Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori

BACKGROUND: Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (Amx) H. pylori isolates, and is mediated by mutations in penicillin-binding protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional Amx H. pylori isolates has been established. METHODS: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven Amx H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained Amx transformants were determined. RESULTS: Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the Amx H. pylori isolates resulted in an increased MIC (0.5-1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402-404) and third (KTG555-557) conserved penicillin-binding protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. CONCLUSION: In naturally occurring Amx H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests.     

Differentiation between isolates of Helicobacter pylori by PCR-RFLP analysis of urease A and B genes and comparison with ribosomal RNA gene patterns

Abstract Genetic diversity amongst 21 human gastric isolates of Helicobacter pylori was investigated by polymerase chain reaction amplification and Hae III digest (restriction fragment length polymorphism) analysis of an internal 2.4-kb segment of the urease A and urease B genes. H. pylori from 11 independent individuals yielded nine distinct restriction fragment patterns but only one pattern was common to H. pylori from two individuals. By contrast, multiple isolate sets of H. pylori from two patients each had common urease gene patterns. Most strains with the same urease gene patterns were distinguishable in their ribosomal RNA gene patterns. The study demonstrated diversity amongst H. pylori and established that PCR analysis of urease genes provided a novel method of identifying isolates. The profiles were reproducible and convenient to obtain and analyse, and were almost as discriminatory as Hae III ribopatterns.     

Characterization of a Single-Spore Isolate Population of Plasmodiophora brassicae Resulting from a Single Club

The single‐spore isolates ‘e₃’ and ‘e₆’ of Plasmodiophora brassicae with different virulence patterns were distinguished by restriction fragment length polymorphisms in fingerprint‐like patterns and by electrophoretic karyotypes using repetitive fragments as hybridization probes. These molecular tools were used to characterize a set of isolates originated from an infected root, which was inoculated with a mixture of the two single‐spore isolates (e₃ and e₆). Spores harvested from mixed‐infected roots were used to establish 53 new single‐spore isolates. All these single‐spore isolates revealed parental patterns according to their molecular fingerprints and their virulence pattern. No sexual recombination could be detected with repetitive molecular probes. However, one isolate (M₃₆ES49) showed the same fingerprint pattern and virulence pattern but different sizes of small chromosomes than the parental type ‘e₆’, which is taken as an indication of chromosome rearrangement during the infection cycle.     

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.     

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.     

Analysis of the rdxA gene in high-level metronidazole-resistant clinical isolates confirms a limited use of rdxA mutations as a marker for prediction of metronidazole resistance in Helicobacter pylori

Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32-192 microg ml(-1)) and high-level (MIC>/=256 microg ml(-1)) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs>/=256 microg ml(-1)). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.     

球形与螺旋形幽门螺杆菌基因表达差异的研究

目的：从基因转录水平了解球形幽门螺杆菌(Helicobacter pylori,Hp)基因表达的变化。方法：幽门螺杆菌标准株NCTC ll637和2株临床分离株,经抗生素—灭滴灵诱导球变。提取等菌量螺旋形和球形Hp的RNA,RT—PCR扩增Hp25种基因,这些基因包括毒力基因(kat、aphA、rdxA、frxA、cysS、ureB glmM、cagA、vacA、fldA、iceAl、hpaA、fliD、napA、oipA、alpAB、babB、hopZ);看家基因(scoB、atpD、g1nA、recA)和功能不明的外膜蛋白基因(hopW、hopX)。扩增产物经分子定量成像系统进行扫描定量。结果和结论：等菌量的球形HpRNA量比螺旋形Hp减少。NCTC ll637、F44和F49菌株的螺旋菌分别有25、20、19个基因RT—PCR阳性,对应的球形菌相应的基因RT—PCR也为阳性。定量分析结果表明：被检测的基因在球形菌中的表达均比螺旋菌降低。提示球形菌致病性的降低与基因的低表达有关。3株球形菌不同基因表达的变化并不完全一致,表达量变化较小且各菌间较一致的是g1mM、oipA、g1nA;表达量变化较大且菌问较一致的基因是：napA、sodB、recA、fliD。     

cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groups

Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3' region of cagA gene were examined among 192 Malaysian H. pylori cagA-positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621-651bp), type B (732-735bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p>0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific-cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p<0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p<0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection.     

