Helicobacter. 2020;00:e12684. wileyonlinelibrary.com/journal/hel
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1 of 8 https://doi.org/10.1111/hel.12684© 2020 John Wiley & Sons Ltd Received: 6 November 2019
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Revised: 25 December 2019|
Accepted: 4 February 2020DOI: 10.1111/hel.12684
O R I G I N A L A R T I C L E
Antibiotic resistance and clonal relatedness of Helicobacter pylori strains isolated from stomach biopsy specimens in northeast of Iran
Sara Hamidi
1| Farzad Badmasti
2| Fatemah Sadeghpour Heravi
3|
Mohammed Hossein Safapoor
4| Amir Mohammad Ali Tabrizi
1| Mohammad Ghorbani
5| Omid Azizi
1,61Department of Laboratory Sciences, School of Paramedical Sciences, Torbat Heydariyeh University of Medical sciences, Torbat Heydariyeh, Iran
2Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran
3Surgical Infection Research Group, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia
4Department of Internal Medicine, 9 Dey Educational Hospital, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
5Department of Public Health, School of Health, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
6Health Sciences Research Center, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
Correspondence
Omid Azizi, Assistant Professor, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran.
Emails: [email protected];
[email protected] Funding information
This research was supported by the Torbat Heydariyeh University of Medical Sciences.
Abstract
Background: Resistant Helicobacter pylori to commonly used antimicrobial agents are associated with severe upper gastrointestinal disorders. To provide an epidemiologi- cal picture of H pylori and characterize the resistance pattern and genetic variation of clinical isolates, stomach biopsies from patients with functional dyspepsia were evaluated in northeast of Iran.
Materials and methods: In this study, 80 patients were recruited. Finally, fifty H pylori strains were isolated from antrum and corpus biopsies by culturing on Columbia agar.
All strains were identified by standard laboratory procedures. Susceptibility testing of antibiotics was performed using minimum inhibitory concentration test. Allele- specific primer (ASP)-PCR of 23S rRNA which associated with clarithromycin resist- ance was done among resistant strains. Moreover, cagA gene and polymorphism in vacA were detected. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied to investigate the genetic variations among all strains.
Results: Antibiotic resistance pattern of H pylori strains was as follows: 68% (34/50) to metronidazole, 50% (25/50) to rifampicin, 30% (15/50) to amoxicillin, 28% (14/50) to levofloxacin, 22% (11/50) to clarithromycin, and 16% (8/50) to tetracycline.
Multidrug-resistant strains were observed in 19 strains (38%). ASP-PCR of 23S rRNA showed four strains had A2143G mutation, six strains had A2142G mutation, and one strain had a Wt+A2143G mutation. Amplification of virulence-associated genes revealed that cagA was present in 27 isolates (54%) and vacA in 36 isolates (72%). The most common genotype of H pylori was vacA s1am2 (40%) followed by vacA s2m2 (14%), vacA s1am1 (12%), vacA s1bm1 (4%), and vacA s1bm2 (2%). DNA fingerprinting pattern indicated a high heterogeneity among isolated strains.
Conclusion: An alarming level of resistance to metronidazole and rifampicin and high heterogeneity among H pylori isolates highlighted the importance of continued moni- toring of antimicrobial susceptibility and epidemiological surveillance of this pathogen.
K E Y W O R D S
antibiotic resistance and clonal relatedness, Helicobacter pylori
1 | INTRODUCTION
Helicobacter pylori is a gram-negative bacterium that colonizes the stomach and leads to one of the most prevalent infectious diseases.1 It has been estimated that more than 50% of the adult population is affected by H pylori infection. More importantly, based on the clas- sification of the World Health Organization (WHO), H pylori is con- sidered in group 1 known as a definite carcinogen associated with gastritis, peptic ulcer, and gastric cancer.2
Many different virulence factors are involved in initial coloni- zation and tissue destruction and are linked to the pathogenesis of H pylori such as flagella-mediated motility, urease, cytotoxin-associ- ated gene A (CagA) and vacuolating cytotoxin A (VacA).3,4 Vacuolation in the epithelium of the stomach may have varying consequences depending on the composition of the VacA toxin. The vacA gene exhibits allelic variation in three major regions known as the signal (s1a, s1b, s1c, and s2), intermediate (i1, i2, and i3), and the middle regions (m1, m1T, and m2).5 Depending on the arrangement of the aforementioned regions, different subtypes can form different vacA genotypes. Based on previously published findings, the expression of VacA with genotype s1/m1 may result in more severe damage to stomach tissue compared with other subtypes.3 Identification of abundant H pylori genotypes may have a great impact on diagnostic and therapeutic approaches.
