Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria

Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria Abstract Objectives In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Methods Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. Results No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Conclusions Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria. Introduction In Algeria, Helicobacter pylori infection was estimated to be higher than 80% in adults at the end of the last century.1,2 Current data concerning the prevalence of H. pylori infection and its implication in different gastric disorders or carcinogenesis in the Algerian population are scarce. However, the low socioeconomic status of the population, the limited development of the healthcare system and the lack of its use in the case of digestive health symptoms may allow evolution of H. pylori infection towards severe diseases including malignancies.3 A triple therapy of two antibiotics and a proton pump inhibitor (PPI) administered for 7 days has been recommended for the treatment of this infection. This regimen has shown a decreased effectiveness in recent years, with eradication rates lower than 80%.4,5 Eradication treatments fail for a significant proportion of patients in Algeria and so it is of prime importance to know the role of bacterial resistance, especially to clarithromycin, in these failures. Despite the fact that tetracycline resistance remains relatively uncommon, it is important to assess it in regions where tetracycline is more widely used.6,7 Susceptibility-guided treatment strategies have shown their benefit. Due to the fact that culture is difficult to carry out (special care is required in the transport of samples, H. pylori is a fastidious bacterium and there is need for appropriate culture media, which may not be available), molecular methods are increasingly used for bacterial detection and determination of antibiotic resistance.8 The aim of the present prospective study was to evaluate the prevalence of primary resistance to key antibiotics in the treatment of this infection, by means of antimicrobial susceptibility testing (AST), and to look for mutations associated with clarithromycin and tetracycline resistance by means of an antimicrobial gradient agar diffusion assay and sequencing for fluoroquinolones. Methods Patients Between January 2014 and December 2016, 270 patients suffering from upper abdominal pain, who had to stop taking PPI or H2-receptor antagonists 4 weeks prior to endoscopy and had never been treated with clarithromycin, tetracycline, rifampicin or ciprofloxacin, were enrolled in the study. The patients were Algerian citizens and provided written informed consent before endoscopy at four different hospital gastroenterology units in four Algerian cities (Mustapha Pacha University Hospital in Algiers, Halouche Clinic of Gastroenterology in Chlef, First November 1954 University Hospital in Oran and University Hospital in Sidi Bel Abbes), during which two gastric biopsies were obtained, placed in 1 mL of sterile PBS, maintained at 4°C and transported immediately to the Bioresources Laboratory (Chlef) for the isolation of H. pylori. Total genomic DNA was extracted from biopsy samples, using the commercial MagaZorb DNA Mini-Prep Kit (Promega, Madison, WI, USA) according to the manufacturer’s guidelines, and was stored at −20°C. The molecular analysis was performed in the Bioresources Laboratory and in the National Reference Centre for Campylobacters and Helicobacters (Bordeaux, France). Bacterial culture and AST Ground gastric biopsy samples were plated on brain heart infusion (BHI) agar (bioMérieux, Marcy-l’Étoile, France) supplemented with 10% defibrinated horse blood, IsoVitaleX (0.4%), trimethoprim (5 mg/L), cefsulodin (5 mg/L), vancomycin (10 mg/L) and amphotericin B (8 mg/L). The plates were incubated at 37°C under microaerobic conditions for more than 7 days. H. pylori was identified by colony aspect, microscopic morphology and positive urease, catalase and oxidase tests. Isolates were stored at −80°C in BHI broth (bioMérieux) with 20% glycerol.9 AST to clarithromycin, ciprofloxacin, tetracycline and rifampicin was performed using an antimicrobial gradient agar diffusion assay (Etest, bioMérieux) as recommended by EUCAST. An isolate was considered resistant to clarithromycin when the MIC was >0.5 mg/L, resistant to ciprofloxacin when the MIC was >1 mg/L, resistant to rifampicin when the MIC was >1 mg/L and resistant to tetracycline when the MIC was >1 mg/L.10 Detection of point mutations in the 23S rRNA and 16S rRNA genes of H. pylori by real-time PCR To detect point mutations conferring resistance to clarithromycin, a real-time fluorescence resonance energy transfer (FRET)-melting curve analysis assay, as described by Oleastro et al.,11 was used directly on DNA obtained from gastric biopsies. Briefly, the real-time FRET-PCR included amplification of a 267 bp fragment of the 23S rRNA gene of H. pylori coupled with simultaneous