Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.     

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.     

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.     

Characterization of Multiple Helicobacter bizzozeronii Isolates From a Finnish Patient With Severe Dyspeptic Symptoms and Chronic Active Gastritis

Background:  Helicobacter pylori is the primary cause of gastritis and peptic ulceration in humans. In a minority of patients with upper gastrointestinal symptoms, long tightly coiled spiral bacteria, provisionally named " Helicobacter heilmannii, " are observed in gastric biopsies. These bacteria are extremely fastidious and only one previous study has succeeded in obtaining an isolate in vitro.
Materials and Methods:  We used two different selective media to isolate " H. heilmannii " from the gastric mucosa of a Finnish patient presenting with severe dyspeptic symptoms. The isolates were characterized by testing for urease and catalase activity, by using light and electron microscopy, and by sequencing of the partial 16S rRNA and ureAB genes. Single-enzyme amplified fragment length polymorphism (sAFLP) was used to analyze the genetic diversity among the isolates.
Results:  We obtained 15 isolates from different gastric biopsies prior and three after unsuccessful treatment of the patient. The isolates were identified as Helicobacter bizzozeronii . Eradication therapy was unsuccessful most probably due to high level of resistance to metronidazole. Persistent colonization by the same H. bizzozeronii clone was confirmed by sAFLP, however, small differences between the profiles suggested long-term colonization of the patient.
Conclusions:  Helicobacter bizzozeronii remains the only " H. heilmannii " species isolated from human gastric mucosa although it has been an infrequent observation among " H. heilmannii "-infected patients in PCR-based screening studies. The relevance of H. bizzozeronii and other potentially zoonotic gastric Helicobacter spp. in human disease remains to be determined.     

Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence.

The infection rate of 60 pigs (10 pigs from each of six farms) by Helicobacter species was studied by two techniques. Histological examination of the cardiac area of the stomach yielded a 58% positive result versus an 80% positive result by PCR with genus-specific primers. Eighty percent of the 16S rRNA gene was amplified, classified in four groups by PCR-restriction fragment length polymorphism, and sequenced. Isolates from all farms except one (farm C) were identified as Helicobacter heilmannii type 1, while those from farm C were identified as H. heilmannii type 2. Attempts to culture this organism in vitro failed. Helicobacter pylori was not found in these animals.     

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.     

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.     

Non-pylori Helicobacteraceae in the upper digestive tract of asymptomatic Venezuelan subjects: detection of Helicobacter cetorum-like and Candidatus Wolinella africanus-like DNA

BACKGROUND: The spectrum of human non-pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non-pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. MATERIALS AND METHODS: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter-like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18-68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. RESULTS: We detected DNA sharing 99-100% sequence identity in over 300-400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). CONCLUSIONS: Our results suggest that non-pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.     

Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.     

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.     

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.     

Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.     

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.     

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.     

Distinction of gastric Helicobacter spp. in humans and domestic pets by scanning electron microscopy

Background. A number of different Helicobacter spp. can colonize the stomach of humans and domestic pets. Difficulties encountered with primary isolation of these spiral microorganisms and their unusual inertia with respect to biochemical reactions still represent considerable obstacles to their characterization with classic tools. In addition, the high degree of similarity in the 16S rRNA sequence hampers differentiation of Helicobacter spp. using routine molecular biological assays.
Materials and Methods. Samples from experimentally monoinfected mice, of naturally infected hosts, and of cultured strains were examined by scanning electron microscopy (SEM). In parallel, all samples were analyzed by molecular techniques to ascertain the Helicobacter spp. involved.
Results. Using the mouse samples as a reference, microorganisms found in naturally infected hosts were identified by SEM as belonging to H. pylori , H. felis , or a group consisting of H. bizzozeronii and H. heilmannii. A further spiral microorganism with unique morphology was found in a dog that was positive for H. salomonis , but the organism could not be recovered from experimentally infected mice. In culture, most Helicobacter strains lost their ultrastructural characteristics.
Conclusions. When gastric Helicobacter spp. were collected from their natural habitat and examined by SEM, relevant differences could be detected between H. felis , H. bizzozeronii and H. heilmannii , and H. salomonis , respectively. SEM, therefore, seems to be a useful auxillary tool for the distinction of various gastric Helicobacter spp. as based on their ultrastructure.     

Isolation of tetracycline-resistant clinical Helicobacter pylori without mutations in 16S rRNA gene in Bangladesh

The occurrence of 16S rRNA gene mutations associated with resistance to tetracycline in H. pylori isolated in Bangladesh was investigated. Tetracycline susceptibility was determined by the agar dilution method. The 16S rRNA genes of these isolates were sequenced and analyzed. A tetracycline accumulation assay was performed. DNA sequence and transformation tests of nine tetracycline-resistant (MIC = 2 microg/ml) Bangladeshi H. pylori clinical isolates showed that in no case was the resistance due to mutations in the 16S rRNA gene, the only known cause of tetracycline resistance in this pathogen. Tetracycline accumulation assays implicated altered uptake or efflux.     

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.     

Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.