Helicobacter pylori infections have been treated empirically with one proton-pump inhibitor (PPI) and two alternative antibi- otics (such as amoxicillin, tetracycline, metronidazole, and clari- thromycin). However, the eradication rate of H pylori infection is decreasing in children and adults—as low as 60% in some coun- tries—indicating the increased rate of antibiotic resistance all over the world.6 The prevalence of clarithromycin-resistant H py- lori has increased and remained as one of the main challenges of healthcare systems in industrialized and developing countries.7 Clarithromycin-resistant H pylori can lead to about 70% decrease in the efficiency of antibiotic therapy. The rate of antibiotic resis- tance of H pylori may vary according to the geographical location of patients. In this regard, continuous monitoring of antibiotic resis- tance patterns of H pylori strains is crucial to assess the efficiency of empirical therapy and replacement of current therapy with tar- geted therapy.8 Therefore, in the present study, we analyzed the antimicrobial resistance and clonal relatedness of H pylori strains from stomach biopsy specimens in the northeast of Iran.
2 | MATERIAL AND METHODS
2.1 | Sample collection
A cross-sectional study on 80 patients undergoing gastroenterol- ogy and endoscopy at educational Hospital (northeast of Iran) was conducted during a 5-month period from November 2017 to March 2018. Patients with functional dyspepsia who had not received oral antibiotics, proton-pump inhibitors (PPIs), or bismuth compounds in
the 2 weeks prior to the endoscopy were eligible for enrollment in this study. Patients who underwent endoscopy were biopsied based on the standards of practice committee of the American Society for gastrointestinal endoscopy.9,10 This study was approved by the Ethics Committee of Torbat Heydariyeh University of Medical Sciences (Reference number: IR.THUMS.REC.1396.21).
2.2 | Sample preparation and bacterial culture
Gastrointestinal endoscopy was performed by a gastroenterolo- gist using Pentax EG-2990K Video Gastroscope (Pentax Medical Company). Two gastric mucosae taken from the antrum and corpus were transported at 4°C in a 0.9% saline solution. Antrum and cor- pus biopsies were mixed and homogenized. Rapid urease testing (RUT) was performed on 50 μL of the homogenized biopsy according to the standard procedure, and the remaining biopsy was subjected to the bacterial culture on Columbia agar medium (Merck) enriched with 10% human blood, 10% fetal bovine serum (FBS; Gibco), and selective supplemented (10 µg/mL of vancomycin, 5 µg/mL of tri- methoprim lactate, 5 µg/mL of cefsulodin, and 5 µg/mL of ampho- tericin B) (Sigma-Aldrich, USA) at pH 6.8-7.0 and incubated at 37°C for 5-10 days.11 Bacterial colonies exhibiting typical morphology of H pylori were isolated and verified by Gram staining (−), rapid urease (+), oxidase (+), and catalase (+) tests.11,12
2.3 | Antimicrobial susceptibility testing
Antibiotic susceptibility of isolated H pylori was performed using minimum inhibitory concentrations (MICs) by the agar dilution method13 using Mueller-Hinton agar (Merck) with 5% horse blood for the concentration of the following antibiotics: 8-256 μg/mL of metronidazole, 0.25-8 μg/mL of clarithromycin, 0.125-2 μg/mL of amoxicillin, 1-8 μg/mL of levofloxacin, 1-8 μg/mL of tetracycline, and 1-8 μg/mL of rifampicin (Sigma). MICs' interpretation was carried out based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Helicobacter pylori ATCC 43504 was used as the control strain. The breakpoints were defined as follows: amoxicil- lin, MIC > 0.125 μg/mL; clarithromycin, MIC > 0.5 μg/mL; metroni- dazole, MIC > 8 μg/mL; tetracycline, MIC > 1 μg/mL; levofloxacin, MIC > 1 μg/mL; and rifampicin, MIC > 1 μg/mL which were inter- preted by European Committee on Antimicrobial Susceptibility Testing (UCAST) guidelines (version 8.0). Resistance to at least three classes of antibiotics was considered as a multidrug resistance iso- late (MDR).14
