The dominant Australian community-acquired methicillin-resistant Staphylococcus aureus clone ST93-IV [2B] is highly virulent and genetically distinct

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.

Figure 1. SmaI pulsed field gel electrophoresis analysis.

PFGE of index isolate, S. aureus JKD6159 and isolates from three family members, FM1, FM2, FM3.

Figure 2. Virulence characteristics of S. aureus isolates.

S. aureus JKD6159 (ST93-IV [2B], CA-MRSA) compared to four other MRSA strains. (A) Galleria mellonella virulence assay. Kaplan-Meier plot showing the percent survival of larvae injected with S. aureus strains JKD6159, JKD6009 (ST239-III [3A], hospital-associated MRSA), JKD6272 (ST1-IV [2B], CA-MRSA), JKD6177 (ST30-IV [2B], CA-MRSA), FPR3757 USA300 (ST8-IV [2B], CA-MRSA) up to 6 days post injection. The reduced survival time for larvae infected with JKD6159 compared to all other strains was significant (p<0.0001). (B) BALB/c mouse skin infection assay. Weight loss induced by intradermal infection with S. aureus strains is demonstrated as percentage loss of weight over 5 days. The difference in percentage weight loss between JKD6159 and all other strains was significant (p<0.05). At least 15 mice were used for each bacterial strain. Data shown are mean weight loss and SEM. (C) BALB/c mouse skin infection assay. Skin lesion area (mm²) at 5 days after infection was greatest with JKD6159 infected mice (p<0.05). Data shown are mean area and SEM. (D) BALB/c mouse skin infection assay. Recovery of S. aureus (log CFU) from infected tissues at 5 days after infection was greatest with JKD6159 infected mice (p<0.05). Data shown are mean CFU and SEM. (E-I) BALB/c mouse skin infection assay. Representative H&E stained histology sections from infected tissues at 100× magnification from JKD6009, JKD6272, JKD6177, USA300 and JKD6159 respectively. There was no significant difference in degree of inflammation observed in the CA-MRSA strain infections. However, mice infected with JKD6009, the hospital-associated MRSA strain had less marked inflammation and only mild myositis. (J-N) BALB/c mouse skin infection assay with representative macroscopic appearance of lesions resulting from infection with JKD6009, JKD6272, JKD6177, USA300 and JKD6159 respectively.

Figure 3. Whole genome sequence analysis and comparison of JKD6159 with other S. aureus strains.

(A) Artemis linear view of JKD6159 chromosome, with vertical red bars identifying the position of accessory genome elements as determined by read mapping against 19 publicly available completed genomes and 62 unpublished, partially assembled genome sequences. Increasing height of vertical red lines indicates increasing specificity for JKD6159. Shown above the accessory genome analysis are the mapped positions of the short-reads obtained from Illumina sequencing CA-MRSA strains JKD6177, JKD6272 and JKD6260. Depicted also is an example of the synthetic reads obtained from completed whole genome sequences (USA300 shown here) to facilitate the comprehensive read mapping approach described in the methods. (B) Circular diagram of the JKD6159 chromosome showing (from inner to outer), % G+C, GC skew and the homology based on BLASTn+ analysis of JKD6159 to 19 completed S. aureus genomes (refer color-coded legend). Outer circle shows the location of accessory elements (grey) and the 12 regions of difference (blue) not present in the other S. aureus genomes examined.

Figure 4. Phylogenetic analysis of S. aureus strains.

Phylogenetic analysis based on the core genomes of S. aureus strains and selected accessory elements from CA-MRSA strains. (A) S. aureus phylogeny inferred by split decomposition analysis from pairwise comparisons of the 78,967 variable nucleotide positions identified from the core chromosome sequences of 20 completed S. aureus chromosomes and the 62 S. aureus strains for which unassembled reads were available, including the CA-MRSA strains sequenced in this study, JKD6177, JKD6260, JKD6272. CA-MRSA strains are indicated in colour (red JKD6159, pink JKD6177, orange FPR3757 USA300, blue MW2, light green JKD6260, dark green JKD6272) (B-D), Phylogenetic relationship of φSA2, pMW2 and SCCmecIV inferred by split decomposition analysis of nucleotide differences for each of these accessory elements between CA-MRSA strains. Scale bar indicates the number of nucleotide substitutions per site. All nodes have 100% bootstrap support except those indicated by asterisk (*) which have >60% support (1000 replicates). CC: clonal complex. ST: sequence type.