detection of the product by probe hybridization and analysis of the melting curve. The amplified product was detected with two probes: the sensor probe, which hybridized with the region containing the mutation sites; and the anchor probe. The existence of a nucleotide mismatch between the sequence and the hybridization probe produced a melting temperature (Tm) lower than the Tm of the WT sequence, where there was 100% homology between the probe and the target. For the detection of the 16S rRNA gene mutations associated with tetracycline resistance, the method included the amplification of the 16S rRNA gene fragment of H. pylori12 and the simultaneous detection of the PCR product by two hybridization probes, a 16S-Anc anchor probe and a 16S-AGA-sensor mutation probe, as well as an additional probe, 16S-TTC-sensor, which matched the sequence of the TTC mutant. The mutant genotypes were sequenced to confirm the type of mutation involved in the resistance to tetracycline. PCR and DNA sequencing of the QRDR of the gyrA gene To amplify the QRDR of gyrA, the pair of primers F-QRDR-Hpylo (5′-GCGTATTTTGTATGCGATGC-3′) and R-QRDR-Hpylo (5′-ACAAAATCAATGGTGTCT TTATCA-3′) were used. The PCR products were sequenced on both strands using the same primers and the BigDye Terminator Cycle Sequencing Kit. Statistical analysis The data were analysed using SPSS software (Version 17, SPSS Inc., Chicago, IL, USA) and P values were calculated using the χ2 test to find any significant relationship. P < 0.05 was considered statistically significant. Ethics This work is registered in a national research project entitled University research project (CNEPRU), financed by the Algerian Research Ministry with the following registration code: D01N01UN020120150001. This study was approved by the Ethics Committees of all hospitals where the endoscopies were performed and all patients provided written informed consent before endoscopy. Results AST From the 270 gastric biopsies included in the study, culture was positive for H. pylori in 212 cases (78.5%) among which 134 cases were defined as having gastritis, 66 cases were defined as having duodenal ulcers and 12 cases were defined as having gastric ulcers. The clinical symptoms and endoscopy results are presented in Tables 1 and 2. Table 1. Distribution of patients according to clinical symptoms Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 More than two symptoms might be present simultaneously in the same patient. Table 1. Distribution of patients according to clinical symptoms Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 More than two symptoms might be present simultaneously in the same patient. Table 2. Distribution of patients according to endoscopy results and hospitals in Algeria Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Table 2. Distribution of patients according to endoscopy results and hospitals in Algeria Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Susceptibility to ciprofloxacin, clarithromycin, rifampicin and tetracycline was tested. According to EUCAST breakpoints, the resistance rates were as follows: clarithromycin, 25%; ciprofloxacin, 17.9%; tetracycline, 0.94%; and no resistance to rifampicin. No significant difference in the prevalence of antibiotic resistance was observed between the four cities or during the study period (P > 0.5). Detection of point mutations in the 23S rRNA and 16S rRNA genes of H. pylori by real-time PCR The real-time FRET-PCR was positive for H. pylori in 232 cases (86%) including the 212 biopsies positive by culture. Melting curve analysis of DNA from the positive control strains produced three different curves, with Tm of approximately 60, 56 and 52°C for the WT strain and mutants A2142C and A2142/43G, respectively. The real-time PCR described above was able to detect the mutations associated with clarithromycin resistance in 69 of the 232 biopsies (29.7%) tested. In 53 cases corresponding to the resistance phenotypes, the results of the real-time PCR correlated perfectly with the clarithromycin resistance profile determined by Etest (Table 1). Of the 212 biopsies positive by culture, 159 produced a melting peak characteristic of the WT genotype, which perfectly matched with phenotypic susceptibility testing results. Among resistant isolates, the transition A to G, either in position 2142 or 2143, was detected in all 53 samples; the transversion A to C in position 2142 was not detected. In 20 additional cases, while there was no positive culture, the real-time PCR detected 16 resistant genotypes with the transition (A2142/43G) and 4 WT genotypes. In 42 biopsies the WT genotype was present simultaneously with the resistant genotype (Table 3). Table 3. Mutations in 23S rRNA of clarithromycin-susceptible and -resistant H. pylori isolates Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 a Includes 42 cases with mixed WT and mutant genotypes. Table 3. Mutations in 23S rRNA of clarithromycin-susceptible and -resistant H. pylori isolates Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 