Table 1.
Clinical and bacteriological information on MRSA isolated in Krasnoyarsk between 2007 and 2011.
Table 2.
List of MRSA strains characterized at molecular levels in the present studya.
Table 3.
Molecular characterization of MRSA strains isolated in Krasnoyarska.
Fig 1.
Pulsed-field gel electrophoresis (PFGE) analysis (right and left) and plasmid carriage patterns (center) of MRSA strains isolated in Krasnoyarsk.
The MRSA strains shown are those described in Table 2. Group A (A1 and A2) and group B are described in Table 3. The color of the strain name indicates fatal pneumonia (red), possible sepsis (brown), and carrier cases (green). Of the cases of fatal pneumonia, OC3, OC8C, OC11, OC76 were from hospital-acquired pneumonia (HAP), while OC8, OC22, OC23, and OC59 were from community-acquired pneumonia (CAP). Of the carrier cases, OC14C and OC52 were from hospital workers and OC217 was from a student.
Fig 2.
Genome information for the ST239Kras strain OC3, in comparison with the ST239 MRSA strain TW20.
The ST239Kras OC3 genome contigs (including filled contigs and complete structures; total 2.91-Mb) were mapped on the 3,043,210-bp TW20 genome (GenBank accession number FN433596); in the figure, the two genome structures were drawn as two circles on a common genome map, outside OC3 and inside TW20. Genome information included staphylococcal cassette chromosome mec (SCCmec), other drug resistance structures (such as a transposon, Tn, plasmid-related structure, and gene mutations), characteristic virulence genes, phages, S. aureus pathogenicity islands (SaPIs), genomic islands (νSa), and characteristic insertion sequences (ISs). SCCmec: SCCmecIIIA (in OC3), SCCmecIII.1.1.2; SCCmecIII (in TW20), SCCmecIII.1.1.1 connected to SCCHg. Drug resistance (gene mutations): Lvxr, levofloxacin resistance; Rifr, rifampicin resistance; Sur, sulfamethoxazole resistance. Virulence genes (region): tst, toxic shock syndrome toxin-1 gene; hld, δ-hemolysin gene; cna, collagen adhesin gene; spa, protein A gene; psmα, phenol-soluble modulin (PSM) gene; hla, α-hemolysin (α-toxin) gene; IEC, immune evasion cluster. The CC30 and CC8 genome sections are from Holden et al. [19], and the genetic element IEC6013 is from [60]. The plasmid pOC3 (2,908 bp; contig 75) of strain OC3 is not shown in the figure. The location of pSK41-related structure (with two IS431 repeats at both ends) currently remains uncertain.
Fig 3.
SCCmecIII.1.1.2 structure of the ST239Kras strain OC3, in comparison with the three structures: SCCHg/SCCmecIII.1.1.1 of the strain TW20, SCCmecIII.1.1.2 of the strain HU25, and SCCmecIII.1.1.4 of the strain 16K.
Isolation of ST239 strains: OC3, Krasnoyarsk; TW20, London; HU25, Brazil; 16K, Vladivostok. Homologous regions are shaded in each comparison. In A, when compared with SCCHg/SCCmecIII.1.1.1 (of TW20), SCCmecIII.1.1.2 (of OC3 and HU25) lacked the middle IS431②-IS431④ region. The J3 region of SCCmecIII.1.1.2 corresponded to the bulk of SCCHg. SCCmecIII.1.1.4 (of 16K) lacked SCCHg. In B, the primer set Hgreg (F)/attM (R) detected attM, and the primer set Hgreg (F)/rec2/4 (R) identified recombination between IS431 copies ② and ④.
Fig 4.
Structure of a phage φSa7-like (W) of the ST239Kras strain OC3.
φSa7-like (W) was compared with φSa7 of the strain Newman for the phage structure, the integration site (att) sequence, and integration site. Homologous regions are shaded in the comparison. The figure at the lower right side indicates each integration site on the S. aureus chromosome. The target huNaDC-1 sequence of φSa7-like (W), shown at the top of the figure (in blue and purple), was also present in the huNaDC-1 gene of other S. aureus strains.
Fig 5.
Analysis of the tst+ SaPI (SaPI2R) structure and tst nucleotide and deduced amino acid sequences of the ST239Kras strain OC3.
In A, the integration site (att) and att sequences of SaPI2R of the ST239Kras strain OC3 are shown. In B, the SaPI2R structure was compared with those of tst- SaPI (ATCC25923) and tst+ SaPI2 (RN3984). Homologous regions between SaPI structures are shaded with color. Genes: tst, toxic shock syndrome toxin-1 gene; eta, S. hyicus exfoliatin A gene; ter, terminase gene (which cleaves multimeric DNA); rep, replication initiator gene; int, the integrase gene. In C, the nucleotide sequences of tst+ SaPIs and tst- SaPI (ATCC25923) were analyzed for phylogenetic diversity. In D-a, the nucleotide sequences of the tst genes were analyzed for phylogenetic diversity. In this figure, each GenBank record year is also shown. In D-b, the deduced amino acid sequences of the tst gene products were analyzed for phylogenetic diversity. The origin (reported source) of each isolate is indicated by the color of the isolate name: red, Russia; yellow, United Kingdom (UK); blue, United States (USA); dark red, Korea; light blue, Argentine; purple, Japan; green, those for animal isolates. In C and D, the scale bar represents substitutions per single-nucleotide polymorphism site. In E, the representative tst gene sequences were compared with the reference sequences (of pRN6100). Arrows indicate the positions of the nucleotide and amino acid changes for the representative tst genes. At the bottom of the figure (green), different amino acids from the amino acid sequences of purified TSST-1 (MN8; GenBank accession number EFH95768) and the deduced amino acid sequence of the tst gene (pRN6100) are indicated in red letters.
Fig 6.
mRNA expression levels of cytolytic peptide genes (psmα and hld) in ST239Kras and ST8Kras strains (A) and of regulatory genes in ST239Kras strains (B), in comparison with CA- and HA-MRSA reference strains and other ST239 MRSA strains.
In A, in the right-side control MRSA box, CA-MRSA, which shows high expression levels, is marked in red; and HA-MRSA, which shows low expression levels, is marked in light blue. In the left-side MRSA box, CA-MRSA (strain RS08), which showed high expression levels as expected, is marked in red; CA-MRSA ST8Kras (ST8/SCCmecIVc) strains, including OC8, also showed high expression levels (dark blue box on the right side). ST239 HA-MRSA strains, which showed low expression levels as expected, are marked in light blue. However, ST239Kras (ST239/SCCmecIII/spa3/tst+), including OC3, unexpectedly showed high expression levels, similar to CA-MRSA; this box is marked in dark blue on the left side. Regarding psmα: *1, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *3, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *5, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *6, P < 0.05 vs. Russian ST239/SCCmecIII/spa3 and spa351. Regarding hld: *2, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *4, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *7, P < 0.05 vs. control ST239/SCCmecIII and ST5/SCCmecII; *8, P < 0.05 vs. Russian ST239/SCCmecIII/spa3 and spa351. In B, the psmα expression levels of ST239 MRSA strains were examined. spa3/tst+, ST239Kras. No significant difference was observed between ST239Kras and other ST239 MRSA for sarA gene expression. *1, P < 0.05 vs. spa351; *2, P < 0.05 vs. spa3; *3, P < 0.05 vs. spa351; *4, P < 0.05 vs. spa3; *5, P < 0.05 vs. spa351; *6, P < 0.05 vs. spa3.