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Cited by 174Helicobacter pylori-related gastroenterology studiesAntibiotic Resistance in BacteriaBacterial Genetics and Biotechnology

Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

Open access
Trieber, Catharine A., Taylor, Diane E.·2002·Journal of Bacteriology
DOI 10.1128/jb.184.8.2131-2140.2002PMC PMC134973
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Abstract

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H.pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H.pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H.pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H.pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H.pylori.

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Written from the paper’s full text.

TL;DR

Tetracycline resistance in two clinical *Helicobacter pylori* isolates is mediated by mutations in the 16S rRNA genes, especially the triple mutation AGA965-967TTC at the primary tetracycline binding site. Transfer of these mutated 16S rRNA genes by natural transformation confers tetracycline resistance on susceptible strains, while metronidazole resistance is genetically separate.

Key findings

  • The two tetracycline-resistant clinical isolates 108 and 109 had tetracycline MICs of 64 μg/ml; their 16S rRNA genes contained four substitutions in 108 and four substitutions plus two deletions in 109, with both sharing G360A and AGA965-967TTC, and 109 additionally carrying ΔG771 and ΔG942.
  • Natural transformation with the 16S rRNA gene from strain 109 conferred tetracycline resistance on susceptible *H. pylori* 26695, yielding transformants with MICs of 4 to 16 μg/ml; the triple mutation AGA965-967TTC alone conferred resistance, with MICs of 32 to 64 μg/ml in 26695 and 128 μg/ml in NCTC 11639.
  • Metronidazole resistance was not linked to tetracycline resistance: strains 108 and 109 were metronidazole resistant (MICs 64 to 128 μg/ml), but tetracycline-resistant transformants remained metronidazole susceptible, and transfer of *rdxA* conferred metronidazole resistance without tetracycline resistance.
  • Serial passage on increasing tetracycline yielded only modest resistance increases to MICs of 4 to 8 μg/ml, and all such mutants had a deletion of G942; no high-level tetracycline-resistant mutants comparable to the clinical isolates were isolated.

Methods

  • Tetracycline-resistant clinical isolates 108 and 109, susceptible strains 26695 and NCTC 11639, and metronidazole-resistant derivative A12 were grown under microaerobic conditions; MICs were determined by agar dilution and confirmed by the Jeong et al. method.
  • The 16S rRNA genes were PCR amplified, sequenced, cloned into pOK12, and mutated individually by recombinant PCR; mutant genes were introduced into *H. pylori* by natural transformation or electroporation, with selection on tetracycline.
  • A 16S rRNA gene deletion strain, UA1374 (26695 *rrs*::*cat*), was constructed, and several strains were serially passaged on increasing tetracycline concentrations to select resistant mutants.

Limitations

  • Stated: the exact mechanism by which the h31 loop mutations mediate tetracycline resistance was not determined; the authors propose altered tetracycline binding but did not measure ribosome affinity directly.
  • Stated: serial passage did not select high-level tetracycline-resistant mutants comparable to the clinical isolates, and no spontaneous tetracycline resistance mutations were isolated.
  • Evident: the study is based on only two tetracycline-resistant clinical isolates, and initial transformation with the strain 109 16S rRNA gene produced mixed 16S rRNA sequences, complicating interpretation of intermediate resistance levels.

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Crossref · 10.1128/jb.184.8.2131-2140.2002

Cites

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Claims the paper makes

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  • Findingown data
    § Abstract

    The metronidazole resistance trait of the two original isolates was not linked to the tetracycline resistance phenotype.

    “The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype.”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Abstract

    The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates.

    “The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695.

    “Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains.

    “Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains.”
    Quote in the paperSubstantial claim
  • Findingown data
    § RESULTS

    Strain 109 had additional deletions of G771 in domain II and G942 in domain III of the 16S rRNA gene.

    “An additional two nucleotides were deleted in strain 109, G771 in domain II and G942 in domain III.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 43
  • Classified as probably not a substantial claim. 26
  • The quote only partly states this claim. 3

  • May combine two claims.

