Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori

Abstract The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincamatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.     

Helicobacter pylori antimicrobial resistance in a pediatric population

Background

The increasing prevalence of Helicobacter pylori (H. pylori) antimicrobial resistance, primarily for clarithromycin decreases the success of treatment. The aim of this study is to determine the local pattern of first‐line antimicrobials resistance and the eradication rate.

Material and Methods

Prospective cohort study of H. pylori infected patients (positive histological or cultural exams) treated at Centro Materno‐Infantil do Norte from January of 2013 to October of 2017. Susceptibility to 4 antibiotics: amoxicilin, metronidazole, clarithromycin, and levofloxacin were analyzed by E‐test (phenotypic resistance). The E‐test was chosen because it is simple and cost‐effective for routine susceptibility testing. Point mutations that confer clarithromycin resistance were surveyed (genotypic resistance). Eradication of H. pylori infection was defined by a negative urea breath test or fecal antigen 6‐8 weeks after the end of treatment.

Results

Of a total of 74 H. pylori infected patients, 16 were excluded because they had previous H. pylori treatment or severe systemic disease. Median age of infection cases was 15 years (3‐17 years). Eradication regimen used in all patients combined the use of 3 antibiotics (amoxicillin and metronidazole or clarithromycin) and proton pump inibhitor for 14 days and was tailored according antimicrobial susceptibility. 79.5% of the patients completed the treatment. The resistance rate for metronidazole and clarithromycin was 3.3% and 23.3%, respectively. There was no resistance for amoxicilin and levofloxacin. The rate of genotypic resistance to clarithromycin was 37.2%. The eradication rate was 97.8%.

Conclusions

The authors found a high resistance rate of H. pylori for clarithromycin in this northern portuguese pediatric center. This factor should determine a change in local current treatment, contraindicating the use of clarithromycin as a first‐line treatment for H. pylori infection in children. The high eradication rate maybe explained for the eradication treatment tailored according antimicrobial susceptibility.     

Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with BsaI and BbsI restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).     

Ranitidine bismuth citrate can help to overcome Helicobacter pylori resistance to clarithromycin in vivo

Background. Helicobacter pylori eradication usually fails when clarithromycin is used against resistant strains.
Objective. The objective of this study was to test whether the apparent synergy found in vitro between ranitidine bismuth citrate (RBC) and clarithromycin also exists in vivo against resistant strains.
Methods. H. pylori was cultured and clarithromycin susceptibility was determined before and after treatment, from duodenal ulcer patients receiving RBC and clarithromycin or omeprazole and clarithromycin for 2 weeks in a multicenter randomized clinical trial.
Results. The overall eradication rate was 88.7% in the RBC group (71 patients) and 52.7% in the omeprazole group (74 patients). The demographic characteristics of the two groups were not different. Clarithromycin-resistant strains were isolated in 22 cases (15.1%). A difference between the eradication rates of susceptible and resistant strains was found in the omeprazole group but not in the RBC group. After treatment, resistance to clarithromycin developed in three of the seven strains (42.3%) cultured from the patients of the RBC group, compared with 11 of the 26 strains (42%) of the omeprazole group. That is, clarithromycin-resistant strains were found in 6% and 27% in the RBC group and the omeprazole group, respectively, on considering the global results.
Conclusion. A synergy between RBC and clarithromycin may exist in vivo and, while clarithromycin resistance is increasing, it is an argument for using RBC in triple therapies.     

Detection of primary clarithromycin resistance of Helicobacter pylori and association between cagA + status and clinical outcome

Helicobacter pylori was examined in 110 patients (82 (74.5) with gastritis, 18 (16.4) with duodenitis, six (5.5) with duodenal ulcer and gastroesophageal reflux, and four (3.6 %) with normal) with gastrointestinal problems living in rural area, no history of macrolide use, and detected by culture (71.8) or direct detection from gastric biopsies by PCR (82.7 %). Also, cagA gene was identified using PCR and was found positive in 68/91 (74.7 %) strains. The prevalence of clarithromycin-resistant H. pylori was investigated by two methods including PCR–RFLP (7.7 (A2142G 1.1 and A2143G 6.6 %)) and twofold agar dilution (8.9 %) to detect phenotypic and genotypic status simultaneously. Among all the H. pylori positive patients, eight (8.8 %) isolates were found to be resistant to clarithromycin by at least one of the AD and/or PCR–RFLP methods. H. pylori positive rates were significantly correlated with patients' sex, age, and endoscopic findings (p?=?0.040, <0.001 and <0.001, respectively). There were no differences in gender or endoscopic findings related to cagA ⁺ and cagA ^? patients. The gene of cagA was not significantly helpful in predicting the clinical outcome of H. pylori infection alone. In conclusion, we revealed that there was a low prevalence of primer clarithromycin resistance in patients living in rural area with no history of macrolide use. The prevalence of mutant strains among the macrolide-resistant H. pylori varies even geographically between close provinces.     