a Includes 42 cases with mixed WT and mutant genotypes. Of the 212 H. pylori isolates, only 2 were resistant to tetracycline with an MIC of 1.5 mg/L. The analysis of the melting curves of all tetracycline-resistant and -susceptible isolates showed two Tms corresponding to two different genotypes: 230 biopsies showed a Tm of 61°C and exhibited an AGA926–928 WT in agreement with our AST results. For two isolates, we found a Tm of 56.8°C and sequencing consequently revealed an A926G mutation of the 16S rRNA gene for these two mutants. Detection of mutations associated with fluoroquinolones in the QRDR of gyrA Mutations in fluoroquinolone-resistant isolates were present in the QRDR at positions 87 and 91. There was perfect concordance between phenotypic and genotypic tests. The most common resistant substitution was N87K (present in 44.73%) followed by D91N (present in 23.68%) then N87I (present in 18.42%); the least common mutation was D91G (present in 7.89%). Two of the patients with fluoroquinolone-resistant isolates (MICs, 1.5 and 2 mg/L) did not present any of the expected mutations. Isolates presenting mutations at position 87 had an MIC >8 mg/L (Table 4). None of the susceptible isolates presented any mutation in the QRDR, but we found 42 susceptible isolates with the N87T (ACC) sequence. Table 4. Mutations in the QRDR of gyrA from resistant isolates Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Table 4. Mutations in the QRDR of gyrA from resistant isolates Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Discussion In Algeria, surveillance of H. pylori antibiotic resistance is necessary in order to adapt the antibiotic combination to local resistance patterns. This is the first known study conducted in Algeria in which H. pylori resistance to several key antibiotics (clarithromycin, ciprofloxacin, rifampicin and tetracycline) was evaluated in different cities in the country (Algiers, Chlef, Oran and Sidi Bel Abbes) both by culture and molecular methods. We did not find any significant difference in the prevalence of H. pylori resistance between regions; the sample size was too small for each region to detect a significant difference. In developing countries, clarithromycin resistance and potential re-infections are factors that contribute to the failure of H. pylori eradication. In our study, the clarithromycin resistance rate was 29.7%; this result is lower than that found in a study conducted in Mustapha Pacha University Hospital, Algiers (33%), but higher than that found in another study conducted in three different hospitals in the same city (23%).13,14 The rate of resistance to clarithromycin is also high in Morocco (29%), Egypt (57.7%) and France (22.2%).15–17 However, the rate of resistance is much lower in Tunisia (14.6%), Nigeria (14.4%) and Senegal (1%).18–20 A2143G was the most prevalent point mutation, and studies have claimed that this mutation plays a major role in clarithromycin resistance.4,20 This result is similar to that of patients in Tunisia. We did not find the A2142C mutation, which is less common.21 One of the resistance mechanisms acquired by macrolides, like clarithromycin, is modifications to the ribosome binding site, which may consist of changes in the base sequence of 23S rRNA. Mutations are selected during treatment and can be observed when a macrolide is used in H. pylori eradication.22 On the other hand, previous reports have shown that the presence of the A2143G point mutation, rather than the A2142G or A2142C mutation, reduces the H. pylori eradication rate.23 This emergence of clarithromycin resistance might be due to the increased prescription of this antibiotic because clarithromycin is widely used in Algeria as an antimicrobial drug to treat infections in other organ systems such as the respiratory system. Furthermore, and especially due to cross-resistance in the macrolide family, the prevalence of clarithromycin-resistant H. pylori is continuously increasing. Concerning tetracycline resistance in our study, we found only two resistant isolates; moreover, with only one mutation of the concerned nucleotide triplet in agreement with a limited increase of the MICs. In a Nigerian study they also found a reduced rate of resistance to tetracycline (4.5%),19 and in Morocco and in Senegal they did not find any resistant isolates.15,20 According to Gerrits et al.,24 resistance to the tetracycline antibiotic group has been associated with mutations in the 16S rRNA gene. They found that transformation of the H. pylori strain 26695 (MIC, 0.19 mg/L) with genomic DNA from strain 181 (MIC, 8 mg/L) resulted in tetracycline-resistant colonies. In their study, a Light Cycler assay successfully distinguished two genotypes: a WT and 16S rRNA gene mutations associated with resistance and/or reduced susceptibility to tetracycline. Our study revealed that out of the 232 H. pylori-positive isolates, two with reduced susceptibility to tetracycline had the A926G mutation in the 16S rRNA gene and MICs were 1.5 mg/L. This