Phenotypic and Genotypic Analysis of Resistant Helicobacter pylori Strains Isolated from Children with Gastrointestinal Diseases

Antibiotic resistance of Helicobacter pylori is currently a global issue. The aim of this study was to analyze actual antibiotic resistance rates of H. pylori strains isolated from children with primary infections and to compare the incidence of mutations that determine resistance to clarithromycin (CH) and metronidazole (MET) in children with different clinical diagnoses. A total of 91 H. pylori strains were isolated from 108 children with primary infections. Drug susceptibility testing of the strains was performed using E-test method. Classical sequencing of DNA fragments was used to detect point mutations for CH and MET resistance. Resistance to CH was detected in 31% of isolated strains (28/91), while resistance to MET and CH was detected in 35% (32/91) of strains. A2143G was the most frequently detected mutation and was dominant among strains isolated from children with peptic ulcer disease (80%). Mutations in the rdxA gene were found significantly more frequently among MET-resistant strains than MET-sensitive strains (p = 0.03, Chi2 = 4.3909). In children, a higher frequency of H. pylori multiresistant strains was observed compared with the previous study in the same area. Differences were found in the occurrence of point mutations among H. pylori strains resistant to CH isolated from children with different clinical diagnoses.

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A Next-Generation Sequencing-Based Approach to Identify Genetic Determinants of Antibiotic Resistance in Cambodian Helicobacter pylori Clinical Isolates

Journal of Clinical Medicine ◽

10.3390/jcm8060858 ◽

2019 ◽

Vol 8 (6) ◽

pp. 858 ◽

Cited By ~ 11

Author(s):

Vo Phuoc Tuan ◽

Dou Narith ◽

Evariste Tshibangu-Kabamba ◽

Ho Dang Quy Dung ◽

Pham Thanh Viet ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Genetic Resistance ◽

23S Rrna ◽

Next Generation ◽

Genetic Determinants ◽

Resistance Determinants ◽

Genotypic Analysis ◽

H Pylori ◽

Resistant Genotypes

We evaluated the primary resistance of Helicobacter pylori (H. pylori) to routinely used antibiotics in Cambodia, an unexplored topic in the country, and assessed next-generation sequencing’s (NGS) potential to discover genetic resistance determinants. Fifty-five H. pylori strains were successfully cultured and screened for antibiotic susceptibility using agar dilution. Genotypic analysis was performed using NGS data with a CLC genomic workbench. PlasmidSeeker was used to detect plasmids. The correlation between resistant genotypes and phenotypes was evaluated statistically. Resistances to metronidazole (MTZ), levofloxacin (LVX), clarithromycin (CLR), and amoxicillin (AMX) were 96.4%, 67.3%, 25.5%, and 9.1%, respectively. No resistance to tetracycline (TET) was observed. Multi-drug resistance affected 76.4% of strains. No plasmids were found, but genetic determinants of resistance to CLR, LVX, and AMX were 23S rRNA (A2146G and A2147G), GyrA (N87K and D91Y/N/G), and pbp1 (P473L), respectively. No determinants were genetically linked to MTZ or TET resistance. There was high concordance between resistant genotypes and phenotypes for AMX, LVX, and CLR. We observed high antibiotic resistance rates of CLR, MTZ, and LVX, emphasizing the need for periodic evaluation and alternative therapies in Cambodia. NGS showed high capability for detecting genetic resistance determinants and potential for implementation in local treatment policies.

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Trend of changes in antibiotic resistance in Helicobacter pylori from 2013 to 2019: a multicentre report from Taiwan

Therapeutic Advances in Gastroenterology ◽

10.1177/1756284820976990 ◽

2020 ◽

Vol 13 ◽

pp. 175628482097699

Author(s):

