Widespread distribution of a tet W determinant among tetracycline-resistant isolates of the animal pathogen Arcanobacterium pyogenes

Abstract

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.

FIG. 1.

Restriction maps of plasmids containing the tet(W) genes of strains 4 (pJGS259) and BBR1 (pJGS279). pJGS264, the NotI deletion derivative of pJGS259 that does not confer tetracycline resistance in E. coli, is also shown. Only the A. pyogenes-derived insert is shown for each plasmid. The large arrows indicate the positions of ORFs, which are labeled. The oriT upstream of mob is indicated by the solid rectangle. Restriction enzyme sites are as follows: B, BglII; Bm, BamHI; C, ClaI; E, EagI; N, NotI; Nd, NdeI; P, PstI; Sa, Sau3AI; S, SacI; Sc, ScaI; V, EcoRV; and X, XhoI. Coordinates (in kilobases) are given below the pJGS279 map.

FIG. 2.

Dot blot hybridization of tetracycline-resistant (Tet^r) and tetracycline-susceptible (Tet^s) isolates with a tet(W)-specific probe. Approximately 500 ng of genomic DNA from 20 tetracycline-resistant and 10 tetracycline-sensitive isolates was spotted onto a nylon membrane in the arrangement indicated and hybridized with the 1,246-bp tet(W)-specific probe.

FIG. 3.

(A) Amino acid sequence alignment of the A. pyogenes Mob protein (Ap Mob) with the clostridial TnpZ proteins from Tn4451 of Clostridium perfringens (Cp TnpZ) (11) and Tn4453a of C. difficile (Cd TnpZ) (20). Amino acids identical to those in Mob are boxed. Amino acid numbers are shown on the right. (B) Nucleotide sequence comparison of the oriT upstream of mob with the oriT of Tn4451. The oriT of Tn4451 and that of Tn4453a are identical (11, 20). Identical nucleotides are boxed, and the region of dyad symmetry is indicated by the divergent arrows.

FIG. 4.

Dot blot hybridization of tetracycline-resistant (Tet^r) and tetracycline-susceptible (Tet^s) strains with a mob-specific probe. Approximately 500 ng of genomic DNA from 20 tetracycline-resistant and 10 tetracycline-sensitive isolates was spotted onto a nylon membrane in the arrangement indicated and hybridized with the 728-bp mob-specific probe.