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Sir, During the course of a study on transferable antimicrobial resistance in uropathogenic Escherichia coli from humans in Nigeria, an 8.4 kb plasmid, designated pTOJO1, was identified (by transformation experiments) to mediate resistance to tetracyclines, sulphonamides and streptomycin. PCR and sequence analysis identified the resistance genes tet(A), sul2 and strA on this plasmid.1,2 Restriction mapping revealed that—except for the tetracycline resistance gene region—pTOJO1 closely resembled the sulphonamide/streptomycin resistance plasmid pSSOJO1 (Figure 1a), which had been detected in E. coli isolates from different hospitals at different locations in the south-western states of Nigeria.3 Based on the comparison of the two plasmids, it was assumed that pTOJO1 might have developed from a pSSOJO1-like ancestor plasmid into which a Tn1721-like tetR-tet(A) area had integrated. For confirmation of this hypothesis, the sequences of Tn1721 and plasmids carrying Tn1721-like tetR-tet(A) genes1 were compared with the tetR-tet(A) gene area of plasmid pTOJO1. The sequence downstream of tetR showed closest homology to that of RP1, a conjugative 56 kb plasmid from E. coli.4 Furthermore, the 0.96 kb EcoRV-DraI fragment of pSSOJO1 (Figure 1a), which contained the potential integration sites for the tet gene area, was determined. Comparison of the pTOJO1 sequence with the corresponding sequences of RP1 and pSSOJO1 revealed the presence of two potential recombination sites of 13 bp (recombination site A) and 16 bp (recombination site B) at the junctions of RP1-homologous and pSSOJO1-homologous sequences in pTOJO1 (Figure 1a). Similarity between pTOJO1 and RP1 was restricted to the 2354 bp segment located between the recombination sites A and B, whereas that between pTOJO1 and pSSOJO1 was seen upstream of recombination site A and downstream of recombination site B. This observation strongly supported the hypothesis that plasmid pTOJO1 was the product of a recombination between an RP1-like tetracycline resistance plasmid and a pSSOJO1-like sulphonamide/streptomycin resistance plasmid. A 206 bp segment that was located between the two recombination sites in pSSOJO1 was not detectable in the pTOJO1 sequence and, therefore, was assumed to be lost during this recombination process. Molecular analysis of the tetracycline resistance gene area of pTOJO1 revealed that the recombination site B was located in the 3′-terminal part of the tet(A) gene. As a consequence, the final nine codons were changed and an alternative translational termination codon was generated. The pTOJO1 sequence was the first tet(A) sequence deposited in the databases that described such a variation in the 3′ end of the tet(A) gene. Since then three more sequences obtained from plasmid-borne tet(A) genes of Shigella sonnei (database accession numbers AF497970, AF502943 and AF534183) showing exactly the same alteration have been deposited in the databases. This alteration of the 3′-end of the tet(A) reading frame had no negative effect on the biological activity of the TetA protein, as assumed from MICs of tetracycline ≥128 mg/L seen in the original E. coli isolate and the pTOJO1-carrying transformant. Similar observations were made with the truncated, but functionally active tet(H) gene of plasmid pPAT1, where the tet(H) reading frame was shortened by 24 bp due to a recombinational event.5 Comparative analysis of the deduced amino acid sequences of TetA proteins deposited in the databases revealed that the TetA protein from pTOJO1 consisted of 398 amino acids instead of the TetA proteins of 399 amino acids previously found in E. coli (X00006, X61367, L27758, AJ313332 and AJ419171), Aeromonas salmonicida (AJ517790), Pseudomonas aeruginosa (X75761), Acinetobacter baumannii (AY196695) or Salmonella Enteritidis (AF542061). However, the reading frames of the tet(A) genes from S. sonnei were reported to have a 75 bp extension at their 5′-end. Since there are no sequences that resemble promoter structures or ribosome binding sites upstream of these alternative translational start codons, it is most likely that the tet(A) genes of S. sonnei will also start with the common GTG start codon and that the corresponding TetA proteins will consist of 399 amino acids instead of 424 amino acids. The loss of one codon in TetA of pTOJO1 resulted from the deletion of three single base pairs in the region between codons 194–198 of TetA from Tn1721 (Figure 1b). The deletion of the first two base pairs caused frame shifts in the tet(A) reading frame, whereas the deletion of the third base pair reconstructed the original reading frame, however, with the loss of one codon. A similar alteration that affected the three consecutive codons at positions 201–203 had been detected in TetA of RP1 (Figure 1b).4 The Tet proteins of Gram-negative bacteria (including TetA) that code for membraneassociated efflux systems are composed of two domains, each consisting of six transmembrane regions that are connected by an interdomain region of variable length.6 The codons affected by the sequence alterations seen in the TetA proteins of pTOJO1 and RP1 are both located in this interdomain region. This might explain why such alterations have no effect on the activity of the respective Tet proteins.

the journal of antimicrobial chemotherapy/journal of antimicrobial chemotherapy

Structural analysis of the tetracycline resistance gene region of a small multiresistance plasmid from uropathogenic Escherichia coli isolated in Nigeria