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J Med Microbiol. 2014 Nov;63(Pt 11):1454-9. doi: 10.1099/jmm.0.078063-0. Epub 2014 Aug 7.

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

Author information

1
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China Institute of Biomedical Science, Fudan University, Shanghai, PR China.
2
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.
3
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China Institute of Biomedical Science, Fudan University, Shanghai, PR China mgwang@fudan.edu.cn.

Abstract

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons.

PMID:
25102906
DOI:
10.1099/jmm.0.078063-0
[Indexed for MEDLINE]

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