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Research Article

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

[version 1; peer review: 2 approved]
PUBLISHED 13 Apr 2016
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This article is included in the Pathogens gateway.

This article is included in the Antimicrobial Resistance collection.

Abstract

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.

Keywords

Helicobacter pylori, antimicrobial resistance, 23S rRNA, mutation, gastric ulcer

Introduction

Helicobacter pylori is a Gram-negative bacterium that plays a causative role in the development of gastric adenocarcinoma, peptic ulcer disease and chronic gastritis1,2. 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 States3,4. In Vietnam, the prevalence of H. pylori is approximately 80% in adults and 26%–71.4% in children57.

Eradication therapy of symptomatic H. pylori infection substantially prevents the recurrence and reduces the risk of developing gastroduodenal-associated diseases811. 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 bacteria1214. However, H. pylori antimicrobial resistance is increasing worldwide, contributing to the main factor that affects the efficacy of current therapeutic regimens15,16. Resistance to clarithromycin is believed to be the main factor in treatment failure16,17. In Vietnam, many studies showed that H. pylori is highly resistant to clarithromycin; 33%–34% primary and 74% secondary resistance1820. The majority of clarithromycin-resistant strains are identified based on point mutations in the peptidyltransferase region of domain V of 23S rRNA, which affects the binding of macrolides to the bacterial ribosome2123.

The common 23S rRNA point mutations (e.g. A2143G, A2142C/G and T2182C) are recommended for rapid routine diagnostic procedures, as compared to the time-consuming bacterial culture. A plethora of studies have evidently reported the association of minimum inhibitory concentrations (MICs) of clarithromycin-resistance strains to the respective point mutation2427. For example, A2142C/G mutations are associated with MIC >256 μg/mL, and mutations such as A2143G and T2182C are associated with MIC >0.5 μg/mL27,28. However, it is unclear whether such association between point mutation and MIC can be utilised as predictors for strains resistant to clarithromycin23,2931. Here, the present study evaluated the antimicrobial resistance of H. pylori strains isolated from patients in Vietnam with the following antimicrobial agents: amoxicillin, metronidazole, tetracycline, levofloxacin and clarithromycin. The strains resistant to clarithromycin were further investigated to assess the point mutations in the 23S rRNA gene and MIC values as predictors for screening H. pylori strains. The overall findings addressed the issues of using 23S rRNA mutations in clinical diagnosis.

Materials and methods

Study samples

The present work was designed as a prospective randomised clinical study across 13 hospitals (Children's Hospital 2, Children's Hospital 1, Trieu An Hospital, Tam Nhat Clinic, Dai Phuoc Clinic, Hoan My Hospital, DH Y Duoc Hospital, Phap Viet Hospital, Yersin International Clinic, Dong Nai International Hospital, Nguyen Tri Phuong Hospital, Van Hanh General Hospital, Gia Dinh People's Hospital) in Ho Chi Minh City, Vietnam, from July 2015 to January 2016 (Data availability). The study was approved by Nam Khoa Biotek Diagnostic Ethics Committee (ID: NCKH 04/02-15/NK). Written informed consent was obtained from each patient or the patient’s parents for the use of this study. Biopsy specimens of the gastric mucosa were obtained from 193 patients, including 136 children (3–15 years of age) and 57 adults (19–69 years of age). These patients showed indication of endoscopy for the examination of dyspeptic symptoms (i.e. gastric ulcer).