AP—PCR结合进化树分析在幽门螺杆菌地域起源特征研究中的价值

目的探讨随机引物PCR（Arbitrary primerPCR,AP—PCR）结合克隆测序及生物信息学分析等方法在研究幽门螺杆菌（Helicobacter pylori,Hpylori）菌株地域起源特征中的价值和意义。方法针对临床分离培养的Hpylori菌株的基因组DNA,采用一组10nt的寡核苷酸引物进行随机PCR扩增,选取相对保守的片段进行回收、克隆及测序,测序的基因序列提交GenBank数据库进行序列相似性的BLAST比对,收集BLAST比对得到的同源性较高不同地域来源螺杆菌的对应序列,用ClustalX软件进行排序,采用Mega4．0软件中的邻位相连法（Neighbor-joining）和最大简约法（Maximum—parsimony）进行进化树分析。结果随机引物扩增及筛选克隆测序得到的基因产物为NADH脱氢酶G和H亚单位的部分编码序列,与27株不同地域来源H．pylori及1株猫科动物来源螺杆菌菌株的同源性均高达90％以上,表明Hpylori中NADH脱氢酶基因序列为保守结构,进化树分析显示：采用AP．PCR方法得到的Hpylori临床菌株的基因序列,显示出东亚菌株来源的遗传特征,与具有东亚菌株特征的美洲秘鲁Sat464和Shi470菌株、韩国的52、51菌株、日本的1757菌株遗传距离较近,与南亚、欧洲菌株距离较远,与非洲的SouthAffica7菌株和猫科动物来源的Sheeba菌株的遗传距离最远。结论不仅某些特殊基因可以反映地域差异,随机定位相对保守的基因片段同样可以反映Hpylori的地域起源特征。AP—PCR、测序等技术方法与进化树分析相结合是探讨Hpylori地域起源特征的一种更为便捷有效的新方法。     

Conservation and Diversity of the Helicobacter pylori Copper-Transporting ATPase Gene (copA) Sequence Among Helicobacter Species and Campylobacter Species Detected by PCR and RFLP

Background Helicobacter pylori is a causative pathogen of such human stomach diseases as chronic type B gastritis, ulcer, and possibly gastric carcinoma. As a co-factor in various redox enzymes and an essential trace metal required for the synthesis of metalloproteins, copper might play a role in the pathogenesis of H. pylori. A gene, copA , associated with copper transport, has been isolated from H. pylori UA802. In this study, conservation and diversity of this gene were analyzed among some Helicobacter and Campylobacter species.
Materials and Methods. Twenty-one clinical isolates and strains of helicobacters and campylobacters were used in this study. Methods including polymerase chain reaction (PCR) amplification, restriction fragment-length polymorphisms (RFLPs), and hybridization were employed to carry out this work.
Results. The copA gene was highly conserved in all the H. pylori isolates tested ( Helicobacter nemestrinae and Helicobacter felis but not in Helicobacter mustelae and the Campylobacter species), whereas the sequence downstream of the copA appears to diverge among H. pylori isolates. In addition, two restriction patterns of the PCR-amplified copA fragments from seven H. pylori isolates and H. nemestrinae were identified, and the RFLP of H. nemestrinae was identical to that of one of the H. pylori isolate group.
Conclusions. The adenosine triposphatase-derived copper-transporting mechanism is employed by various H. pylori strains, H. nemestrinae, H. felis , and perhaps by other Helicobacter species. The nucleotide mutations have risen in the copA gene. It appears that there is a genetic relatedness of the copA gene to H. pylori and H. nemestrinae.     

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.     

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.     

PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori.

DNA sequence diversity among 60 independent isolates of the gastric pathogen Helicobacter pylori was assessed by testing for restriction fragment length polymorphisms (RFLPs) in several PCR-amplified gene segments. 18 Mbol and 27 HaeIII RFLPs were found in the 2.4 kb ureA-ureB (urease) segment from the 60 strains; this identified 44 separate groups, with each group containing one to four isolates. With one exception, each isolate not distinguished from the others by RFLPs in ureA-ureB was distinguished by Mbol digestion of the neighboring 1.7 kb ureC-ureD segment. The 1.5 kb flaA (flagellin) gene, which is not close to ure gene cluster, was also highly polymorphic. In contrast, isolates from initial and followup biopsies yielded identical restriction patterns in each of the three cases tested. The potential of this method for detecting population heterogeneity was tested by mixing DNAs from different strains before amplification: the arrays of restriction fragments obtained indicated co-amplification from both genomes in each of the five pairwise combinations tested. These results show that H. pylori is a very diverse species, that indicate PCR-based RFLP tests are almost as sensitive as arbitrary primer PCR (RAPD) tests, and suggest that such RFLP tests will be useful for direct analysis of H. pylori in biopsy and gastric juice specimens.     

Lack of a relationship between Lewis antigen expression and cagA, CagA, vacA and VacA status of Irish Helicobacter pylori isolates

The cagA gene, vacA gene, CagA (cytotoxin-associated gene A product) and VacA (vacuolating cytotoxin) status of a collection of Helicobacter pylori isolates from the geographically distinct Irish population was determined, the potential association of these traits with Lewis (Le) antigen expression was assessed, and the relationship between these bacterial properties and the pathology associated with H. pylori infection was evaluated. Of the 57 isolates, a higher proportion from ulcer than from non-ulcer patients expressed VacA (71% vs. 53%). H. pylori isolates which were cagA-positive were no more significantly associated with peptic ulcers than non-ulcer disease (71% vs. 67%, P = 0.775), nor were CagA-positive isolates (57% vs. 50%, P = 0.783), but 80% of the isolates from duodenal ulcer patients were cagA-positive. Thirty-seven of the 57 isolates were tested for Le antigen expression. No statistically significant relationship (P > 0.05) was found between the occurrence and level of expression of Le(x) or Le(y) and cagA, vacA, or VacA status. This lack of an association in the Irish H. pylori isolates contrasts with that previously reported for predominantly North American isolates, and may be attributable to the adaptation of H. pylori strains with differing attributes to different human populations.     

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.