Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences

Abstract

A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates--those that comprise the epidemic USA300 clone--were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.

FIG. 1.

Phylogenetic analyses of 3-kb plasmids from USA300 isolates. (A) Comparison of open reading frames in LAC-p01 and pUSA01 (an FPR3757 plasmid). (B) Comparison of open reading frames in 19321-p01 and pWBG738. Arrows and arrowheads indicate ORFs. Green arrows indicate ermC, an antibiotic resistance element. Common elements and ORFs are connected by gray shading. (C) Neighbor-joining phylogenetic trees of 3-kb plasmids from USA300 isolates and selected S. aureus isolates/strains was performed as described in Materials and Methods. Plasmids sequenced in this study are shown in red text. The numbers at each node are the percentages of bootstraps that support the branch. The scale bar represents branch length as it relates to the number of substitutions per base position. (D) Extended phylogenetic analysis (including plasmids from S. aureus strains not related to USA300) indicating the relationship between 3-kb plasmids of the closely related USA300 isolates and small plasmids from other sequenced S. aureus isolates/strains. 18805-p01 was used as a representative of the newly sequenced plasmids from the epidemic USA300 clone.

FIG. 2.

Gene contents of USA300 plasmids. (A) Comparison of ORFs carried by the large (>26-kb) plasmids from USA300 strains. Antibiotic resistance elements are shown in green, transposase or DNA integration/recombination/inversion elements are in red, conjugal transfer (tra) elements are in yellow, and enterotoxins-encoding elements are in pink. ORFs with no reported function are in white. Selected common DNA elements are connected by light-gray shading. All other ORFs with putative or proven functions are shown in blue. Underlines and accompanying asterisks represent possible homopolymer sequencing errors resulting in frameshifts or truncations of a single ORF. (B) Comparison of ORFs present in plasmid 18813-p04 and those of other sequenced plasmids. TCS, two-component gene regulatory system.

FIG. 3.

Phylogenetic analyses of large plasmids from USA300 isolates. (A) Neighbor-joining phylogenetic trees of large plasmids from USA300 isolates were generated as described in Materials and Methods. All strains are multilocus sequence type 8 (ST8). Plasmids sequenced in this study are shown in red text. (B) Phylogenetic analysis of large plasmids from diverse S. aureus isolates/strains. The pulsed-field type, common name, and/or multilocus sequence type of each strain (if available) are in parentheses next to the plasmid name. 18805-p03 was used as a representative of the newly sequenced plasmids from the epidemic USA300 clone.