JAC Advance Access originally published online on December 21, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):453-455; doi:10.1093/jac/dkm500
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Research letters |
Identification of tet(S) gene area in tetracycline-resistant Streptococcus dysgalactiae subsp. equisimilis clinical isolates
1 Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, 1 Chang-Te Street, Taipei 100, Taiwan 2 Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine, 1 Jen-Ai Road, Taipei 100, Taiwan 3 Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan 4 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
* Corresponding author. Tel: +886-2-23123456 ext. 6918; Fax: +886-2-23711574; E-mail: ljteng{at}ntu.edu.tw
Keywords: IS1216 , truncated tnpA , chromosome location
While screening the association of IS1216 with antibiotic resistance genes using primers IS1216F(55)1 and IS1216_upR (5'-CATCGTCATTCCTCCTGCTATC-3'), we found a 4.5 kb product from a tetracycline-resistant Streptococcus dysgalactiae subsp. equisimilis clinical isolate, NTUH-3089. DNA sequencing revealed the presence of the tet(S) gene flanked by IS1216 and other open reading frames (ORFs). Since the tet(S) gene has not been previously reported in this species, we further analysed the presence and the genetic environment of tet(S) in clinical isolates of S. dysgalactiae subsp. equisimilis.
First, we screened the presence of tet(S) by PCR from 188 clinical isolates of tetracycline-resistant S. dysgalactiae subsp. equisimilis that were obtained from 1998 to 2004 at the National Taiwan University Hospital, a 2000-bed teaching hospital in Northern Taiwan. Each isolate was from a different patient. The gene tet(S) was present in 12 isolates. PFGE analysis with ApaI indicated that these 12 tet(S)-carrying isolates represented 6 strains. The presence of tet(M), tet(O), tet(L) and tet(K)2,3 was also tested in these six strains but none was positive.
In addition, we identified the tet(S) flanking sequences by the long and accurate (LA)-PCR as described previously.4 Briefly, the digoxigenin-labelled tet(S) amplicon was used as a probe to determine the most suitable restriction enzyme for LA-PCR. After ligating the restriction enzyme-digested DNA fragments with the corresponding cassette adapters, the amplification was performed with cassette primers and target-specific primers. The amplified products were cloned and sequenced. Nucleotide and amino acid sequences were analysed by tools available from the National Center for Biotechnology Information (www.ncbi.nih.gov).
The analysis of tet(S) and its flanking sequences among the six tet(S)-containing strains revealed three types of structure, similar to a fragment located on plasmid pK214 from Lactococcus lactis subsp. lactis5 (Figure 1). The gene structure and contents of these three types are described below:
- Type A element (GenBank accession no. EF679789). This type of structure, found in strain NTUH-3089, consists of two terminal insertion sequences (IS1216), the tet(S) gene and two ORFs as revealed by sequence analysis of a 4.5 kb DNA fragment obtained from amplification with primers IS1216F(55) and IS1216_upR (Figure 1). As seen in plasmid pK214 in L. lactis subsp. lactis, a full-length orf27 was located immediately downstream of the tet(S) gene followed by orf26 orientated in the opposite direction. In comparison with pK214, orf25 is absent and orf26 is truncated in NTUH-3089. The sequence between tet(S) and the right-hand IS1216 in NTUH-3089 was identical to the corresponding region of pK214, except that a 241 bp deletion was found in the former. Further sequencing of the regions outside of the two IS1216s identified two potential ORFs, ORF1 (378 bp) and ORF2 (801 bp), upstream of the left-hand IS1216. The function of ORF1 is unknown whereas ORF2 is predicted to encode a protein with 49% amino acid identity to a hypothetical protein in Streptococcus agalactiae NEM316 (GenBank accession no. CAD46647
[GenBank]
). However, the 3'-terminal sequence of ORF2 extends into the inverted repeat of the left-hand IS1216.
- Type B element (GenBank accession no. EF682210). The type B element was identified in three strains. Like type A, the type B element also contained a full-length orf27. orf26 was less truncated, and there was a truncated tnpA gene upstream of orf26 (Figure 1). No orf25 was detected. Compared with type A, the length of the region between tet(S) and the right-hand IS1216 is slightly shorter due to a larger deletion (437 bp) in type B. Upstream of the truncated tnpA gene, a putative partial gamma gene (5' end is partially sequenced and 3' end is truncated) was found. The predicted peptide shows 98% identity (94% DNA sequence identity) to that of the Streptococcus pyogenes plasmid pSM19035 (GenBank accession no. AY357120
[GenBank]
). A truncated aadE gene (coding for a putative aminoglycoside 6-adenyltansferase) and a reading frame for a putative protein were found downstream of the right-hand IS1216.
- Type C element (GenBank accession no. EF682209). Type C was identified in two strains (NTUH-1743 and 8940). It contained full-length orf25, orf26 and orf27. The sequence in the region between tet(S) and the right-hand partial tnpA was mostly conserved, with a 20 bp deletion, compared with the corresponding region in pK214 (Figure 1). Both tnpA gene copies in this type were truncated. Upstream of the left-hand truncated tnpA, we found a partially sequenced aadE gene. A 147 bp orfA was identified downstream of the right-hand truncated tnpA. This orfA was predicted to encode a putative ATPase protein sharing 44% amino acid identity with a chromosome segregation ATPase of S. pyogenes MGAS2096 (GenBank accession no. YP_600067).
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The nucleotide sequences of the tet(S) genes in these six strains were identical, and were nearly identical to that in pK214 with only a two nucleotide difference (G and C at positions 100 and 101, respectively), resulting in an amino acid exchange of arginine in pK214 versus alanine in S. dysgalactiae subsp. equisimilis. Diversity in the length of IS1216 or the transposase gene was found.
The chromosomal location of the tet(S) element in these strains was shown by hybridization of I-CeuI digested DNA with a 16S rRNA gene probe and the tet(S) probe. The bands recognized by the tet(S) probe were also recognized by the 16S rRNA probe and thus suggested a chromosomal location.
The nucleotide sequences of the three types of tet(S)-containing structure were deposited in GenBank with accession nos EF679789, EF682210 and EF682209.
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This work was supported by a grant NSC 94-2320-B-002-095 from the National Science Council of Taiwan.
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None to declare.
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1 Tsai JC, Hsueh PR, Chen HJ, et al. The erm(T) gene is flanked by IS1216V in inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus. Antimicrob Agents Chemother (2005) 49:4347–50.
2
Brenciani A, Ojo KK, Monachetti A, et al. Distribution and molecular analysis of mef(A)-containing elements in tetracycline-susceptible and -resistant Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance. J Antimicrob Chemother (2004) 54:991–8.
3 Ng LK, Martin I, Alfa M, et al. Multiplex PCR for the detection of tetracycline resistant genes. Mol Cell Probes (2001) 15:209–15.[CrossRef][Web of Science][Medline]
4
Teng LJ, Hsueh PR, Wang YH, et al. Determination of Enterococcus faecalis groESL full-length sequence and application for species identification. J Clin Microbiol (2001) 39:3326–31.
5 Perreten V, Schwarz F, Cresta L, et al. Antibiotic resistance spread in food. Nature (1997) 389:801–2.[Medline]
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