Genotypes of Helicobacter pylori in Polish population

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.     

Prevalence of Helicobacter pylori resistance to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone in Brazil

Background. Helicobacter pylori infection is associated with a wide range of digestive diseases and is very prevalent in developing countries, although few data exist on the susceptibility of H. pylori to antimicrobials commonly used in eradication schedules in these countries. The aim of this study was to evaluate the resistance of H. pylori to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone in dyspeptic Brazilian patients.
Material and Methods. Ninety consecutive H. pylori –positive patients were enrolled. Resistance was evaluated by an agar dilution test.
Results. Resistance to metronidazole was detected in 38 patients (42%); to amoxicillin in 26 individuals (29%); to clarithromycin in 6 patients (7%); to tetracycline in 6 patients (7%); and to furazolidone in 4 individuals (4%). Thirteen strains were resistant to two agents, and eight strains were resistant to three antimicrobials.
Conclusions. These results confirm the need for culture and susceptibility testing to define H. pylori resistance patterns in particular geographical areas before the general use of an eradication schedule. They also suggest the possibility of resistance to such antimicrobials as amoxicillin or tetracycline in geographical areas with a high prevalence of H. pylori infection and still not fully evaluated for antimicrobial susceptibility.     

Gene mutations of 23S rRNA associated with clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients

Although resistance of Helicobacter pylori to clarithromycin is a major cause of failure of eradication therapies, little information is available regarding gene mutations of clarithromycin-resistant primary and secondary H. pylori isolates in Korea. In the present study, we examined gene mutations of H. pylori 23S rRNA responsible for resistance to clarithromycin. DNA sequences of the 23S rRNA gene in 21 primary clarithromycin-resistant and 64 secondary clarithromycin-resistant strains were determined by PCR amplification and nucleotide sequence analyses. Two mutations of the 23S rRNA gene, A2143G and T2182C, were observed in primary clarithromycin-resistant isolates. In secondary isolates, dual mutation of A2143G+T2182C was frequently observed. In addition, A2143G+T2182C+ T2190C, A2143G+T2182C+C2195T, and A2143G+T2182C +A2223G were observed in secondary isolates. Furthermore, macrolide binding was tested on purified ribosomes isolated from T2182C or A2143C mutant strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the mutant strains. These results indicate that secondary isolates show a greater variety of 23S rRNA gene mutation types than primary isolates, and triple mutations of secondary isolates are associated with A2143G+T2182C in H. pylori isolated from Korean patients.     

Whole genome sequencing reveals novel genetic mutations of Helicobacter pylori associating with resistance to clarithromycin and levofloxacin

Background

Detection of mutations in one or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of Helicobacter pylori to clarithromycin (CLA) and levofloxacin (LVX). We aimed to perform whole genome sequencing to explore novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests.

Methods

Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based antibacterial susceptibility test, H. pylori strains were divided into three groups, with nine strains in each group: CLA single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all antibacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and amino acid mutations were screened, and potential candidate genes related to CLA and LVX resistance were identified.

Results

The median age of study subjects was 35 years (IQR: 31–40), and 17 (63.0%) were male. All nine CLA-resistant strains had A2143G mutations in 23S rRNA, while none of nine sensitive strains had the mutation. Six of nine strains in group L and six of nine strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the three groups, we identified five mutated positions belonging to four genes related to CLA resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CLA resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated.

Conclusion

Whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CLA and LVX including fliJ and cheA. Further studies to correlate these findings with treatment outcome should be performed.     

