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Antimicrobial Agents and Chemotherapy, September 2003, p. 2984-2986, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.2984-2986.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Monique M. Gerrits, Marco Berning, Arnoud H. M. Van Vliet, Ernst J. Kuipers, and Johannes G. Kusters*

Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands

Received 3 February 2003/ Returned for modification 12 May 2003/ Accepted 18 June 2003


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ABSTRACT
 
The triple-base-pair 16S rDNA mutation AGA926-928->TTC 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.


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TEXT
 
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 (Tetr) carried the triple-base-pair mutation AGA926-928->TTC in both copies of the 16S rRNA genes (4, 12), whereas strains with low-level Tetr 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 AGA926-928-> 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). Tetr H. pylori colonies were selected on plates containing tetracycline (1 µg/ml). For each possible base pair mutation or combination thereof, eight Tetr 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.


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  		TABLE 1. Primers used in this study

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 µg/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 (t0) and after passage 20 (t20), the 16S rRNA genes were sequenced. The stability of the various types of mutations and their effects on the levels of resistance are summarized in Table 2.


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  		TABLE 2. Characterization of mutants construction by using site-directed mutagenesis

At t0, the single- and double-base-pair mutations did not result in tetracycline resistance with levels of clinical relevance, as the MIC of tetracycline was below 4 µg/ml. With the exception of those for the AGC mutants, the MICs of tetracycline at t20 were similar to those at t0 (Table 2). The MIC of tetracycline for the AGC mutants increased from 1.5 to 6.0 µg/ml. Subsequent analysis of the 16S rRNA gene sequences of all mutants at t0 and t20 revealed that the TGA and AGC mutations were unstable. Already during the two subculturing steps needed for propagation of the colonies to obtain sufficient material for storage at -80°C, the sequences of both 16S rRNA genes of the TGA mutants had changed to TGC, and while stable during the initial propagation steps, after 20 rounds of subculturing both AGC mutants contained a TTC sequence at positions 926 to 928 within both 16S rRNA genes instead of only in the rrnB gene. Apart from these TGC and AGC mutations, all other mutations were stable as the complete 16S rRNA gene sequences did not reveal any other sequence changes except for the desired mutations.

To determine whether the 16S rRNA mutations affected the growth rates of the mutants in the presence or absence of tetracycline, all mutants from t0 were cultured in duplicate in brucella broth supplemented with 3% newborn calf serum (Life Technologies Ltd., Auckland, New Zealand). One culture of each mutant was supplemented with tetracycline (1 µg/ml), whereas the other culture was kept unsupplemented. Growth was monitored by measuring the optical density at 600 nm each 24 h for a period of 72 h. In the absence of tetracycline, the growth of the mutants did not differ from that of the H. pylori wild-type strain 26695 (Table 2). However, in the presence of tetracycline the growth of the wild-type strain and the mutants with single- and double-base-pair mutations was clearly reduced compared to that of the TTC mutant. The reduction was most pronounced in the wild-type strain and the AGC, ATA, and TGC mutants (Table 2).

From this study it is apparent that for high-level tetracycline resistance, H. pylori requires the triple-base-pair mutation AGA926-928->TTC in both copies of the 16S rRNA genes. Such high-level tetracycline resistance is most likely generated by a stepwise process that is driven by selection that depends on both the duration of exposure to and the dose of tetracycline. For this reason, it was not surprising that the previously described H. pylori strains with low-level Tetr contained only single- or double-base-pair mutations in the exact same region as the TTC mutation (2). In this report (2) it was also suggested that mutant H. pylori strains with even a small increase in tetracycline resistance have an advantage whenever inhibitory concentrations of tetracycline are encountered. However, it could not be excluded that the observed differences were due to strain differences or secondary mutations. The data presented here have been obtained from mutants with identical genetic backgrounds, thus excluding the effect of strain differences. Taken together, the data indicate that the preference in H. pylori for particular 16S rRNA gene mutations mediating tetracycline resistance results not only from the differences in MICs but also from the differences in growth rates in the presence of tetracycline and from the stability of the mutations.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Gastroenterology and Hepatology, Rm. L459, Erasmus MC-University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 31-10-4632982. Fax: 31-10-4632793. E-mail: j.g.kusters{at}erasmusmc.nl. Back


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Antimicrobial Agents and Chemotherapy, September 2003, p. 2984-2986, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.2984-2986.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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