Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam

Helicobacter pylori is a gastric pathogen that causes several gastroduodenal disorders such as peptic ulcer disease and gastric cancer. Eradication efforts of H. pylori are often hampered by antimicrobial resistance in many countries, including Vietnam. Here, the study aimed to investigate the occurrence of antimicrobial resistance among H. pylori clinical isolates across 13 hospitals in Vietnam. The study further evaluated the clarithromycin resistance patterns of H. pylori strains. In order to address the study interests, antimicrobial susceptibility testing, epsilometer test and PCR-based sequencing were performed on a total of 193 strains isolated from patients, including 136 children (3–15 years of age) and 57 adults (19–69 years of age). Antimicrobial susceptibility testing showed that the overall resistance to amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline was 10.4%, 85.5%, 24.4%, 37.8%, and 23.8% respectively. The distribution of minimum inhibitory concentrations (MICs) of clarithromycin-resistant strains was 85.5% with MIC >0.5 μg/mL. The majority of the clarithromycin resistant isolates (135 of 165 subjects) have MICs ranging from 2 μg/mL to 16 μg/mL. Furthermore, sequencing detection of mutations in 23S rRNA gene revealed that strains resistant and susceptible to clarithromycin contained both A2143G and T2182C mutations. Of all isolates, eight clarithromycin-resistant isolates (MIC >0.5 μg/mL) had no mutations in the 23S rRNA gene. Collectively, these results demonstrated that a proportion of clarithromycin-resistant H. pylori strains, which are not related to the 23S rRNA gene mutations, could be potentially related to other mechanisms such as the presence of an efflux pump or polymorphisms in the CYP2C19 gene. Therefore, the present study suggests that providing susceptibility testing prior to treatment or alternative screening strategies for antimicrobial resistance is important for future clinical practice. Further studies on clinical guidelines and treatment efficacy are pivotal for successful eradication of H. pylori infection.

Helicobacter pylori is a Gram-negative bacterium that plays a causative role in the development of gastric adenocarcinoma, peptic ulcer disease and chronic gastritis 1,2 . The prevalence of H. pylori infection is more than half of the world's population, comprising of >80% in developing countries and approximately 40% in the United States 3,4 . In Vietnam, the prevalence of H. pylori is approximately 80% in adults and 26%-71.4% in children [5][6][7] .
Eradication therapy of symptomatic H. pylori infection substantially prevents the recurrence and reduces the risk of developing gastroduodenal-associated diseases [8][9][10][11] . Recommended therapy, triple-therapy regimen, composed of two antimicrobial agents (e.g. amoxicillin, metronidazole, tetracycline, levofloxacin, and clarithromycin) in combination with a proton pump inhibitor (PPI), has been widely used to eliminate the bacteria 12-14 . However, H. pylori antimicrobial resistance is increasing worldwide, contributing to the main factor that affects the efficacy of current therapeutic regimens 15,16 . Resistance to clarithromycin is believed to be the main factor in treatment failure 16,17 . In Vietnam, many studies showed that H. pylori is highly resistant to clarithromycin; 33%-34% primary and 74% secondary resistance [18][19][20]

Statistical analysis
Mann-Whitney t-test, unpaired two-tailed was used to compare resistance rate between different patient groups. All analyses were performed using SPSS Statistics version 20 (SPSS, Chicago, USA) and Prism version 5.0 (GraphPad, San Diego, USA). Antimicrobial resistance of Helicobacter pylori isolates To assess the antimicrobial resistance of H. pylori in Vietnam, susceptibility testing was performed and the resistance rate of each antimicrobial is listed in Table 1. The prevalence of antimicrobial resistance was detected in the following order, from highest to lowest: clarithromycin, metronidazole, levofloxacin, tetracycline and amoxicillin. Of all the antimicrobial agents, the majority of isolates were resistant to clarithromycin as shown in 85.5% of all patients (84.6% in children and 87.7% in adults). The occurrence of metronidazole resistance was lower than clarithromycin (overall 37.8% vs. 85.5%) in this study, as compared to the other published reports 18-20 . Antimicrobial resistance in adults is predominately higher than children, except for amoxicillin resistance which occurred in 12.5% of children and 5.3% of adults without statistical significance. A statistically significant difference was observed in the resistance rate of levofloxacin (p = 0.0103) between children and adults in Figure 1.

Results
Minimum inhibitory concentration values of clarithromycinresistant isolates predominately range from 2 μg/ml to 16 μg/ml To validate the clarithromycin resistant isolates, MIC values were obtained from a total of 193 clinical isolates using an E-test. Based on EUCAST proposed breakpoints, the respective occurrence of clarithromycin susceptible and resistant isolates was 24 (12.4%) and 165 (85.5%) of the total number of isolates used in this study. The distribution of MICs showed that the majority of clinical isolates resistant to clarithromycin (135 of 165 isolates, 81.8%, including 97 children and 38 adults) ranged from 2 μg/mL to 16 μg/mL ( Figure 2). Of all isolates, only five subjects (including four children and one adult) showed a MIC of 24 μg/mL, and one adult subject had a MIC >256 μg/mL (Figure 2).