Helicobacter pylori culture and antimicrobial susceptibility testing

The H. pylori culture and susceptibility testing were performed as described in previous studies32,33. Briefly, biopsy specimens were homogenised in 500 µL transport medium (20% glycerol; 0.9% NaCl in Milli-Q water), and were subsequently inoculated onto H. pylori selective agar plates at 37°C in a microaerophilic atmosphere. Biochemical identification of H. pylori was performed using Gram stain (Gram negative), oxidase test (oxidase positive), catalase test (catalase positive) and urease test (urease positive). Susceptibility testing was performed on Muller-Hinton agar plates supplemented with 10% lysed horse blood for the following antibiotics: amoxicillin (0.25 μg/mL), clarithromycin (0.75 μg/mL), levofloxacin (1 μg/mL), metronidazole (8 μg/ml), and tetracycline (2 μg/mL). The MIC values were obtained by the epsilometer test (E-test; bioMerieux, Marcy I’Etoile, France) for clarithromycin in accordance with the manufacturer’s protocol using 10% lysed horse blood supplemented in Mueller-Hinton Z agar (bioMerieux). Bacterial suspensions were prepared in Mueller-Hinton broth and adjusted to a McFarland turbidity of three. Resistance criteria for clarithromycin was defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST); susceptible (MIC ≤0.25 μg/mL) and resistance (MIC >0.5 μg/mL)34.

PCR amplification and sequence detection of 23S rRNA mutation

The PCR mixture (20-µL final volume) contained HotStar Taq master mix (Qiagen, Hilden, Germany) and 10 pmol of forward DP1 (5’-GTAAAACGACGGCCAGTACGGCGGCCGTAACTATA-3’) and reverse ZGE23 (5’-TATTTAGGTGACACTATAGACAGGCCAGTTAGCTA-3’) primers. These primers contain sequences (written in bold-faced type) that are specific for SP6 (DP1) and M13 (ZGE23), and underlined sequences indicate 23S rRNA amplicon of 308 bp comprising of 2142, 2143 and 2182 positions. H. pylori colonies on selective medium was added to 1× TE buffer (10 mM Tris-HCL, 1 mM EDTA, pH 7.6) and heated up to 100°C for 5 min, followed by centrifugation at 8000 rpm. 1 µL of supernatant was added to the PCR mix to amplify 23S rRNA gene. The PCR cycling conditions were 95°C for 15 min to activate HotStart Taq DNA polymerase, followed by 40 cycles of 94°C for 15 sec, 57°C for 30 sec, 72°C for 30 sec, and final extension at 72°C for 5 min. The PCR products were purified prior to sequencing by Illustra ExoStar 1-Step (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) according to manufacturer’s instructions, and followed by Big-Dye (Perkin-Elmer Applied Biosystems, Foster City, USA) amplification using SP6 and M13 primers. Sequencing was then performed using ABI 3130XL sequencer. In total, 193 sequences were obtained and analysed using MEGA version 5.035 against wild-type 23S rRNA gene available in the GenBank36 database (Accession number: U27270). Sequence data can be downloaded from GenBank database (Accession numbers: KU904824-KU905015).

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).

