Phenotypic and genomic analysis of the hypervirulent ST22 methicillin-resistant Staphylococcus aureus in China

ABSTRACT ST22 MRSA (methicillin-resistant Staphylococcus aureus) strains are only sporadically reported in China. Through the phylogenetic reconstruction of 30 ST22 strains from China and 480 ST22 strains from global sources, we found that the global ST22 strains can be divided into three clades (I, II, and III). The China ST22 strains were found primarily in clade II (IIb and IIc) and also in clade III, indicating that the China ST22-MRSA clones have different origins. The China subclade IIb strains (SCCmec Vb-t309) may evolve from the native ST22 MSSA clone, while the China IIc strains may have spread from other countries. Subclade IIc (SCCmecIVa-t309) strains exhibited particularly strong lethality and invasiveness in Galleria mellonella infection and mouse skin abscess models in comparison to USA300 and other dominant China HA-MRSA (ST5 and ST239) or CA-MRSA (ST59) strains. This study described the emergence of a highly virulent ST22 MRSA subclade and improved our insight into the molecular epidemiology of ST22 strains in China. IMPORTANCE ST22 is a successful hospital-associated MRSA lineage which first appeared in the United Kingdom as EMRSA-15. At present, ST22 MRSA clones are spreading rapidly around the world and even replaced other dominant clones in some regions. We placed the Chinese ST22 in the worldwide phylogeny of ST22, demonstrating a distinctive molecular epidemiology and to our knowledge, this is the first time that a novel clade of ST22 has been found in China. Among the 15 ST22 MRSA strains belonging to the novel clade, 14 ST22 SCCmecIVa strains from different regions carried both pvl and tst and displayed significantly higher in vitro and in vivo virulence in comparison to other clade/subclade ST22 strains as well as other common China HA-MRSA or CA-MRSA strains. The further spread of this subclade of strains could pose a serious threat to the health system in China and other regions.


Phylogenetic analysis and Bayesian evolutionary analysis
In order to investigate the evolutionary dynamics of ST22 MRSA isolates in China, genomes of additional 480 ST22 strains were downloaded from NCBI RefSeq or SRA database for comparison. A previous described method was used to infer time scaled phylogeny of ST22 strains(4). In brief, Snippy v4.6.0 was used to identify core SNPs for the ST22 genome and the BactDating R package was used to estimate node dates of ST22 strains. The recombination-corrected tree from Gubbins output and the isolation dates were used as the inputs in BactDating v1.1.

Mouse skin abscess model
BALB/c female mice aged four to six weeks old were selected for the mouse skin abscess model. S. aureus strains were grown for nine hours (the post-exponential phase) in fresh TSB at 37°C with shaking (220rpm), and washed twice in sterile phosphate-buffered saline (PBS) solution. Then, the mice were subcutaneously inoculated with 100μL PBS containing 1×10 8 CFU live S. aureus in the back skin, or injected with 100μL sterile PBS solution as the negative control (six mice in each group). The abscess length (L) and width (W) dimensions were measured by a vernier caliper daily, and the size of the abscesses was calculated using the formula: A =(L×W). Wilcoxon tests or unpaired two-tailed Student's t-tests were performed to analyze statistical significance. After measuring and recording skin abscesses for six days, all the mice were sacrificed with euthanasia. One representative mouse was selected from each group to examine the histopathological section of the skin abscess, while the other five mice were used to evaluate the bacterial burden in the skin abscess site. were injected in the right hind paw with 10μL containing 3×10 8 CFU live S. aureus bacterial suspension. Subsequently, the G. mellonella were placed in a clean petri dish, and incubated in a constant temperature incubator at 37°C for three days. The number of dead G. mellonella was recorded every 12 hours and a survival curve was drawn. This assay was repeated in three times.

Analysis of Hemolytic activities
Lysis of erythrocytes tests were carried out as described before (5). S. aureus strains were cultivated for 16 h in fresh TSB at 37°C with shaking (220rpm) and centrifuged. The hemolytic activities were identified by adding 200μL supernatant samples to 800μL PBS solution containing 3% sterile rabbit red blood cells (RRBCs) and incubating at 37°C for one hour. Hemolytic capacity was determined by measuring the optical density at 600 nm using Micro ELISA Autoreader. Every sample was performed in triplicate.

Biofilm semi-quantitative assay
Biofilm semi-quantitative assays were performed as described before (5

Quantitative Enzyme-Linked Immunosorbent Assay (ELISA) for α-Toxin
The α-toxin was detected by a staphylococcal α-toxin Elisa kit (Sigma-Aldrich, St. Louis, MO, United States). Overnight S. aureus cultures were diluted 1:200 into 4ml TSB for an additional 24 h at 37°C and adjusted to a same absorbance at OD600. Thereafter, the supernatants were collected by centrifugation for two minutes at 12,000 g and then followed the kit instructions for the ELISA. The samples were tested in triplicate and this assay was repeated three times.
Each reaction was performed in triplicate.

Statistical analysis
Unpaired two-tailed Student's t-tests and Wilcoxon tests were performed to analyze statistical significance. All data in this study were analyzed using GraphPad Prism 8.0.2 and the error bars in all graphs represented the standard deviation (mean ± SD). P values <0.05 were considered statistically significant.