Mutations of 23S rRNA gene in Helicobacter pylori isolates
To investigate the point mutations in the 23S rRNA gene of clarithromycin-resistant isolates, mutations at position 2142 (A2142G or A2142C), 2143 (A2143G), and 2182 (T2182C) were analysed in this study. Sequence analyses showed the point mutations in the 23S rRNA gene were detected not only in clarithromycin-resistant isolates, but also in clarithromycin-susceptible isolates. In Table 2, both A2143G and T2182C mutations were predominantly detected in 91.7% (n = 177) of the clarithromycinsusceptible and -resistant isolates. Only two clarithromycinresistant isolates in adults had the A2142G and T2182C mutations with a respective MIC value of 8 μg/mL and >256 μg/mL. In addition, a total of 10 clarithromycin-resistant and -susceptible isolates had no mutations in the 23S rRNA gene. The present study also identified four isolates with both A2143G and T2182C mutations at MIC values ranging from 0.38 to 0.5 μg/mL, which are considered to be intermediate resistance strains 34 .

Discussion
Antimicrobial resistance in H. pylori has become a global health problem because the prevalence of infection and incidence is increasing worldwide 37-39 . The increasing H. pylori resistance to antimicrobial agents, such as clarithromycin, is considered the main

Figure 1. Antimicrobial resistance rate of Helicobacter pylori isolates from Vietnamese children and adults.
The graph displays the resistance rate of amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline in both children and adults. Among the antimicrobial agents, clinical isolates resistant to levofloxacin is significantly higher (p = 0.0103) in adults than in children.

Figure 2. Minimum inhibitory concentration values of clarithromycin susceptible and resistant isolates in children and adults.
The graph shows the number of isolates across a range of minimum inhibitory concentration values of clarithromycin. The total number of clarithromycin susceptible and resistant isolates is 24 and 165, respectively. Majority of clinical isolates resistant to clarithromycin have MIC values ranging from 2 μg/mL to 16 μg/mL. Our present study showed that the overall resistance rate for clarithromycin and metronidazole was 85.5% and 37.8%, respectively.
The high incidence of H. pylori strains resistant to clarithromycin and metronidazole in Vietnam might be attributed to the following: (i) unregulated or widespread over-the-counter use of antibiotics, (ii) clarithromycin is prescribed frequently for treatment due to its high bactericidal effect, and (iii) antibiotics are often used to treat H. pylori infection and other infections including respiratory tract infections (clarithromycin) and intestinal parasites (metronidazole) 18,45,46 . Of note, this study highlighted that clarithromycin resistance was the highest among the 193 H. pylori clinical isolates collected in 2015−2016, as compared to the other studies in which metronidazole has the highest resistance rate (69.9%−76.1%) in Vietnam 18-20 . The observation of high clarithromycin resistance rate from our data suggested the increasing occurrence of resistant strains among other antimicrobial agents. Therefore, constant surveillance for antimicrobial resistance rates is necessary to gain insights into effective eradication therapy of H. pylori infection.
Another interest of this study was to assess the variations of MIC values obtained from the clarithromycin-resistant strains. Our representative clinical isolates obtained from the gastric mucosa revealed that the majority of strains resistant to clarithromycin conferred MIC values ranging from 2 μg/mL to 16 μg/mL. There is also a degree of variation on the MIC range between studies 19,32,[47][48][49] (Table 2). Further investigation on other nucleotide positions of the 23S rRNA region should be performed on these resistant strains 58,59 . Additionally, we suggested that a proportion of these resistant strains, which are not related to the 23S rRNA gene sequence, could be potentially related to other mechanisms such as the presence of an efflux pump (e.g. outer membrane protein hefA) or polymorphisms in the CYP2C19 gene 60-62 .

Conclusions
In conclusion, our present results confirm that MIC values are critical for accurate identification of antimicrobial resistant strains. Susceptibility tests prior to treatment are necessary to select the optimal H. pylori therapy regimens in Vietnam. Further studies on other resistance mechanisms, particularly the mutations of the host genes, will provide additional insights into the development of diagnostic biomarkers and therapeutic drugs.

Consent
Written informed consent for publication of their clinical details was obtained from the parents of the patients.

Competing interests
The authors declare that they have no competing interests.

Grant information
This work was supported by the Nam Khoa-Biotek and Nguyen Tri Phuong hospital.