The erm(T) gene is flanked by IS1216V in inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus

Abstract

We investigated the sequence and the genetic context of the erm(T) gene in six inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus (formerly S. bovis biotype II.2) isolates. In all isolates, the erm(T) genes were flanked by two IS1216V-like elements with the same polarity and were found to be inserted in the chromosome.

FIG. 1.

Genetic organization of erm(T) resistance element and flanking regions in S. gallolyticus subsp. pasteurianus NTUH-7421. (A) Southern blot hybridization of erm(T) probe to S. gallolyticus subsp. pasteurianus NTUH-7421 genomic DNA after digestion with restriction enzymes ClaI, DdeI, HaeIII, HindII, and EcoRI (lanes 1 to 5, respectively). Lane M, DNA marker (digoxigenin-labeled DNA Molecular Weight Marker II′ [Roche]). (B) Genetic organization of erm(T) resistance element and flanking regions in S. gallolyticus subsp. pasteurianus NTUH-7421. Arrows represent putative open reading frames. The restriction sites are also shown.

FIG. 2.

Alignment of the leader peptide-encoding sequences of erm(T) in S. gallolyticus subsp. pasteurianus NTUH-7421, erm(GT) in Lactobacillus species (GenBank accession number M64090), and staphylococcal erm(C) (GenBank accession number M17990). A dash indicates an identical nucleotide and a dot indicates a gap. TAA (stop codon) is underlined.

FIG. 3.

Southern blot hybridization of erm(T) and IS1216V probe to S. gallolyticus subsp. pasteurianus strains. (A) Hybridization with an erm(T)-specific probe. Lanes 1 to 6 show erm(T)-positive isolates. Lane 1, NTUH-7421; lane 2, NTUH-8819; lane 3, NTUH-7499; lane 4, NTUH-3004; lane 5, NTUH-1043; lane 6, NTUH-4807; lanes 7 and 8, erm(T)-negative clinical isolates NTUH-1443 and NTUH-4046, respectively; lane 9, S. gallolyticus subsp. pasteurianus ATCC 43144; lane M, DNA marker (digoxigenin-labeled DNA Molecular Weight Marker II′ [Roche]). (B) Hybridization with IS1216V-specific probe. Lanes 1 to 6 and 8 to 13, erm(T)-positive isolates, as in lanes 1 to 6 in panel A. Lanes 7 and 14, erm(T)-negative clinical isolate NTUH-1443.

FIG. 4.

Localization of erm(T) on I-CeuI-generated chromosome fragments of S. gallolyticus subsp. pasteurianus isolates. (A) I-CeuI fragment restriction patterns separated by pulsed-field gel electrophoresis. (B) Hybridization with a probe specific for the erm(T) gene. (C) Hybridization with a 16S rRNA gene-specific probe. Lane M, molecular size standard (Saccharomyces cerevisiae chromosomal DNA). Lanes 1 to 6 show erm(T)-positive isolates. Lane 1, NTUH-7421; lane 2, NTUH-8819; lane 3, NTUH-7499; lane 4, NTUH-3004; lane 5, NTUH-1043; lane 6, NTUH-4807; lanes 7 and 8, erm(T)-negative clinical isolates NTUH-1443 and NTUH-4046, respectively.