Emergence of amoxicillin resistance and identification of novel mutations of the pbp1A gene in Helicobacter pylori in Vietnam

Abstract

Background: Amoxicillin-resistant Helicobacter pylori (H. pylori) strains seem to have increased over time in Vietnam. This threatens the effectiveness of H. pylori eradication therapies with this antibiotic. This study aimed to investigate the prevalence of primary resistance of H. pylori to amoxicillin and to assess its association with pbp1A point mutations in Vietnamese patients.

Materials and methods: Naive patients who presented with dyspepsia undergoing upper gastrointestinal endoscopy were recruited. Rapid urease tests and PCR assays were used to diagnose H. pylori infection. Amoxicillin susceptibility was examined by E-tests. Molecular detection of the mutant pbp1A gene conferring amoxicillin resistance was carried out by real-time PCR followed by direct sequencing of the PCR products. Phylogenetic analyses were performed using the Tamura-Nei genetic distance model and the neighbor-joining tree building method.

Results: There were 308 patients (46.1% men and 53.9% women, p = 0.190) with H. pylori infection. The mean age of the patients was 40.5 ± 11.4 years, ranging from 18 to 74 years old. The E-test was used to determine the susceptibility to amoxicillin (minimum inhibitory concentration (MIC) ≤ 0.125 μg/ml) in 101 isolates, among which the rate of primarily resistant strains to amoxicillin was 25.7%. Then, 270 sequences of pbp1A gene fragments were analysed. There were 77 amino acid substitution positions investigated, spanning amino acids 310-596, with the proportion varying from 0.4 to 100%. Seven amino acid changes were significantly different between amoxicillin-sensitive (Amox^S) and amoxicillin-resistant (Amox^R) samples, including Phe₃₆₆ to Leu (p < 0.001), Ser₄₁₄ to Arg (p < 0.001), Glu/Asn_464-465 (p = 0.009), Val₄₆₉ to Met (p = 0.021), Phe₄₇₃ to Val (p < 0.001), Asp₄₇₉ to Glu (p = 0.044), and Ser/Ala/Gly_595-596 (p = 0.001). Phylogenetic analyses suggested that other molecular mechanisms might contribute to amoxicillin resistance in H. pylori in addition to the alterations in PBP1A.

Conclusions: We reported the emergence of amoxicillin-resistant Helicobacter pylori strains in Vietnam and new mutations statistically associated with this antimicrobial resistance. Additional studies are necessary to identify the mechanisms contributing to this resistance in Vietnam.

Keywords: Amoxicillin resistance; Helicobacter pylori; Mutation; pbp1A gene, Vietnam.

Fig. 1

MIC range of isolates sensitive (blue) and resistant (red) to amoxicillin

Fig. 2

Amino acid substitution proportion of deduced PBP-1A discovered in the population compared to the Amx^S H. pylori strain 26,695. Red represents amino acid substitution differences with statistical significance from Amx^S and Amx^R samples by the E-test. A proportion lower than 1% was not included in the picture

Fig. 3

Phylogenetic tree of 85 sequences of the pbp1A gene obtained in this study. Scale bars indicate the numbers of nucleotide substitutions per site. Red nodes/tips represent pbp1A nucleotide sequences from the clinical specimens in which H. pylori isolates showed resistance to amoxicillin