Results

Supplementary Table 1. A summary of patient information, antimicrobial susceptibility and clarithromycin resistance patterns.
Antimicrobial susceptibility testingMIC values (?g/mL)Point mutation (23S rRNA gene)
AgeAdult or ChildrenGenderHospitalCulturePCRAmoxicillinMetronidazoleClarithromycinLevofloxacinTetracyclineClarithromycinA2142G/CA2143GT2182C
9ChildrenMaleChildren's Hospital 2(+)(+)SRRSS8AGC
14ChildrenMaleChildren's Hospital 1(+)(+)SSSSS<0.016AGC
35AdultFemaleTam Nhat Clinic(+)(+)SRRSS16AGC
15ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS3AGC
8ChildrenFemaleChildren's Hospital 2(+)(+)SSSSS0.25AGC
15ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
11ChildrenMaleChildren's Hospital 1(+)(+)SRSSR<0.016AGC
37AdultFemalePhap Viet Hospital(+)(+)SSRSS16AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS24AGC
8ChildrenFemale Children's Hospital 2(+)(+)SSRSS1AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SSSRS0.38AGC
12ChildrenMaleChildren's Hospital 1(+)(+)SRSSR0.19AGC
39AdultMalePhap Viet Hospital(+)(+)SSRSR16AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SRRSS0.75AGC
10ChildrenMaleChildren's Hospital 2(+)(+)SRRSS4AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR12AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR24AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)SRRSS16AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)RRRSS16AGC
14ChildrenMaleChildren's Hospital 1(+)(+)SSSSS<0.016AGC
33AdultFemaleTrieu An Hospital(+)(+)SRRSR3AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SSRSR16AGC
5ChildrenFemaleChildren's Hospital 2(+)(+)SSRRS3AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SRRSR12AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SRRSR6AGC
7ChildrenMaleChildren's Hospital 1(+)(+)SSRSR8AGC
8ChildrenMaleChildren's Hospital 2(+)(+)SSSSS0.19AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SRRRS16AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)SSSSS0.125AGC
14ChildrenMaleChildren's Hospital 2(+)(+)RRRSS2AGC
6ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
11ChildrenMaleChildren's Hospital 1(+)(+)SSRSS6AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SRRSS6AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)SRRSR16AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SRSRS<0.016AGC
13ChildrenMaleChildren's Hospital 1(+)(+)SSRSR12AGC
31AdultFemaleDong Nai International Hospital(+)(+)SRRSR12AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)RSSSS<0.016AGC
14ChildrenMaleChildren's Hospital 1(+)(+)SRRSS3AGC
51AdultFemaleTam Nhat Clinic(+)(+)SRRSR8AGC
4ChildrenMaleChildren's Hospital 2(+)(+)SSRRS3AGC
51AdultMalePhap Viet Hospital(+)(+)SRRRS8AGC
5ChildrenFemaleChildren's Hospital 2(+)(+)RSRSR8AGC
8ChildrenMaleChildren's Hospital 2(+)(+)SSRSS2AGC
12ChildrenMaleChildren's Hospital 2(+)(+)SRRSS16AGC
31AdultFemaleTam Nhat Clinic(+)(+)SSRSR3AGC
41AdultFemaleTam Nhat Clinic(+)(+)RSRRS4AGC
40AdultMalePhap Viet Hospital(+)(+)RSRSS4AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)RRRSS12AGC
32AdultFemaleTam Nhat Clinic(+)(+)SRRSS2AGC
35AdultFemaleTam Nhat Clinic(+)(+)SRRSS16AGC
40AdultFemaleTam Nhat Clinic(+)(+)SRRSR16AGC
10ChildrenMaleChildren's Hospital 2(+)(+)SSRSR12AGC
14ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR6AGC
8ChildrenFemaleTam Nhat Clinic(+)(+)SSRRS6AGC
5ChildrenFemaleChildren's Hospital 2(+)(+)SSRRS8AGC
26AdultFemaleTam Nhat Clinic(+)(+)SRRSS2AGC
50AdultMaleTrieu An Hospital(+)(+)RSRRS4AGC
57AdultMalePhap Viet Hospital(+)(+)SRRRS16AGC
37AdultMaleNguyen Tri Phuong Hospital(+)(+)SRRSS3AGC
