Transfer of vancomycin resistance transposon Tn1549 from Clostridium symbiosum to Enterococcus spp. in the gut of gnotobiotic mice

Abstract

The vancomycin resistance vanB2 gene cluster is disseminated worldwide and has been found in phylogenetically remote bacterial genera. The vanB2 operon is part of conjugative transposons Tn1549/Tn5382, but conjugative transposition of these elements has not been demonstrated. We have obtained transfer of a Tn1549-like element (referred to herein as "Tn1549-like") from Clostridium symbiosum MLG101 to Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 in the digestive tract of gnotobiotic mice and to E. faecium 64/3 in vitro. Retransfer of Tn1549-like from an E. faecium transconjugant also containing Tn916 to E. faecium BM77 was obtained in vitro, albeit at a very low frequency. Transfer efficiency was found to be both donor and recipient dependent. Pulsed-field gel electrophoresis analysis of total SmaI-digested DNA of 48 transconjugants indicated in 27 instances the acquisition of ca. 34 kb of DNA. Two transconjugants harbored two copies of the transposon. Sequencing of the flanking regions of Tn1549-like in 48 transconjugants revealed 29 integration events in 26 loci in the E. faecium genome, and two hot spots for insertion were identified. Integration of the transposon was associated with the acquisition of 5 (n = 18) or 6 (n = 7) bp of donor DNA or with 5-bp duplications of target DNA in the remaining transconjugants. These data demonstrate functionality of the Tn1549-like element and attest that the transfer of the vanB operon between enterococci and human commensal anaerobes occurs in the intestinal environment.

FIG. 1.

Bacterial counts in fecal samples from gnotoxenic mice receiving vancomycin in drinking water. Vancomycin was added step-wise in incrementally increasing amounts (15 μg/ml on day 8 to 120 μg/ml on day 24).

FIG. 2.

Target DNA sequences at the integration sites of Tn1549-like in C. symbiosum MLG101-1, E. faecalis JH2-2, E. faecium 64/3, and E. faecium BM77. Insertion target rows: A, C. symbiosum MLG101-1; T1, E. faecalis JH2-2; T2 to T28, E. faecium 64/3; T29, E. faecium BM77. Coupling sequences are colored, each color corresponds to a specific sequence, and target duplication is indicated by yellow boxes.

FIG. 3.

SmaI PFGE profiles of Enterococcus recipient strains and of the transconjugants. Lanes: 1, 48.5- to 970-kb lambda ladder (Bio-Rad); 2, E. faecalis JH2-2; 3, T1 (JH2-2::Tn1549-like); 4, E. faecium BM77; 5, T29 (BM77::Tn1549-like); 6, E. faecium 64/3; 7, T2 (64/3::Tn1549-like); 8, T10; 9, T7; 10, T4; 11, T5; 12, T6; 13, T25; 14, T11; 15, T12; 16, T13; 17, T14; 18, T21; 19, T17; 20, T19; 21, T20; 22, T24; 23, T15; 24, lambda ladder. Arrows indicate the fragments that varied in size by acquisition of the ca. 34-kb Tn1549-like element. In some cases, comigrating fragments were observed. For T1 and T11, two copies of Tn1549-like were detected.