2.4 | PCR detection of cagA and vacA genes
Bacterial DNA was extracted using the DNA Mini Kit (Qiagen) ac- cording to the manufacturer's instruction. The purity and quantity of extracted DNA were determined by Thermo Scientific™ NanoDrop™
One. Genes cagA, vacA s- and m-region alleles (s1a, s1b, s1c, s2, m1, and m2) were amplified using PeqSTAR Thermocycler (PEQLAB).
DNA amplification was performed under the following program: an initial temperature of 95°C for 15 minutes followed by 40 cycles of 95°C for 30 seconds, annealing for 30 seconds, and a further 72°C for 10 minutes. Primer's properties and size of PCR products are shown in Table 1.
2.5 | Allele-specific primers (ASP)-PCR of 23S rRNA associated with clarithromycin resistance
Determination of the susceptibility of H pylori to clarithromycin was carried out by the genotyping of the A2142G and A2143G muta- tions of 23S rRNA using four different primers under allele-specific primer-polymerase chain reaction (ASP-PCR) technique as described previously.15 ASP-PCR was performed with AccuPOL™ DNA poly- merase (Ampliqon) and obtained a band of 320 bp for both wild type (wt) and mutant. An amplicon size with 238 bp band showed the A2142G mutation and 118 bp showed the A2143G mutation.15 DNA amplification was conducted under the following conditions: 1 cycle at 94°C for 2 minutes, 40 cycles of 98°C for 10 seconds, 65°C for 30 seconds, and 68°C for 20 seconds, with a final extension at 72°C for 2 minutes.16
2.6 | Random amplification of polymorphic DNA (RAPD)
To study the clonal lineage of the H pylori isolates, genomic DNA was extracted for RAPD typing test using primer combinations includ- ing 1281 (5′-AACGCGCAAC-3′) and 1254 (5′-CCGCAGCCAA-3′) as described previously.17 DNA Banding patterns were analyzed using the GelCompar II software version 4.0 (Applied Maths, Sint- Martens-Latem). Degrees of homology were determined by Dice
coefficient, and clustering was calculated by the UPGMA (un- weighted pair group method with arithmetic averages) with 80%
similarity cutoff.
3 | RESULTS
3.1 | Demographic data of Patient population
A total of 80 patients with the average age of 50.2 ± 12.94 (mean ± SD) with required criteria (functional dyspepsia, not receiv- ing oral antibiotics, proton-pump inhibitors, and bismuth compounds during the last 2 weeks of endoscopy) were recruited in this study.
Finally, 50 consecutive non-duplicate H pylori (62.5%) strains (RUT+) were isolated (Table 2).
3.2 | Helicobacter pylori antibiotic resistance pattern
Based on the minimum inhibitory concentration test (MIC), nineteen isolated H pylori strains (38%) were resistant to at least three classes of antibiotics and considered as multidrug resistance isolates (MDR), while seven isolated strains (14%) were sensitive to all the tested antibiotics (metronidazole, clarithromycin, amoxicillin, levofloxacin, tetracycline, and rifampicin). The highest (n = 34/50, 68%) and the lowest (n = 8/50, 16%) number of resistant isolated strains were ob- served in antibiotics metronidazole and tetracycline, respectively (Table 2). Also, resistance to clarithromycin was observed in 22%
(n = 11/50) of isolated strains, of which four strains had A2143G mutation (118 bp band), six strains had A2142G mutation (238 bp band), and one strain had mixed of Wt DNA band + A2143G muta- tion (320 bp + 118 bp band) (Figure 1). The MIC values for clarithro- mycin of the strains with mutations in 23S rRNA were distributed at 1-8 µg/mL. Six isolated strains (12%) were resistant to both clarithro- mycin and metronidazole.
