Wound secretion specimens were submitted to the clinical laboratory of Zhejiang local hospital for slide microscopy and bacterial culture using 5% sheep blood agar plates. A GAS strain was obtained and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonics, Bremen, Germany) (14). Antimicrobial susceptibility testing (AST) of levofloxacin, chloramphenicol, clindamycin, ceftriaxone, erythromycin, penicillin, tetracycline, and vancomycin against the identified GAS strain, named SHZ-1, was performed using a disk diffusion method, and the results were interpreted according to the Clinical and Laboratory Standards Institute (15).

Genomic DNA of strain SHZ-1 (single colony) was prepared using a DNA mini kit (Qiagen, Valencia, CA, United States) and submitted to next-generation sequencing (NGS) and long-read sequencing using the Illumina HiSeq2000TM (Illumina Inc., San Diego, CA, United States) and Oxford Nanopore MinION platforms (Oxford Nanopore Technologies, Oxford, United Kingdom), respectively. The Illumina sequencing generated 9.1M reads with an average of ∼150 bp per read and 200 X coverage. The nanopore sequencing of this strain generated 139 contigs with an N50 of 1329619 bp and a GC content of 39.22%. Both short and long reads were used for de novo assembly via Unicycler version 1.0 (15) in a hybrid assembly model with default parameters. The genome was then used as input for in silico multilocus sequence typing (MLST) via BLAST against the PubMLST database (16), emm typing against the CDC emm type and subtype database tsemm (17). The annotation of SHZ-1 was performed via Prokka against S. pyogenes database (18). The annotated genome was then used for virulence factor detection against the VFDB database (19). Both SNP and cgMLST strategies were performed in the BacWGSTdb server (20) for bacterial source tracking. An ad hoc cgMLST scheme for S. pyogenes (1,170 target genes) was designed to characterize the gene-by-gene allelic profile of S. pyogenes strains. The identified S. pyogenes strain SHZ-1 was used as the reference genome in this analysis. SNP distance matrices were calculated using snp-dists 0.6.3 (21).

A 37-year-old man injured his left foot at work 4 days prior to admission. He immediately felt pain, but it was tolerable and he had no open wounds; hence, he ignored the symptoms. The injured foot developed skin redness, swelling, and obvious tenderness 1 day later. The patient developed a conscious fever accompanied by chills, dizziness, abdominal pain, and distension. The symptoms worsened after another 2 days, and the patient was then admitted to the local hospital, Zhejiang, China. His lab results showed leukocyte counts of 22.3 × 10⁹/L with 92.3% neutrophils, and an X-ray showed no fracture. Based on this, the diagnoses of severe skin and soft tissue infection (SSTI) was made. Three days of sulbenicillin (4 g/q12 h) and levofloxacin (0.5 g bid) treatment showed no improvement. After debridement, postoperative anti-infection treatment with vancomycin (1.0/q8 h) was applied. Leukocyte counts were still high with elevated levels of procalcitonin, and the patient was diagnosed with septicemia. X-ray confirmed no migrating lung lesions. On the fourth day of admission, a specimen culture was confirmed to be positive for GAS. AST showed that the bacterial isolate was susceptible to all tested drugs (Supplementary Table 2). Antimicrobial therapy was then applied with linezolid (0.6 g q12 h) and amoxicillin/clavulanate potassium (1.2 g q6 h). Declines in all inflammatory indices were then observed. Thereafter, the patient improved from GAS infection after 11 days of treatment and was transferred to debridement, suture, and skin grafting. The patient was treated with amoxicillin/clavulanate potassium (1.2 g q8 h) for another 6 days since Day 37 due to GAS culture positivity. The patient fully recovered and was discharged from the hospital on Day 43 (Figure 1).

Hypervirulent invasive GAS infections are rare in China. To better understand the pathogen in the current case, the strain was sent for whole-genome sequencing. The complete chromosome data of SHZ-1 were submitted to NCBI (Genbank No.CP072523.1). After the hybrid assembly, we obtained a 1819973 bp complete circular genome of GAS strain SHZ-1 with a GC content of 38.6%. We identified SHZ-1 as an emm3/ST15-type GAS strain via emm typing and MLST analysis (Supplementary Figure). Then we downloaded the complete genome of emm3/ST15 GAS strains that were isolated from different regions. Using BacWGSTdb to determine the clonal relationship among these isolates according to the pairwise comparison of the cgMLST alleles or SNP differences. In the end, the phylogenetic relatedness showed that the closest strain to SHZ-1 was MGAS315 from the United States, which has 53 core gene differences (Figure 2A). The SNP calling results indicated that the smallest SNP number of 93 was determined between strains SHZ-1 and M3-b from Japan (Figure 2B). Virulence factor detection against VFDB showed that SHZ-1 had all the virulence factors found in the other two emm3/ST15 strains (MGA315 and M3-b) except the streptococcal superantigen SSA-encoding gene ssa (Supplementary Table 2).