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Abstract

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in e, are encoded by (A) and (A)- 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 () determinants (A) and (A)-, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of (A)- was analysed. The start codons in class A 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 10-148 containing (A)- plasmid pHS27 to J53 by conjugation. The transformants harbouring recombinant plasmids that carried (A) or (A)- exhibited tetracycline MICs of 256–512 µg ml, with or without (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 following GTG mutation, and to ≤4 µg ml following mutation of both GTG and ATG. It was presumed that class A 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 determinants can confer high-level tetracycline resistance and have two start codons.

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2014-11-01
2021-10-16
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