mutation was previously reported in the Congo,25 in one German isolate with an MIC of 1 mg/L (Etest method)12 and in seven UK isolates with MICs of 0.75–4 mg/L (Etest method).26 The 16S rRNA gene polymorphisms are excellent targets for the real-time PCR detection assays, which allow access to susceptibility testing directly from biopsy specimens without culture. This method could be especially useful in patients who have experienced treatment failure. Fluoroquinolone resistance was found in 17.9% of the study population. This result was higher than that found in Morocco (11%),15 in Senegal (15%),20 in France (15.4%),17 in Europe (14.1%)27 and in Japan (14.9%),28 but lower than that found in the Congo (50%)25 and in China (28%).22 In a single Italian study29 and in Korea the ciprofloxacin resistance rate of H. pylori was reported to be 33.8%.30 However, in Sweden a low ciprofloxacin resistance rate (3.3%) was reported.31 This increasing resistance rate could be explained by the use of fluoroquinolones for urinary infections and several other infectious diseases in Algeria and throughout the world. QRDR mutations in fluoroquinolone-resistant H. pylori isolates were previously reported. For the strains isolated in our study we did not find any correlation between the mutation present and the level of resistance. All 34 fluoroquinolone-resistant isolates harboured one mutation already found in the QRDR of gyrA. N87K and D91N were the most common mutations in this region. The major mutation in position 87 was detected in other studies.32 D91N is a well-known mutation detected worldwide.33 As two isolates with ciprofloxacin MICs of 1.5 and 2 mg/L displayed no mutations in the QRDR of gyrA, we suggest that another mutation or another mechanism such as reduced drug accumulation could be involved. However, higher levels of resistance were most likely related to isolates with N87I and D91G mutations, with MIC values of 32 mg/L. Our data are in agreement with reports from Asia and Europe.34,35 The Maastricht IV/Florence Consensus Report recommended that empirical use of levofloxacin should be abandoned when the prevalence of resistance reaches 15%.36 In this study, we did not look for mutations (in rdxA and frxA) associated with metronidazole resistance, which are frequent but not always correlated to the resistance pattern. In conclusion, the mean primary H. pylori clarithromycin resistance rate in Algeria has crossed the borderline (15%) for applying the standard triple therapy. The primary fluoroquinolone resistance rate is also of growing concern. Depending on local circumstances the logistical problems associated with getting gastric biopsies into the laboratory on time in order to ensure successful culture of the pathogens might be impossible to overcome; hence genotyping susceptibility testing might be the preferred option. Furthermore, they can be used for continuous surveillance of the proper drug selection for anti-H. pylori therapy in different regions. Acknowledgements We thank Samir Halouche (Gastroenterology Clinic, Chlef, Algeria) and Professor Bouasria (First November 1954 University Hospital, Oran, Algeria) for their technical assistance, as well as the clinicians involved in this study. We thank Hacene Mahmoudi and Ahmad Aichouni for their moral support. Funding This work was financed by the Bioresources Laboratory, Department of Biology, Faculty of Natural and Life Sciences, Hassiba Ben Bouali University of Chlef, Chlef, Algeria.  In Algeria, all research projects are financed by the Algerian Research Ministry through research laboratories in each university. Transparency declarations None to declare. Author contributions Conceived and designed the experiments: M. B., R. A., L. B., A. T. and M. M. Performed the experiments: M. B., A. T., A. E.-M. D. and M. M. Analysed the data: M. B., A. T. and L. B. Contributed reagents/materials/analysis tools: M. B., M. M., F. M. and K. T. D. Wrote the paper: M. B., R. A. and F. M. All authors read and approved the final manuscript. References 1 Mégraud F , Brassens-Rabbé MP , Denis F et al. Seroepidemiology of Campylobacter pylori infection in various populations . J Clin Microbiol 1989 ; 27 : 1870 – 3 . Google Scholar PubMed 2 Bachir M , Allem R , Tifrit K et al. 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Management of Helicobacter pylori infection–the Maastricht V/Florence Consensus Report . Gut 2017 ; 66 : 6 – 30 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria

Journal of Antimicrobial Chemotherapy , Volume Advance Article (8) – May 11, 2018

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1460-2091
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10.1093/jac/dky167
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Abstract

