Analysis of epidemiology and novel mutation of Helicobacter pylori antibiotic resistance in South Korea

Abstract

BackgroundHelicobacter pylori is a Gram‐negative bacterium associated with upper gastrointestinal disease, peptic ulcer, mucosa‐associated lymphoid tissue lymphoma and eradication treatment is recommended because it is one of the causes of gastric cancer. However, resistance to some antibiotics used for treatment has developed, the eradication rate is declining worldwide. This study focused on antibiotic susceptibility test (AST) of H. pylori isolated from patient clinical specimens. The aim of this study is to identify the resistance epidemiology of H. pylori and to elucidate the resistance mechanism through gene analysis.methodsH. pylori isolated from patient clinical specimens is identified and AST for amoxicillin (AMX) and clarithromycin (CLR) are performed, and the results are analyzed through Whole Genome Sequencing (WGS) and MultiLocus Sequence Typing (MLST), respectively. The strain type of the MLST result is compared with the CLR resistance minimum inhibitory concentration (MIC) value to analyze the significant points.H. pylori is cultured on EYE Agar plate and incubated in Anaerobic Jar for 2~5 days with microaerophilic air condition (5% O2, 10% CO2, 85% N2). Cultured isolates are identified by MALDI‐TOF system and performed AST using Epsilometer test to determining MIC.Results366 H. pylori were isolated from 1952 clinical patients, and 36 AMX‐resistant H. pylori and 101 CLR‐resistant H. pylori were identified. Among the identified H. pylori, 9 AMX high MIC strains were selected and WGS was performed, and 12 CLR high MIC strains were selected and MLST was performed.As the WGS result of 9 clinical strains resistant to AMX, single nucleotide polymorphism (SNP) of the potential target gene was analyzed with reference to ATCC 26695. Six genes, Penicillin binding protein1A (PBP1), PBP2, NhaC, hofH, hofC, and hefC, were selected as potential target genes. As a result of the analysis, 8, 7, 4, 9, 16, and 2 SNPs were found, respectively, and it was specifically confirmed that there were many SNPs in the outer membrane component Gene. However, No correlation was found between AMX MIC value and target SNP.12 For the CLR resistance strain, MLST was performed using 7 housekeeping genes (atpA, efp, trpC, ppa, mutY, yphC, Ure1). In 12 strain, 10 CLR‐resistance H. pylori are not identified strain type(ST) to The National Center for Biotechnology Information(NCBI) database because they have a lots of mutation in 7 housekeeping gene. 2 CLR‐resistance H. pylori are identified the new type strains.ConclusionIn this study, we conclude that hofC, hofH related to outer membrane protein component may be associated to the AMX resistance. And additional research is continued to need about other resistance potential gene, especially hopC.