Phenotypic and Genomic Comparison of Staphylococcus aureus Highlight Virulence and Host Adaptation Favoring the Success of Epidemic Clones

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) of the sequence type 59 (ST59) and ST398 lineages has emerged in hospitals and displayed a higher virulent potential than its counterparts ST5 and ST239. However, the mechanism of the host cell-pathogen interaction and specific determinates that contribute to the success of epidemic clones remain incompletely understood. In the present study, 142 S. aureus strains (ST59, ST398, ST239, and ST5) were selected from our 7-year national surveillance of S. aureus bloodstream infections (n = 983). We revealed that ST59 and ST398 had a higher prevalence of the protease-associated genes hysA^VSaβ, paiB, and cfim and enhanced proteolytic activity than the other lineages. ST59 and ST398 showed a higher expression of RNAIII and psmα and greater proficiency at causing cell lysis than other lineages. Furthermore, ST59 and ST398 were strongly recognized by human neutrophils and caused more cell apoptosis and neutrophil extracellular trap degradation than the other lineages. In addition, these strains differed substantially in their repertoire and composition of intact adhesion genes. Moreover, ST398 displayed higher adaptability to human epidermal keratinocytes and a unique genetic arrangement inside the oligopeptide ABC transport system, indicating functional variations. Overall, our study revealed some potential genomic traits associated with virulence and fitness that might account for the success of epidemic clones. IMPORTANCE Considerable efforts have been exerted to identify factors contributing to the success of epidemic Staphylococcus aureus clones, however, comparative phenotypic studies lack representation owing to the small number of strains. Large-scale strain collections focused on the description of genomic characteristics. Moreover, methicillin-resistant S. aureus infections constitute 30% to 40% of S. aureus bloodstream infections, and recent research has elucidated highly virulent methicillin-susceptible S. aureus strains. However, comprehensive research on the factors contributing to the success of epidemic S. aureus clones is lacking. In this study, 142 S. aureus strains were selected from our 7-year national surveillance of S. aureus bloodstream infections (n = 983) accompanied by a rigorous strain selection process. A combination of host cell-pathogen interactions and genomic analyses was applied to the represented strains. We revealed some potential genomic traits associated with virulence and fitness that might account for the success of epidemic clones.

Keywords: Staphylococcus aureus; adhesion and invasion; lineage replacement; neutrophil; virulence determinants.

FIG 1

Epidemic and phylogenetic features for ST59 and ST398. (A) The dynamic changes of epidemic S. aureus clones from bloodstream infections of inpatients in China from 2013 to 2020. (B, C) Phylogenetic analysis of the epidemic clones ST59 and ST398. The trees were constructed by IQ-TREE, based on the core gene alignment generated by Roary, annotated by the iTOL Web tool. Strain informations were mapped on the tree, from inner to outer circle: 1) The represented 142 strains selected in ST59 and ST398 were painted as filled yellow stars. 2) The represented 32 strains were painted as filled blue dots. 3) The filled red square represented the presence of mecA. 4) The circle colors distinguished the different collection years. 5) The circle colors distinguished the different locations.

FIG 2

Genetic traits among the represented 142 S. aureus strains. (A) Heat map of the enzyme, cytolytic, and superantigen-associated genes; each cell in the heat map indicates the percentage of the virulence gene in specific clones. The aureolysin (aur) ClpP protease (clpP), hyaluronidase (hysA, located on chromosome), hla, and hlgA were conserved among the different clones. (B) The SCCmec type, spa_type, agr_type, and immune evasion cluster (IEC) type for the 142 strains grouped by different resistance and clonal type combinations; the numbers in brackets represent the number of strains.

