165.4 and ΦHKU.vir, and arrows indicate predicted insertion dates within the emm12 population.

Supplementary Figure 6 Comparative analysis and temporal distribution of speC- and spd1-harboring phage variants within the GAS emm12 population.

ΦSF370.1 (black) from the M1 GAS strain SF370 is a closely related variant of ΦNS488.1 and is represented at the top of the figure. Another five major variants are present within the emm12 population. The yellow and purple coding sequences refer to the deoxyribonuclease spd1 and the streptococcal pyrogenic exotoxin speC, respectively. Red blocks indicate homologous sections of genome sequence as defined by BLASTN. The different prophage variants are color coded, and their distribution relative to the emm12 Bayesian phylogeny is indicated to the left of the figure. An asterisk indicates that ΦHKU43.1 and ΦHKU81.4 are partial genome sequences, with the location of the assembly break indicated by a gray box. With the exception of ΦHKU81.4, which is located at the ΦHKU.vir attachment site, all represented prophages are located at the same genomic location as ΦSF370.1 (ΦMGAS9429.1).

Supplementary Figure 7 Comparative analysis and temporal distribution of antibiotic resistance integrative and conjugative elements (ICEs) within the GAS emm12 population.

(a) Comparative genomics of eight ICE variants identified within the GAS emm12 population. Tetracycline (tetM and tetO) and macrolodide (ermB and ermTR) resistance coding sequences are indicated in blue, yellow, purple and brown, respectively. Red blocks indicate homologous sections of genome sequence as defined by BLASTN. Each variant is color coded, and distributions relative to the emm12 Bayesian phylogeny are indicated in b. Presence (black box) and absence (white block) of tetM, tetO, ermB and ermTR are indicated. Color-coded branches indicate clones possessing ICE variants, and arrows indicate predicted insertion times within the emm12 population.

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Supplementary Figures 1–7. (PDF 1780 kb)

Supplementary Table 1

Details of S. pyogenes M12 strains used in this study and associated distribution of toxin and antibiotic resistance genes. (XLSX 65 kb)

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Davies, M., Holden, M., Coupland, P. et al. Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance. Nat Genet 47, 84–87 (2015).

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