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Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 2003 Sep;47(9):2984–2986. doi: 10.1128/AAC.47.9.2984-2986.2003

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

Monique M Gerrits 1, Marco Berning 1, Arnoud H M Van Vliet 1, Ernst J Kuipers 1, Johannes G Kusters 1,*
PMCID: PMC182601  PMID: 12937008

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.


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.

TABLE 1.

Primers used in this study

Primer Sequence (5′-3′)a Position relative to 16S rRNA geneb 16S rRNA mutationc
Sequencing, cloning, and PCR
    Hp16S-F1 CTGACGCTGATTGCGCGAAA 711-730, forward NA
    Hp16S-F2 CCTGCTGGAACATTACTGAC 696-715, forward NA
    Hp16S-F3 TTTATGGAGAGTTTGATCCT 1-20, forward NA
Hp16S-F4 TAGTTGTTGGAGGGCTTAGT 789-808, forward NA
    Hp16S-R1 TCGTTGCGGGACTTAACCCA 1071-1052, reverse NA
    Hp16S-R2 TGGCTCCACTTCGCAGTATT 1245-1226, reverse NA
    Hp16S-R3 AGGAGGTGATCCAACCGCA 1499-1480, reverse NA
    Hp16S-rrnA CCCAAATCCTTGAGCGTTTA 1840-1821, reversed NA
    Hp16S-rrnB CGCATTCATAATCAGCTCAG 1826-1807, reversee NA
Mutagenesis
    Hp16S-mutF1 TAATTCGATGATACACGAAG 918-937, forward TGA
    Hp16S-mutF2 TAATTCGAATATACACGAAG 918-937, forward ATA
    Hp16S-mutF3 TAATTCGAAGCTACACGAAG 918-937, forward AGC
    Hp16S-mutF4 TAATTCGATTATACACGAAG 918-937, forward TTA
    Hp16S-mutF5 TAATTCGATGCTACACGAAG 918-937, forward TGC
    Hp16S-mutF6 TAATTCGAATCTACACGAAG 918-937, forward ATC
    Hp16S-mutF7 TAATTCGATTCTACACGAAG 918-937, forward TTC
    Hp16S-mutR1 CTTCGTGTATCATCGAATTA 937-918, reverse TGA
    Hp16S-mutR2 CTTCGTGTATATTCGAATTA 937-918, reverse ATA
    Hp16S-mutR3 CTTCGTGTAGCTTCGAATTA 937-918, reverse AGC
    Hp16S-mutR4 CTTCGTGTATAATCGAATTA 937-918, reverse TTA
    Hp16S-mutR5 CTTCGTGTAGCATCGAATTA 937-918, reverse TGC
    Hp16S-mutR6 CTTCGTGTAGATTCGAATTA 937-918, reverse ATC
    Hp16S-mutR7 CTTCGTGTAGAATCGAATTA 937-918, reverse TTC
a

Primers used for amplification were based on the published genome sequence of H. pylori strain 26695 (11). The substituted residues are underlined.

b

Positions of oligonucleotides are given relative to the start point of the rrnA gene of H. pylori strain 26695 (11).

c

16S rRNA mutations at positions 926 to 928 (numbering according to sequence of the rrnA gene of H. pylori strain 26695) (11). The substituted residues are underlined. NA, not applicable.

d

The rrnA-specific oligonucleotide is located outside the 16S rRNA gene at positions 1207242 to 1207261 of the H. pylori 26695 genome sequence (4, 11).

e

The rrnB-specific oligonucleotide is located outside the 16S rRNA gene at positions 1510809 to 1510828 of the H. pylori 26695 genome sequence (4, 11).

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.

TABLE 2.

Characterization of mutants construction by using site-directed mutagenesis

Strain or mutanta MIC (μg/ml) of TET atb:
Growth rate
t0c t20
On TET-free medium On TET (1μg/ml) On TET-free mediumd On TET (1 μg/ml)e
Wild-type strain 26695 0.19 0.19 0.19 +++ No growth
TGAf ND ND ND ND ND
ATA 1.5 1.5 2.0 +++ +
AGCg 1.5 6.0 6.0 +++ +
TTA 1.5 2.0 2.0 +++ ++
TGC 1.0 1.5 2.0 +++ +
ATC 2.0 2.0 3.0 +++ ++
TTC 6.0 6.0 8.0 +++ +++
a

The substituted residues are underlined.

b

MIC were determined by using E-test for two independent mutants. MIC shown are means of results from four to six independent experiments. TET, tetracycline; ND, not determined.

c

At t0 all mutants were already subcultured twice in order to obtain sufficient bacteria for storage at −80°C.

d

Growth rates of the isogenic mutants relative to the growth rate of the wild-type strain 26695 (100%). +++, 95 to 100%; ++, 70 to 95%; +, 50 to 70%.

e

Growth rates of the isogenic mutants containing single- and double-base-pair mutations relative to the growth rate of the isogenic mutants containing the triple-base-pair mutation TTC (100%). +++, 95 to 100%; ++, 70 to 95%; +, 50 to 70%.

f

The TGA sequence had already changed into the TGC sequence during subculturing before storage.

g

Each of the tested mutants contained an AGC mutation in the rrnA gene and a TTC mutation in the rrnB gene.

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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