Pylori 23S rRNA Gene Research Articles

Novel Genotypes in Helicobacter pylori Involving Domain V of the 23S rRNA Gene

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. 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. 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. 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.

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian- cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian- cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western- cagA positive and 53.7% (22/41) were East Asian- cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.

Possibility for the presence of Helicobacter pylori in drinking well water

Forty three well waters which are currently used as drinking water were studied for the presence of Helicobacter pylori. Using magnetic-beads purification and PCR amplification of H. pylori-specific gene, 4 of the 43 samples were positive for H. pylori-ureA gene (9.3%) and 1 of the 43 samples was positive for H. pylori-16SrRNA gene (2.3%). The presence of H. pylori-specific amplified product did not correlate with the type, depth and location of the wells. This study demonstrated that H. pylori can be transmitted via drinking water, especially well water in Japan.

Prevalence and rapid identification of clarithromycin-resistant Helicobacter pylori isolates in children.

Little is known about the prevalence of antibiotic-resistant Helicobacter pylori infection in children. Culture and antimicrobial susceptibility testing are generally time-consuming and not a routine in many hospitals. To investigate the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. Enrolled were 245 children investigated for H. pylori infection by endoscopic examination. The gastric antral specimens were subjected to DNA extraction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with primers specific to the H. pylori 23S rRNA gene. Conventional bacterial cultures were performed simultaneously as the diagnostic standard. Minimal inhibitory concentrations of clarithromycin and metronidazole were determined by E test. This was used as a standard to determine the sensitivity and specificity of the above PCR-RFLP assay. The specimens were processed for histologic examination and evaluated by the updated Sydney system. H. pylori was isolated in 67 of the 245 children; 12 (18%) of them were clarithromycin-resistant and 6 (9%) were metronidazole-resistant. No difference in histologic examinations was noted between the antibiotic-resistant and -susceptible strains. We performed PCR-RFLP with all 12 clarithromycin-resistant isolates: 10 had a 23S ribosomal RNA A2144G point mutation; 1 had a mixture of an A2143G point mutant and susceptible strains; and 1 had neither of the 2 mutations. The prevalence of clarithromycin-resistant H. pylori isolates in Taiwanese children is 18%. PCR-RFLP had a high sensitivity (92%) and specificity (100%) for the clarithromycin resistance gene mutation determination. The dominant mutation is A2144G. PCR-RFLP provides a rapid and accurate approach to detect clarithromycin-resistant strains within 24 h.

Helicobacter pylori in water systems for human use in Mexico City

Helicobacter pylori infection is associated with peptic ulcers and gastric cancer in humans. Transmission of H. pylori is still not certain with some epidemiological data suggesting water as a possible transmission route. The objective of this study was to detect H. pylori 16S rRNA gene in five water systems in the Mexico City area. Samples were taken between 1997 and 2000 from extraction wells (system 1), from dams used as water sources, both pre- and post-treatment (systems 2 and 3), treated wastewater (system 4) and non-treated wastewater (system 5). Detection of the H. pylori 16S rRNA gene in water samples was carried out using nested PCR in 139 water samples and confirmed by using cagA gene detection by PCR-hybridisation. The results showed the presence of H. pylori in 58 (42%) of the water samples in total with a prevalence of 68% in system 1, 100% in system 2, 0% in system 3, 17% in system 4 and 20% in system 5. This first stage showed the presence of H. pylori in the tested water systems; nevertheless, viability of the microorganism in water and vegetables needs to be confirmed as well as demonstration of a relationship between human and environmental strains.

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases.

Our aim was to detect Helicobacter pylori (H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/m1 (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases

OBJECTIVE: Our aim was to detect Helicobacter pylori ( H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. METHODS: A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. RESULTS: Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/ml (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. CONCLUSIONS: Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.

Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance

Background—Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5–10% of strains are reported to be resistant. It has been shown that one point mutation in...

Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.