Amoxicillin resistance in H. pylori is one of the greatest concerns of clinicians because anti-H. pylori regimens often consist of amoxicillin in addition to other antibiotics and proton pump inhibitors [20, 21]. Recent studies have suggested that amoxicillin resistance in H. pylori results from alterations in PBP1A [12, 13, 15, 22]. One of the main purposes of this study was to investigate the molecular mechanism of amoxicillin resistance in H. pylori strains collected from endoscopic biopsies in Vietnam.
The amoxicillin-resistant H. pylori proportion discovered in this study (25.7%) was similar to the finding by Saniee et al. in Iran (27.1%; p = 0.423) in 2018  but significantly higher than that in other studies, such as Zerbetto et al. in Argentina (7.6%, p < 0.001) in 2017 , Manal et al. in Egypt (18.3%; p = 0.035) in 2018 , Ortis et al. in Central America (10%; p < 0.001) in 2019 , Azzaya et al. in Mongolia (11.9%; p < 0.001), Aumpan et al. in Cambodia (0%; p < 0.001) in 2020 [26, 27], Calinga-Ponce et al. in Mexico (1.8%; p < 0.001) and Li et al. in China (0%; p < 0.001) in 2021 [28, 29]. Compared to previous studies in Vietnam, the amoxicillin resistance proportion has been trending up significantly, for instance, from 0% (p < 0.001) in 2013 , 1.1% (p < 0.001) in 2015 , 10.4% (p < 0.001) in 2016 , 15% (p = 0.002) in 2019  and to 25.7% in 2020 for this study. Although the previous local studies were different from each other regarding geographical areas, sample sizes, study period and antimicrobial testing methods, an increasing proportion of amoxicillin resistance in H. pylori in Vietnam has been generally demonstrated, suggesting that this is a serious emerging threat to the success of amoxicillin-based regimens. The high rate of amoxicillin-resisting H. pylori in our study might be explained by the fact that the combination of amoxicillin and clavulanate potassium is often empirically prescribed for various infectious diseases . The incorrect use of antimicrobials can accelerate the selection of drug-resistant strains .
Multiple strains of H. pylori can coinfect the same patient . The coinfection detected by vacA genotype in our study presented a rate of 12.3%. A similar proportion in terms of coinfection determination was obtained when confirmed by random amplified polymorphic DNA (RAPD) fingerprinting (12.5%; p = 0.500)  or through vacA and iceA genotyping (11.0%; p = 0.255) . Therefore, coinfection needs to be excluded before evaluating the relationship between H. pylori genotypes and other factors, such as disease status and clinical symptoms, to maintain accuracy. Moreover, coinfection could undermine the success of eradication therapy and should be considered when interpreting the results of antimicrobial susceptibility tests .
Based on the H. pylori genotype analysis, our data showed an association between the vacAm1 genotype and gastric ulcers. This result suggests that individuals colonised with vacAm1-positive H. pylori strains are at an increased risk of developing gastric ulcers. These results confirmed the vacAm1 genotype is associated with an increased risk of peptic ulcers, which has been reported by Nguyen et al.  and Trang et al.  in Vietnam. Conversely, Milad et al. revealed that the vacAm2 genotype was significantly higher in patients with peptic ulcer disease than in patients with gastritis in Iran . On the other hand, several previous studies published by Godoy et al. in Brazil and Loivukene et al. in Estonia did not find any association between virulence factors such as vacAm1 genotype and clinical outcomes or bacterial resistance to metronidazole, although the coinfection by multiple strains has been well considered in these studies [33, 37]. The difference in cagA and vacA genotype proportions as well as the association of these genotypes with the clinical outcomes might result from the fact that various populations have been evaluated with different ethnic groups and that a high genetic variability of strains in different countries exists [38, 39]. However, there is universal agreement regarding the role of the vacAm1 genotype among studies carried out in Vietnam at different time points. In addition, we did not find any association between the virulence factors and resistance to amoxicillin.
Amino acid substitutions in the acyl transpeptidase domain of PBP1A are required for resistance to amoxicillin [12, 13]. However, other mechanisms could be involved in increasing the MIC value and contributing to the levels of high AmxR strains, such as decreasing membrane permeability due to altered porin proteins (HopC, HopH), increasing the activity of efflux pumps to eject antibiotics from the periplasm, or even reducing the binding of antibiotics to other PBPs, especially PBP2 [19, 40].
In this study, we discovered a high rate of resistance to amoxicillin. To identify the mutations of pbp1A responsible for amoxicillin resistance, sequences of the pbp1A gene were analysed. We found seven amino acid changes possibly linked to amoxicillin resistance in clinical samples. Among them, the Ser414 to Arg substitution has been proven to be the main factor in amoxicillin resistance of the Hardenberg strain by site-directed mutagenesis  and it was also common in clinical AmxR strains by natural transformation [11, 13]. The Phe366 to Leu alteration was reported to be present in the clinical AmxR strain SZ79 in combination with the Ser414 to Arg substitution . Phe473 was recognised in strains sensitive to but less susceptible to amoxicillin but also in AmxR transformants in the absence of Ser414 to Arg substitutions , while Val473 was common in our AmxR samples in the context of Ser414 to Arg changes. Although it has become clear that amino acid variations conferring resistance vary by the geographical origins of the strains , these data have confirmed the combination of amino acid substitutions or mutations in multiple loci to amoxicillin resistance .
To investigate the relatedness, as well as the combination of different mutations of pbp1A sequences in the resistance to amoxicillin, we created a phylogenetic tree of 85 obtained pbp1A gene fragments. The grouping of the pbp1A gene sequences suggested that there were other mechanisms in addition to the mutations in the pbp1A gene contributing to amoxicillin resistance in H. pylori in Vietnam. Other studies have also shown that distinct mechanisms of antimicrobial resistance also play important roles in the resistance to amoxicillin in H. pylori .
Our study had several limitations. First, resistant mutations were not identified directly from isolates but only from gastric biopsy specimens. We excluded coinfection cases from the data analysis, and this approach might not be ideal for identifying the molecular mechanisms related to amoxicillin resistance. Second, the role of other genes that could have synergistic effects in amoxicillin resistance could not be excluded. We did not evaluate coinfection by multiple H. pylori strains by fingerprinting methods such as random amplified polymorphic DNA (RAPD) or multilocus sequence typing (MLST). Last but not least, resistance has not been confirmed to have a direct correlation with eradication efficacy in real-life practice.
However, the strength of this study is that it was conducted at one of the largest hospitals in southern Vietnam, which usually admits patients from Ho Chi Minh City and many nearby areas. This is the first study that reported mutations related to AmxR H. pylori in Vietnamese patients and it has identified some novel mutations, especially insertion mutations Glu/Asn464−465 and Ser/Ala/Gly595−596 in the pbp1A gene and other nearby mutations, which are likely specific to H. pylori strains in Vietnamese. Further studies are required to validate the role of these novel mutations in conferring amoxicillin resistance. In addition to the emerging prevalence of amoxicillin-resistant H. pylori strains, direct detection of pbp1A gene mutations from H. pylori-positive biopsy specimens may lead to novel diagnostic strategies for amoxicillin resistance determination and would be useful in clinical practice. More importantly, other mechanisms, such as the acquisition or expression of β-lactamase or changes in other proteins involved in cell wall synthesis, such as PBP2, PBP3, and PBP4, should also be evaluated.