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.     

A2144G is the main mutation in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance

To detect point mutations A2115C, A2143G/C, and A2143G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.     

Novel genotypes in Helicobacter pylori involving domain V of the 23S rRNA gene

BACKGROUND: Helicobacter pylori is a pathogenic bacterium that infects a half of the human population. In Chile, between 55% and 79% of people are colonized by H. pylori. At present, therapeutic strategies to eradicate the bacterium depend on the knowledge of its resistance to antibiotics. The clarithromycin resistance in H. pylori is associated with point mutations in the 23S rRNA. This study analyzes 23S rRNA gene mutations and minimum inhibitory concentration (MIC) for clarithromycin in H. pylori isolates from patients of the metropolitan region of Chile. MATERIALS AND METHODS: H. pylori isolates from 50 dyspeptic patients with no history of clarithromycin exposure were tested for clarithromycin resistance by agar dilution method. Resistant strains were analyzed for mutations in the 23S rRNA gene by polymerase chain reaction-based restriction fragment length polymorphism and sequencing. RESULTS: Primary resistance was observed in 10 isolates (20%). A single mutation was detected in four of the 10 isolates and two or more mutations in the other six cases. The C2147G transversion and G1939A, T1942C, and A2142G transitions in the peptidyltransferase region of domain V were novel. CONCLUSIONS: The study shows: 1, novel variants of the H. pylori 23S rRNA gene; and 2, a high prevalence of H. pylori displaying primary clarithromycin resistance with low level of MIC in an urban area of the Metropolitan Region of Chile.     

Primary resistance of Helicobacter pylori to antimicrobial agents in Polish children

Helicobacter pylori resistance to antimicrobial agents is an important factor compromising the efficacy of treatment. Therefore the aims of our study were: to determine the prevalence of H. pylori resistance to clarithromycin, metronidazole, amoxycillin and tetracycline in children prior to eradication therapy, to compare different methods of susceptibility testing and to detect mutations responsible for clarithromycin resistance. During 1996-2000, 259 H. pylori strains were isolated from antral gastric biopsies. Susceptibility to antimicrobials was determined by the agar dilution method and the Etest. Mutations in the 23S rRNA gene associated with clarithromycin resistance were analysed by PCR-RFLP and direct sequencing. Overall, ninety-six strains (37%) were resistant to metronidazole, 50 strains (19.3%) were resistant to clarithromycin, and 20 strains (7.7%) were simultaneously resistant to both drugs. All cultured isolates were sensitive to amoxycillin and only one isolate (0.4%) was resistant to tetracycline. The agar dilution method and the Etest showed a perfect category correlation for clarithromycin and 4% discrepancies for metronidazole. Primary resistance to clarithromycin was mainly associated with an A2143G mutation in the 23S rRNA gene of H. pylori. The study highlights the high prevalence of H. pylori primary resistance to clarithromycin in Polish children, which implies a need for pretreatment susceptibility testing.     

Identification of a novel mutation affecting domain V of the 23S rRNA gene in Helicobacter pylori

BACKGROUND: This study analyzes clarithromycin resistance status and 23S rRNA gene mutations in Helicobacter pylori strains from Central Italian patients. MATERIALS AND METHODS: H. pylori strains from 235 dyspeptic patients (205 with no history of clarithromycin exposure and 30 referred for failure of eradication therapy) were tested for clarithromycin resistance by screening agar method and E-test. Resistant strains were analyzed for mutations of the 23S rRNA gene by PCR-RFLP and sequencing. RESULTS: Primary resistance was observed in strains from 43/205 (21%) patients with no history of clarithromycin exposure and secondary resistance in 30/30 (100%) strains from previously treated patients. A single mutant strain was detected in 54/73 (74%) cases, a mixture of one or more mutant(s) plus the wild type in the remaining 19/73 (26%) cases. One 23S rRNA gene mutation (A-->T transversion at nucleotide 2144) in the peptidyltransferase region of domain V was novel. CONCLUSIONS: This study shows: (a) a high prevalence of H. pylori strains with primary or secondary clarithromycin resistance in an urban area of Central Italy; (b) colonization by both mutant and wild-type H. pylori in the same patient; (c) a novel variant of the H. pylori 23S rRNA gene.     

Genotypic characterization of clarithromycin-resistant Helicobacter pylori strains

Helicobacterpylori (Hp) resistance to clarithromycin, one of the antibiotics most used to eradicate infection, is connected with the presence of a point mutation on the level of adenine at position 2143 or 2144 of 23S rRNA. AIM: The aim of the study is to evaluate of the presence of these mutation vs control clarithromycin resistant Hp strains present in North Sardinia; to verify the real association between the type of mutation and the resistance-level; to use easier molecular biology methods to quickly locate the resistance-associated mutations beginning with the bioptic material. The clarithromycin susceptibility of Hp isolates was tested by the E-test method (antibiotic assay). Genomic DNA of Hp strains was amplified using specific primers for the domain V. of ribosomic 23S rRNA and sequenced after the reaction with a primer within the fragment 23S. At the same time PCR-RFLP reliability was examined underlining the presence of these mutations with BsaI, BbsI, MboII restriction enzymes. Two mutations in 2143 (A- - G) and 2144 (A- - G) were found by domain V sequencing. The strains with mutation 2143 are characterized by a greater resistance level (MIC>64 g/ml) than those with mutation 2144 (MIC <64 g/ml). Restriction endonucleases BbsI and MboII recognise the site containing the mutation 2143 (A- - G), while BsaI recognise the mutation 2144 (A- - G). These methods might enable us to identify the presence of Hp directly from bioptic material and possible clarithromycin resistance and plan a suitable therapeutic strategy and consequently a better control of the infection.     

Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia

Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.     

Clarithromycin resistance in Helicobacter pylori strains isolated from French children: prevalence of the different mutations and coexistence of clones harboring two different mutations in the same biopsy

BACKGROUND: The aim of our study was to assess the different mutations involved in clarithromycin-resistant Helicobacter pylori strains isolated from French children and their temporal trends. METHODS: The point mutations of H. pylori were detected by PCR followed by RFLP technique in 50 clarithromycin-resistant strains collected between 1993 and 2004 in France. RESULTS: Clarithromycin resistance was observed in 23% (50/217) of H. pylori isolates. Two mutations A2143G and A2142G in the 23S rRNA genes of H. pylori were detected. The former was found in 45/50 (90%) of isolates. The rate of resistance increased with time from 18.6% in the period 1993-1996 to 41.6% in 2001-2004. No significant difference was observed in the distribution of mutations during the same periods. No correlation was found between any mutation and age, sex, and ethnic origin of children. Furthermore, no significant differences in minimal inhibitory concentrations level were observed according to the different point mutations. In all cases, only one point mutation was present, except in two cases where two different mutations were found in two different clones from the same biopsy. CONCLUSION: The mutation A2143G is predominant in clarithromycin-resistant H. pylori strains isolated from children in France. We report for the first time the presence of two clarithromycin-resistant clones harboring two different mutations of the 23S rRNA genes present in the same biopsy specimen and genotypically identical as demonstrated by RAPD fingerprinting.     

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).     

Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains

The use of phenylethynylpyrene excimer forming pair in the design of specific fluorescent probes for determination of A2144G (A2143G and/or A2143C) mutations in 23S rRNA gene of Helicobacter pylori is described. Analysis of fluorescence spectra of model duplexes revealed optimal positions of fluorophore residues in the probe sequences for maximum efficiency of SNP detection. Application of excimer forming probes for analysis of DNA samples isolated from natural bacterial strains of H. pylori was demonstrated.     

Dual-Priming Oligonucleotide-Based Multiplex PCR for the Detection of Helicobacter pylori and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens

Background:  Assessment of Helicobacter pylori ( H. pylori ) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori.
Materials and Methods:  Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests.
Results:  Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens.
Conclusions:  DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.     

Identification of shiga toxin-producing bacteria by a new immuno-capture toxin gene PCR

Mechanisms and occurrence of macrolide resistance in the periodontal pathogen Treponema denticola have received little attention. In this study, erythromycin resistance due to mutations in the genes encoding T. denticola 23S rRNA was investigated. The T. denticola genome was shown to contain two copies of 23S rDNA. 23S rRNA genes of nine erythromycin-resistant isolates derived from T. denticola were amplified and sequences were analyzed. All the erythromycin-resistant strains had at least one A-->G transition mutation at the 23S rRNA gene sequence cognate to position A2058 in Escherichia coli 23S rDNA. This suggests that antibiotic pressure is sufficient to select for point mutations that confer resistance in this organism.     

Functional interactions within 23S rRNA involving the peptidyltransferase center.

A molecular genetic approach has been employed to investigate functional interactions within 23S rRNA. Each of the three base substitutions at guanine 2032 has been made. The 2032A mutation confers resistance to the antibiotics chloramphenicol and clindamycin, which interact with the 23S rRNA peptidyltransferase loop. All three base substitutions at position 2032 produce an erythromycin-hypersensitive phenotype. The 2032 substitutions were compared with and combined with a 12-bp deletion mutation in domain II and point mutations at positions 2057 and 2058 in the peptidyltransferase region of domain V that also confer antibiotic resistance. Both the domain II deletion and the 2057A mutation relieve the hypersensitive effect of the 2032A mutation, producing an erythromycin-resistant phenotype; in addition, the combination of the 2032A and 2057A mutations confers a higher level of chloramphenicol resistance than either mutation alone. 23S rRNAs containing mutations at position 2058 that confer clindamycin and erythromycin resistance become deleterious to cell growth when combined with the 2032A mutation and, additionally, confer hypersensitivity to erythromycin and sensitivity to clindamycin and chloramphenicol. Introduction of the domain II deletion into these double-mutation constructs gives rise to erythromycin resistance. The results are interpreted as indicating that position 2032 interacts with the peptidyltransferase loop and that there is a functional connection between domains II and V.     

Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.     

Chloramphenicol-erythromycin resistance mutations in a 23S rRNA gene of Escherichia coli.

