Helicobacter pylori Primary and Secondary Genotypic Resistance to Clarithromycin and Levofloxacin Detection in Stools: A 4-Year Scenario in Southern Italy

Antibiotic resistance has become an emerging problem for treating Helicobacter pylori (H. pylori) infection. Clarithromycin and levofloxacin are two key antibiotics used for its eradication. Therefore, we reviewed our experience with genotypic resistance analysis in stools to both clarithromycin and levofloxacin in the last four years to evaluate time trends, both in naive and failure patients. Patients collected a fecal sample using the THD fecal test device. Real-time polymerase chain reaction was performed to detect point mutations conferring resistance to clarithromycin (A2142C, A2142G, and A2143G in 23S rRNA) and levofloxacin (substitutions at amino acid position 87 and 91 of gyrA). One hundred and thirty-five naive patients were recruited between 2017–2020. Clarithromycin resistance was detected in 37 (27.4%). The time trend did not show any significant variation from 2017 to 2020 (p = 0.33). Primary levofloxacin resistance was found in 26 subjects (19.2%), and we observed a dramatic increase in rates from 2017 (10%) to 2018 (3.3%), 2019 (20%), and 2020 (37.8%). Ninety-one patients with at least one eradication failure were recruited. Secondary resistance to clarithromycin and levofloxacin was found in 59 (64.8%) and 45 patients (59.3%), respectively. In conclusion, our geographic area has a high risk of resistance to clarithromycin. There is also a progressive spreading of levofloxacin-resistant strains.

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The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02011-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 6

Author(s):

Xiwen Geng ◽

Wen Li ◽

Zhenghong Chen ◽

Sizhe Gao ◽

Wei Hong ◽

...

Keyword(s):

Helicobacter Pylori ◽

Point Mutations ◽

23S Rrna ◽

Bifunctional Enzyme ◽

Key Factors ◽

Content Type ◽

Bacterial Drug Resistance ◽

Resistant Strains ◽

H Pylori

ABSTRACT Clarithromycin (CLA) is a commonly recommended drug for Helicobacter pylori eradication. However, the prevalence of CLA-resistant H. pylori is increasing. Although point mutations in the 23S rRNA are key factors for CLA resistance, other factors, including efflux pumps and regulation genes, are also involved in the resistance of H. pylori to CLA. Guanosine 3′-diphosphate 5′-triphosphate and guanosine 3′,5′-bispyrophosphate [(p)ppGpp)], which are synthesized by the bifunctional enzyme SpoT in H. pylori, play an important role for some bacteria to adapt to antibiotic pressure. Nevertheless, no related research involving H. pylori has been reported. In addition, transporters have been found to be related to bacterial drug resistance. Therefore, this study investigated the function of SpoT in H. pylori resistance to CLA by examining the shifts in the expression of transporters and explored the role of transporters in the CLA resistance of H. pylori. A ΔspoT strain was constructed in this study, and it was shown that SpoT is involved in H. pylori tolerance of CLA by upregulating the transporters HP0939, HP1017, HP0497, and HP0471. This was assessed using a series of molecular and biochemical experiments and a cDNA microarray. Additionally, the knockout of genes hp0939, hp0471, and hp0497 in the resistant strains caused a reduction or loss (the latter in the Δhp0497 strain) of resistance to CLA. Furthermore, the average expression levels of these four transporters in clinical CLA-resistant strains were considerably higher than those in clinical CLA-sensitive strains. Taken together, our results revealed a novel molecular mechanism of H. pylori adaption to CLA stress.

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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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Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2724 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2724-2728 ◽

Cited By ~ 129

Author(s):

A Occhialini ◽

M Urdaci ◽

F Doucet-Populaire ◽

C M Bébéar ◽

H Lamouliatte ◽

...

Keyword(s):

Helicobacter Pylori ◽

Point Mutations ◽

Restriction Enzymes ◽

The United States ◽

Drug Binding ◽

Macrolide Resistance ◽

Rrna Genes ◽

23S Rrna ◽

Dependent Manner ◽

H Pylori

Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.

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Genotypic Resistance inHelicobacter pyloriStrains Correlates with Susceptibility Test and Treatment Outcomes after Levofloxacin- and Clarithromycin-Based Therapies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01131-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 1123-1129 ◽

Cited By ~ 42

Author(s):

Jyh-Ming Liou ◽

Chi-Yang Chang ◽

Wang-Huei Sheng ◽

Yu-Chi Wang ◽

Mei-Jyh Chen ◽

...

