Genetic Characterization of Streptococcus pyogenes emm 89 Strains Isolated in Japan From 2011 to 2019

Abstract

Invasive infection caused by Streptococcus pyogenes emm89 strains has been increasing in several countries linked to a recently emergent clade of emm89 strains, designated clade 3. In Japan, the features of emm89 S. pyogenes strains, such as clade classification, remains unknown. In this study, we collected emm89 strains isolated from both streptococcal toxic shock syndrome (STSS) (89 STSS isolates) and noninvasive infections (72 non-STSS isolates) in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, invasive disease-associated factors were found among emm89 strains, including mutations of control of virulence sensor, and absence of the hylP1 gene encoding hyaluronidase. These findings provide new insights into genetic features of emm89 strains.

Keywords: Streptococcus pyogenes; clade 3; emm89; streptococcal toxic shock syndrome.

Figure 1

Classification criteria for clade, and genetic relationships among various S. pyogenes strains. A. Schematic diagram showing nga promoter region sequences for each clade. B. Schematic alignment of hasABC operon for clade 1, 2, and 3. hasABC was not found in clade 3. C. Genetic relationships between emm89 strains in Japan and various emm/M protein serotype strains. Genetic relationships were inferred among 49 GAS strains of 23 emm/M types based on 73,030 concatenated core chromosomal SNPs using the maximum likelihood method. Three closed genomes (clade 1, MGAS11027; clade 2, MGAS23530; clade 3, MGAS27061) were used as references. S. pyogenes emm89 strain clade 1, 2, and 3 are shaded in blue, green, and pink, respectively. Various emm/M protein serotypes except emm89 strains are shaded in purple. 20xx indicates year of isolation. Scale bar indicates nucleotide substitutions per site. FS: regions containing Sapporo City, Iwate Prefecture, Fukushima Prefecture, Sendai City, and Niigata City; TY; Toyama Prefecture; TK: Tokyo Prefecture; YH: Yokohama City; OS: regions containing Shiga Prefecture, Kyoto City, Osaka Prefecture, and Hyogo Prefecture; YG; Yamaguchi Prefecture; SS; isolates from STSS patients; NS: pharyngeal or asymptomatic isolates.

Figure 2

Genetic relationships among emm89 strains in Japan and their clades. Genetic relationships between emm89 strains in Japan and emm89 reference strains (clade 1, MGAS11027; clade 2, MGAS23530; clade 3, MGAS27061) are shown. Genetic relationships were inferred among 164 strains based on 8866 concatenated core chromosomal SNPs using the maximum likelihood method. Strains are colored based on clade, as indicated in the index. Scale bar indicates nucleotide substitutions per site.

Figure 3

Hyaluronidase activity of emm89 S. pyogenes. A. Hyaluronidase HylA active or inactive sequence. Serotype M4 strain was shown to harbor HylA activity and serotype M1 strain possession of Asp to Val substitution at position 199 of the HylA sequence (D199V), which abolishes hyaluronidase activity. B. Hyaluronidase activity on hyaluronan agar plate. Dotted circles indicate colony locations before incubating with 2 N acetic acid solution. Although 161 emm89 strains in this study harbored D199V in the HylA sequence, 117 of the strains were indicated to have hyaluronidase activity.