Abstract Objectives In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Methods Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. Results No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Conclusions Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria. Introduction In Algeria, Helicobacter pylori infection was estimated to be higher than 80% in adults at the end of the last century.1,2 Current data concerning the prevalence of H. pylori infection and its implication in different gastric disorders or carcinogenesis in the Algerian population are scarce. However, the low socioeconomic status of the population, the limited development of the healthcare system and the lack of its use in the case of digestive health symptoms may allow evolution of H. pylori infection towards severe diseases including malignancies.3 A triple therapy of two antibiotics and a proton pump inhibitor (PPI) administered for 7 days has been recommended for the treatment of this infection. This regimen has shown a decreased effectiveness in recent years, with eradication rates lower than 80%.4,5 Eradication treatments fail for a significant proportion of patients in Algeria and so it is of prime importance to know the role of bacterial resistance, especially to clarithromycin, in these failures. Despite the fact that tetracycline resistance remains relatively uncommon, it is important to assess it in regions where tetracycline is more widely used.6,7 Susceptibility-guided treatment strategies have shown their benefit. Due to the fact that culture is difficult to carry out (special care is required in the transport of samples, H. pylori is a fastidious bacterium and there is need for appropriate culture media, which may not be available), molecular methods are increasingly used for bacterial detection and determination of antibiotic resistance.8 The aim of the present prospective study was to evaluate the prevalence of primary resistance to key antibiotics in the treatment of this infection, by means of antimicrobial susceptibility testing (AST), and to look for mutations associated with clarithromycin and tetracycline resistance by means of an antimicrobial gradient agar diffusion assay and sequencing for fluoroquinolones. Methods Patients Between January 2014 and December 2016, 270 patients suffering from upper abdominal pain, who had to stop taking PPI or H2-receptor antagonists 4 weeks prior to endoscopy and had never been treated with clarithromycin, tetracycline, rifampicin or ciprofloxacin, were enrolled in the study. The patients were Algerian citizens and provided written informed consent before endoscopy at four different hospital gastroenterology units in four Algerian cities (Mustapha Pacha University Hospital in Algiers, Halouche Clinic of Gastroenterology in Chlef, First November 1954 University Hospital in Oran and University Hospital in Sidi Bel Abbes), during which two gastric biopsies were obtained, placed in 1 mL of sterile PBS, maintained at 4°C and transported immediately to the Bioresources Laboratory (Chlef) for the isolation of H. pylori. Total genomic DNA was extracted from biopsy samples, using the commercial MagaZorb DNA Mini-Prep Kit (Promega, Madison, WI, USA) according to the manufacturer’s guidelines, and was stored at −20°C. The molecular analysis was performed in the Bioresources Laboratory and in the National Reference Centre for Campylobacters and Helicobacters (Bordeaux, France). Bacterial culture and AST Ground gastric biopsy samples were plated on brain heart infusion (BHI) agar (bioMérieux, Marcy-l’Étoile, France) supplemented with 10% defibrinated horse blood, IsoVitaleX (0.4%), trimethoprim (5 mg/L), cefsulodin (5 mg/L), vancomycin (10 mg/L) and amphotericin B (8 mg/L). The plates were incubated at 37°C under microaerobic conditions for more than 7 days. H. pylori was identified by colony aspect, microscopic morphology and positive urease, catalase and oxidase tests. Isolates were stored at −80°C in BHI broth (bioMérieux) with 20% glycerol.9 AST to clarithromycin, ciprofloxacin, tetracycline and rifampicin was performed using an antimicrobial gradient agar diffusion assay (Etest, bioMérieux) as recommended by EUCAST. An isolate was considered resistant to clarithromycin when the MIC was >0.5 mg/L, resistant to ciprofloxacin when the MIC was >1 mg/L, resistant to rifampicin when the MIC was >1 mg/L and resistant to tetracycline when the MIC was >1 mg/L.10 Detection of point mutations in the 23S rRNA and 16S rRNA genes of H. pylori by real-time PCR To detect point mutations conferring resistance to clarithromycin, a real-time fluorescence resonance energy transfer (FRET)-melting curve analysis assay, as described by Oleastro et al.,11 was used directly on DNA obtained from gastric biopsies. Briefly, the real-time FRET-PCR included amplification of a 267 bp fragment of the 23S rRNA gene of H. pylori coupled with simultaneous detection of the product by probe hybridization and analysis of the melting curve. The amplified product was detected with two probes: the sensor probe, which hybridized with the region containing the mutation sites; and the anchor probe. The existence of a nucleotide mismatch between the sequence and the hybridization probe produced a melting temperature (Tm) lower than