Keyword(s):

Treatment Outcomes ◽

Triple Therapy ◽

Direct Sequencing ◽

Point Mutations ◽

Kappa Coefficient ◽

23S Rrna ◽

Genotypic Resistance ◽

Phenotypic Resistance ◽

Therapeutic Outcomes ◽

H Pylori

ABSTRACTThe accuracy of genotypic resistance to levofloxacin (gyrAmutations) and its agreement with treatment outcomes after levofloxacin-based therapy have not been reported. We aimed to assess the correlation.Helicobacter pyloristrains isolated from patients who received levofloxacin-based and clarithromycin-based triple therapies in a previous randomized trial were analyzed for point mutations ingyrAand 23S rRNA. PCR followed by direct sequencing was used to assess thegyrAand 23S rRNA mutations. An agar dilution test was used to determine the MICs of clarithromycin and levofloxacin. We found that the agreement between genotypic and phenotypic resistance to levofloxacin was best when the MIC breakpoint was >1 μg/ml (kappa coefficient, 0.754). The eradication rates in patients with and withoutgyrAmutations were 41.7% and 82.7%, respectively (P= 0.003). The agreement between genotypic and phenotypic resistance to clarithromycin was best when the MIC breakpoint was >2 μg/ml (kappa, 0.694). The eradication rates in patients with and without 23S rRNA mutations were 7.7% and 93.5%, respectively (P< 0.001). The agreements (kappa coefficient) between therapeutic outcomes after clarithromycin-based triple therapy and genotypic and phenotypic resistance were 0.671 and 0.356, respectively. The agreements (kappa coefficient) between therapeutic outcomes after levofloxacin-based triple therapy and genotypic and phenotypic resistance were 0.244 and 0.190, respectively. In conclusion,gyrAand 23S rRNA mutations inH. pyloristrains appeared to be better markers than phenotypic resistance in the prediction of treatment outcomes. The optimal breakpoints for levofloxacin and clarithromycin resistance appeared to be >1 μg/ml and >2 μg/ml, respectively.

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Rapid screening of clarithromycin resistance in Helicobacter pylori by pyrosequencing

Journal of Medical Microbiology ◽

10.1099/jmm.0.47371-0 ◽

2007 ◽

Vol 56 (10) ◽

pp. 1370-1376 ◽

Cited By ~ 11

Author(s):

Karen-Anja Moder ◽

Franziska Layer ◽

Wolfgang König ◽

Brigitte König

Keyword(s):

Helicobacter Pylori ◽

Point Mutations ◽

Rapid Screening ◽

23S Rrna ◽

Resistance Testing ◽

Resistance Mutations ◽

Additional Time ◽

Clarithromycin Resistance ◽

H Pylori ◽

Domain V

Helicobacter pylori infections can be effectively treated with clarithromycin, a macrolide, in combination with other antibiotics, such as amoxicillin, tetracycline or metronidazole. The failure of H. pylori eradication is mainly associated with macrolide-resistant strains. Three point mutations (A2142G/C, A2143G, T2182C) in the peptidyltransferase region of domain V of the 23S rRNA have been described as being associated with clarithromycin resistance. Therefore, the determination of clarithromycin resistance by pyrosequencing was evaluated. H. pylori from 81 gastric biopsies was cultured and clarithromycin resistance was determined by Etest, as well as by pyrosequencing technology (PSQ 96 system; Biotage). The respective mutations were set in relation to the MIC measured in μg ml−1 by Etest. In this study, point mutations in positions 2142 and 2143 were associated with clarithromycin resistance. Mutations in position 2182 did not contribute to clarithromycin resistance. In addition, from 22 out of the 81 biopsies, clarithromycin resistance was determined directly without culturing H. pylori to save additional time. Identical results were obtained as compared to resistance testing with pure H. pylori strains. All results obtained by pyrosequencing were evaluated by Sanger sequencing. The data show that pyrosequencing to detect point mutation is a fast and reliable method for determining clarithromycin resistance in H. pylori, and provides the same results as the Etest.

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Rapid Detection, by PCR and Reverse Hybridization, of Mutations in the Helicobacter pylori 23S rRNA Gene, Associated with Macrolide Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1779 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1779-1782 ◽

Cited By ~ 51

Author(s):

Leen-Jan van Doorn ◽

Yvette J. Debets-Ossenkopp ◽

Armelle Marais ◽

Ricardo Sanna ◽

Francis Mégraud ◽

...

Keyword(s):

Helicobacter Pylori ◽

Fragment Length Polymorphism ◽

Simultaneous Detection ◽

Point Mutations ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Reverse Hybridization ◽

23S Rrna Gene ◽

H Pylori

ABSTRACT A PCR-based reverse hybridization system (research prototype kit INNO-LiPA for H. pylori resistance) was developed and evaluated for simultaneous detection of 23S ribosomal DNA point mutations, associated with macrolide resistance in Helicobacter pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains) were tested by PCR-LiPA (detecting mutations A2115→G, G2141→A, A2142→G, A2142→C, A2143→G, A2143→C, and A2143→T), DNA sequencing, restriction fragment length polymorphism, and/or hybridization to oligonucleotide probes. Results were highly concordant, but PCR-LiPA appears to be more sensitive for the simultaneous detection of multiple mutants.