45AdultFemaleTrieu An Hospital(+)(+)SSSSR0.38AGC
12ChildrenMaleChildren's Hospital 2(+)(+)SRRSS8AGC
11ChildrenMaleChildren's Hospital 1(+)(+)SRSSR<0.016AGC
15ChildrenMaleChildren's Hospital 2(+)(+)SSRSR12AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR3AGC
10ChildrenFemaleChildren's Hospital 1(+)(+)SRRSS2AGC
10ChildrenFemalePhap Viet Hospital(+)(+)SRRRS8AGC
51AdultFemaleYersin International Clinic(+)(+)SRRSS1.5AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SRRSS6AGC
10ChildrenMaleYersin International Clinic(+)(+)SRRSS3AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)RSRRS8AGC
8ChildrenFemaleChildren's Hospital 2(+)(+)RSRSS8AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SRRSR8AGC
8ChildrenMaleChildren's Hospital 1(+)(+)RRRSS4AGC
9ChildrenFemaleChildren's Hospital 1(+)(+)SSRSR3AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SSRSR2AGC
14ChildrenMaleChildren's Hospital 2(+)(+)SRRSS12AGC
11ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR12AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)RSSSS0.064AGC
12ChildrenMaleChildren's Hospital 2(+)(+)SRRSR16AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)RSRSR6AGC
9ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)RSRSS2AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SSRRS3AGC
11ChildrenMaleChildren's Hospital 2(+)(+)SSRSS3AGC
7ChildrenMaleChildren's Hospital 2(+)(+)SSRRR4AGC
11ChildrenMaleChildren's Hospital 2(+)(+)SSRSS12AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS6AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)RRRSS1.5AGC
15ChildrenFemaleChildren's Hospital 1(+)(+)SSRRS24AGC
5ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS3AGC
9ChildrenMaleChildren's Hospital 2(+)(+)SRRSS6AGC
6ChildrenMaleChildren's Hospital 1(+)(+)SSRSS2AGC
8ChildrenMaleChildren's Hospital 1(+)(+)SSRSS1.5AGC
24AdultMaleDH Y Duoc Hospital(+)(+)SRRSS6AGC
35AdultFemaleDH Y Duoc Hospital(+)(+)SRRRS1AGC
6ChildrenMaleChildren's Hospital 1(+)(+)RSSRS<0.016AGC
13ChildrenMaleChildren's Hospital 1(+)(+)SSSSS<0.016AGC
50AdultFemaleDH Y Duoc Hospital(+)(+)SSRRS>256GAC
37AdultFemaleDong Nai International Hospital(+)(+)SRRSS1AGC
12ChildrenFemaleChildren's Hospital 2(+)(+)SSSSS<0.016AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS2AGC
8ChildrenFemaleChildren's Hospital 1(+)(+)RSRSS6AGC
8ChildrenFemaleChildren's Hospital 1(+)(+)SRRSS16AGC
5ChildrenFemaleChildren's Hospital 2(+)(+)RSRSS2AGC
5ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS4AAT
9ChildrenMaleChildren's Hospital 2(+)(+)RSRSS6AAT
8ChildrenMaleChildren's Hospital 2(+)(+)SSRSS2AGC
41AdultFemaleTrieu An Hospital(+)(+)SSRRS2AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS4AGC
10ChildrenMaleChildren's Hospital 2(+)(+)SRRSS12AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS4AGC
9ChildrenMaleChildren's Hospital 2(+)(+)SSRSS4AGC
7ChildrenMaleChildren's Hospital 2(+)(+)SSRRS4AGC
13ChildrenFemaleChildren's Hospital 2(+)(+)SRRRS1AGC
6ChildrenMaleChildren's Hospital 2(+)(+)SSRRS4AGC
6ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS16AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)SRRSS1.5AGC
14ChildrenMaleChildren's Hospital 1(+)(+)SSSRS<0.016AGC
51AdultFemaleTrieu An Hospital(+)(+)SRRSR2AGC
41AdultFemalePhap Viet Hospital(+)(+)SRRRS0.75AGC
43AdultFemaleTrieu An Hospital(+)(+)SSSRS<0.016AGC
13ChildrenFemaleChildren's Hospital 2(-)(-)SSRRR3AGC
4ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS24AGC
43AdultFemaleDH Y Duoc Hospital(+)(+)SSRRR8AAT
13ChildrenFemaleChildren's Hospital 2(-)(-)SSRRR3AGC
11ChildrenFemaleChildren's Hospital 2(+)(+)SSRSR3AGC
9ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
9ChildrenMaleYersin International Clinic(+)(+)SRRSS3AGC
3ChildrenMaleChildren's Hospital 2(+)(+)SRRSS4AGC
8ChildrenFemaleChildren's Hospital 2(+)(+)SSRRS0.75AGC
8ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
9ChildrenFemaleChildren's Hospital 2(+)(+)SRRSR2AGC
8ChildrenMaleChildren's Hospital 2(+)(+)SSRSR12AGC
5ChildrenFemaleChildren's Hospital 1(+)(+)SSRSS6AGC
35AdultFemaleGia Dinh People's Hospital(+)(+)SSRSS8AGC
13ChildrenMaleChildren's Hospital 2(+)(+)SSRSS6AGC
12ChildrenMaleChildren's Hospital 2(+)(+)SSRSS2AGC
65AdultFemalePhap Viet Hospital(+)(+)SSRRS16AGC
31AdultFemaleDH Y Duoc Hospital(+)(+)SSRSR2AGC
14ChildrenMaleChildren's Hospital 2(+)(+)SRRSS2AGC
56AdultMaleDH Y Duoc Hospital(-)(+)SSRSS8AAT
7ChildrenMalePhap Viet Hospital(+)(+)SSRSS2AGC
9ChildrenMaleChildren's Hospital 2(+)(+)SSRSS3AGC
49AdultFemaleDH Y Duoc Hospital(+)(+)SRRSR6AGC
3ChildrenMaleChildren's Hospital 2(+)(+)SSRSR8AGC
8ChildrenMaleChildren's Hospital 2(+)(+)SSRSR3AGC
12ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS4AGC
3ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS0.75AGC
10ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS2AGC
35AdultFemaleVan Hanh General Hospital(+)(+)SSRRS6AGC
11ChildrenFemaleChildren's Hospital 2(+)(+)SSRSS6AGC
4ChildrenMaleChildren's Hospital 2(+)(+)SRRRS4AGC
11ChildrenMaleChildren's Hospital 2(+)(+)SSRRS6AGC
60AdultFemaleDH Y Duoc Hospital(+)(+)SSRSS3AGC
29AdultMaleDH Y Duoc Hospital(+)(+)SSRRS4AGC
39AdultMaleTrieu An Hospital(+)(+)SRRSS4AGC
12ChildrenMaleTrieu An Hospital(+)(+)SSRRS2AGC
19AdultMaleTam Nhat Clinic(+)(+)SRSSS<0.016AGC
24AdultFemaleDai Phuoc Clinic(+)(+)SSSSS<0.016AGC
8ChildrenFemaleChildren's Hospital 2(+)(+)SSSSS<0.016AGC
7ChildrenMaleChildren's Hospital 2(+)(+)SSRSS1AGC
12ChildrenMaleChildren's Hospital 2(+)(+)SSSSS<0.016AGC
55AdultFemaleDH Y Duoc Hospital(+)(+)SSRSS1.5AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SSRRS0.75AGC
7ChildrenFemaleChildren's Hospital 2(+)(+)SRRRS6AGC
39AdultFemalePhap Viet Hospital(+)(+)SRRRS3AGC
13ChildrenMaleChildren's Hospital 1(+)(+)SSSSS<0.016AGC
11ChildrenFemaleChildren's Hospital 1(+)(+)SRRRS12AGC
39AdultMaleTrieu An Hospital(+)(+)SRRSS8AGC
43AdultFemaleDai Phuoc Clinic(+)(+)SSRSS1AGC
31AdultMalePhap Viet Hospital(+)(+)SSRSR2AGC
42AdultFemaleDH Y Duoc Hospital(+)(+)SSSSR<0.016AAT
8ChildrenMalePhap Viet Hospital(+)(+)SSRSS0.75AGC
8ChildrenMalePhap Viet Hospital(+)(+)SSRSS2AGC
33AdultFemaleHoan My Hospital(+)(+)SSSSS<0.016AAT
34AdultMalePhap Viet Hospital(-)(+)SRRSS1AAT
27AdultFemalePhap Viet Hospital(+)(+)SRRRS1AGC
33AdultMaleDH Y Duoc Hospital(+)(+)SRRRS12AAT
51AdultFemaleDH Y Duoc Hospital(+)(+)SSRRS8GAC
12ChildrenMaleChildren's Hospital 1(+)(+)SSSSR<0.016AGC
69AdultFemaleYersin International Clinic(+)(+)SSRSS0.75AAT
12ChildrenMaleChildren's Hospital 1(+)(+)SSRSS1AGC
8ChildrenMaleChildren's Hospital 1(+)(+)RSSSS0.5AGC
57AdultFemaleTam Nhat Clinic(+)(+)SSRSS1.5AGC
33AdultFemaleDH Y Duoc Hospital(+)(+)SRSRS0.5AGC
46AdultFemaleDH Y Duoc Hospital(+)(+)SSRSS2AGC
10ChildrenMalePhap Viet Hospital(+)(+)SSRSS1AAT
10ChildrenFemaleChildren's Hospital 1(+)(+)SSRSR1.5AGC
43AdultMaleDH Y Duoc Hospital(+)(+)SRRRS2AGC
40AdultFemaleHoan My Hospital(+)(+)SSRRS2AGC
Dataset 1.A summary of patient information, antimicrobial susceptibility and clarithromycin resistance patterns.
The table summarised individual patient records such as age, gender, hospital, and presence of H. pylori strains. Antimicrobial susceptibility testing and clarithromycin resistance patterns of each patient is also included in the data.

