Genomic Analysis of Vancomycin-Resistant Staphylococcus aureus Isolates from the 3rd Case Identified in the United States Reveals Chromosomal Integration of the vanA Locus

Abstract

Vancomycin-resistant Staphylococcus aureus (VRSA) is a human pathogen of significant public health concern. Although the genome sequences of individual VRSA isolates have been published over the years, very little is known about the genetic changes of VRSA within a patient over time. A total of 11 VRSA, 3 vancomycin-resistant enterococci (VRE), and 4 methicillin-resistant S. aureus (MRSA) isolates, collected over a period of 4.5 months in 2004 from a patient in a long-term-care facility in New York State, were sequenced. A combination of long- and short-read sequencing technologies was used to obtain closed assemblies for chromosomes and plasmids. Our results indicate that a VRSA isolate emerged as the result of the transfer of a multidrug resistance plasmid from a coinfecting VRE to an MRSA isolate. The plasmid then integrated into the chromosome via homologous recombination mediated between two regions derived from remnants of transposon Tn5405. Once integrated, the plasmid underwent further reorganization in one isolate, while two others lost the staphylococcal cassette chromosome mec element (SCCmec) determinant that confers methicillin-resistance. The results presented here explain how a few recombination events can lead to multiple pulsed-field gel electrophoresis (PFGE) patterns that could be mistaken for vastly different strains. A vanA gene cluster that is located on a multidrug resistance plasmid that is integrated into the chromosome could result in the continuous propagation of resistance, even in the absence of selective pressure from antibiotics. The genome comparison presented here sheds light on the emergence and evolution of VRSA within a single patient that will enhance our understanding VRSA genetics. IMPORTANCE High-level vancomycin-resistant Staphylococcus aureus (VRSA) began to emerge in the United States in 2002 and has since then been reported worldwide. Our study reports the closed genome sequences of multiple VRSA isolates obtained in 2004 from a single patient in New York State. Our results show that the vanA resistance locus is located on a mosaic plasmid that confers resistance to multiple antibiotics. In some isolates, this plasmid integrated into the chromosome via homologous recombination between two ant(6)-sat4-aph(3') antibiotic resistance loci. This is, to our knowledge, the first report of a chromosomal vanA locus in VRSA; the effect of this integration event on MIC values and plasmid stability in the absence of antibiotic selection remains poorly understood. These findings highlight the need for a better understanding of the genetics of the vanA locus and plasmid maintenance in S. aureus to address the increase of vancomycin resistance in the health care setting.

Keywords: Staphylococcus aureus; VRSA; antibiotic resistance; vancomycin; vancomycin-resistant Staphylococcus aureus; whole-genome sequencing.

FIG 1

Schematic representation of the 78.6 kb plasmid pWC79. A more detailed view of the vanA locus is shown in Fig. 2. Outer ring, genes in the sense orientation; inner ring, genes in antisense orientation; red, antibiotic resistance genes; blue, transposase genes; green, replicons and the axe/txe toxin antitoxin system; gray, other genes.

FIG 2

Schematic representation of the vanA locus and adjacent region in VRSA and VRE isolates (e.g., position 48,466 to 61,950 in pWC79 from VMRSA-WC052, CP092559). The left inverted repeat, transposase, and resolvase (res, ORF2 in Arthur et al. [6]) of Tn1546 has been replaced by an IS6-like element IS1216 family transposase. As in Tn1546, the locus includes the vanRSHAXYZ genes and the right inverted repeat, but unlike Tn1546, the locus also includes an ISL3 family transposase. The locus is identical in all VRSA isolates and in VRE-WC072, while VRE-WC031 and VRE-084 differ by 5 SNPs each (indicated by the isolate name and the number of SNPs in parentheses). HTH, helix-turn-helix transcriptional regulator; AbrB, AbrB family transcriptional regulator; RelE, type II toxin-antitoxin system RelE/ParE family toxin; rec, recombinase family protein.