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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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Variability in Prevalence ofHelicobacter pyloriStrains Resistant to Clarithromycin and Levofloxacin in Southern Poland

Gastroenterology Research and Practice ◽

10.1155/2012/418010 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Elżbieta Karczewska ◽

Karolina Klesiewicz ◽

Iwona Skiba ◽

Izabela Wojtas-Bonior ◽

Edward Sito ◽

...

Keyword(s):

Helicobacter Pylori ◽

Statistical Analysis ◽

Antimicrobial Agents ◽

Test Method ◽

Secondary Resistance ◽

Significance Level ◽

Southern Poland ◽

Resistant Strains ◽

Therapeutic Problem ◽

H Pylori

Background. An increasing resistance ofHelicobacter pyloristrains to antimicrobial agents is the serious therapeutic problem. The aim of this study was to compare the primary and secondary resistance ofH. pyloristrains isolated between 2006–2008 (data published) and 2009–2011 to clarithromycin and levofloxacin.Material and Methods. 220 dyspeptic patients (153 before treatment, 67 after), were enrolled in the study. 51H. pyloristrains were isolated. MIC values of clarithromycin and levofloxacin were determined by theE-test method. The statistical analysis was conducted with theχ2test with Yates correction at the 0.05 significance level (P≤0.05).Results. Between 2006 and 2008, 34% (39/115) ofH. pyloristrains were resistant to clarithromycin (primary 21% (19/90), secondary 80% (20/25)). 5% (6/115) of strains were resistant to levofloxacin (primary 2% (2/90), secondary 16% ((4/25); data published) Between 2009–2011, 22% (11/51) ofH. pyloristrains were resistant to clarithromycin (primary 19% (8/43), secondary 38% (3/8)). 16% (8/51) of strains were resistant to levofloxacin (primary 12% (5/43), secondary 38% (3/8)).Conclusion. The present study has shown the increasing amount of resistantH. pyloristrains isolated from patients in Southern Poland to levofloxacin and decreasing number of resistant strains to clarithromycin.

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PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1901 ◽

2014 ◽

Vol 61 (2) ◽

Cited By ~ 22

Author(s):

Karolina Klesiewicz ◽

Paweł Nowak ◽

Elżbieta Karczewska ◽

Iwona Skiba ◽

Izabela Wojtas-Bonior ◽

...

Keyword(s):

Helicobacter Pylori ◽

Point Mutations ◽

Rflp Analysis ◽

23S Rrna ◽

Rrna Gene ◽

Efficient Management ◽

Clarithromycin Resistance ◽

Pcr Rflp ◽

23S Rrna Gene ◽

H Pylori

The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.

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Resistotype of Helicobacter pylori isolates: the impact on eradication outcome

Journal of Medical Microbiology ◽

10.1099/jmm.0.069781-0 ◽

2014 ◽

Vol 63 (5) ◽

pp. 703-709 ◽

Cited By ~ 16

Author(s):

Hanafiah Alfizah ◽

Ahmad Norazah ◽

Razlan Hamizah ◽

Mohamed Ramelah

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Eradication Therapy ◽

Fluoroquinolone Resistance ◽

23S Rrna ◽

Nonsense Mutations ◽

Metronidazole Resistance ◽

Resistant Strains ◽

H Pylori ◽

The Impact

Antibiotic resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. The aim of this study was to determine the phenotype and genotype of antibiotic-resistant strains of H. pylori in the Malaysian population and to evaluate the impact of antibiotic resistance to eradication outcome. One hundred and sixty-one H. pylori isolates were analysed in this study. Metronidazole, clarithromycin, fluoroquinolone, amoxicillin and tetracycline susceptibilities were determined by Etest. PCR followed by DNA sequencing was carried out to determine mutations. The medical records of the patients infected with resistant strains were reviewed to determine the eradication outcome. Metronidazole resistance was encountered in 36.6 % of H. pylori isolates, whereas clarithromycin and fluoroquinolone resistance was observed in 1.2 and 1.9 % of isolates, respectively. All strains tested were susceptible to amoxicillin and tetracycline. Frameshift and nonsense mutations in rdxA and frxA genes resulting in stop codons contributed to metronidazole resistance, which leads to reduced eradication efficacy. A2142G and A2143G mutations of 23S rRNA were identified as causing failure of the eradication therapy. Mutation at either codon 87 or 91 of the gyrA gene was identified in fluoroquinolone-resistant strains. However, the effect of resistance could not be assessed. This study showed that frameshift and nonsense mutations in rdxA or frxA genes and point mutations in the 23S rRNA affected the efficacy of H. pylori eradication therapy.

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