Effect of Temperature on Metronidazole Resistance in Helicobacter pylori

Abstract

Efficacy of <i>Helicobacter pylori (H. pylori)</i> eradication therapy has declined due to rapid rises in antibiotic resistance. We investigated how increased temperature affected <i>H. pylori</i> (NCTC 11637) growth and its sensitivity to metronidazole <i>in vitro</i>. 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 <i>H. pylori</i> 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 <i>rdxA, frxA</i> nor <i>fdxB.</i> Our findings suggest that temperature increase can inhibit the growth and reduce <i>H. pylori</i> 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 <i>H. pylori</i>.