Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Abstract

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

FIG. 1.

eBURSt diagram (26) showing the relationships between the S. agalactiae bovine strains (appearing as black triangles on the figure) analyzed by MLST (31).

FIG. 2.

Nested-PCR analysis of the excision of two ICEs detected in bovine S. agalactiae strains. The principle of the detection of the circular form of ICEs by nested PCR using two sets of primers (one set of outer primers in black and one set of inner primers in gray) is indicated (A), as well as the results of nested PCR to detect the circular form of ICE-tRNA^Lys in 9 strains in parallel with a positive control (S. agalactiae strain 515) (B) and of ICE-rplL in 18 strains in parallel with a positive control (S. agalactiae strain 2603 V/R) (C).

FIG. 3.

Characterization of triple-resistant (rifampin, streptomycin, and tetracycline) transconjugant clones obtained by conjugal transfer of Tn916 from the different donor cells tested to recipient strain S. agalactiae Nem316. TC Sag1667 and TC Sag2584, transconjugants obtained using S. agalactiae strains 1667 and 2584, respectively, as donor cells; TC Sub20549 and TC Sdy20402, transconjugants obtained using S. uberis 20549 and S. dysgalactiae 20402, respectively, as donor cells. PCRs were done with primers specific for the S. agalactiae species (first lane), primers specific for a genetic element found in the Nem316 recipient strain (second lane), primers specific for the tet(M) gene of Tn916 (third lane), and five sets of primers for the multiple-locus variant repeat assay (fourth lane).