Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

Abstract

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.

FIG. 1.

PCR detection of tet resistance markers in E. coli clinical isolates. The PCR primer pairs for the detection of tet(A) (A) and tet(M) (B) are presented in Table 1. Templates were prepared from GAR3139 (lane 2), GAR3141 (lane 3), GAR7071 (lane 4), GAR7090 (lane 5), GC7939 (lane 6), GC7940 (lane 7), GC7941 (lane 8), GC7942 (lane 9), GC2270 as a positive control (lane 10), and DH5α (lane 11). As a control for lysate preparation, gel loading, and the PCR conditions, primers for 16S rRNA were included in the assay. Molecular weight standards were loaded in lane 1 for reference.

FIG. 2.

Cloning strategy and schematic diagram of the E. coli tet(M) gene and flanking regions. Locations of primers are indicated with arrows. The IS26 and ISVs1 insertions are also shown diagrammatically along with the tet(M) promoter region.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG.3.

Nucleotide sequence of the E. coli tet(M) gene and flanking regions. The encoded amino acid sequence of tet(M) and the putative transposase are presented by the single-letter code under the respective nucleotide sequence. The deduced amino acid sequence of the IS26 transposase tnpA encoded on the noncoding strand of IS26 is shown in italics for clarity. Transcriptional control elements for tet(M) and the start point for the Tet(M) leader peptide are indicated in boldface. The terminal repeat features for IS26 and ISVs1 are also shown in bold and underlined, and the 8-bp direct repeat sequence for IS26 is presented in bold. The silent point mutation at position 2570 that differentiates the tet(M) sequence from those of strains GAR3139 and GAR3141 is indicated in bold and underlined. DR, direct repeat; ITR-R, right inverted terminal repeat; ORF, open reading frame; ITR-L, left inverted terminal repeat; IRL, inverted repeat left.

FIG. 4.

Phylogenetic tree. An unrooted phylogenetic tree was created from a ClustalW (10) alignment of 24 unique Tet(M) protein sequences. A neighbor-joining tree was drawn by PhyloDraw 0.8 (11). The bar indicates an evolutionary distance of 0.01 amino acid substitution per position. Sequences are derived from Bacillus sp. (GenBank accession no. AAM19211), Clostridium difficile (accession no. AAO24820), Clostridium perfringens (accession no. AAK17952), Clostridium septicum (accession no. BAB71968), E. coli (this paper), E. faecalis 1 (accession no. CAA63530), E. faecalis 2 (accession no. CAA39796), E. faecalis 3 (accession no. CAA27977), Enterococcus faecium (accession no. EAN10521), Erysipelothrix rhusiopathiae (accession no. BAB82500), Gardnerella vaginalis 1 (accession no. AAB05245), Gardnerella vaginalis 2 (accession no. AAB05246), Lactococcus lactis (accession no. AAY62599), L. plantarum (accession no. AAN40886), Neisseria meningitidis (accession no. CAA52967) S. agalactiae (accession no. AAM99809), S. aureus 1 (accession no. AAA26678), S. aureus 2 (accession no. BAB56560), Streptococcus mitis 1 (accession no. CAE46077), S. mitis 2 (accession no. CAE46076), Streptococcus oralis (accession no. CAE46078), Streptococcus pneumoniae 1 (accession no. AAS45561), S. pneumoniae 2 (accession no. AAR22397), and Ureaplasma urealyticum (accession no. AAA73978).

FIG.5.

Amino acid alignment. E. coli Tet(M) was aligned with Tet(M) from L. plantarum (GenBank accession no. AAN40886), N. meningitidis (accession no. CAA52967), S. agalactiae (accession no. AAM99809), E. faecalis (accession no. CAA39796), and S. aureus (accession no. AAA26678). Amino acid residues differing from those of the E. coli protein are shown in boxes in the alignment.

FIG.5.

Amino acid alignment. E. coli Tet(M) was aligned with Tet(M) from L. plantarum (GenBank accession no. AAN40886), N. meningitidis (accession no. CAA52967), S. agalactiae (accession no. AAM99809), E. faecalis (accession no. CAA39796), and S. aureus (accession no. AAA26678). Amino acid residues differing from those of the E. coli protein are shown in boxes in the alignment.