    Findingown data
    § RESULTS

    Both tetracycline-resistant strains had a mutation of G360 to A in domain I and a triplet mutation of AGA965-967TTC in domain III of the 16S rRNA gene.

    “Both strains had a mutation of G360 to A in domain I and a triplet mutation of AGA965-967TTC in domain III.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    In all cases there was no sequence heterogeneity, indicating that the mutant DNA had recombined and replaced both 16S rRNA genes.

    “In all cases there was no sequence heterogeneity, indicating that the mutant DNA had recombined and replaced both 16S rRNA genes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § RESULTS

    When heavier inocula were tested, the MIC of metronidazole increased two- to fourfold.

    “When heavier inocula were tested, the MIC of metronidazole increased two- to fourfold (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    After serial passage on increasing concentrations of tetracycline, the MICs of tetracycline increased fourfold for all strains (eightfold for 11639) and would go no further.

    “The MICs of tetracycline increased fourfold for all the strains (eightfold for 11639) and would go no further.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION

    Several unique sequence variations in the 16S rRNA genes of strain 109 were identified and placed on a secondary structure model of the H. pylori 16S rRNA.

    “We identified several unique (among H. pylori strains) sequence variations in the 16S rRNA genes of this strain, and these have been placed on a secondary structure model of the H. pylori 16S rRNA in Fig. 2 (indicated by thick arrows).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The deletion strain UA1374 was twofold-more susceptible to tetracycline; the MIC of tetracycline for UA1374 was 0.25 μg/ml, reduced from 0.5 μg/ml for strain 26695.

    “The deletion strain was also twofold-more susceptible to tetracycline; the MIC of tetracycline for UA1374 was 0.25 μg/ml, reduced from 0.5 μg/ml for strain 26695.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data
    § RESULTS

    Only DNA encoding the triple AGA965-967TTC mutation conferred tetracycline resistance on susceptible H. pylori strains (26695 and NCTC 11639).

    “At 4 μg of tetracycline/ml only DNA encoding the triple mutation AGA965-967TTC was able to confer resistance on 26695.”
    • Also stated in: § RESULTS · plain
      “Only DNA containing the AGA965-967TTC mutation was able to confer tetracycline resistance on NCTC 11639 (Table 2).”
    • Also stated in: § DISCUSSION · plain
      “Of the several sequence variations present in the 16S rRNA genes of strain 109 only DNA encoding the triple AGA965-967TTC mutation was found to confer tetracycline resistance on a susceptible strain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    A single sequence was obtained for the 16S rRNA genes of each tetracycline-resistant strain, indicating that the genes in these strains were homogeneous.

    “A single sequence was obtained for the 16S rRNA genes of each tetracycline-resistant strain, indicating that the genes in these strains were also homogeneous.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data
    § RESULTS

    Both the standard method and the method proposed by Jeong et al. gave similar results, with the second method typically giving a value twofold less than the standard method.

    “Both our standard method and the one proposed by Jeong et al. (11) gave similar results, with the second method typically giving a value twofold less than that of our standard method (Table 2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Jeong, J. Y., A. K. Mukhopadhyay, J. K. Akada, D. Dailidiene, P. Hoffman, and D. E. Berg. 2001. Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole. J. Bacteriol. 183:5155-5162. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Findingown data
    § RESULTS

    When plates were incubated for 3 additional days, colonies appeared on the 32-μg/ml tetracycline plates and faint growth was present on plates containing 64 to 128 μg/ml.

    “When plates were incubated for 3 additional days, colonies appeared on the 32-μg/ml tetracycline plates and it appeared that faint growth was present on plates containing 64 to 128 μg/ml.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Abstract

    The two original isolates were also metronidazole resistant.

    “The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data
    § RESULTS

    Strain 109 had six unique nucleotide changes in its 16S rRNA gene, comprising four substitutions and two deletions.