TA B L E 1 List of primers for genotyping used in the present study Gene or DNA region
amplified Primer sequence (5′→3′) Annealing temperature Product size (bp) Reference
cagA F: GATAACAGGCAAGCTTTTGAGG
R: CTGCAAAAGATTGTTTGGCAGA
56°C 349 42
vacAs1a F: GTCAGCATCACACCGCAAC
R: CTGCTTGAATGCGCCAAAC
52°C 190 43
vacAs1b F: AGCGCCATACCGCAAGAG
R: CTGCTTGAATGCGCCAAAC 52°C 187 44
vacAs1c F: TTAGTTTCTCTCGCTTTAGTRGGGYT
R: CTGCTTGAATGCGCCAAAC 52°C 220 45
vacAs2 F: ATGGAAATACAACAAACACAC
R: CTGCTTGAATGCGCCAAAC
56°C 286 43
vacAm1, vacAm2
F: CAATCTGTCCAATCAAGCGAG R: GCGTCTAAATAATTCCAAGG
56°C m1:570
m2: 645
43
TA B L E 2 Data on 50 non-duplicated H pylori isolates recovered from gastric biopsies
No. Strain Age Gender
Clinical profile
Antibiotic resistance (MIC, µg/mL)
23S rRNA
mutation CagA VacA type
AMX RIF TET CLR LVX MTZ
1 HP.3 35 F AP S S S S S S ND Neg. s1am2
2 HP. 6 55 M AP S R(4) S R(8) S R(64) A2143G Pos. s1am1
3 HP.8 72 F V R(0.25) R(2) S S S S ND Neg. s2m2
4 HP.10 33 M G R(0.25) S S S S R(64) ND Neg. s1bm2
5 HP.32 65 M SP R(0.25) S S R(8) R(8) S A2143G Pos. s1am1
6 HP.34 30 M G S S S S S R(128) ND Pos. s1am2
7 HP.36 44 M PU S R(4) S S R(8) S ND Pos. s1am2
8 HP.39 75 F AP S R(8) S S S R(64) ND Pos. s1am2
9 HP.41 21 M AP S S S S S S ND Pos. s1am1
10 HP.45 55 F D S S S S S S ND Pos. s1bm1
11 HP.48 62 M D S R(8) R(2) S S R(16) ND Pos. s1am2
12 HP.49 58 F D S S S S S R(32) ND Neg. s2m2
13 HP.50 52 F AP S S S S S S ND Neg. Neg.
14 HP.51 62 F PU S R(4) S S S R(64) ND Neg. s1am2
15 HP.55 39 F V R(0.5) S S S R(4) S ND Pos. s1am1
16 HP.58 58 F G S R(2) S S S S ND Pos. s1am2
17 HP.59 69 F G R(0.25) S S S S R(16) ND Neg. Neg.
18 HP.60 70 F G S S S R(2) S S A2142G Neg. s1am2
19 HP.61 50 M D S S S S R(8) R(64) ND Pos. Neg.
20 HP.62 38 F V S R(2) S S R(8) R(64) ND Neg. s1am2
21 HP.63 61 M D R(0.25) S R(2) S S S ND Pos. s1am2
22 HP.64 55 F G R(0.25) R(2) S S S R(64) ND Neg. Neg.
23 HP.68 56 F V S R(2) S S S S ND Pos. s1am2
24 HP.70 56 F V S S S S S S ND Neg. Neg.
25 HP.74 59 F G S S R(8) S S R(32) ND Neg. Neg.
26 HP.75 52 F DU R(0.5) R(2) S S R(8) R(64) ND Pos. Neg.
27 HP.79 48 F G S R(2) S R(8) R(8) R(128) A2143G Pos. s1am2
28 HP.82 65 M AP S S S R(1) S R(32) A2142G Neg. s1am2
29 HP.87 38 M D S R(8) S S S R(64) ND Neg. Neg.
30 HP.95 37 F PU R(0.5) S S S R(8) R(16) ND Pos. s1am2
31 HP.101 50 F G S R(4) S S R(8) R(64) ND Pos. Neg.
32 HP.105 55 F D S S S S S R(64) ND Neg. s1am2
33 HP.106 41 M AP S S R(2) R(1) S S A2142G Pos. s1am1
34 HP.111 49 F G R(0.25) S S S S R(16) ND Pos. Neg.
35 HP.113 33 F G R(0.25) R(2) S S S R(64) ND Pos. s1am2
36 HP.118 41 F AP S R(2) S R(8) S R(64) A2142G Pos. Neg.
