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.
Highlights
Helicobacter pylori (H. pylori) infection has been established as the main cause of various gastroduodenal diseases including chronic gastric inflammation, peptic ulcer disease and gastric cancer (GC) and it is classified as a class I carcinogen (Ishaq and Nunn, 2015)
We investigated the effect of increased temperature on sensitivities of the metronidazole-resistant H. pylori NCTC 11637 strain to metronidazole using the Epsilometer test (E-test) strip
After culturing at 41◦C for 3 days, minimum inhibitory concentration (MIC) of H. pylori to metronidazole decreased to 8 μg/ml (Figure 1B), which is the breakpoint of metronidazole resistance
Summary
Helicobacter pylori (H. pylori) infection has been established as the main cause of various gastroduodenal diseases including chronic gastric inflammation, peptic ulcer disease and gastric cancer (GC) and it is classified as a class I carcinogen (Ishaq and Nunn, 2015). The standard triple therapy, consisting of a proton pump inhibitor combined with clarithromycin and amoxicillin or metronidazole, has been the mainstay of treatment for H. pylori infection over the last two decades. Resistance to clarithromycin and metronidazole varies between regions across the world and have hampered the elimination of H. pylori (Kim et al, 2015). Various mechanisms have been shown to affect H. pylori antibiotic resistance under physiological conditions (Hu et al, 2016; Alba et al, 2017). Point mutations in 23S rRNA and changes in efflux pump systems have been shown to confer resistance to macrolides such as clarithromycin (Francesco et al, 2011). Reduced activities in nitroreductase (rdxA), flavinoxido reductase (frxA) and ferrodoxinlike enzymes (frxB) lead to reduced activation of metronidazole (Francesco et al, 2011)
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