Chih-Ming Liang ◽

Wei-Chen Tai ◽

Pin-I Hsu ◽

Deng-Chyang Wu ◽

Chao-Hung Kuo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Linear Trend ◽

Eradication Therapy ◽

Resistance Rate ◽

Test Method ◽

Success Rates ◽

Primary Resistance ◽

Secondary Resistance ◽

H Pylori

Background: Antibiotic resistance plays a crucial role in the treatment failure of Helicobacter pylori (H. pylori) infection. This study aimed to determine the trend of changes in the primary, secondary and tertiary antibiotic resistance of H. pylori in Taiwan over the last 7 years. Methods: We retrospectively analysed H. pylori-infected isolates from patients with primary resistance ( n = 1369), secondary resistance ( n = 196) and tertiary resistance ( n = 184) from January 2013 to December 2019. The H. pylori strains were tested for susceptibility to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline using the Epsilometer test method. Results: A progressively higher primary resistance rate was observed for clarithromycin (11.8–20.4%, p = 0.039 in χ2 test for linear trend), levofloxacin (17.3–38.8%, p < 0.001) and metronidazole (25.6–42.3%, p < 0.001) among naïve patients who received first-line eradication therapy. The dual primary resistance to clarithromycin and metronidazole also progressively increased in a linear trend (2.4–10.4%, p = 0.009). For secondary resistance, an increase was observed for levofloxacin (30.5–64.7%, p = 0.006) and metronidazole (40.5–77.4%, p < 0.001). For tertiary resistance, the observed increase was even more significant for levofloxacin (65.9–100.0%, p = 0.106) and metronidazole (44.4–88.2%, p < 0.001). The resistance to amoxicillin and tetracycline remained very low in Taiwan regardless of primary, secondary and tertiary resistance. Conclusion: Primary, secondary and tertiary antibiotic resistance to clarithromycin, levofloxacin and metronidazole for H. pylori has been increasing in Taiwan since 2013. Treatment should be targeted for eradication success rates of more than 90%. Third-line treatment should be based on antibiotic susceptibility.

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Molecular Assessment of Resistance to Clarithromycin in Helicobacter pylori Strains Isolated from Patients with Dyspepsia by Fluorescent In Situ Hybridization in the Center of Iran

BioMed Research International ◽

10.1155/2020/2304173 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Jina Vazirzadeh ◽

Jamal Falahi ◽

Sharareh Moghim ◽

Tahmineh Narimani ◽

Rahmatollah Rafiei ◽

...

Keyword(s):

Helicobacter Pylori ◽

In Situ Hybridization ◽

Point Mutations ◽

23S Rrna ◽

Rrna Gene ◽

Clarithromycin Resistance ◽

Genotypic Analysis ◽

23S Rrna Gene ◽

H Pylori

Background and Aims. Helicobacter pylori is a common infectious bacterium mostly found in gastroduodenal diseases. The increased prevalence of clarithromycin-resistant H. pylori strains is a major challenge in the successful treatment of infections caused by this organism. The present study is aimed at detecting the clarithromycin resistance pattern of H. pylori strains isolated from gastric biopsies and evaluating point mutations of the 23S rRNA gene. Patients and methods. In the present descriptive cross-sectional study, 165 patients with gastrointestinal disorders, who were referred to the Endoscopy Center of Dr. Shariati Hospital of Isfahan, Iran, were enrolled from April to July 2018. H. pylori infection was diagnosed by culture, and susceptibility of the isolates to clarithromycin was assessed by the E-test. Minimum inhibitory concentration (MIC) values were obtained based on EUCAST recommendations. Also, fluorescence in situ hybridization (FISH) was used to determine point mutations associated with clarithromycin resistance. Results. By using culturing, H. pylori was isolated from 50.3% (83/165) gastric biopsy specimens. The overall frequency of resistance to clarithromycin was 25.3% (21/83) by the E-test. In the resistance genotypic analysis, 19 isolates had mutations. The prevalence of A2143G and A2144G mutations was 68.4% (13/19) and 31.5% (6/19), respectively. A2143C mutation was not tracked in any isolate. Two isolates with MIC>0.5 μg/mL had no mutations that could be related to other mechanisms of resistance. Conclusion. As presented in the study, the high prevalence of clarithromycin-resistant H. pylori due to point mutations of the 23S rRNA gene indicates the necessity of revising the standard treatment regimen based on antibiotic susceptibility pattern of each region.

