Effects of 16S rRNA Gene Mutations on Tetracycline Resistance in Helicobacter pylori

Abstract

The triple-base-pair 16S rDNA mutation AGA 926-928 3TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single-and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence of tetracycline, explaining the preference for the TTC mutation in tetracycline-resistant H. pylori isolates.Tetracycline is a cheap and effective antibiotic for the treatment of Helicobacter pylori infections (7,8), but in the past few years the incidence of tetracycline resistance has significantly increased (1,5,6,9,13). The only known mechanism mediating tetracycline resistance in H. pylori involves mutations at positions 926 to 928 in both the 16S rRNA genes (2,4,12). H. pylori strains with high-level tetracycline resistance (Tet r ) carried the triple-base-pair mutation AGA 926-928 3TTC in both copies of the 16S rRNA genes (4, 12), whereas strains with low-level Tet r contained only single-and double-base-pair mutations in the exact same region (2). As the different mutations were present in unrelated strains, it is still unclear whether high-level tetracycline resistance requires the AGA 926-928 3 TTC mutation or whether single-or double-base-pair mutations at these positions may suffice for high-level tetracycline resistance. Therefore, we have created all possible combinations of single, double, and triple mutations at the 16S rRNA gene positions 926 to 928 in H. pylori strain 26695 and have determined the effects of the mutations on levels of tetracycline resistance, stability, and growth rate.Site-directed mutagenesis at positions 926 to 928 was carried out by using a three-step PCR approach (3, 10) with primers listed in Table 1, followed by natural transformation to H. pylori reference strain 26695 (4). Tet r H. pylori colonies were selected on plates containing tetracycline (1 g/ml). For each possible base pair mutation or combination thereof, eight Tet r transformants from at least two independent transformation experiments were selected. Both alleles of the 16S rRNA genes were amplified by PCR (4) to confirm the presence of the desired base pair mutations. With the exception of the AGC mutants, all mutants contained the desired mutations in both alleles of the 16S rRNA genes. All AGC mutants were heterozygous and contained the AGC mutation in the rrnA gene and a TTC mutation in the rrnB gene.The effects of the 16S rRNA mutations on both the stability and the level of tetracycline resistance were determined by subculturing two mutants of each type for 20 passages on Columbia agar plates supplemented with 7% lysed horse blood (BioTrading, Mijdrecht, The Netherlands) in either the presence or absence of tetracycline (1 g/ml). After each five rounds of subculturing, the MIC of tetracycline was determined by using E-test (AB Biodisk, Solna, Sweden) (4). In addition, for all mutants at time point zero (t 0 ) and after passage 20 (t 20 ), the 16S rRNA genes were sequenced. The stability of the various types of mutations and their effects on t...

16S rRNA Mutations That Confer Tetracycline Resistance inHelicobacter pyloriDecrease Drug Binding inEscherichia coliRibosomes

“…The incidence of tetracycline-resistant H. pylori has increased recently (12,13,15,16,21,25). All high-level resistant strains have triple nucleotide substitutions at the same positions (965 to 967) in helix 31 of the 16S rRNA, which forms a part of primary tetracycline-binding sites within the 30S subunit.…”

Tetracyclines in veterinary medicine and bacterial resistance to them

“…Recently, other substitution mutations of this triple-nucleotide at the same position in the 16S-rRNA have been detected: AGA → GTA or GGC; and AGA → GGA or AGC as the examples of double or single base change, respectively. However, these single and double substitutions mediated only low-levels of tetracycline resistance (Dailidiene et al, 2002;Gerrits et al, 2003).…”

Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

“…The resistance of H. pylori to tetracyclines is reported to be caused by mutations in the 16S rRNA. H. pylori isolates exhibiting AGA 926-928 3 TTC triple-base-pair mutations (4,5,22) show MICs higher than 4 mg/liter and probably represent the clinically most relevant strains, whereas single-or double-base-pair mutations were, rather, associated with MICs between 1 mg/liter and 4 mg/liter (3, 5).The susceptibility of H. pylori to tetracycline is routinely examined by agar diffusion (Etest) or agar dilution tests, which are accepted to be the "gold standards" (10). These methods are slow and time-consuming, and they sometimes fail due to a lack of growth of the infecting H. pylori strain or due to overgrowth with contaminating bacteria.…”