    “For the tetracycline-resistant strains, there were four unique nucleotide changes in strain 108 and six in 109.”
    • Also stated in: § RESULTS · plain
      “An additional two nucleotides were deleted in strain 109, G771 in domain II and G942 in domain III.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Metronidazole was added at 0.5 μg/ml, but no tetracycline-resistant mutants were isolated.

    “Metronidazole was added at 0.5 μg/ml, a concentration just below the MIC (1 to 2 μg/ml) for the strains 26695, 11639, and UA1374, but no tetracycline-resistant mutants were isolated.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    H. pylori 26695 was metronidazole susceptible, with the MIC being 1 to 2 μg/ml.

    “H. pylori 26695 was metronidazole susceptible, with the MIC being 1 to 2 μg/ml.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The triple mutant grew as well as wild-type 26695, and the mutation and tetracycline resistance were retained over several months, several passages, and storage at −80°C.

    “The triple mutant grew as well as did the wild-type 26695, and the mutation and tetracycline resistance were retained over several months, several passages, and storage at −80°C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Transfer of the rdxA genes from strains 108 and 109 to H. pylori 26695 conferred metronidazole resistance on transformants but did not confer tetracycline resistance.

    “Transfer of the rdxA genes from strains 108 and 109 to H. pylori 26695 did confer metronidazole resistance on transformants but did not confer tetracycline resistance.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The MICs of tetracycline for the 16S AGA965-967TTC transformants were 32 to 64 μg/ml; some clones were twofold-more resistant than others.

    “The MICs of tetracycline for the 16S AGA965-967TTC transformants were determined to be 32 to 64 μg/ml; some clones were twofold-more resistant than others were.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given
    § DISCUSSION

    The 16S rRNA triple mutation was not deleterious to the organism's growth and was stably maintained.

    “The 16S rRNA triple mutation was not deleterious to the organism's growth and was stably maintained.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given
    § DISCUSSION

    Mutation of the residues in h31 did not appear to affect the growth rate of H. pylori, but replacement of one of the 16S rRNA genes with the cat gene did.

    “Mutation of the residues in h31 did not appear to affect the growth rate of H. pylori, but replacement of one of the 16S rRNA genes with the cat gene did.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation
    § DISCUSSION

    The residue identified in tetracycline-resistant P. acnes mutants is directly adjacent to the primary binding site, but this mutation did not mediate tetracycline resistance in H. pylori.

    “The residue identified in the tetracycline-resistant P. acnes mutants is directly adjacent to the primary binding site (4, 23, 25), but this mutation did not mediate tetracycline resistance in H. pylori.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [4] Brodersen, D. E., W. M. Clemons, Jr., A. P. Carter, R. J. Morgan-Warren, B. T. Wimberly, and V. Ramakrishnan. 2000. The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103:1143-1154. [DOI] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • cites [23] Pioletti, M., F. Schlünzen, J. Harms, R. Zarivach, M. Glühmann, H. Avila, A. Bashan, H. Bartels, T. Auerbach, C. Jacobi, T. Hartsch, A. Yonath, and F. Franceschi. 2001. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 20:1829-1839. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • cites [25] Ross, J. I., E. A. Eady, J. H. Cove, and W. J. Cunliffe. 1998. 16S rRNA mutation associated with tetracycline resistance in a gram-positive bacterium. Antimicrob. Agents Chemother. 42:1702-1705. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

  • May combine two claims.

    Findingown data
    § DISCUSSION

    Although the MIC of tetracycline for a metronidazole-resistant mutant could be increased to 8 μg/ml, no resistant mutants comparable to strains 108 and 109 were isolated.

    “However, although we could increase the MIC of tetracycline for a metronidazole-resistant mutant to 8 μg/ml, we did not isolate any resistant mutants comparable to strains 108 and 109.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation
    § DISCUSSION

    The triple mutation changed the rRNA from AGA to UUC; the first and last bases were changed to the eubacterial preferred nucleotides, but the middle substitution U966 is found in only 0.15% of eubacterial 16S rRNA genes.