FIG 3

Proteolysis-associated phenotypic and genetic environment analysis. (A) Proteolytic activity is assessed on TSA containing 2% skim milk among the different clones. (B) Genomic island νSaβ carrying the operon spl cluster that is complete in ST239 and ST5, hysA^VSaβ is specifically presented in ST59 and ST398 with different sequences, and ST59 has a disrupted νSaβ structure with prophage-associated genes inserted. (C) Genomic region encompassing the gene paiB, which is missing in ST239 and ST5 genomes. (D) The CPBP family intramembrane metalloprotease gene (cfim) and surrounding genes among the four clones. Statistical significance of proteolysis abilities was determined by using the unpaired Student’s t test. Data are shown as mean ± SEM. Each dot represents a methicillin-resistant (MRSA, filled dot) or methicillin-susceptible (MSSA, hollow circle) isolate. ****, P < 0.0001; ns, not significant (P ≥ 0.05). Genes are indicated by arrowed boxes and colored based on gene function classification. The gray boxes with no function annotations represent hypothetical protein-encoding genes.

FIG 4

In vitro cytolytic capacity comparison. (A) In vitro growth curve among the different STs; ST239-MRSA and ST5-MRSA showed impaired fitness during growth in vitro. (B) Toxicity for each isolate was determined by incubating the bacterial supernatant with human red blood cells (RBCs), using absorbance at 540 nm to quantify hemolysis ability relative to the hypervirulent strain 8325. (C, D) Eight strains from each clone were selected to compare the RNAIII (C) and psmα (D) expression levels with qPCR. The results were normalized to gyrB expression with RN4220 as the negative control. The Wilcoxon signed-rank test was used to compare differences of the growth curve. Statistical significance of cytolytic abilities and gene expression was determined by using the unpaired Student’s t test and Mann-Whitney U test. Data are shown as mean ± SEM. Each dot represents a methicillin-resistant (MRSA, filled dot) or methicillin-susceptible (MSSA, hollow circle) isolate. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 5

ST59 and ST398 are more potent microbial invaders when confronted with neutrophils. (A) Bacterial survival rate of different clones after incubating with freshly isolated neutrophils for 30 min. (B) Apoptosis and necrosis of neutrophils were confirmed by annexin V-propidium iodide (PI) staining and flow cytometry analysis; bacterial supernatants were 10-fold diluted. Statistical significance of the bacterial survival rate was determined by using the unpaired Student’s t test. Data are shown as mean ± SEM. Each dot represents a methicillin-resistant (MRSA, filled dot) or methicillin-susceptible (MSSA, hollow circle) isolate. **, P < 0.01; ****, P < 0.0001; ns, not significant (P ≥ 0.05).

FIG 6

NETs formation and degradation abilities among the represented strains. (A) The CCK8 assay was used to compare the neutrophil NAD⁺ release ability after bacterial infection; eight representative strains are presented from each clone. (B) The proportion of NETs per total amount of neutrophils was calculated to compare NETs formation in five individual images per sample. (C) Representative picture for NETs formation for one isolate from each clone, visualized by DNA and MPO stain. Statistical significance of NAD⁺ release and NETs formation rate were determined by using the unpaired Student’s t test. Data are shown as mean ± SEM. Each dot represents a methicillin-resistant (MRSA, filled dot) or methicillin-susceptible (MSSA, hollow circle) isolate. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant (P ≥ 0.05).

FIG 7

ST398 has unique Opp system, adhesion-associated genes, and higher adhesion and invasion capacity with keratinocytes. (A) Variation of the surface protein-encoding genes among the different clones in 142 isolates (cna in 32 strains, because of the analysis of cna depends on nanopore sequencing results) * refers to the percentage of gene absent. (B) Different structures within the Opp system between ST398 and the other clones, compared by the Easyfig tool. Genes are indicated by arrowed boxes and colored based on the Opp system gene function classification. The gray boxes with no function annotations represent hypothetical protein-encoding genes. (C) Cell adhesion and invasion capacity in keratinocytes among the 32 strains, separated by different clones. Statistical significance of adhesion and invasion abilities was determined by using the Mann-Whitney U test. Data are shown as mean ± SEM. Each dot represents a methicillin-resistant (MRSA, filled dot) or methicillin-susceptible (MSSA, hollow circle) isolate. *, P < 0.05; ns, not significant (P ≥ 0.05).