37 HP.119 52 F AP S R(8) S S R(8) R(64) ND Neg. Neg.
38 HP.120 36 M AM S R(8) S S S R(64) ND Pos. s1am1
39 HP.121 44 F G S R(4) S S S R(64) ND Neg. s2m2
40 HP.123 45 F D S R(2) S S S R(64) ND Neg. s2m2
41 HP.124 59 M AP S S R(8) R(1) R(4) R(64) A2142G Neg. Neg.
42 HP.125 50 F G R(0.25) R(4) S S R(8) R(64) ND Pos. s1bm1
43 HP.128 47 F G R(2) S R(8) S S R(32) ND Neg. s2m2
44 HP.133 43 M AP S R(2) S R(8) S R(64) Wt + A2143G Pos. s1am2
(Continues)
3.3 | PCR detection of cagA and vacA genes
Polymerase Chain Reaction demonstrated positive results in 54%
(27/50) of strains for cagA and 72% (36/50) of strains for vacA gene.
Based on ASP-PCR result, among vacA positive strains (n = 36), s1am2 was the most common genotype in H pylori strains (n = 22/36), followed by s2m2 (n = 7/36). In contrast, s1bm1 (n = 2/36) and s1bm2 (n = 1/36) were detected only in one or two strains, respectively (Table 2).
3.4 | Genetic diversity
DNA fingerprinting pattern of H pylori isolates was assessed using RAPD method. The number of DNA fragments amplified in the RAPD ranged from 8 to 16 bands. Among 50 H pylori strains, four different RAPD patterns, each one contained two isolates, were found with an 80% similarity cutoff. Other strains were singleton.
The RAPD patterns of H pylori strains confirmed a high heterogene- ity among isolates (Figure 2).
4 | DISCUSSION
Helicobacter pylori is implicated in the development of many gastro- intestinal disorders such as gastritis, mucosa-associated lymphoma and gastric adenocarcinoma.18 The prevalence of H pylori infections is more common in less industrialized countries (90%) compared with industrialized countries (40%).19 In Iran, H pylori infection is present in nearly 90% of the adult population.20 The standard antibiotic regimen for H pylori infection is comprised of a combination of a proton-pump inhibitor with two antibiotics such as clarithromycin and metronida- zole or amoxicillin. This regimen has been reported to be successful in 70%-90% of cases in the absence of multidrug-resistant strains.21 Due to the low MIC value of clarithromycin and its stability in low pH as well as a high binding affinity to H pylori ribosomes, it is one of the main prescribed macrolide antibiotics for H pylori infection. However, overuse of this antibiotic in some countries has changed the pheno- type of clarithromycin susceptible strains to resistant phenotypes.22 Therefore, identification of clarithromycin resistance pattern, as well as other commonly used antibiotics in circulating strains may prevent unnecessary antibiotic consumption in similar cases.