    “The triple mutation changed the rRNA from AGA to UUC; the first and last bases were changed to the eubacterial preferred nucleotides, but the middle substitution (U966) is found in only 0.15% of eubacterial 16S rRNA genes (www.rna.icmb.utexas.edu/CSI).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The eubacterial 16S rRNA sequence conservation database is representative and applicable to H. pylori.

  • May combine two claims.

    Findingown data
    § DISCUSSION

    Once exposed to DNA containing the mutations, H. pylori easily took it up and recombined it into the chromosome, thus acquiring resistance.

    “However, once exposed to DNA containing the mutations they easily took it up and recombined it into the chromosome, thus acquiring resistance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    In one recent study of clinical isolates from Shanghai, China, the level of tetracycline resistance was 59%.

    “In one recent study of clinical isolates from Shanghai, China, the level of tetracycline resistance was an astonishing 59% (39).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Wu, H., X. D. Shi, H. T. Wang, and J. X. Liu. 2000. Resistance of Helicobacter pylori to metronidazole, tetracycline and amoxycillin. J. Antimicrob. Chemother. 46:121-123. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    Tetracycline resistance in H. pylori is relatively rare, with the vast majority of studies reporting no tetracycline resistance in clinical isolates.

    “Tetracycline resistance in H. pylori is relatively rare, with the vast majority of studies reporting no tetracycline resistance in clinical isolates.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] Kim, J. J., R. Reddy, M. Lee, J. G. Kim, F. A. El-Zaatari, M. S. Osato, D. Y. Graham, and D. H. Kwon. 2001. Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J. Antimicrob. Chemother. 47:459-461. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [14] Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A. K. El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3203-3205. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Abstract

    The two Australian isolates were highly resistant to tetracycline, with MICs of ≥256 μg/ml for the antrum isolate and 32 μg/ml for the corpus isolate as determined by E test.

    “These isolates were highly resistant to tetracycline, with the MICs of tetracycline for the strains being ≥256 μg/ml (antrum isolate) and 32 μg/ml (corpus isolate) as determined by E test.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Midolo, P. D., M. G. Korman, J. D. Turnidge, and J. R. Lambert. 1996. Helicobacter pylori resistance to tetracycline. Lancet 347:1194-1195. [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    With the use of tetracycline in therapy, tetracycline-resistant H. pylori isolates have started to emerge at low levels, on average 5 to 7% of isolates.

    “However, with the use of tetracycline in therapy, tetracycline-resistant isolates have started to emerge at low levels, on average 5 to 7% of isolates (12, 14).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] Kim, J. J., R. Reddy, M. Lee, J. G. Kim, F. A. El-Zaatari, M. S. Osato, D. Y. Graham, and D. H. Kwon. 2001. Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J. Antimicrob. Chemother. 47:459-461. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [14] Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A. K. El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3203-3205. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § RESULTS

    H. pylori strains are naturally competent and take up exogenous DNA, incorporating it into the chromosome by homologous recombination.

    “H. pylori strains are naturally competent and take up exogenous DNA, incorporating it into the chromosome by homologous recombination (8).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Ge, Z., and D. E. Taylor. 1997. H. pylori DNA transformation by natural transformation and electroporation, p. 145-152. In C. L. Clayton and H. L. T. Mobley (ed.), Methods in molecular medicine: Helicobacter pylori protocols. Humana Press Inc., Totowa, N.J. [DOI] [PubMed]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given
    § DISCUSSION

    Nucleotide 967 was a C in 85% of sequences and an A in only 13%.

    “Nucleotide 967 was a C in 85% of sequences and an A in only 13%.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The eubacterial 16S rRNA sequence conservation database is representative and applicable to H. pylori.

  • May combine two claims.

    Interpretationcitation
    § RESULTS

    An A118T mutation was seen previously for a metronidazole-susceptible strain, and so the Y141 deletion may be responsible for metronidazole resistance.

