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Effect of Temperature on Metronidazole Resistance in Helicobacter pylori.

ABSTRACT: Efficacy of Helicobacter pylori (H. pylori) eradication therapy has declined due to rapid rises in antibiotic resistance. We investigated how increased temperature affected H. pylori (NCTC 11637) growth and its sensitivity to metronidazole in vitro. We performed transcriptomic profiling using RNA-sequencing to identify differentially expressed genes (DEGs) associated with increased temperature. Transcriptional pathways involved in temperature-driven metronidazole resistance changes were analyzed through bioinformatic and literature curation approaches. We showed that H. pylori growth was inhibited at 41°C and inhibition was more apparent with prolonged incubation. Resistance to metronidazole was also reduced-minimum inhibitory concentration for metronidazole decreased from > 256 μg/ml at 37°C to 8 μg/ml at 41°C after culturing for 3 days. RNA-sequencing results, which were highly concordant within treatment conditions, revealed more than one third of genes (583/1,552) to be differentially expressed at increased temperatures with similar proportions up and down-regulated. Quantitative real-time PCR validation for 8 out of 10 DEGs tested gave consistent direction in gene expression changes. We found enrichment for redox and oxygen radical pathways, highlighting a mechanistic pathway driving temperature-related metronidazole resistance. Independent literature review of published genes associated with metronidazole resistance revealed 46 gene candidates, 21 of which showed differential expression and 7 out of 9 DEGs associated with "redox" resistance pathways. Sanger sequencing did not detect any changes in genetic sequences for known resistance genes rdxA, frxA nor fdxB. Our findings suggest that temperature increase can inhibit the growth and reduce H. pylori resistance to metronidazole. Redox pathways are possible potential drivers in metronidazole resistance change induced by temperature. Our study provides insight into potential novel approaches in treating antibiotic resistant H. pylori.

SUBMITTER: Gong M

PROVIDER: S-EPMC8170400 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Metronidazole (MNZ) is administered as first-line antibiotic for Helicobacter pylori eradication therapy; however, increasing resistance to MNZ impaired the efficacy. Increasing the dose of MNZ was recommended to overcome low-level resistance, but it was difficult to determine MNZ resistance level simply based on the rdxA gene mutation. In this study, the rdxA sequences of 511 clinical H. pylori strains were analyzed to assess the genotypes associated with MNZ resistance. We observed that the prevalences of rdxA sequences with missense, nonsense, and frameshift mutations were 70.25, 11.35, and 17.03%, respectively. Regarding the amino acid substitutions, T31E, H53R, D59N, L62V, S88P, G98S/N, R131K, and V172I were present in most strains regardless of the resistance phenotype. The correlation analysis showed R16H/C, Y47C, A67V/T, and V204I substitutions were associated with MNZ resistance. The mutation resulting in RdxA truncation was observed in 36.29% of the resistant strains, and 83.45% of these strains displayed high-level MNZ resistance (MIC > 256 μg/mL). Moreover, all strains with truncated mutation positions before amino acid 70 expressed high-level MNZ resistance. Our results indicated that most amino acid mutations probably contributed to the sequence diversity of RdxA, while R16H/C, Y47C, A67V/T, and V204I were potentially helpful to identify resistant strains. Although it was difficult to determine the mutations associated with MNZ resistance, the prediction of high-level resistance based on truncated characteristics of RdxA might be an important approach, which can effectively avoid H. pylori eradication therapy with unreasonable of MNZ dose increases for patients with high-level drug resistance. IMPORTANCE The increasing resistance to metronidazole impaired the efficacy of Helicobacter pylori eradication, and increasing the dose of metronidazole was recommended to overcome low-level resistance. For patients infected with highly resistant strains, the current empirical treatments, which generally used metronidazole in double doses or more, appeared impossibly to overcome the resistance and would only increase the incidence of adverse effects. Our results indicated that high-level metronidazole resistance was predominant, and almost half of the patients with high-level drug resistance could avoid usage of metronidazole based on the truncated mutations of RdxA sequences, which can effectively avoid H. pylori eradication therapy with unreasonable increases in the metronidazole dose.

Project description:Background:The prevalence of Helicobacter pylori resistance to metronidazole and clarithromycin is high in Indonesia. Moreover, the increasing levofloxacin resistance rates in the absence of bismuth treatment in Indonesia has led to the use of other antibiotics as alternative regimens. Methods:We determined the minimum inhibitory concentrations (MICs) of five alternative antibiotics for H. pylori (rifaximin, rifabutin, furazolidone, garenoxacin, and sitafloxacin) using the agar dilution method and assessed mutations associated with antibiotic resistance using next-generation sequencing. Result:Analysis of 106 strains isolated from 1039 adult dyspeptic patients revealed that none of the strains were furazolidone-resistant. All strains were also sensitive to rifabutin and sitafloxacin. In contrast, the rates of resistance to rifaximin and garenoxacin were high (38.9% and 6.7%, respectively). The strains isolated from patients on Java Island had the highest resistance rates to garenoxacin and rifaximin. In addition, the resistance was distributed evenly among the ethnic groups, ranging between 25.0% and 69.2%. Except for rifaximin, for which the resistance rate was 38.9%, the other four antibiotics could be successfully employed to eradicate levofloxacin- and metronidazole-resistant H. pylori infections in vitro. Interestingly, garenoxacin-sensitive strains were found in regions with high clarithromycin resistance rates such as Bali and Papua Islands. In contrast, rifaximin might not be considered as an alternative antibiotic in regions with high clarithromycin resistance. There was an inconsistent association between gyrA and gyrB mutations and garenoxacin resistance. We confirmed that the I837V (replacement of isoleucine at position 837 with valine), A2414T/V, Q2079K and K2068R were the predominant rpoB point mutations. There was an association between vacA genotypes of H. pylori and rifaximin resistance (P = 0.048). Conclusion:furazolidone-, rifabutin-, and sitafloxacin-based therapies might be considered as alternative regimens to eradicate H. pylori in Indonesia, including regions with high metronidazole and clarithromycin resistance rates. Moreover, sitafloxacin but not garenoxacin should be considered for eradication of levofloxacin-resistant strains.

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Effect of Temperature on Metronidazole Resistance in Helicobacter pylori.

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