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Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy

Journal of Medical Microbiology ◽

10.1099/jmm.0.067942-0 ◽

2014 ◽

Vol 63 (3) ◽

pp. 453-457 ◽

Cited By ~ 30

Author(s):

Vincenzo De Francesco ◽

Angelo Zullo ◽

Floriana Giorgio ◽

Ilaria Saracino ◽

Cristina Zaccaro ◽

...

Keyword(s):

Helicobacter Pylori ◽

Real Time ◽

Real Time Pcr ◽

Single Point ◽

Point Mutations ◽

Clarithromycin Resistance ◽

Genotypic Analysis ◽

Previously Treated ◽

Resistant Strains ◽

H Pylori

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8 %), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88 % (P<0.001; odds ratio 6.1, 95 % confidence interval 2−18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

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Random Mutagenesis of Helicobacter pylori vacA To Identify Amino Acids Essential for Vacuolating Cytotoxic Activity

Infection and Immunity ◽

10.1128/iai.00915-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6188-6195 ◽

Cited By ~ 5

Author(s):

Mark S. McClain ◽

Daniel M. Czajkowsky ◽

Victor J. Torres ◽

Gabor Szabo ◽

Zhifeng Shao ◽

...

Keyword(s):

Helicobacter Pylori ◽

Cytotoxic Activity ◽

Point Mutations ◽

Random Mutagenesis ◽

Single Amino Acid ◽

Hydrophobic Region ◽

Ulcer Disease ◽

Amino Terminal ◽

Selective Membrane ◽

H Pylori

ABSTRACT VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.

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Application of Visual Gene Clip-Based Tailored Therapy for the Eradication of Helicobacter pylori

BioMed Research International ◽

10.1155/2021/6150628 ◽

2021 ◽

Vol 2021 ◽

pp. 1-6

Author(s):

Lili Yang ◽

Ao Zou ◽

Huihua Wu ◽

Hai Guo ◽

Fangting Zhang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Confidence Interval ◽

Acid Secretion ◽

Antimicrobial Agents ◽

Point Mutations ◽

Eradication Therapy ◽

23S Rrna ◽

Tailored Therapy ◽

H Pylori

Background. Helicobacter pylori eradication with therapies employing a proton pump inhibitor (PPI) and antimicrobial agents is mainly achieved via bacterial susceptibility to antimicrobial agents and the magnitude of acid secretion inhibition. However, annual eradication rates have greatly declined in Mainland China, and therefore, tailored H. pylori eradication regimens that inhibit acid secretion and employ optimal antimicrobial agents determined based on gene clip testing may improve eradication rates. This study was aimed at evaluating the efficacy of tailored H. pylori eradication therapy guided by visual gene clip testing for antibiotic resistance and PPI metabolism genotypes. Methods. This prospective study included 244 patients (141 men and 103 women aged 20–79 years) receiving initial treatment for H. pylori infection. Visual gene clip testing using gastric mucosal specimens was performed to detect antibiotic resistance to clarithromycin conferred by the A2142G and A2143G point mutations of the H. pylori 23S rRNA gene and to levofloxacin conferred by the Asn87 and Asp91 point mutations of the H. pylori gyrA gene. Patients received a 14-day bismuth quadruple therapy regimen guided by testing for antibiotic resistance and CYP2C19 polymorphisms, and primary H. pylori eradication was assessed at least 4 weeks after therapy. Results. H. pylori strains were successfully isolated from the gastric mucosa tissues of 244 patients. Antibiotic resistant isolates were identified in 63 patients, with clarithromycin resistance observed in 50 patients, levofloxacin resistance in 7 patients, and dual resistance in 6 patients. The PPI metabolic genotype of CYP2C19 was detected in 242 of 244 cases, and 97 cases were categorized as extensive metabolizers, 141 as intermediate metabolizers, and 4 as poor metabolizers. Among the 242 patients who received tailored therapy, the H. pylori eradication rate was 90.9% (95% confidence interval 87.3%~94.6%) in the intention-to-treat analysis and 96.9% (95% confidence interval 94.7%~99.2%) in the per protocol analysis. Conclusions. Tailored therapy for H. pylori infection guided by determination of antibiotic resistance and CYP2C19 polymorphism using visual gene chip technology may provide high clinical effectiveness as initial H. pylori eradication therapy.