the Tm of the WT sequence, where there was 100% homology between the probe and the target. For the detection of the 16S rRNA gene mutations associated with tetracycline resistance, the method included the amplification of the 16S rRNA gene fragment of H. pylori12 and the simultaneous detection of the PCR product by two hybridization probes, a 16S-Anc anchor probe and a 16S-AGA-sensor mutation probe, as well as an additional probe, 16S-TTC-sensor, which matched the sequence of the TTC mutant. The mutant genotypes were sequenced to confirm the type of mutation involved in the resistance to tetracycline. PCR and DNA sequencing of the QRDR of the gyrA gene To amplify the QRDR of gyrA, the pair of primers F-QRDR-Hpylo (5′-GCGTATTTTGTATGCGATGC-3′) and R-QRDR-Hpylo (5′-ACAAAATCAATGGTGTCT TTATCA-3′) were used. The PCR products were sequenced on both strands using the same primers and the BigDye Terminator Cycle Sequencing Kit. Statistical analysis The data were analysed using SPSS software (Version 17, SPSS Inc., Chicago, IL, USA) and P values were calculated using the χ2 test to find any significant relationship. P < 0.05 was considered statistically significant. Ethics This work is registered in a national research project entitled University research project (CNEPRU), financed by the Algerian Research Ministry with the following registration code: D01N01UN020120150001. This study was approved by the Ethics Committees of all hospitals where the endoscopies were performed and all patients provided written informed consent before endoscopy. Results AST From the 270 gastric biopsies included in the study, culture was positive for H. pylori in 212 cases (78.5%) among which 134 cases were defined as having gastritis, 66 cases were defined as having duodenal ulcers and 12 cases were defined as having gastric ulcers. The clinical symptoms and endoscopy results are presented in Tables 1 and 2. Table 1. Distribution of patients according to clinical symptoms Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 More than two symptoms might be present simultaneously in the same patient. Table 1. Distribution of patients according to clinical symptoms Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 Symptoms n % Epigastralgia 170 62.9 Nausea 120 44.4 Pyrosis 105 38.8 Dyspepsia (other than epigastralgia) 90 33.3 Vomiting 60 22.2 Digestive haemorrhage 50 18.5 Odynophagia 10 3.7 More than two symptoms might be present simultaneously in the same patient. Table 2. Distribution of patients according to endoscopy results and hospitals in Algeria Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Table 2. Distribution of patients according to endoscopy results and hospitals in Algeria Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Hospital Gastritis Gastric ulcer Duodenal ulcer Total Mustapha Pacha University Hospital, Algiers (n) 44 32 22 98 Halouche Clinic of Gastroenterology, Chlef (n) 35 24 13 72 First November 1954 University Hospital, Oran (n) 26 12 14 52 University Hospital of Sidi Bel Abbes (n) 24 13 11 48 Proportion (%) 47.7 30 22.2 100 Susceptibility to ciprofloxacin, clarithromycin, rifampicin and tetracycline was tested. According to EUCAST breakpoints, the resistance rates were as follows: clarithromycin, 25%; ciprofloxacin, 17.9%; tetracycline, 0.94%; and no resistance to rifampicin. No significant difference in the prevalence of antibiotic resistance was observed between the four cities or during the study period (P > 0.5). Detection of point mutations in the 23S rRNA and 16S rRNA genes of H. pylori by real-time PCR The real-time FRET-PCR was positive for H. pylori in 232 cases (86%) including the 212 biopsies positive by culture. Melting curve analysis of DNA from the positive control strains produced three different curves, with Tm of approximately 60, 56 and 52°C for the WT strain and mutants A2142C and A2142/43G, respectively. The real-time PCR described above was able to detect the mutations associated with clarithromycin resistance in 69 of the 232 biopsies (29.7%) tested. In 53 cases corresponding to the resistance phenotypes, the results of the real-time PCR correlated perfectly with the clarithromycin resistance profile determined by Etest (Table 1). Of the 212 biopsies positive by culture, 159 produced a melting peak characteristic of the WT genotype, which perfectly matched with phenotypic susceptibility testing results. Among resistant isolates, the transition A to G, either in position 2142 or 2143, was detected in all 53 samples; the transversion A to C in position 2142 was not detected. In 20 additional cases, while there was no positive culture, the real-time PCR detected 16 resistant genotypes with the transition (A2142/43G) and 4 WT genotypes. In 42 biopsies the WT genotype was present simultaneously with the resistant genotype (Table 3). Table 3. Mutations in 23S rRNA of clarithromycin-susceptible and -resistant H. pylori isolates Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 a Includes 42 cases with mixed WT and mutant genotypes. Table 