Helicobacter pylori Infection and Recurrent Abdominal Pain in Turkish Children

Background. Helicobacter pylori infection is primarily acquired in childhood. However, the association between H. pylori infection and recurrent abdominal pain (RAP) remains unclear. Materials and methods. One hundred and forty-one children with and 21 without RAP underwent upper gastrointestinal endoscopy. At least five antral gastric biopsies were obtained from each patient and the presence of H. pylori infection was accepted when at least two out of four tests (histology, direct antral smear, culture, and rapid urease test) were positive. Patients with H. pylori infection underwent triple therapy with omeprazole, clarithromycin, and metronidazole. Results. Eighty-five out of 141 (60.3%) patients with RAP were H. pylori positive whereas 5 out of 21 (20.8%) patients without RAP were (p = .0037). Symptoms were disappeared in 87% of children whose H. pylori infection was eradicated compared with 41% of those in whom the infection was not eradicated (p = .0035). Conclusions. It was concluded that children with RAP and H. pylori infection appear to benefit from eradication therapy in Turkey.     

High Helicobacter pylori resistance to metronidazole but zero or low resistance to clarithromycin, levofloxacin, and other antibiotics in Malaysia

Objective: Bacterial resistance to antibiotics is the single most important determinant of treatment success. The objective of this study was to determine the prevalence of Helicobacter pylori resistance to clarithromycin, amoxicillin, metronidazole, tetracycline, levofloxacin, rifabutin, and furazolidone in our local bacterial strains. Methods: Samples from consecutive ninety patients were obtained for culture and sensitivity testing. Resistance to individual antibiotics were tested using the E‐test and MIC₉₀ read from the strips. Resistance to rifampicin and nitrofurantoin were used as a surrogate for rifabutin and furazolidine. Results: There was a high prevalence of resistance to metronidazole 68/90 (75.5%). No male (34/45 (75.5%) versus female (35/45 (77.7%) difference in frequency of metronidazole resistance was noted (p = 1.000). There was zero resistance ₀ to clarithromycin, levofloxacin, amoxicillin, and nitrofurantoin/furazolidone. Resistance to rifampicin/rifabutin was for breakpoints of 1 and 4 μg/mL of 14.4 and 2.2% respectively. Conclusions: Although there was high bacterial resistance to metronidazole, the absence of resistance particularly to the key antibiotics used in H. pylori eradication therapy: clarithromycin and levofloxacin is reassuring to note. Continued monitoring of antibiotic resistance should be carried out.     

Antimicrobial effects of antioxidants with and without clarithromycin on Helicobacter pylori

Increasing resistance to currently used antimicrobials has resulted in the evaluation of other agents that have antimicrobial activity against Helicobacter pylori. H. pylori American Type Culture Collection (ATCC) strain 49503 (a toxin-producing strain known to be associated with gastric cancer) was grown, a cell suspension prepared in 2 mL PBS and diluted 10-fold. One hundred L of this cell suspension was added to vitamin C 0.5%, vitamin E 0.5%, garcinol 100 g/mL, Protykin® (containing 50% rans-resveratrol) 100 g/mL and garcinol + Protykin® 100 g/mL in Lennox broth, and incubated for 16 h under microaerophilic conditions. Three replicates of 10 L from each 10^–7 dilution tube were plated, colonies were counted after 16 h, and growth of H. pylori was confirmed by the CLO® test. These colony counts were compared to control cultures without the addition of any antioxidants. The experiments were then repeated with the addition of 15 g/mL of clarithromycin to experimental and control samples. Enhanced killing of H. pylori by 37.6% was noted when vitamin C was added, which increased to 66% when clarithromycin was added, compared to controls (p < 0.05). With garcinol and Protykin® alone there was 91.4 and 87% killing of H. pylori, respectively, while a combination of garcinol + Protykin® resulted in 90.8% killing compared to controls (p < 0.05). When clarithromycin was added, there was 76.3% increased killing with garcinol alone, 55.3% with Protykin® alone, and 73.7% with garcinol + Protykin® compared to controls (containing clarithromycin) (p < 0.05). Vitamin E had no effect on H. pylori growth compared to controls. We conclude from this study that some antioxidants such as vitamin C, garcinol and Protykin®, but not vitamin E, may have potential as antimicrobial agents against H. pylori. (Mol Cell Biochem 270: 125–130, 2005)