Cloning and nucleotide sequence of a chromosomally encoded tetracycline resistance determinant, tetA(M), from a pathogenic, methicillin-resistant strain of Staphylococcus aureus.

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription. Images

We have studied MRSA101, a methicillin-resistant strain of Staphylococcus aureus which was obtained from the peripheral blood of a pediatric patient with acquired immune deficiency syndrome and staphylococcal sepsis in a New York City hospital. We report here the isolation of a tetracycline resistance determinant from MRSA101 and show that this gene belongs to the tet(M) class of tetracycline resistance determinants. We have also demonstrated by Northern (RNA) blot analysis that the tet(M) gene is inducible by subinhibitory concentrations of tetracycline at the level of transcription.
Plasmid and chromosomal DNAs were isolated as described elsewhere (13), and three plasmid bands of 3.0, 8.0, and >20 kb were visible on agarose gels (results not shown). Protoplast transformation of S. aureus 8325-4 (10) revealed that chloramphenicol resistance was associated with the 3.0-kb plasmid, which had the same restriction enzyme digestion pattern as pC194 (2). The >20-kb plasmid encoded penicillin, cadmium, and arsenate resistances, phenotypes associated with the S. aureus penicillinase plasmid p1524 (11); the 8.0-kb plasmid was apparently cryptic. The remaining resistances are therefore chromosomally encoded.
Cloning and sequencing the tetracycline gene from MRSA101. Plasmids used in this study are listed in Table 1.   tionated fragments between 2 and 5 kb were obtained from a 5 to 20% neutral sucrose gradient and cloned into the Escherichia coli cloning vehicle pUC8 at the BamHI site and transformed into E. coli JM107 (21). The pUC9-MRSA101 library was screened for tetracycline-resistant transformants, and one of several resistant colonies was isolated. This strain carried a recombinant plasmid, pMVN6, which carried a 2.9-kb insert. Cleavage with MboI yielded insert fragments of 1.6 and 1.3 kb. These two fragments were gel purified (7), pooled, and used as a probe for Southern blot hybridizations (17) of MRSA101 whole-cell DNA to verify that the cloned DNA actually originated from MRSA101. The probe hybridized to a single EcoRI fragment, two HindlIl fragments, and two MboI fragments (Fig. 1, lanes 4  through 6, respectively). However, this probe did not hybridize to pT181 (Fig. 1, lanes 2), a staphylococcal plasmid which carries the tet(K) gene (nor did the probe hybridize to any of the plasmids from MRSA101 [data not shown]). The probe did hybridize to pAM120, a plasmid carrying Tn916 (Fig. 1, lanes 3) a conjugative transposon from Enterococcus faecalis which carries a tet(M) tetracycline resistance determinant (16). The cloned insert from pMVN6 was isolated on a 2.9-kb SmaI-Hindll fragment (sites in pUC8) and ligated (with T4 ligase from U.S. Biochemical, Cleveland, Ohio) into the ClaI site of the S. aureus plasmid pE194 by blunt-end ligation (5). Protoplasts of S. aureus RN4220 were transformed with selection for tetracycline resistance. As predicted, the tetracycline-resistant transformants recovered carried recombinant plasmids that carried the pMVN6 DNA inserted at the ClaI site of pE194. The 1.3-and 1.6-kb MboI fragments were cloned into M13mpl9 in both orientations (9) and sequenced by the dideoxy-chain termination method (15) with Klenow fragment and/or Sequenase (U.S. Biochemical). Initial sequence data were obtained with an M13mpl9 sequencing primer (U.S. Biochemical). The sequence for each strand was completed by using synthetic oligonucleotide primers based on the previously determined sequences. The entire cloned segment consisted of 2,899 nucleotides (Fig. 2). An open reading frame of 1,917 nucleotides is preceded by a grampositive ribosome-binding site (8). Comparison of the amino acid and nucleotide sequences with those of the tet(M) gene from the streptococcal transposon Tn1545 (6) revealed 92.3% similarity at both amino acid and nucleotide sequence levels. Additionally, there was 95% homology to the tet(M) gene from Ureaplasma urealyticum (14). Therefore, the tetracycline resistance gene from MRSA101 belongs to the tet(M) class, and we have designated it tetA(M) (4). The sequence of the tetA(M) gene is 68.7% AT, which matches the base composition of S. aureus DNA (18).
The possibility that tetA(M) is associated with a Tn916like element was tested by Southern blotting. When a recombinant plasmid carrying Tn916 (pAM120) (16) was used to probe digests of MRSA101 DNA, the probe hybridized to fragments of the same size as the probe (described above) from the S. aureus tetA(M) gene. Additionally, two small MboI fragments of about 800 and 600 bp were observed. This indicates that while the tetA(M) gene may be common to MRSA101 and Tn9O6, at most, 1,400 bp of Tn9O6 DNA is present on MRSA101 in addition to the tetA(M)specific DNA. This amount of Tn916-specific DNA would not be sufficient to encode transposition functions, which require at least 2.5 kb (16). Therefore, if the tetA(M) gene is associated with a transposon, it is probably not similar to Tn916.
Induction of tet4(M) by tetracycline. The MIC of tetracycline for MRSA101 was 25 tig/ml. However, when cells grown in broth were first treated with 0.05 p,g of tetracycline per ml, the MIC increased to 65 ,ug/ml. Therefore, MRSA101 tetracycline resistance was inducible by the antibiotic. To test whether induction involved transcription, the level of tetA(M) mRNA after induction was determined by Northern blot analysis by the method of Kornblum et al. (3). In Figure  3, lane 1 shows RNA from the culture prior to induction (time zero), lane 2 shows RNA from a culture 1 h after induction with 0.05 jig of tetracycline per ml, and lane 3 shows RNA from a noninduced culture grown in parallel with the lane 2 culture. A strand-specific probe was prepared by using the tetA(M) MboI fragment from position 1571 to 2997. The greatly increased amount of tetA(M)-specific RNA in the induced culture indicates that this gene is induced at the level of the transcript. In the uninduced culture, a single, faint band is visible while the induced culture shows two intense bands, suggesting either two transcriptional starts or two transcripts which share a common start but terminate at different places.
FIG. 2. Nucleotide sequence of MRSA101 tet(M). Restriction sites are indicated above the sequence. The amino acid sequence as determined by the DNA sequence is shown under the DNA sequence. Nucleotides which differ from the Tn1545 tet(M) sequence are indicated above the sequence; amino acid residue differences are shown under the amino acid sequence. The opposing arrows indicate a palindromic region in the sequence 5' to the tetA(M) coding sequence. This potential hairpin has a 22-bp perfect inverted repeat and has a predicted free energy of -40.3 kcal (ca. -168.6 kJ) based on the Tinoco rules (19). ANTIMICROB. AGENTS CHEMOTHER.
Nucleotide sequence accession number. The sequence described in this paper has been assigned GenBank accession number M21136.