Study on the susceptibility genes that mediate the resistance of Helicobacter pylori to clarithromycin and levofloxacin based on comprehensive antibiotic research database comparison / 中华消化杂志

ABSTRACT

Objective:Taking clinical strains of Helicobacter pylori ( H. pylori) with different antimicrobial resistance as the research object, to explore the new genes related to the resistance of H. pylori to clarithromycin (CLA) and levofloxacin (LVX) based on whole-genome sequencing. Methods:From September 1st, 2016 to August 31st, 2019, 1 749 patients with upper gastrointestinal symptoms and positive 13C urea breath test who visited the Department of Gastroenterology and Hepatology, the University of Hong Kong-Shenzhen Hospital were enrolled. After gastric mucosal biopsy, H. pylori was isolated and cultured from gastric mucosa. Ninety H. pylori strains were successfully preserved. According to the results of in vitro drug sensitivity test, a total of 40 strains including 10 strains with single-drug resistance to CLA (CLA group), 10 strains with single-drug resistance to LVX (LVX group), 10 strains with dual-resistance to CLA and LVX (dual resistance group) and 10 strains sensitive to CLA, LVX, amoxicillin, furazolidone, tetracycline and metronidazole (all sensitive group) were screened out. By whole-genome sequencing and comparison to the comprehensive antibiotic research database (CARD), single nucleotide variations (SNV) and indels were analyzed, genes related to H. pylori resistance to CLA and LVX were screened out and the differences of new genes among 4 groups were analyzed. Independent sample t test, one-way analysis of variance, least significant difference method and chi-square test were used for statistical analysis. Results:Among the 4 groups there were no statistically significant differences in the number of SNV (74 952.00±8 755.21, 77 128.10±3 191.35, 78 639.90±601.23 and 77 474.60±2 421.05) and Indels (2 582.20±265.45, 2 653.60±108.37, 2 667.10±43.82 and 2 641.10±80.25) (all P>0.05). Compared to CARD, a total of 223 drug resistance-related genes were detected, of which 19 genes related to CLA mono-resistance in CLA group, 24 genes related to LVX mono-resistance in LVX group, 16 genes related to CLA mono-resistance, 14 genes related to LVX mono-resistance, and 12 dual resistance-related genes in dual resistance group, and 11 genes related to CLA mono-resistance, 17 genes related to LVX mono-resistance, and 13 dual resistance-related genes in all sensitive group. Among the genes related to CLA mono-resistance, the detection rates of erythromycin esterase gene ( ere)B in CLA group, LVX group, dual resistance group and all sensitive group were 0/10, 0/10, 3/10, 0/10, respectively, and the difference was statistically significant( χ2=5.79, P=0.049). The detection rate of erythromycin ribosomal methylase gene ( erm) family in CLA group and dual resistance group was higher than that in LVX group and all sensitive group (45.0%, 9/20 vs. 10.0%, 2/20), and the difference was statistically significant ( χ2=6.14, P=0.013). The detection rates of free methionine-(R)-sulfoxide reductase gene ( msrC) in CLA group, LVX group, dual resistance group and all sensitive group were 10/10, 7/10, 6/10, 4/10, respectively, and the difference was statistically significant ( χ2=8.97, P=0.030). Among the genes related to LVX mono-resistance, the detection rate of quinolone resistance pentapeptide repeat protein gene ( qnr) family in LVX group and dual resistance group was higher than that in CLA group and all sensitive group (60.0%, 12/20 vs. 25.0%, 5/20), and the difference was statistically significant ( χ2=5.01, P=0.025). The detection rates of qnrB4 in CLA group, LVX group, dual resistance group and all sensitive group were 1/10, 3/10, 7/10, 1/10, respectively, and the difference was statistically significant ( χ2=10.17, P=0.010). The number of efflux transporter genes related to CLA mono-resistance in 4 groups were less than those of LVX mono-resistance and dual drug resistance (11 vs. 29 and 11 vs. 23), and the differences were statistically significant ( χ2=11.87, 5.80; P=0.001, 0.016). The detected numbers of LVX resistance-related efflux transport genes in CLA group, LVX group, dual resistance group and all sensitive group were 28, 40, 24 and 27, respectively, and the difference was statistically significant ( χ2=10.26, P=0.016). Conclusions:Erm family and msrC may be important genes that mediate the resistance of H. pylori to CLA, and qnr family is related to mediating the resistance of H. pylori to LVX. Efflux transport genes may play a synergistic role in the process of drug efflux, and are more likely to mediate H. pylori resistance to LVX.