In this study, we tested the susceptibility of 50 H pylori isolated strains to empirically used antibiotics (metronidazole, clarithromycin, amoxicillin, levofloxacin, tetracycline, and rifampicin) using minimum inhibitory concentration method and evaluated the clonal relatedness of isolated strains by means of Random Amplification of Polymorphic DNA (RAPD). Our findings showed a high rate of multidrug-resistant H pylori isolates to at least three classes of tested antibiotics (38%, n = 50). In contrast, seven H pylori isolates were sensitive to all the tested antibiotics (n = 50, 14%). The highest number of isolated strains in this study were resistant to metronidazole (68%, n = 50) as one of the main prescribed antibiotics to prevent H pylori infection. Overuse of metronidazole is associated with higher antibiotic resistance rate in developing countries.23 Increased rate of resistance to metronidazole was also previously noted in different studies conducted in different No. Strain Age Gender
Clinical profile
Antibiotic resistance (MIC, µg/mL)
23S rRNA
mutation CagA VacA type
AMX RIF TET CLR LVX MTZ
45 HP.134 40 F AP S S S S S R(64) ND Neg. s1am2
46 HP.142 41 F G S S S R(8) S S A2142G Neg. s2m2
47 HP.145 45 F G R(1) R(2) R(8) S R(8) R(256) ND Pos. s1am2
48 HP.150 23 M D R(0.25) R(4) S R(8) R(8) S A2143G Pos. s2m2
49 HP.151 68 F AP R(0.5) S R(8) S S R(32) ND Pos. s1am2
50 HP.165 78 M AP S R(4) S S S R(64) ND Neg. Neg.
Abbreviations: AM, anemia; AMX, amoxicillin; AP, abdominal Pain; CLR, clarithromycin; D, dyspepsia; DU, duodenal ulcer; F, female; G, gastritis; LVX, levofloxacin; M, male; MTZ, metronidazole; ND, not determined; PU, peptic ulcer; R, resistant; RIF, rifampicin; S, sensitive; SP, stomach polyp; TET, tetracycline; V, vomiting.
TA B L E 2 (Continued)
F I G U R E 1 Allele-specific primer-polymerase chain reaction (ASP-PCR) agarose gel electrophoresis of H pylori stains isolated from clinical samples. In wild-type (Wt) strains of H pylori, the 320 bp band is observed. In the case of 2143G mutation, a 118 bp band, and in the case of 2142G mutation, a 238 bp band is observed. In the case of wt + 2143G mutation, there are two DNA bands (320 bp + 118 bp band). These mutations showed clarithromycin resistance. NC, negative control; Wt, wild type
cities in Iran such as 30% of 80 isolates21 and 34.2% of 120 isolates,24 and 64.6% of patients.25 However, the metronidazole resistance rate varies geographically from 14.4% to 93.2% around the world.26 For instance, in the south and east of Europe, the prevalence of H pylori was estimated as high as 50%, and 11.4-100% in China, which led to the unsuccessful treatment of first-line therapy in 70% of the infected population.23,27 Although the prevalence of clarithromycin-resistant strains is still lower than bacterial resistance to metronidazole, it can be a serious problem in the near future.28 Based on previous findings, the rate of clarithromycin resistance was variable geographically from 9.9% to 43.5% in Europe,29 10-15% in the USA,30 4% in Canada,31 and 11-12% and 8-17% in Iran.32 In our study, the lowest number of isolated strains showed resistance to clarithromycin which was consistent with the previous study in Iran (n = 11, 22%).25
Dual bacterial resistance to clarithromycin and metronidazole is another major challenge in H pylori infection eradication. In this study, only six isolated strains (n = 12, 12%) with dual resistance to both clarithromycin and metronidazole were isolated which was rel- atively higher than previous findings in Iran (7.31%).25 It is recom- mended not to prescribe the combination of both metronidazole and clarithromycin as the first-line treatment particularly in countries with a high prevalence of resistance to these antibiotics.
