Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s

Abstract

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the β-lactamase transposon Tn552 An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS257 likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive S. aureus (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.

Keywords: CA-MRSA; IS257; MITEs; Staphylococcus aureus; Tn552; conjugation; mupirocin; plasmid; resolvase; trimethoprim.

FIG 1

The 72-kb conjugative multiresistance plasmid pWBG731. Map of the pWBG731 sequence highlighting sequence features. The outer ring shows positions of predicted genes and key cis-acting sequences likely involved in its evolutionary construction. Asterisks before or after a gene name indicate a 5′ or 3′ gene truncation, respectively. The gray ring shows the sequence ruler (in kilobase pairs), and positions of ClaI sites (corresponding to ClaI-digested DNA in Fig. 4B) are shown as red lines. The next inner ring highlights positions of IS257 and MITESau1 elements. All IS257 copies except IS257#5 carry an intact transposase gene. Yellow, blue, and gray sectors indicate regions nearly identical to those present on plasmids pWBG745, pWBG753, and pWBG715. TIR, terminal inverted repeat; MTase, predicted class I SAM-dependent methyltransferase domain-encoding gene.

FIG 2

Tn552-derived regions on pWBG753 and pWBG731. (A) Gene maps of the Tn552 and Tn552-like sequences on pI9789, pWBG753, and pWBG731, showing the positions of the right and left terminal inverted repeats (TIR_R and TIR_L, respectively), resL sites, and other genes or insertion sequences. (B) Sequence alignment of the resL sites on the same three plasmids. The region of the resL site is the main recombination site of the resL region “subsite I.” Accessory subsites II and III are identifiable upstream of the binL and res3 coding sequences.

FIG 3

Alignment of MITESau1-like elements. Shown is an alignment (M-Coffee [60]) of distinct MITESau1-like sequences from pWBG731, pSW49 (GenBank accession number AM040730.1), pWBG759 (GenBank accession number GQ900401.1), SaPIbov2 (GenBank accession number AY220730.1), and pAvX (GenBank accession number MH785253.1). Nonidentical nucleotides are shown in the alignment with MITESau1. The terminal inverted repeats of the elements are indicated by horizontal arrows, and the ends of pWBG731 IS257#1 are presented at the top for comparison, with nonmatching nucleotides shown in lowercase type. MITESau1 of pWBG731 is identical to ISLE39 (GenBank accession number AF535086.1).

FIG 4

Stepwise evolution of pWBG753, pWBG715, and pWBG731. (A) Plasmid maps of sequenced plasmids pWBG753 and pWBG731 and map for the predicted sequence of pWBG715 (not to scale relative to each other). The outermost rings on each plasmid map indicate percent nucleotide identity to contigs from a BLASTN query of the de novo sequence assembly of WBG7565, which carries pWBG715b, against each plasmid sequence. The gray sectors indicate the pWBG753 backbone conserved on all three plasmids, the yellow region on pWBG753 represents the region replaced with the orange region on pWBG715, and the red sector indicates the pT181 region on pWBG715 that was replaced by the blue pWBG745-like conjugative plasmid on pWBG731. (B) Agarose electrophoresis of ClaI-digested pWBG731 (left) and pWBG715b (right) DNA. Fragment sizes for pWBG731 are indicated on the left, and recognizable corresponding fragments in the pWBG715 digest are indicated on the right.

FIG 5

Comparison of oriT sequences on pWBG749 family plasmids. Shown is an alignment of the oriT sequences (excluding upstream AR1 to AR3 regions) on conjugative plasmids pWBG745, pWBG731, pC02, and pWBG749, along with oriT mimic sequences of subtypes OT49, OT45, and OTUNa present on pWBG762. Comparison of the IR2 regions reveals the similarity between the IR2 regions of the OT45 and OTUNa oriT sequences in contrast to the IR2 region of OT49 sequences.