16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori

Abstract

Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet(r)) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tet(s) strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tet(r) strain 181, the Tet(s) strain 26695, and four Tet(r) 26695 transformants showed that a single triple-base-pair substitution, AGA(926-928)-->TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet(r) H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet(r) strain 181.

FIG. 1.

Schematic representation of the 16S rRNA genes of H. pylori. Alignment of the 16S rRNA genes (rrnA and rrnB) from the Tet^r strain 181, the Tet^s strain 26695, and four Tet^r 26695 transformants is shown. Sequence analysis of the 16S rRNA genes revealed only a few base pair substitutions (numbering according to 16S rrnA of H. pylori strain 26695) in the Tet^r strain 181 that did not occur in the Tet^s strain 26695. For the identification of the 16S rRNA region required for tetracycline resistance, the Tet^s strain 26695 was transformed with smaller overlapping PCR fragments of the 16S rRNA gene of the Tet^r strain 181 (only fragments containing mutations are shown). The transformants were selected on tetracycline (2 μg/ml)-containing Dent plates. Primers used for the amplification of the smaller overlapping PCR fragments started at the outside of the fragment and each had a length of 20 bp.

FIG. 2.

Both 16S rRNA genes are mutated in H. pylori tetracycline resistance. rrnA- and rrnB-specific sequences were amplified using specific primers based on sequences which are found outside the two 16S rRNA genes. The rrnA-specific primers, F1 and F2, are located at position 1207020 and 1207242 (numbers corresponding to the H. pylori 26695 sequence [23]), respectively, and the rrnB-specific primers, F3 and F4, are located at position 1510569 and 1510809, respectively. For amplification, primer R1 (located at position 1208293 and 1511828) was used in combination with one of the other primers. All primers had a length of 20 bp.

FIG. 3.

Schematic representation of the primary binding site of tetracycline, based on the 16S rRNA structure of Thermus thermophilus proposed by Wimberly et al. (29). The primary binding pocket for tetracycline is formed by the 16S rRNA residues 1054 to 1056 (box A) and residues 1196 to 1200 (box B) of helix 34 and residues 964 to 967 of helix 31 (box C). The interactions between tetracycline and this pocket are formed by hydrophobic interactions, hydrogen bonds, and salt bridges (3). The triple-base-pair substitution AGA_926-928→TTC (corresponding to bp 965 to 967 of E. coli 16S rRNA) is located in box C and is indicated by asterisks.