FIG 3

Comparison between pWC79 (78.6 kb) from VMRSA-WC052, pWC91 (90.9 kb) from VRE-WC031, and pWC84 (83.6 kb) from VRE-WC072. Regions of the same color are shared among the plasmids; regions that are depicted inside the plasmid are in the antisense orientation relative to pWC79. Genes of interest are indicated for reference; an asterisk (*) denotes that the gene is a pseudogene.

FIG 4

Schematic representation of the chromosome and the vancomycin resistance-conferring plasmid pWC79 in S. aureus. Sections of the chromosome are indicated by black bars with hashed ends. A gray-black gradient marks a region of the chromosome that underwent an inversion. Plasmid pWC79 is indicated by a blue background; a dark green background indicates that the plasmid has undergone significant rearrangements in strain VMRSA-WC082. Colored boxes indicate various antibiotic resistance genes. AG, aminoglycoside resistance genes. Arrows show the orientation of the ant(6)-sat4-aph(3′) loci L1 to L3; L2′ and L3′ indicate loci after integration of plasmid pWC79; L2″ and L3″ denote loci after undergoing further rearrangements.

FIG 5

Schematic representations of transposon Tn5405 and the ant(6)-sat4-aph(3′) loci in VMRSA-WC052 and VMRSA-WC062. (a) Reconstituted composite transposon Tn5405 at the L3′ site where integration of plasmid pWC79 into the chromosome resulted in the combination of the two halves of the transposon that had been separated on the chromosome (locus L2) and the plasmid (locus L3). [IS1182] denotes the location of the missing second IS1182 element to complete Tn5405. Open reading frames orfX-orfZ were initially described by Derbise (28–30) and encode a nucleotidyltransferase domain-containing protein, a class I SAM-dependent methyltransferase, and a RibD family protein. Open reading frames orfA-orfC encode a single-stranded DNA-binding protein, an HTH domain-containing protein, and a WYL domain-containing protein. (b) Schematic depiction of the three ant(6)-sat4-aph(3′) loci, L1 to L3, in VMRSA-WC052, representing a strain with a free pWC79 plasmid. While L1 remained unaffected in VMRSA-WC062, L2′ and L3′ represent the ant(6)-sat4-aph(3′) loci after integration of pWC79 into the chromosome. In L1 and L2/L2′, the ant(6)-Ia gene is truncated relative to the allele provided by the plasmid in L3/L3′. (c) Schematic representation of the three ant(6)-sat4-aph(3′) loci that result in an indirect repeat (IR) and a direct repeat (DR) in S. aureus isolates with chromosomal integration of the pWC79 plasmid (with the exception of VMRSA-WC082). [1], the mph(C)-containing region that is inverted in some of the S. aureus isolates; [2], the remaining sequence of pWC79.

FIG 6

Minimum spanning tree. Illumina reads were mapped to VMRSA-WC052 as a common reference. Numbers indicate the number of single nucleotide polymorphisms (SNP) between a pair of isolates. Lines connect isolate pairs that are most similar to each other. Isolates in the same bubble have no differentiating SNPs. Green, MRSA isolates; blue, VMRSA isolates with pWC79 as a free plasmid; orange and magenta, VMRSA and VRMSSA isolates, respectively, with chromosomally integrated pWC79. An asterisk (*) denotes the isolate from the patient’s roommate.

FIG 7

Schematic representation of how three recombination events between inverted repeats could have led to the rearrangements observed in isolate VMRSA-WC082 after integration of plasmid pWC79 into the chromosome. Isolates VMRSA-WC052, VMRSA-WC083, and VMRSA-WC102 are shown in panel a. Isolate VMRSA-WC082, after having undergone multiple inversion events, is schematically depicted in panel e. The remaining VMRSA and VRMSSA isolates, having an integrated copy of pWC79, are represented in panel b. Colored lines indicate the regions undergoing inversion. The IS257 transposase that is unique to this isolate is highlighted in yellow. The ant(6)-sat4-aph(3′) loci are designated as L2/L2′ and L3/L3′, in Fig. 4. Key genes are indicated. chromo., chromosome.