The genetic feature and virulence determinant of highly virulent community-associated MRSA ST338-SCCmec Vb in China

Abstract

ST59 is the predominant pathotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in China. As a variant of ST59, there is relatively little known about the detailed information of ST338. To address this issue, here, we described thirteen ST338 CA-MRSA strains isolated from severe bloodstream infection cases, and focused on their epidemiology, genetic features and virulence potential. Phylogenetic analysis showed the earliest isolated strain of this study is likely a predecessor of recent ST338 lineage (after year of 2014). Furthermore, the phylogenetic reconstruction and time estimation suggested that ST338 evolved from ST59 in 1991. Notably, the carrying patten of virulence factors of all ST338 strains were similar, and the genomic islands νSaα, νSaγ and SaPI and the core virulence factors like hla and psm were detected in ST338 isolates. However, all ST338 isolates lacked some adhesion factors such as clfA, clfB, eap, cna and icaD. Additionally, among these ST338 strains, one PVL-negative ST338 isolate was detected. Experiment on mice nose and human alveolar epithelial cell showed that the nasal colonization ability of ST338 was weaker than that of CA-MRSA MW2. In a mouse bloodstream infection model and skin infection model, PVL+ and PVL- strains had the similar virulence, which was dependent on upregulation of toxin genes rather than the presence of mobile genetic elements such as ΦSa2 carrying PVL. Our findings provide important insight into the epidemiology and pathogenicity of the novel and highly virulent ST338-SCCmec Vb clone.

Keywords: CA-MRSA; Panton–Valentine leucocidin; ST338; bloodstream infection; core-virulence factors.

Figure 1.

Relationships among ST338 isolates in this study. (a). Geographic distribution of clinical cases of ST338 infection in China from 2014 to 2019. Different colours represent areas where the strains were isolated. (b). MST of ST338 isolates. The genome sequences of 13 ST338 isolates were aligned. Each circle represents a ST338 strain, and each colour represents a different geographic region. Numbers on the connecting lines indicate the number of single nucleotide polymorphisms between two strains.

Figure 2.

Phylogeny of ST338 isolates. We aligned 43 ST338 genome sequences (13 from our study, 30 from that by NCBI database) and 63 ST59 sequence (33 from our database and 30 from NCBI). Isolate characteristics are shown on the right, including the location, host and Spa type.

Figure 3.

Comparison of virulence factors between ST338, ST59 isolates, MW2 and USA300 strains. Red and white blocks represent the presence and absence of genes, respectively. The horizontal coloured bar represents (from left to right) genes associated with adhesion, serine protease, immune evasion, secretion system, enterotoxin, exfoliative toxin, leukocidin, toxic shock syndrome toxin, exotoxin, and pathogenicity islands.

Figure 4.

Comparison of the vSaα and SaPI genomic islands between ST338 isolates (ST338-PVL+ and ST338-PVL-), M013 and rh10 (ST59), MW2 and USA300 strains. a, b. Comparison of vSaα (a) and SaPI (b) structure between ST338, rh10, M013, MW2 and USA300. Arrows and arrowheads represent open reading frames (ORFs) and their direction of transcription.

Figure 5.

Nasal colonization and cell adhesion capacities of ST338 isolates. (a). Nasal colonization capacity of ST338 isolates compared to MW2 and SA113 strains in mice. Data for the thirteen ST338 isolates in five mice were averaged. (b). Adhesive capacity of ST338, MW2, and SA113 in A549 human alveolar epithelial cells. *P<0.05, **P<0.01.

Figure 6.

The virulence potential of ST338 isolates in vivo. (a). Comparison of invasive capacity among ST338 isolates, MW2, HA-MRSA ST239 strain and the MW2Δagr mutant. Abscess area on day 3 after infection. **P<0.01 (q test). (b). Survival analysis of mice (n=10 per isolate) injected with 2×10⁹ CFU or NaCl solution. Survival curves were compared with the log-rank (Mantel–Cox) test. (c). CFUs in kidneys. *P<0.05 (q test). (d). Haematoxylin and eosin staining of lung tissue at 48 h post infection. Inflammatory cell infiltration and tissue damage were greater in ST338 isolates than in HA-MRSA ST239 and the MW2Δagr mutant. A thickened alveolar septum, oedema and congestion, inflammatory cuffs of blood vessels, and leukocyte influx were observed.

Figure 7.

Expression of virulence toxins and cytotoxicity of ST338 isolates. a. Expression of hla, psmα, agrA, and RNAIII in ST338 isolates compared to USA300 and ST239 strains. b. α-Toxin activity and production in ST338 isolates compared to USA300 and ST239 strains. c. Cytotoxicity of ST338 in human neutrophils compared to USA300 and ST239 strains. Values represent mean±SD of 3 independent experiments. *P<0.05, **P<0.01.