    “An A118T mutation was seen previously for a metronidazole-susceptible strain (30), and so it appears that the Y141 deletion may be responsible for resistance.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Solca, N. M., M. V. Bernasconi, and J. C. Piffaretti. 2000. Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains. Antimicrob. Agents Chemother. 44:2207-2210. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

    Study design and conductown data
    § RESULTS

    MIC determinations for strains 108 and 109 were repeated several times and remained stable.

    “MIC determinations were repeated several times and remained stable.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § RESULTS

    Negative controls in which no DNA was added to the cells did not give rise to any colonies.

    “Negative controls in which no DNA was added to the cells did not give rise to any colonies.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The base G966 directly contacts the anticodon loop of the tRNA in the P site.

    “In particular, the base G966 directly contacts the anticodon loop of the tRNA in the P site (40).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Yusupov, M. M., G. Z. Yusupova, A. Baucom, K. Lieberman, T. N. Earnest, J. H. D. Cate, and H. F. Noller. 2001. Crystal structure of the ribosome at 5.5 A resolution. Science 292:883-896. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given
    § RESULTS

    Mutant 16S rRNA genes were cloned and then sequenced to ensure that only the desired mutation was present.

    “Mutant 16S rRNA genes were cloned and then sequenced to ensure that only the desired mutation was present.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § RESULTS

    The reported MICs of tetracycline were >256 μg/ml for strain 108 and 32 μg/ml for strain 109, but these had originally been determined by an alternate method, the E test.

    “The reported MICs of tetracycline were >256 μg/ml for 108 and 32 μg/ml for 109, but these had originally been determined by an alternate method, the E test (18).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Midolo, P. D., M. G. Korman, J. D. Turnidge, and J. R. Lambert. 1996. Helicobacter pylori resistance to tetracycline. Lancet 347:1194-1195. [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § RESULTS

    Previous studies have linked tetracycline and metronidazole resistance and their transfer to susceptible organisms.

    “Previous studies have linked tetracycline and metronidazole resistance and their transfer to susceptible organisms (12, 14, 18).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [12] Kim, J. J., R. Reddy, M. Lee, J. G. Kim, F. A. El-Zaatari, M. S. Osato, D. Y. Graham, and D. H. Kwon. 2001. Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J. Antimicrob. Chemother. 47:459-461. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [14] Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A. K. El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3203-3205. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [18] Midolo, P. D., M. G. Korman, J. D. Turnidge, and J. R. Lambert. 1996. Helicobacter pylori resistance to tetracycline. Lancet 347:1194-1195. [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § RESULTS

    The 16S rRNA genes from strains 108 and 109 differed from the rest of the H. pylori 16S sequences, a strong indication that they are both derivatives of the same strain.

    “The 16S rRNA genes from strains 108 and 109 were identical along their entire length, with the exception of the two extra deletions in 109, and differed from the rest of the H. pylori 16S sequences, a strong indication that they are both derivatives of the same strain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § DISCUSSION

    The triple AGA965-967TTC mutations overlap the primary tetracycline binding site and part of the P site (G966).

    “These mutations overlap the primary tetracycline binding site (Fig. 2B) and part of the P site (G966).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § RESULTS

    Metronidazole becomes mutagenic when reduced, so spontaneous mutations are observed and complicate MIC determinations.

    “As metronidazole becomes mutagenic when reduced, spontaneous mutations are observed and complicate MIC determinations (11, 28).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Jeong, J. Y., A. K. Mukhopadhyay, J. K. Akada, D. Dailidiene, P. Hoffman, and D. E. Berg. 2001. Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole. J. Bacteriol. 183:5155-5162. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [28] Sisson, G., J.-Y. Jeong, A. Goodwin, L. Bryden, N. Rossler, S. Lim-Morrison, A. Raudonikiene, D. E. Berg, and P. S. Hoffman. 2000. Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori rdxA+ (nitroreductase) gene. J. Bacteriol. 182:5091-5096. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § MATERIALS AND METHODS

    Both methods of MIC determination gave consistent results within one MIC level.

    “Both methods gave consistent results within one MIC level.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § MATERIALS AND METHODS

    The quality control strain ATCC 43504 had a tetracycline MIC of 64 μg/ml, in agreement with literature values.