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Unraveling the Novel Effect of Patchouli Alcohol Against the Antibiotic Resistance of Helicobacter pylori

Frontiers in Microbiology ◽

10.3389/fmicb.2021.674560 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuanzun Zhong ◽

Liyao Tang ◽

Qiuhua Deng ◽

Li Jing ◽

Jiao Zhang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Underlying Mechanism ◽

Gastrointestinal Diseases ◽

Bactericidal Effect ◽

Patchouli Alcohol ◽

H Pylori

The long-term colonization of Helicobacter pylori can cause various gastrointestinal diseases, and its high genetic variability is prone to antibiotic resistance and leads to failure of clinical treatment. Intracellular survival also contributes to the drug tolerance of H. pylori. Patchouli alcohol (PA) shows a highly efficient activity against H. pylori in vitro and in vivo. And this study aims to explore whether PA can reduce the resistance of H. pylori and determine the underlying mechanism. Checkerboard and time–kill bactericidal curve assay reveal that the combination of PA and clarithromycin (CLR) promoted the inhibition and bactericidal effect against H. pylori. Stimulation of CLR leads to the internalization of H. pylori, but PA can effectively inhibit the invasion induced by CLR. Compared with antibiotics, PA remarkably eradicated the intracellular H. pylori, and this intracellular sterilized ability was further improved in combination with antibiotics (CLR and metronidazole). The expression of H. pylori efflux pump genes (hp0605, hp1327, and hp1489) was dose-dependently downregulated by PA. Digital droplet PCR indicated that the H. pylori mutant of A2143G can be inhibited by PA. Cellular uptake and transport assays showed that PA is rapidly absorbed, which promotes its activity against intracellular bacteria. Therefore, PA can act synergistically with CLR as a candidate treatment against drug-resistant H. pylori.

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Geographic distribution of the cagA, vacA, iceA, oipA and dupA genes of Helicobacter pylori strains isolated in China

Gut Pathogens ◽

10.1186/s13099-021-00434-4 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Zhijing Xue ◽

Hong Yang ◽

Dongxing Su ◽

Xiangfeng Song ◽

Xin Deng ◽

...

Keyword(s):

Helicobacter Pylori ◽

Clinical Outcomes ◽

Regional Distribution ◽

Gastrointestinal Diseases ◽

Geographic Variations ◽

Ulcer Disease ◽

Geographic Diversity ◽

Polymerase Chain ◽

Non Ulcer Dyspepsia ◽

H Pylori

Abstract Background There are geographic variations in the genotypes of Helicobacter pylori (H. pylori) cagA, vacA, iceA, oipA and dupA. The aim of the study was to investigate the distribution of these genotypes among H. pylori strains from five regions of China and their association with clinical outcomes. Materials and methods Gastric biopsy specimens were obtained from 348 patients with different gastrointestinal diseases in the five regions of China. The regional distribution was 89 patients from Shandong, 91 from Guangxi, 57 from Hunan, 58 from Qinghai and 53 from Heilongjiang. The presence of cagA, vacA, iceA, oipA and dupA genotypes was determined by polymerase chain reaction (PCR) from H. pylori DNA. Results A total of 269 H. pylori isolates were obtained, of which 74 isolates were from Shandong, 78 from Guangxi, 46 from Hunan, 33 from Qinghai and 38 from Heilongjiang. The cagA-positive status was predominant in the five regions. The predominant vacA genotypes were s1c (73.4%), m2 (70.6%) and i1 (92.9%). In strains from Shandong, s1a and m1 were dominant. By contrast, s1c was dominant in Guangxi and i1 was dominant in Hunan and Heilongjiang. The prevalence of m2 subtype in Qinghai (78.8%) was significantly higher than that in other regions (P < 0.05). The predominant iceA genotype was iceA1 and the frequency of iceA1 was significantly more prevalent in Hunan than in other regions (P < 0.05). The oipA status “on” gene was more frequent in Shandong (91.9%) and Guangxi (91%) than in Heilongjiang (71.7%) (P < 0.05). Conversely, the dupA-positive status was less than half in Shandong (31.1%) and Guangxi (15.4%), whereas it was 73.9% in Hunan and 81.8% in Qinghai (P < 0.001). There were no significant associations between the cagA, vacA, iceA, oipA genotypes and clinical outcomes. The dupA-positive strains were more common in peptic ulcer disease (PUD) patients than in non-ulcer dyspepsia (NUD) patients in Shandong and Guangxi (P < 0.05), but the association was not observed in other geographic regions. Conclusions There was significant geographic diversity of H. pylori genotypes in different regions of China and the presence of dupA gene can be considered as a marker for the development of gastroduodenal diseases. However, the cagA, iceA, vacA and oipA genes cannot be regarded for prediction of the clinical presentation of H. pylori infection in China.