Two chloramphenicol resistance mutations were isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Both mutations also confer resistance to 14-atom lactone ring macrolide antibiotics, but they do not confer resistance to 16-atom lactone ring macrolide antibiotics or other inhibitors of the large ribosomal subunit. Classic genetic and recombinant DNA methods were used to map the two mutations to 154-base-pair regions of the 23S RNA genes. DNA sequencing of these regions revealed that chloramphenicol-erythromycin resistance results from a guanine-to-adenine transition at position 2057 of the 23S RNA genes of both independently isolated mutants. These mutations affect a region of 23S RNA strongly implicated in peptidyl transfer and known to interact with a variety of peptidyl transferase inhibitors.     

Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.     

Primary antibiotic resistance of Helicobacter pylori isolated from Beijing children

Background: The antimicrobials resistance of Helicobacter pylori (H. pylori) was able to sharply decline the eradication rate of H. pylori both in adults and children, but there are limited studies about the primary antibiotic resistance and the related gene mutations, specifically in China. Materials and Methods: The primary resistance to 9 antibiotics of 73 H. pylori strains isolated from gastric biopsies of children recruited at Beijing Children’s Hospital was assessed, and the mutations in 23S rRNA gene of 65 macrolide‐resistant strains and in gyrA and gyrB of 12 quinolone‐resistant strains were investigated. Results: The resistance rate to clarithromycin, azithromycin, metronidazole, levofloxacin, moxifloxacin, and rifampicin was 84.9%, 87.7%, 61.6%, 13.7%, 15.1%, and 6.8%, respectively. No resistance to amoxicillin, gentamicin, and tetracycline was observed. Dual, triple, and quadruple antibacterial resistant percentage was 46.6% (34/73), 15.1% (11/73), and 2.7% (2/73), respectively. The gene mutation rate of A2142C, A2142G, and A2143G in 23S rRNA gene was 1.5% (1/65), 6.2% (4/65), and 84.6% (55/65), respectively. The detection rate of mutations of Asn87, Asp91, and Met191 in GyrA was 41.7% (5/12), 25% (3/12), and 25% (3/12), respectively. Conclusion: The high prevalence of primary antibiotic resistance was out of expectation in H. pylori strains isolated from the children in Beijing. Antibiotic susceptibility should be made clear before the antibiotic was used in the anti‐H. pylori therapy in this population. The A2143G was the most populated mutation in macrolide‐resistant strains, and Asn87 and Asp91 of GyrA were the most common mutation points in quinolone resistance strains.     

Prevalence of Helicobacter pylori infection and the incidence of ureA and clarithromycin resistance gene 23S rRNA genotypes status in Saudi Arabia

ObjectivesTo determine the prevalence of Helicobacter pylori ureA and clarithromycin resistance gene 23S rRNA genotypes among H. pylori in Saudi Arabia.MethodsA total of 100 serum and fecal samples from 70 patients and 30 healthy volunteers, from patients who presented with symptoms suggestive of gastritis or peptic ulcer disease, were taken from the main hospital in the Southern region of Saudi Arabia from September 2010 C.E. to March 2011 C.E. corresponding to Shawwal 1431 A.H. to Rabi Al-Thani 1432 A.H. We cultured the samples for H. pylori and a polymerase chain reaction was carried out to check for the presence or absence of ureA gene and clarithromycin resistance gene 23S rRNA genotypes.ResultsAmong the 70 suspected patients, the suspected bacteria isolated from the fecal samples of 60, (85.7%) were positive using the culture techniques. The presence of ureA gene and clarithromycin resistance gene 23S rRNA was determined by using the polymerase chain reaction, Among the 100 fecal specimens, 65 fecal specimens from 70% patients showed positive results to clarithromycin resistance gene 23S rRNA (sensitivity, 93%; Specificity, 100% and Accuracy, 95%), Only 60 fecal specimens were positive with ureA gene (sensitivity, 86%; Specificity, 100% and Accuracy, 90%).Conclusion23S rRNA gene was associated with clarithromycin resistance in H. pylori. There was a high prevalence of H. pylori resistance to clarithromycin in Saudi Arabia. H. pylori is a neutrophilic bacteria that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms as Urease activity that hydrolyzed the Urea producing 2 NH₃ and H₂CO₃.     

Chloramphenicol resistance mutations in the single 23S rRNA gene of the archaeon Halobacterium halobium.

A broad range of antibiotics affecting protein biosynthesis were screened for their ability to inhibit growth of the archaeon Halobacterium halobium. Only a few drugs, including chloramphenicol, produced inhibitory effects. Mutants which showed increased resistance to chloramphenicol were isolated; of the nine tested, eight exhibited a C----U transition at position 2471 and the ninth had an A----C transversion at position 2088 of 23S rRNA. A double mutant containing both C----U (position 2471) and A----C (position 2088) mutations was isolated, but the level of its chloramphenicol resistance did not exceed that of either single-point mutant. Inferences are made concerning the functional significance of the conserved nucleotides in rRNAs.     

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.