    “The quality control strain ATCC 43504 was used as a control, and the MIC for it was found to be 64 μg/ml, in agreement with literature values.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    Tetracycline enhances the chemical reactivity of bases at positions 1052 and 1054 and photolabels bases 1300 and 1338.

    “Tetracycline has been shown previously to enhance the chemical reactivity of bases at positions 1052 and 1054 (19) and to photolabel bases 1300 and 1338 (22).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Moazed, D., and H. F. Noller. 1987. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327:389-394. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [22] Oehler, R., N. Polacek, G. Steiner, and A. Barta. 1997. Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli. Nucleic Acids Res. 25:1219-1224. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION

    The G360A mutation (domain I) and the G771 deletion (domain II) are in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding.

    “In contrast, the G360A mutation (domain I) and the G771 deletion (domain II) are in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation
    § DISCUSSION

    The finding that all characterized tetracycline-resistant H. pylori strains are also metronidazole resistant suggests that there may be a progressive acquisition of resistance where metronidazole resistance may be required before tetracycline resistance can develop.

    “The finding that all characterized tetracycline-resistant H. pylori strains are also metronidazole resistant (14, 18) suggests that there may be a progressive acquisition of resistance where metronidazole resistance may be required before tetracycline resistance can develop.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A. K. El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3203-3205. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [18] Midolo, P. D., M. G. Korman, J. D. Turnidge, and J. R. Lambert. 1996. Helicobacter pylori resistance to tetracycline. Lancet 347:1194-1195. [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § DISCUSSION

    Tetracycline resistance in H. pylori is due to ribosomal mutations and can be spread through natural transformation events.

    “Tetracycline resistance in H. pylori is due to ribosomal mutations and can be spread through natural transformation events.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation
    § DISCUSSION

    The Tet-4 site may contribute to tetracycline inhibition, but it is not the major, high-affinity site of tetracycline action.

    “This site may contribute to tetracycline inhibition, but it is not the major, high-affinity site of tetracycline action.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION

    Organisms with the 16S rRNA sequence UGC at 965 to 967 (such as E. coli) are tetracycline susceptible, so it is likely that the substitution of the middle G residue is primarily responsible for resistance.

    “Organisms with the 16S rRNA sequence of UGC at 965 to 967 (such as E. coli) are tetracycline susceptible, so it is likely that the substitution of the middle G residue is primarily responsible for resistance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given
    § DISCUSSION

    This residue may be too far from the tetracycline binding site to significantly interfere with binding.

    “This residue may be too far from the tetracycline binding site to significantly interfere with binding.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § DISCUSSION

    The G942 residue is located at the Tet-4 binding site identified in one of the 30S structures and is deleted in strain 109.

    “This residue is located at the Tet-4 binding site identified in one of the 30S structures (23) and is deleted in strain 109.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Pioletti, M., F. Schlünzen, J. Harms, R. Zarivach, M. Glühmann, H. Avila, A. Bashan, H. Bartels, T. Auerbach, C. Jacobi, T. Hartsch, A. Yonath, and F. Franceschi. 2001. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 20:1829-1839. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • Rests on an assumption: Structural and biochemical findings from T. thermophilus and E. coli 30S ribosomes apply to H. pylori 16S rRNA and tetracycline binding.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given
    § DISCUSSION

    The presence of the additional mutations may add some aspect of stability or a functional advantage to the rRNA that is lacking with a sequence of AUA.

    “The presence of the additional mutations may add some aspect of stability or a functional advantage to the rRNA that is lacking with a sequence of AUA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given
    § DISCUSSION

    The effects of this mutation were quite varied in P. acnes, with MICs of tetracycline ranging from 2 to 64 μg/ml.

    “The effects of this mutation were quite varied in P. acnes, with MICs of tetracycline ranging from 2 to 64 μg/ml.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION

    These mutations are located at the primary tetracycline binding site and are likely to alter the affinity of the ribosome for this antibiotic.

