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Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori.

ABSTRACT: Background:Metronidazole is one of the first-line drugs of choice in the standard triple therapy used to eradicate Helicobacter pylori infection. Hence, the global emergence of metronidazole resistance in Hp poses a major challenge to health professionals. Inactivation of RdxA is known to be a major mechanism of conferring metronidazole resistance in H. pylori. However, metronidazole resistance can also arise in H. pylori strains expressing functional RdxA protein, suggesting that there are other mechanisms that may confer resistance to this drug. Methods:We performed whole-genome sequencing on 121 H. pylori clinical strains, among which 73 were metronidazole-resistant. Sequence-alignment analysis of core protein clusters derived from clinical strains containing full-length RdxA was performed. Variable sites in each alignment were statistically compared between the resistant and susceptible groups to determine candidate genes along with their respective amino-acid changes that may account for the development of metronidazole resistance in H. pylori. Results:Resistance due to RdxA truncation was identified in 34% of metronidazole-resistant strains. Analysis of core protein clusters derived from the remaining 48 metronidazole-resistant strains and 48 metronidazole-susceptible identified four variable sites significantly associated with metronidazole resistance. These sites included R16H/C in RdxA, D85N in the inner-membrane protein RclC (HP0565), V265I in a biotin carboxylase protein (HP0370) and A51V/T in a putative threonylcarbamoyl-AMP synthase (HP0918). Conclusions:Our approach identified new potential mechanisms for metronidazole resistance in H. pylori that merit further investigation.

SUBMITTER: Chua EG

PROVIDER: S-EPMC6375344 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori.

Chua Eng-Guan EG Debowski Aleksandra W AW Webberley K Mary KM Peters Fanny F Lamichhane Binit B Loke Mun-Fai MF Vadivelu Jamuna J Tay Chin-Yen CY Marshall Barry J BJ Wise Michael J MJ

Gastroenterology report 20190102 1

<h4>Background</h4>Metronidazole is one of the first-line drugs of choice in the standard triple therapy used to eradicate Helicobacter pylori infection. Hence, the global emergence of metronidazole resistance in Hp poses a major challenge to health professionals. Inactivation of RdxA is known to be a major mechanism of conferring metronidazole resistance in H. pylori. However, metronidazole resistance can also arise in H. pylori strains expressing functional RdxA pro ...[more]

PMID: 30792865

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Project description: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.

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.

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Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori.

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Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in <i>Helicobacter pylori</i>.

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