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 reports1820. 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.

Table 1. Prevalence of antimicrobial resistance in Helicobacter pylori isolates.

Antimicrobial
resistance
Children, n = 136
N (%)
Adults, n = 57
N (%)
Total, n = 193
N (%)
Amoxicillin17 (12.5)3 (5.3)20 (10.4)
Clarithromycin115 (84.6)50 (87.7)165 (85.5)
Levofloxacin26 (19.1)21 (36.8)47 (24.4)
Metronidazole46 (33.8)27 (47.4)73 (37.8)
Tetracycline32 (23.5)14 (24.6)46 (23.8)
a0ba3f63-afc0-424b-9cd2-01cac3ed4775_figure1.gif

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.

Minimum inhibitory concentration values of clarithromycin-resistant 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).

a0ba3f63-afc0-424b-9cd2-01cac3ed4775_figure2.gif

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.

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 clarithromycin-susceptible and –resistant isolates. Only two clarithromycin-resistant 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 strains34.

Table 2. Minimum inhibitory concentration values and 23S rRNA mutations of clarithromycin-susceptible and -resistant isolates.

Mutation(s)No. of susceptible
isolates
No. of resistance
isolates
Total
N (%)
MICs
(μg/mL)
ChildrenAdultsChildrenAdults
A2143G + T2182C19311243177 (91.7)≤0.25 (S) and
>0.5 (R)
A2142G + T2182C00022 (1.0)8 and >256
A2143G + T2182C*N.A.N.A.N.A.N.A.4 (2.1)0.38 and 0.5
No mutations023510 (5.2)>0.016 and ≤12
Total193 (100)

‘*’ indicates H. pylori isolates with A2143G and T2182C mutation at MIC values, which are considered to be intermediate resistance strains.

Abbreviations: ‘N.A.’ – not applicable; ‘S’ – susceptible; ‘R’ – resistance.

Discussion

Antimicrobial resistance in H. pylori has become a global health problem because the prevalence of infection and incidence is increasing worldwide3739. The increasing H. pylori resistance to antimicrobial agents, such as clarithromycin, is considered the main factor for reduced treatment success in several countries, including Vietnam and Japan17,18,4042. Therefore, the understanding of geographical region specific prevalence is crucial for treatment of H. pylori infection.