    “These mutations are located at the primary tetracycline binding site and are likely to alter the affinity of the ribosome for this antibiotic.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Structural and biochemical findings from T. thermophilus and E. coli 30S ribosomes apply to H. pylori 16S rRNA and tetracycline binding.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The occupancy of the Tet-4 site by tetracycline in the 30S subunit-tetracycline crystal structure is only half of that of the Tet-1 site.

    “The occupancy of the Tet-4 site by tetracycline in the 30S subunit-tetracycline crystal structure is only half of that of the Tet-1 site (23).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Pioletti, M., F. Schlünzen, J. Harms, R. Zarivach, M. Glühmann, H. Avila, A. Bashan, H. Bartels, T. Auerbach, C. Jacobi, T. Hartsch, A. Yonath, and F. Franceschi. 2001. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 20:1829-1839. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • Rests on an assumption: Structural and biochemical findings from T. thermophilus and E. coli 30S ribosomes apply to H. pylori 16S rRNA and tetracycline binding.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § DISCUSSION

    A decrease in the ribosome's affinity for tetracycline may increase the ability of the tRNA to compete for binding.

    “A decrease in the ribosome's affinity for tetracycline may increase the ability of the tRNA to compete for binding.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    Metronidazole in H. pylori is converted to an active, mutagenic form that could potentially increase mutations in the 16S rRNA genes.

    “Metronidazole in H. pylori is converted to an active, mutagenic form (28) that could potentially increase mutations in the 16S rRNA genes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Sisson, G., J.-Y. Jeong, A. Goodwin, L. Bryden, N. Rossler, S. Lim-Morrison, A. Raudonikiene, D. E. Berg, and P. S. Hoffman. 2000. Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori rdxA+ (nitroreductase) gene. J. Bacteriol. 182:5091-5096. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given
    § DISCUSSION

    The failure to acquire h31 mutations from serial passage may reflect the rarity of the emergence of tetracycline-resistant H. pylori in clinical settings.

    “perhaps reflecting the rarity of the emergence of tetracycline-resistant H. pylori in clinical settings.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation
    § DISCUSSION

    Only the primary or Tet-1 site was fully occupied, and it is the most probable site at which tetracycline inhibits protein synthesis.

    “Only the primary or Tet-1 site was fully occupied, and it is the most probable site at which tetracycline inhibits protein synthesis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Structural and biochemical findings from T. thermophilus and E. coli 30S ribosomes apply to H. pylori 16S rRNA and tetracycline binding.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given
    § DISCUSSION

    The rare emergence of tetracycline resistance in a clinical setting may be due to the circumstances of treatment and the nature of the gastric environment.

    “Their rare emergence in a clinical setting may be due to the circumstances of treatment and the nature of the gastric environment.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § DISCUSSION

    Metronidazole resistance in the overwhelming majority of H. pylori strains is due to mutations in the rdxA gene, which codes for an NADPH nitroreductase, and may involve mutations of an NADPH flavin oxidoreductase, FrxA.

    “Metronidazole resistance in the overwhelming majority of H. pylori strains is due to mutations in the rdxA gene, which codes for an NADPH nitroreductase (11), and may involve mutations of an NADPH flavin oxidoreductase, FrxA (13).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] Jeong, J. Y., A. K. Mukhopadhyay, J. K. Akada, D. Dailidiene, P. Hoffman, and D. E. Berg. 2001. Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole. J. Bacteriol. 183:5155-5162. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [13] Kwon, D. H., K. Hulten, M. Kato, J. J. Kim, M. Lee, F. A. El-Zaatari, M. S. Osato, and D. Y. Graham. 2001. DNA sequence analysis of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant clinical Helicobacter pylori isolates. Antimicrob. Agents Chemother. 45:2609-2615. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § DISCUSSION

    Strains 108 and 109 have a unique deletion of amino acid Y141 in the RdxA protein that is likely the cause of metronidazole resistance.