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The correlation between lncRNAs and Helicobacter pylori in gastric cancer

Pathogens and Disease ◽

10.1093/femspd/ftaa004 ◽

2019 ◽

Vol 77 (9) ◽

Author(s):

Narges Dastmalchi ◽

Seyed Mahdi Banan Khojasteh ◽

Mirsaed Miri Nargesi ◽

Reza Safaralizadeh

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Molecular Mechanisms ◽

Gastrointestinal Diseases ◽

Mechanisms Of Action ◽

Molecular Pathways ◽

Gastric Diseases ◽

Non Coding Rnas ◽

H Pylori ◽

The Relationship

ABSTRACT Helicobacter pylori infection performs a key role in gastric tumorigenesis. Long non-coding RNAs (lncRNAs) have demonstrated a great potential to be regarded as effective malignancy biomarkers for various gastrointestinal diseases including gastric cancer (GC). The present review highlights the relationship between lncRNAs and H. pylori in GC. Several studies have examined not only the involvement of lncRNAs in H. pylori-associated GC progression but also their molecular mechanisms of action. Among the pertinent studies, some have addressed the effects of H. pylori infection on modulatory networks of lncRNAs, while others have evaluated the effects of changes in the expression level of lncRNAs in H. pylori-associated gastric diseases, especially GC. The relationship between lncRNAs and H. pylori was found to be modulated by various molecular pathways.

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Genomic Analysis of Antimicrobial Resistance Genotype-to-Phenotype Agreement in Helicobacter pylori

Microorganisms ◽

10.3390/microorganisms9010002 ◽

2020 ◽

Vol 9 (1) ◽

pp. 2

Author(s):

Tal Domanovich-Asor ◽

Yair Motro ◽

Boris Khalfin ◽

Hillary A. Craddock ◽

Avi Peretz ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antimicrobial Resistance ◽

Point Mutations ◽

Genomic Analysis ◽

23S Rrna ◽

Phenotype Correlation ◽

Bioinformatics Pipeline ◽

Phenotypic Resistance ◽

Commercial Kits ◽

H Pylori

Antimicrobial resistance (AMR) in Helicobacter pylori is increasing and can result in treatment failure and inappropriate antibiotic usage. This study used whole genome sequencing (WGS) to comprehensively analyze the H. pylori resistome and phylogeny in order to characterize Israeli H. pylori. Israeli H. pylori isolates (n = 48) underwent antimicrobial susceptibility testing (AST) against five antimicrobials and WGS analysis. Literature review identified 111 mutations reported to correlate with phenotypic resistance to these antimicrobials. Analysis was conducted via our in-house bioinformatics pipeline targeting point mutations in the relevant genes (pbp1A, 23S rRNA, gyrA, rdxA, frxA, and rpoB) in order to assess genotype-to-phenotype correlation. Resistance rates of study isolates were as follows: clarithromycin 54%, metronidazole 31%, amoxicillin 10%, rifampicin 4%, and levofloxacin 2%. Genotype-to-phenotype correlation was inconsistent; for every analyzed gene at least one phenotypically susceptible isolate was found to have a mutation previously associated with resistance. This was also observed regarding mutations commonly used in commercial kits to diagnose AMR in H. pylori cases. Furthermore, 11 novel point mutations associated with a resistant phenotype were detected. Analysis of a unique set of H. pylori isolates demonstrates that inferring resistance phenotypes from WGS in H. pylori remains challenging and should be optimized further.

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