Molecular Evolutionary Pathways toward Two Successful Community-Associated but Multidrug-Resistant ST59 Methicillin-Resistant Staphylococcus aureus Lineages in Taiwan: Dynamic Modes of Mobile Genetic Element Salvages

Abstract

Clonal complex 59 (CC59) Staphylococcus aureus in Taiwan includes both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). As the most prominent community-associated MRSA (CA-MRSA) in Taiwan, CC59 has two major clones characterized as PVL-negative SCCmec IV (carrying the staphylococcal cassette chromosome mec IV but Panton-Valentine leukocidin-negative) and PVL-positive SCCmec V (5C2&5). We investigated the drug resistance, phylogeny and the distribution and sequence variation of SCCmec, staphylococcal bacteriophage φSA3, genomic island νSaβ and MES (an enterococcal mobile genetic element conferring multidrug resistance) in 195 CC59 S. aureus. Sequencing and PCR mapping revealed that all of the CC59/SCCmec V (5C2&5) MRSA strains had acquired MESPM1 or its segregants, and obtained a φSA3-related fragment in νSaβ. In contrast, MES6272-2 and MES4578, which showed gentamicin resistance that was not encoded by MESPM1, were dominant in SCCmec IVg MRSA. Translocation of a whole φSA3 into νSaβ instead of only a φSA3-related fragment was common in SCCmec IVg MRSA. However, the non-subtype-g SCCmec IV MRSA (SCCmec IVa is the major) still carried MES and νSaβ structures similar to those in SCCmec V (5C2&5) MRSA. A minimum spanning tree constructed by multiple-locus variable-number tandem repeat analysis revealed that SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA grouped respectively in two major clades. The CC59 MSSA was equally distributed among the two clades, while the non-subtype-g SCCmec IV MRSA mostly clustered with SCCmec V (5C2&5) MRSA. Our findings strongly suggest that CC59 MSSA acquired divergent mobile genetic elements and evolved to SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA/non-subtype-g SCCmec IV MRSA independently. The evolutionary history of CC59 S. aureus explains how mobile genetic elements increase the antimicrobial resistance and virulence and contribute to the success of CA-MRSA in Taiwan.

Fig 1. Comparative genomics of 4578 (ST59/SCCmec IVg MRSA) and PM1 (ST59/SCCmec V (5C2&5) MRSA).

Information on the 4578 and PM1 genomes is presented outside and inside the genome circle, respectively. Colored regions: blue, conserved region; gray, deletion in strain 4578; red, distinct mobile genetic elements.

Fig 2. The comparison of MES structures integrated into the sasK gene.

(A) MES₄₅₇₈ in ST59/SCCmec IVg MRSA strain 4578 is compared to MES_PM1 in ST59/SCCmec V (5C2&5) MRSA strain PM1 (accession number AB699882) [23]. (B) MES_6272-2, MES₄₅₇₈ and MES₂₂₅₀ in three ST59/SCCmec IVg MRSA strains 6272–2, 4578 and 2250, respectively, are compared. Homologous regions are shaded in gray. A Greek delta symbol indicates a truncated coding sequence. IS1216V with a transposase gene (tnp) is shaded in blue. The sasK gene is indicated in green arrow and box with dashed line.

Fig 3. Selection for loss of antibiotic resistance in ST59/SCCmec V (5C2&5) MRSA strain PM1.

(A) Cartoon representation of MES structures in PM1 and the generated colonies PM1-1 to PM1-5. (B) SmaI-digested PFGE of PM1 and PM1-1 to PM1-5 and the Southern blot. The SmaI-digested DNA separated by PFGE is shown in the left. The DNA was transferred to a nylon membrane and detected by Southern blot hybridization with DIG-labeled cat-, ermB- and IS1216V-specific probes, which are shown on the right. Abbreviations: E^r, erythromycin resistant; K^r, kanamycin resistant; S^r, streptomycin resistant; C^r, chloramphenicol resistant.

Fig 4. The νSaβ structure of strains 4578, PM1 and 187–4.

The strains 4578 and 187–4 are ST59/SCCmec IVg MRSA carrying IEC type G or no IEC, respectively. Strain PM1 is ST59/SCCmec V (5C2&5) MRSA with IEC type C. Homologous regions are shaded. A Greek delta symbol indicates a truncated coding sequence. The att sequence is indicated by a triangle. Arrows with dashed lines indicate nearly complete coding sequence.

Fig 5. Minimum spanning tree of CC59 S. aureus strains constructed by MLVA.

Each circle represents an MLVA type. Circles or sectors of circles with orange, yellow, blue or green colors denote SCCmec IVg MRSA, non-subtype-g SCCmec IV MRSA, SCCmec V (5C2&5) MRSA or MSSA, respectively. The USA1000, which is ST59 MRSA in the United States, is shown in white. The size of each circle is proportional to the number of strains.

Fig 6. Proposed evolutionary history of CC59 S. aureus in Taiwan.

SCCmec IVg MRSA, non-subtype-g SCCmec IV MRSA, SCCmec V (5C2&5) MRSA or MSSA with the characteristics found in this study are shaded in orange, yellow, blue or green, respectively. Dash line indicates an uncertain evolutionary route. MDR: multidrug resistance; S^r: streptomycin resistance; G^r: gentamicin resistance.