    “Strains 108 and 109 have a unique deletion of amino acid Y141 in the RdxA protein that is likely the cause of metronidazole resistance.”
    Quote in the paperNot a substantial claim
60 of 60

  • The paper relies on this without support.

    Structural and biochemical findings from T. thermophilus and E. coli 30S ribosomes apply to H. pylori 16S rRNA and tetracycline binding.

    “The 30S ribosomal subunits in the crystal structures were purified from Thermus thermophilus and have the sequence AGC at positions 965 to 967 of the 16S rRNA.”

  • The paper relies on this without support.

    The eubacterial 16S rRNA sequence conservation database is representative and applicable to H. pylori.

    “A phylogenic analysis of the 16S rRNA from eubacteria showing the conservation of each base at every position can be found at www.rna.icmb.utexas.edu/CSI.”

  • The paper relies on this without support.

    The presence of only two rRNA operons in H. pylori increases the likelihood of isolating strains with tetracycline resistance mutations in the 16S rRNA by serial passage on increasing concentrations of tetracycline.

    “The presence of only two rRNA operons in H. pylori increases the likelihood of isolating strains with tetracycline resistance mutations in the 16S rRNA by serial passage on increasing concentrations of tetracycline.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • 16S rRNA mutations seen in H. pylori and P. acnes that confer resistance to tetracycline are in close proximity to the primary binding site.

    “(v) 16S rRNA mutations seen in H. pylori (19, 55) and P. acnes (40) that confer resistance to tetracycline are in close proximity to the primary binding site.”

    From Ribosomal Protection Proteins and Their Mechanism ofTetracyclineResistance (2003) · cites this paper as [55]

  • Mutations within helices h31 and h34 of 16S rRNA confer tetracycline resistance in H. pylori and P. acnes.

    “mutations within helices h31 and h34 of 16S rRNA have been reported to confer tetracycline resistance in Helicobacter pylori and Propionibacterium acnes (31, 40).”

    From Target- and resistance-based mechanistic studies with TP-434, a novel fluorocycline antibiotic (2012) · cites this paper as [40]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract's passaged-mutant MIC range conflicts with Table 2

    Minor

    The abstract gives a range of 4 to 8 μg/ml, but Table 2 lists passaged-mutant MICs of 2 or 8 μg/ml, with none at 4 μg/ml.

1 of 1

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Authors
Trieber, C. A., & Taylor, D. E.
Affiliations
  • Departments of Medical Microbiology and Immunology
  • University of Alberta (CA)
Corresponding author
Taylor, Diane E.
ORCID
Venue
Journal of Bacteriology
Publisher
American Society for Microbiology
Published
2002-04-15
Volume
184
Issue
8
Pages
2131-2140
ISSN
  • 0021-9193 (ISSN-L)
  • 1098-5530
DOI
10.1128/jb.184.8.2131-2140.2002
PMC
PMC134973
OpenAlex
W2101376032
Semantic Scholar
e25737f9e654b9759e37e09577830e12f6e0415a
Cited by
174
References
40
Citations by year
2026: 7 · 2025: 7 · 2024: 3 · 2023: 10 · 2022: 7 · 2021: 5 · 2020: 5 · 2019: 2 · 2018: 3 · 2017: 3 · 2016: 15 · 2015: 12 · 2014: 8 · 2013: 3 · 2012: 7
FWCI
14.32
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Journal of Bacteriology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Canadian Association of Gastroenterology
Topics
  • Health Sciences › Medicine › Surgery › Helicobacter pylori-related gastroenterology studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Antibiotic Resistance in Bacteria
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Helicobacter pylori, tetracycline resistance, 16S rRNA mutations, antibiotic resistance mechanisms, natural transformation, metronidazole resistance, serial passage, minimum inhibitory concentration, clinical isolates
MeSH terms
Base Sequence, Humans, Metronidazole, Molecular Sequence Data, Mutation, RNA, Ribosomal, 16S, Tetracycline Resistance, Transformation, Bacterial, Helicobacter pylori

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

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