Genetic diversity and remarkable heterogeneity between H py- lori strains may also lead to phenotypic changes and influence strains susceptibility to antimicrobial agents.33 Despite the extended view of the clonality of H pylori infection, findings vary and are some- times inconsistent depending on patient demographics (eg, coun- try, age, and hygienic conditions).34 For instance, a single H pylori clone has been reported predominantly in the infected population in Singapore35 and Japan,33 while in a close region to Singapore, patients were infected by multiple clones36 indicating the extent of H pylori genetic diversity in the world.
Compared with other epidemiological analysis methods such as pulse-field gel electrophoresis and ribotyping, RAPD-PCR pro- cedure is quick, easy to perform, and cost-effective and a reliable method for H pylori strain differentiation.33 DNA fingerprinting pattern of isolated strains showed high heterogeneity among iso- lated strains in this study. Due to high heterogeneity of H pylori, each infected subject may carry a distinctive strain. This char- acteristic can facilitate the adaptation of H pylori to the host im- mune system.37 For instance, vacuolating toxin (vacA s1m1) has been associated with a higher risk of gastric adenocarcinoma.38 In this study, we evaluated the pathogenicity of H pylori by the detection of cytotoxin-associated gene (cagA) and vacuolating cy- totoxin gene A (vacA). PCR of virulence-associated genes showed that cagA was detected in 27 isolates (54%) and vacA in 36 isolates F I G U R E 2 Computer-assisted random amplified polymorphic DNA (RAPD) typing of 50 isolates of H pylori. Clustering was based on the unweighted pair group method with arithmetic mean. The vertical line shows 80% similarity cutoff. This tree displays the genetic heterogeneity of H pylori isolates based on their RAPD profiles
(72%). The most common genotype of H pylori was vacA s1am2 (40%) in this study. Others identified were, vacA s2m2 (14%), vacA s1am1 (12%), vacA s1bm1 (4%), and vacA s1bm2 (2%). High prev- alence of s1am2 and s2m2 genotypes has also been reported in previous studies which demonstrated the colonization of gastric mucosa by multiple virulent types of H pylori strains and better adaption of genetically diverse bacterial strains to host immune response.39,40
In conclusion, the high prevalence of metronidazole-resistant strains was alarmingly high in this study which may indicate the im- portance of H pylori antibiotic susceptibility monitoring regularly.
The prevalence of bacterial strains resistant to both clarithromycin and metronidazole also requires more attention before it spreads clinically in the region. High bacterial resistance to metronidazole in this study may suggest the careful consideration of this antibiotic as first-line therapy, while lower resistance to clarithromycin and amox- icillin may indicate better clinical outcomes for future cases in this re- gion. There are several guidelines available such as the Maastricht V/
Florence Consensus and the American College of Gastroenterology (ACG) which recommend conducting H pylori antimicrobial suscepti- bility testing after the failure of the second course of therapy. Due to high resistance to metronidazole, antimicrobial susceptibility testing may be recommended if this antibiotic is chosen as the first line of treatment in our region.41
As a limitation in this study, we had a relatively small sample size and only a few H pylori virulence genes were evaluated. Such a study should not be limited to a period of time, and continuous monitoring of H pylori epidemiology is required for the development of thera- peutic approaches on a larger scale.
ACKNOWLEDGEMENTS
We would like to kindly appreciate our colleagues in Central Research Laboratory of Torbat Heydariyeh University of Medical Sciences who provided insight and expertise that greatly assisted the execution of current research.
CONFLIC T OF INTEREST
The authors have no competing interests.
ETHICAL APPROVAL
This study was approved by the Ethics Committee of Torbat Heydariyeh University of Medical Sciences (Reference number:
IR.THUMS.REC.1396.21).
ORCID
Omid Azizi https://orcid.org/0000-0001-7759-8029
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How to cite this article: Hamidi S, Badmasti F, Sadeghpour Heravi F, et al. Antibiotic resistance and clonal relatedness of Helicobacter pylori strains isolated from stomach biopsy specimens in northeast of Iran. Helicobacter.
2020;00:e12684. https ://doi.org/10.1111/hel.12684