Vietnam is categorised as a region with a high prevalence of H. pylori infection and an intermediate risk of gastric cancer43,44. In Vietnam (Ho Chi Minh City and Hanoi), clarithromycin and metronidazole are recommended as a first-line therapy regimen13. 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 Vietnam1820. 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 studies19,32,4749. The variability of MIC values for resistant isolates might be attributed to different gastric sites. The evidence is supported by Borody et al. who demonstrated that the bimodal distribution of clarithromycin resistance of isolates cultured from 4 gastric sites (i.e. antrum, distal body, proximal body and fundus) ranged from <0.016 μg/mL to 256 μg/mL50. The recent studies also demonstrated that MIC values for clarithromycin resistance vary at different gastric sites4749. Therefore, the present results confirm previous studies that multiple gastric biopsies from different sites of the stomach are crucial for accurate diagnosis of H. pylori infection.

Furthermore, antimicrobial susceptibility testing using MIC values is often used to determine the appropriate dosage of antimicrobial for a patient’s prescription. However, the respective antimicrobial resistance rate is based on the defined MIC breakpoints, which are much lower than the achievable tissue concentrations of antimicrobial agents such as clarithromycin (ranging from 5.2 μg/mL to 22.2 μg/mL)51. Only a few studies have reported the eradication rate of H. pylori infection with high MIC values (e.g. >24 μg/mL), highlighting that the significant eradication rate of 50%−80% on MIC-defined resistant strains can be achieved by administering PPI with precise antibiotic dosage and appropriate treatment duration20,32,52,53. Hence, further longitudinal studies on treatment efficacy and treatment guidelines are necessary for successful treatment.

Point mutations at positions 2142, 2143 and 2182 on the 23S rRNA gene were commonly reported2527. Yet it remains unclear whether or not these point mutations could be a strong predictor of clarithromycin resistance23,2931. In some studies, only the A2142G mutation was found to be associated with high MIC values54,55. While other studies showed that mutations at positions 2142 and/or 2143 were associated with clarithromycin resistance53,54,56,57. In addition, mutation T2182C was only reported in one study25. Here, we reported that H. pylori strains with mutations in A2143G and T2182C exhibited not only in clarithromycin-resistant strains, but also in susceptible strains as observed in Table 2. Similar to Phan et al.’s study19, none of the clarithromycin-resistant strains portrayed A2142C mutation in our study. It is important to note that the association of MIC values and point mutations was not identified in our work. Additionally, a proportion of all isolates had no point mutations in the 23S rRNA gene (Table 2). Further investigation on other nucleotide positions of the 23S rRNA region should be performed on these resistant strains58,59. 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 gene6062.

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.

Data availability

F1000Research: Dataset 1. A summary of patient information, antimicrobial susceptibility and clarithromycin resistance patterns, 10.5256/f1000research.8239.d11824963

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Quek C, Pham ST, Tran KT et al. Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam [version 1; peer review: 2 approved]. F1000Research 2016, 5:671 (https://doi.org/10.12688/f1000research.8239.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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Reviewer Report 02 Aug 2016
Simon Cutting, School of Biological Sciences, Royal Holloway, University of London, Egham, UK 
Approved
VIEWS 16
In general this is an interesting paper reporting on resistance ot the antibiotic clarithromycin in Vietnam and the relevance of this resistance to Helicobacter pylori infection. The main outcome of this work is that it demonstrates the need for susceptibility ... Continue reading
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Cutting S. Reviewer Report For: Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam [version 1; peer review: 2 approved]. F1000Research 2016, 5:671 (https://doi.org/10.5256/f1000research.8861.r13392)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 31 May 2016
Duc Trong Quach, Department of Internal Medicine, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam 
Approved
VIEWS 29
This is one among the informative studies about the antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam. The title is appropriate for the content. The methods and analysis of the results are well-described and appropriate.

... Continue reading
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Quach DT. Reviewer Report For: Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam [version 1; peer review: 2 approved]. F1000Research 2016, 5:671 (https://doi.org/10.5256/f1000research.8861.r14071)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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