Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci

Abstract

Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between vanB subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583. Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern. The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception. A U.S. vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element. The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance. The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.

FIG. 1

PCR amplification of the vanS_B-vanY_B intergenic region. Shown is a representative agarose electrophoresis gel of the vanS_B-vanY_B PCR products. Lane 1, φX174 DNA HaeIII digest (Promega); lane 2, isolate TUH7-15 amplicon of 1,098 bp; lane 3, typical 309-bp amplicon, represented by isolate TUH4-64. Molecular sizes shown to the left of the gel (in base pairs) refer to the φX174 DNA HaeIII digest.

FIG. 2

Comparison of DNA sequences of vanS_B-vanY_B amplicons. Shown is a comparison of vanS_B-vanY_B DNA sequences, represented by vanB1 isolate TUH4-64 (identical to the vanS_B-vanY_B region of the V583 vanB gene cluster), vanB2 isolate TUH2-18, and vanB3 isolate TUH7-68. Base pair differences between the vanB1 and the vanB2 and vanB3 strains are shown by single letters below the vanB1 cluster sequence. Gaps are shown by dashes. Nucleotide positions and stop vanS_B and start vanY_B positions, according to the published vanB gene cluster sequence of reference strain V583, are shown above the aligned sequences.

FIG. 3

Restriction pattern and PCR products in the vanB gene cluster. Restriction pattern and PCR products are deduced from the sequence of the reference strain V583. D, DraI; B, BspHI; 1 to 6, BspHI/DraI restriction fragments with decreasing sizes as follows: 1,491, 1,202, 1,086, 960, 654, and 566 bp, respectively. Open arrows represent coding sequences as labeled.

FIG. 4

Analysis of vanB long PCR amplicons. (Top) Representative agarose electrophoresis gel of vanB long PCR amplicons. Lanes 1 and 6, 1-kb ladder (Life Technologies, Gaithersburg, Md.); lane 2, vanB2 isolate TUH2-18; lane 3, vanB3 isolate TUH7-68; lane 4, vanB2 isolate TUH7-15 with a 789-bp insertion; lane 5, vanB1 isolate TUH4-64. (Bottom) Restriction fragment analysis of vanB long PCR amplicons. Shown are BspHI/DraI-digested vanB long PCR amplicons analyzed by agarose gel electrophoresis. Lanes 1 and 6, 1-kb ladder; lane 2, vanB2 isolate TUH2-18 with RFLP-2; lane 3, vanB3 isolate TUH7-68 RFLP-2; lane 4, vanB2 isolate TUH7-15 with a 789-bp enlargement of fragment 4 (RFLP-2*); lane 5, vanB1 isolate TUH4-64 with RFLP-1. Molecular sizes shown to the left of each gel (in base pairs) refer to the 1-kb ladder.