Global Antimicrobial Resistance Gene Study of Helicobacter pylori: Comparison of Detection Tools, ARG and Efflux Pump Gene Analysis, Worldwide Epidemiological Distribution, and Information Related to the Antimicrobial-Resistant Phenotype

Abstract

We conducted a global-scale study to identify H. pylori antimicrobial-resistant genes (ARG), address their global distribution, and understand their effect on the antimicrobial resistance (AMR) phenotypes of the clinical isolates. We identified ARG using several well-known tools against extensive bacterial ARG databases, then analyzed their correlation with clinical antibiogram data from dozens of patients across countries. This revealed that combining multiple tools and databases, followed by manual selection of ARG from the annotation results, produces more conclusive results than using a single tool or database alone. After curation, the results showed that H. pylori has 42 ARG against 11 different antibiotic classes (16 genes related to single antibiotic class resistance and 26 genes related to multidrug resistance). Further analysis revealed that H. pylori naturally harbors ARG in the core genome, called the 'Set of ARG commonly found in the Core Genome of H. pylori (ARG-CORE)', while ARG-ACC-the ARG in the accessory genome-are exclusive to particular strains. In addition, we detected 29 genes of potential efflux pump-related AMR that were mostly categorized as ARG-CORE. The ARG distribution appears to be almost similar either by geographical or H. pylori populations perspective; however, some ARG had a unique distribution since they tend to be found only in a particular region or population. Finally, we demonstrated that the presence of ARG may not directly correlate with the sensitive/resistance phenotype of clinical patient isolates but may influence the minimum inhibitory concentration phenotype.

Keywords: Helicobacter pylori; antimicrobial resistance (AMR); antimicrobial-resistant gene (ARG); clinical implications; genome analysis; global distribution.

Figure 1

ARG detection by several tools, methods, and databases. (a) The ARG detection results are based on well-known ARG detection tools, showing that each tool could yield different results since they have different detection methods or databases. (b) Final ARG detection results in the global H. pylori dataset. A total of 42 ARG were included after collecting the results from all tools and databases, then performing careful curation with Prokka annotation to delete duplication, confirm the presence of the gene, and find undetected ARG.

Figure 2

Antibiotic class, mechanism, and position of ARG identified within the H. pylori genome. (a) Schematic link between the ARG antibiotic class and the ARG resistance mechanism. Most of the MDR ARG were related to transporters or EPs. The inner and outer scales represent the number and proportion of ARG inside each antibiotic class. (b) Position of ARG within the H. pylori genome. The number of ARG represents the gene name that could be found within the antibiotic class. ARG-CORE: Set of ARG commonly found in the Core Genome of H. pylori. ARG-ACC: Set of ARG commonly found in the Accessory Genome of H. pylori. The core genome was defined if the ARG was found in ≥95% of the total global H. pylori dataset (n = 2170 strains).

Figure 3

Summary of efflux pumps (EPs) related to AMR detected from the H. pylori genome. The green circle contains Eps reported by previous H. pylori AMR studies. The sequences of these Eps were collected from H. pylori strain 26695 then used as a query for BLASTN against the global H. pylori sequence dataset (2170 strains). The result showed that most of these EPs were ARG-CORE. The blue circle contains EPs identified by ARG detection tools and databases. Many of these EPs were found in low numbers of H. pylori and were commonly reported in other species of bacteria. This result suggests that some EP genes may have been obtained by HGT via MGE transfer.

Figure 4

The ARG-ACC distribution based on the geographic location and H. pylori population. The ARG-ACC were clustered based on their antibiotic classes. (a) The ARG-ACC distribution tended to be similar between countries. Some areas are magnified to show the distribution more clearly. The magnified regions were displayed in correspondence with their respective numerical designations. (b) The ARG-ACC proportion for each continent. The proportion tended to be the same for each continent. (c) The ARG-ACC distribution is based on the H. pylori population. Each dot outside the phylogenetic tree represents the ARG-ACC distribution, with each color representing its antibiotic class.