Identification of tet(B), encoding high-level tetracycline resistance, in Neisseria meningitidis.

Abstract

A Neisseria meningitidis strain, NIID129, with high-level resistance to tetracycline (Tetr) was isolated from a Japanese woman's nasopharynx; the MIC of tetracycline (Pfizer Pharmaceuticals Inc.) was 32 μg/ml on Muller-Hinton agar (Difco) as determined by the microdilution method performed in accordance with NCCLS guidelines (6) and 24 μg/ml when measured by the Etest (AB Biodisk). In order to detect the gene specifying the Tetr determinant, a chromosomal DNA library of NIID129 was constructed in vector pMW118 with an ampicillin resistance marker (Toyobo) and transformed into Escherichia coli K-12 derivative DH5α to isolate tetracycline-resistant colonies. One tetracycline-resistant transformant that grew on L agar plates containing 15 μg of tetracycline per ml contained plasmid pMW118 with a 2.3-kb insert designated pHT230. The 2.3-kb DNA fragment and its flanking region on the chromosome were sequenced with a DNA analyzer (ABI PRISM 310; Applied Biosystems) (DDBJ accession numbers {type:entrez-nucleotide,attrs:{text:AB084245,term_id:22450181,term_text:AB084245}}AB084245 and {type:entrez-nucleotide,attrs:{text:AB084246,term_id:22450183,term_text:AB084246}}AB084246) (8) and found to contain homologues of tet(B) and tetR(B) of Tn10 (Fig. ​(Fig.1a);1a); the nucleotide sequence of tet(B) of NIID129 was 99.8% identical to that of Tn10, and that of tetR(B) of NIID129 was 97% identical to that of Tn10. The last four deduced amino acids at the C-terminal end of TetR(B) of NIID129 were quite different from those of Tn10 (Fig. ​(Fig.1b).1b). The function of TetR(B) of NIID129 seems to be inactive since the expression of tetracycline resistance was not inducible but constitutive (data not shown). Only a 30-bp sequence downstream of tet(B) was identical to the corresponding sequence of Tn10, and beyond this, the sequence was identical to open reading frame NMB0217 (putative rpoN gene; GenBank accession number {type:entrez-nucleotide,attrs:{text:AE002379,term_id:7225435,term_text:AE002379}}AE002379) of N. meningitidis strain MC58 (Fig. ​(Fig.1c).1c). Downstream of the tetR(B) gene was an open reading frame identical to NMB0216 (GenBank accession number {type:entrez-nucleotide,attrs:{text:AE002379,term_id:7225435,term_text:AE002379}}AE002379) of N. meningitidis strain MC58. Thus, NIID129 contained only tetR(B)-tet(B) genes homologous to Tn10 but not other Tn10-associated genes or insertion elements necessary for transposition. FIG. 1. Genetic structure of a Tetr determinant region of NIID129.(a) Physical map of the tetR(B)-tet(B) genes and their flanking regions. Percentages indicate the nucleotide identity of the tetR(B) and tet(B) genes of NIID129 with those of Tn10. Arrows A and ... Tetracycline-resistant strains of N. gonorrhoeae have been frequently isolated that contained the tet(M) gene located on the 25-MDa plasmid (2, 4, 5). Some N. meningitidis strains and others in related genera have also acquired the plasmid and show tetracycline resistance (1, 3, 7, 9). However, no tetracycline resistance determinants other than tet(M) have been found in neisseriae, including N. meningitidis. As far as we know, this is the first report of high-level tetracycline resistance due to the tet(B) gene on the chromosome of N. meningitidis. Although the example of strain NIID129 may be rare, clinical workers should be aware of the appearance of a new tetracycline resistance determinant, Tet(B), in N. meningitidis.