PCR fragment length polymorphism analysis of vancomycin-resistant Enterococcus faecium

Abstract

In this study, the glycopeptide resistance element, Tn1546, in 124 VanA Enterococcus faecium clinical isolates from 13 Michigan hospitals was evaluated using PCR fragment length polymorphism. There were 26 pulsed-field gel electrophoresis (PFGE) types, which consisted of epidemiologically related and unrelated isolates from separate patients (1992 to 1996). Previously published oligonucleotides specific for regions in the vanA gene cluster of Tn1546 were used to amplify vanRS, vanSH, vanHAX, vanXY, and vanYZ. The glycopeptide resistance element, Tn1546, of E. faecium 228 was used as the basis of comparison for all the isolates in this study. Five PCR fragment length patterns were found, as follows. (i) PCR amplicons were the same size as those of EF228 for all genes in the vanA cluster in 19.4% of isolates. (ii) The PCR amplicon for vanSH was larger than that of EF228 (3.7 versus 2.3 kb) due to an insertion between the vanS and vanH genes (79.2% of isolates). (iii) One isolate in a unique PFGE group had a vanSH amplicon larger than that of EF228 (5.7 versus 2.3 kb) due to an insertion in the vanS gene and an insertion between the vanS and vanH genes. (iv) One isolate did not produce a vanSH amplicon, but when vanS and vanH were amplified separately, both amplicons were the same size as those as EF228. (v) One isolate had a vanYZ PCR product larger than that of EF228 (2.8 versus 1.6 kb). This study shows that in a majority of the VanA E. faecium isolates, Tn1546 is altered compared to that of EF228. A total of 79.2% of the study isolates had the same-size insertion between the vanS and vanH genes. The results of this study show dissemination of an altered Tn1546 in heterologous VanA E. faecium in Michigan hospitals.

FIG. 1

Agarose gel electrophoresis of PCR-amplified vanA genes of EF228 and VanA E. faecium isolates with altered Tn1546. Lanes: 1, HindIII-digested lambda phage DNA; 2, EF228 vanRS (1.8 kb); 3, EF13231 vanRS (3.1 kb); 4, EF228 vanSH (2.3 kb); 5, EF13180 vanSH (3.7 kb); 6, EF13231 vanSH (5.7 kb); 7, EF11456 (vanSH negative); 8, EF11456 vanS and vanH (amplified separately and run in the same lane); 9, EF228 vanHAX (2.6 kb); 10, EF228 vanXY (1.9 kb); 11, EF228 vanYZ (1.6 kb); 12, EF13566 vanYZ (2.8 kb).