Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans

Abstract

The internal areas and the position of integration of the glycopeptide resistance element Tn1546 were characterized by using PCR fragment length polymorphism, sequencing, and DNA hybridization techniques with 38 high-level vancomycin-resistant Enterococcus faecium isolates of human and animal origins from Europe and the United States. Only minor variations in the coding regions within Tn1546 were found, suggesting high genetic stability. The isolates originated from broilers (n = 5), a chicken (n = 1), a duck (n = 1), a turkey (n = 1), pigs (n = 8), a pony (n = 1), and humans (n = 23). A total of 13 different types were defined based on a single-nucleotide difference in the vanX gene, the presence of insertion sequences, and hybridization patterns. For some types more than one isolate were found. For type 1, 10 isolates of both human and animal origins were found. All were indistinguishable from the reference strain, BM4147. For type 2, 11 isolates of human and animal origins were found. Six human isolates from England were all of type 3. Two human isolates from the United States, indistinguishable from each other, were type 9. These results showed that vancomycin-resistant E. faecium of animal and human origins can contain indistinguishable genetic elements coding for vancomycin resistance, indicating either horizontal gene transfer between E. faecium organisms of human and animal origins or the existence of a common reservoir for glycopeptide resistance.

FIG. 1

Map of Tn1546. Numbers at the top represent kilobases. Open arrows represent coding sequences, and letters inside them stand for genes (e.g., R, vanR). Positions of recognition sites for selected restriction enzymes are marked by vertical arrows. Only essential sizes for the hybridizations are indicated.

FIG. 2

PCR amplification of the internal region of the VanA gene cluster. (A) The vanA gene of selected isolates, from position 7266 to 7623. Isolates 12 and 18 (lanes 5 and 10) gave no positive product. (B) The vanX gene of selected isolates, from position 8129 to 8483. Isolates 12 and 18 (lanes 5 and 10) gave no positive product. (C) The vanSH intergenic region of selected isolates, from position 5715 to 6031. Isolates 12 and 18 (lanes 5 and 10) gave no positive product. Isolates 15 through 17 (lanes 7 through 9) gave a positive product of approximately 1,900 bp. The Boehringer IV (Boehringer Mannheim) was used for size determination. Sizes of markers are 2,176, 1,766, 1,230, 1,033, 653, 517, 453, 394, 298, 234, 220, and 154 bp. Results for isolates are shown as follows: lane 1, 5; lane 2, 6; lane 3, 7; lane 4, 11; lane 5, 12; lane 6, 14; lane 7, 15; lane 8, 16; lane 9, 17; lane 10, 18; lane 11, 22; lane 12, 24; lane 13, 25; lane 14, 27; lane 15, 28; lane 16, 29; lane 17, 30; lane 18, 34; lane 19, 37; lane 20, 40.