3. Mutations in 23S rRNA of clarithromycin-susceptible and -resistant H. pylori isolates Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 Phenotype n (%) of biopsies (N = 232) WT (n) A2142/43G (n) A2142C (n) Susceptible 163 (70.3) 163 0 0 Resistant 69 (29.7)a 42 69 0 a Includes 42 cases with mixed WT and mutant genotypes. Of the 212 H. pylori isolates, only 2 were resistant to tetracycline with an MIC of 1.5 mg/L. The analysis of the melting curves of all tetracycline-resistant and -susceptible isolates showed two Tms corresponding to two different genotypes: 230 biopsies showed a Tm of 61°C and exhibited an AGA926–928 WT in agreement with our AST results. For two isolates, we found a Tm of 56.8°C and sequencing consequently revealed an A926G mutation of the 16S rRNA gene for these two mutants. Detection of mutations associated with fluoroquinolones in the QRDR of gyrA Mutations in fluoroquinolone-resistant isolates were present in the QRDR at positions 87 and 91. There was perfect concordance between phenotypic and genotypic tests. The most common resistant substitution was N87K (present in 44.73%) followed by D91N (present in 23.68%) then N87I (present in 18.42%); the least common mutation was D91G (present in 7.89%). Two of the patients with fluoroquinolone-resistant isolates (MICs, 1.5 and 2 mg/L) did not present any of the expected mutations. Isolates presenting mutations at position 87 had an MIC >8 mg/L (Table 4). None of the susceptible isolates presented any mutation in the QRDR, but we found 42 susceptible isolates with the N87T (ACC) sequence. Table 4. Mutations in the QRDR of gyrA from resistant isolates Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Table 4. Mutations in the QRDR of gyrA from resistant isolates Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Patients with gyrA mutations in H. pylori, no./total (%) MIC (mg/L) Nucleotide change gyrA mutation 7/38 (18.42) 8–32 AAC→ATC N87I 17/38 (44.73) 8–32 AAC→AAA N87K 9/38 (23.68) 2–8 GAT→AAT D91N 3/38 (7.89) 2 GAT→GGT D91G Discussion In Algeria, surveillance of H. pylori antibiotic resistance is necessary in order to adapt the antibiotic combination to local resistance patterns. This is the first known study conducted in Algeria in which H. pylori resistance to several key antibiotics (clarithromycin, ciprofloxacin, rifampicin and tetracycline) was evaluated in different cities in the country (Algiers, Chlef, Oran and Sidi Bel Abbes) both by culture and molecular methods. We did not find any significant difference in the prevalence of H. pylori resistance between regions; the sample size was too small for each region to detect a significant difference. In developing countries, clarithromycin resistance and potential re-infections are factors that contribute to the failure of H. pylori eradication. In our study, the clarithromycin resistance rate was 29.7%; this result is lower than that found in a study conducted in Mustapha Pacha University Hospital, Algiers (33%), but higher than that found in another study conducted in three different hospitals in the same city (23%).13,14 The rate of resistance to clarithromycin is also high in Morocco (29%), Egypt (57.7%) and France (22.2%).15–17 However, the rate of resistance is much lower in Tunisia (14.6%), Nigeria (14.4%) and Senegal (1%).18–20 A2143G was the most prevalent point mutation, and studies have claimed that this mutation plays a major role in clarithromycin resistance.4,20 This result is similar to that of patients in Tunisia. We did not find the A2142C mutation, which is less common.21 One of the resistance mechanisms acquired by macrolides, like clarithromycin, is modifications to the ribosome binding site, which may consist of changes in the base sequence of 23S rRNA. Mutations are selected during treatment and can be observed when a macrolide is used in H. pylori eradication.22 On the other hand, previous reports have shown that the presence of the A2143G point mutation, rather than the A2142G or A2142C mutation, reduces the H. pylori eradication rate.23 This emergence of clarithromycin resistance might be due to the increased prescription of this antibiotic because clarithromycin is widely used in Algeria as an antimicrobial drug to treat infections in other organ systems such as the respiratory system. Furthermore, and especially due to cross-resistance in the macrolide family, the prevalence of clarithromycin-resistant H. pylori is continuously increasing. Concerning tetracycline resistance in our study, we found only two resistant isolates; moreover, with only one mutation of the concerned nucleotide triplet in agreement with a limited increase of the MICs. In a Nigerian study they also found a reduced rate of resistance to tetracycline (4.5%),19 and in Morocco and in Senegal they did not find any resistant isolates.15,20 According to Gerrits et al.,24 resistance to the tetracycline antibiotic group has been associated with mutations in the 16S rRNA gene. They found that transformation of the H. pylori strain 26695 (MIC, 0.19 mg/L) with genomic DNA from strain 181 (MIC, 8 mg/L) resulted in tetracycline-resistant colonies. In their study, a Light Cycler assay successfully distinguished two genotypes: a WT and 16S rRNA gene mutations associated with resistance and/or reduced susceptibility to tetracycline. Our study revealed that out of the 232 H. pylori-positive isolates, two with reduced susceptibility to tetracycline had the A926G mutation in the 16S rRNA gene and MICs were 1.5 mg/L. This mutation was previously reported in the Congo,25 in one German isolate with an MIC of 1 mg/L (Etest method)12 and in seven UK isolates with MICs of 0.75–4 mg/L (Etest method).26 The 16S rRNA gene polymorphisms are excellent targets for the real-time PCR detection assays, which allow access to susceptibility testing directly from biopsy specimens without culture. This method could be especially useful in patients who have experienced treatment failure. Fluoroquinolone resistance was found in 17.9% of the study population. This result was higher than that found in Morocco (11%),15 in Senegal (15%),20 in France (15.4%),17 in Europe (14.1%)27 and in Japan (14.9%),28 but lower than that found in the Congo (50%)25 and in China (28%).22 In a single Italian study29 and in Korea the ciprofloxacin resistance rate of H. pylori was reported to be 33.8%.30 However, in Sweden a low ciprofloxacin resistance rate (3.3%) was reported.31 This increasing resistance rate could be explained by the use of fluoroquinolones for urinary infections and several other infectious diseases in Algeria and throughout the world. QRDR mutations in fluoroquinolone-resistant H. pylori isolates were previously reported. For the strains isolated in our study we did not find any correlation between the mutation present and the level of resistance. All 34 fluoroquinolone-resistant isolates harboured one mutation already found in the QRDR of gyrA. N87K and D91N were the most common mutations in this region. The major mutation in position 87 was detected in other studies.32 D91N is a well-known mutation detected worldwide.33 As two isolates with ciprofloxacin MICs of 1.5 and 2 mg/L displayed no mutations in the QRDR of gyrA, we suggest that another mutation or another mechanism such as reduced drug accumulation could be involved. However, higher levels of resistance were most likely related to isolates with N87I and D91G mutations, with MIC values of 32 mg/L. Our data are in agreement with reports from Asia and Europe.34,35 The Maastricht IV/Florence Consensus Report recommended that empirical use of levofloxacin should be abandoned when the prevalence of resistance reaches 15%.36 In this study, we did not look for mutations (in rdxA and frxA) associated with metronidazole resistance, which are frequent but not always correlated to the resistance pattern. In conclusion, the mean primary H. pylori clarithromycin resistance rate in Algeria has crossed the borderline (15%) for applying the standard triple therapy. The primary fluoroquinolone resistance rate is also of growing concern. Depending on local circumstances the logistical problems associated with getting gastric biopsies into the laboratory on time in order to ensure successful culture of the pathogens might be impossible to overcome; hence genotyping susceptibility testing might be the preferred option. Furthermore, they can be used for continuous surveillance of the proper drug selection for anti-H. pylori therapy in different regions. Acknowledgements We thank Samir Halouche (Gastroenterology Clinic, Chlef, Algeria) and Professor Bouasria (First November 1954 University Hospital, Oran, Algeria) for their technical assistance, as well as the clinicians involved in this study. We thank Hacene Mahmoudi and Ahmad Aichouni for their moral support. Funding This work was financed by the Bioresources Laboratory, Department of Biology, Faculty of Natural and Life Sciences, Hassiba Ben Bouali University of Chlef, Chlef, Algeria.  In Algeria, all research projects are financed by the Algerian Research Ministry through research laboratories in each university. Transparency declarations None to declare. Author contributions Conceived and designed the experiments: M. B., R. A., L. B., A. T. and M. M. Performed the experiments: M. B., A. T., A. E.-M. D. and M. M. Analysed the data: M. B., A. T. and L. B. Contributed reagents/materials/analysis tools: M. B., M. M., F. M. and K. T. D. Wrote the paper: M. B., R. A. and F. M. All authors read and approved the final manuscript. References 1 Mégraud F , Brassens-Rabbé MP , Denis F et al. Seroepidemiology of Campylobacter pylori infection in various populations . J Clin Microbiol 1989 ; 27 : 1870 – 3 . Google Scholar PubMed 2 Bachir M , Allem R , Tifrit K et al. 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Management of Helicobacter pylori infection–the Maastricht V/Florence Consensus Report . Gut 2017 ; 66 : 6 – 30 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 11, 2018

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