Different Helicobacter pylori strains colonize the antral and duodenal mucosa of duodenal ulcer patients

Background. We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O –side chains of LPS.
Materials and Methods. Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies.
Results. Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient.
Conclusion. The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

两种PCR方法对木耳属菌株的遗传多样性评价

应用ERIC和RAPD两种PCR方法对木耳属3种29个菌株进行遗传鉴别,其中ERIC方法是首次运用于食用菌的研究领域。在相似系数75％的水平上,ERIC和RAPD分别将供试菌株分为9组和6组。由ERIC所得的聚类图可将黑木耳和毛木耳两个种区分开,而RAPD则不能完全区分两个种,但两种方法得到了一个相似的结果,即琥珀木耳与黑木耳的亲缘关系极其相近。Southern杂交实验进一步证明了ERIC所得到的29个菌株的同源性关系。分析表明,RAPD方法主要在种的水平上进行鉴别,而ERIC则可以在菌株水平上进行鉴别,结果与菌株栽培性状更为一致。研究结果表明ERIC-PCR是一种比RAPD更快捷可靠的分子标记方法,可以替代RAPD应用于木耳属的遗传多样性及遗传分类的研究。     

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.     

Detection of peptidoglycan and β-glucan with silkworm larvae plasma test

Abstract Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.     

副溶血性弧菌肠细菌基因间共有重复序列-PCR分子分型和耐药性、血清型研究

目的对副溶血性弧菌进行ERIC-PCR分子分型、耐药性和血清型相关性研究。方法肠细菌基因间共有重复序列（ERIC）为引物,对40株菌株基因组DNA进行扩增,得到DNA指纹图谱,并利用SPSS13.0统计软件对DNA扩增图谱进行分析,做出聚类图从而分型,并与菌株血清型、耐药性比较分析。结果40株菌用ERIC-PCR分为5个型,分辨力指数为（DI）为0.5;血清分型分为4个型;对8种抗生素中的萘啶酸、头孢噻亏、头孢西丁出现了不同程度的耐药。耐药菌株均出现在ERIC-PCR方法分型A型和血清分型O3型中。结论研究显示ERIC-PCR方法可以用于该菌分型分析,具有较好的分型能力。血清分型与ERIC-PCR方法分型一致。通过ERIC-PCR分型的树状图和血清分型结果推断,血清型O3群菌株很可能起源于血清型O1群菌株,血清型O3群和O1群密切相关。     

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

Molecular analysis of Helicobacter pylori populations in antral biopsies from individual patients using randomly amplified polymorphic DNA (RAPD) fingerprinting

Abstract In the present study, randomly amplified polymorphic DNA (RAPD) fingerprinting has been used to analyse multiple single colony isolates of Helicobacter pylori from antral biopsies in an attempt to ascertain whether or not multiple strains are present in individual patients using single biopsy samples. The RAPD fingerprints derived from single colonies obtained from the same biopsy specimen were in all cases indistinguishable. The previously noted heterogeneity between H. pylori strains from different individuals was confirmed. RAPD fingerprinting, combined with a simple method of template preparation, was shown to be an excellent method for H. pylori strain differentiation. The results of this study indicate that the H. pylori population is homogeneous in individual patients at a single gastric site.     

Molecular epidemiology of Legionella pneumophilia serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting

Abstract Hybridization with acetylaminofluorene-labelled 16 + 23 S rRNA from Escherichia coli was used to detect DNA polymorphism among Legionella pneumophila serogroup 1 isolates. Isolates from unrelated patients showed at least four different rRNA restriction patterns, whereas those from related patients showed a single pattern. Amplification of genomic regions with an arbitrary primer by polymerase chain reaction was used to further analyze the isolates. Related isolates showed closely related patterns while unrelated isolates displayed six distinct patterns. We could differentiate the majority of unrelated isolates with the combination of the patterns obtained with the ribotyping and the PCR fingerprinting, while strains from the same outbreak remained highly related. The ribotyping and the PCR fingerprinting are proposed as useful and easy to perform epidemiological markers of L. pneumophila serogroup 1 infection.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling

Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.     

Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria

Repetitive element sequencebased PCR (rep-PCR) was used to generate DNA fingerprints for Listeria spp. Two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2) used in respectively REP-and ERIC-PCR revealed that bacteria of the genus Listeria possess short repetitive extragenic palindromic elements and enterobacterial repetitive intergenic consensus sequences. Specific band profiles obtained by ERIC-PCR enabled the identification of Listeria species. With both REP-and ERIC-PCR the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3b and 4b could be clearly distinguished from each other. Within the serotype 1/2a, REP-PCR showed a higher discriminative potential than ERIC-PCR and a comparable discriminative potential as RAPD combining 3-4 primers.     

新疆有毒火欣麻内生菌的分离及AP-PCR与ERIC-PCR分析

目的分离火欣麻的内生菌,探讨ERIC-PCR引物在植物内生细菌分型中的优化及其与AP-PCR的结合在植物内生菌分型中的应用价值。方法通过改进的碾碎法对火欣麻的内生菌进行分离,依据形态学与生理生化特性同时对比AP-PCR和ERIC-PCR图谱,对所分离菌进行分类鉴定。结果所筛选的12株内生细菌,2株为奈氏菌属,1株为葡萄球菌属,1株为链球菌属,3株为芽胞杆菌属,2株为短杆菌属,3株为肠杆菌属。优化引物后的ERIC-PCR条带稳定、丰富、清晰。结论ERIC-PCR与AP-PCR结合使用分型效果明显增强。     

Mixed Infection with cagA-Positive and cagA-Negative Strains of Helicobacter pylori

Background Helicobacter pylori infection has been implicated strongly in the pathogenesis of gastritis, peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma, but the reasons for these widely different clinical outcomes are unknown. The aim of this study was to determine whether these differences could be due in part to mixed infection in the same individual, with bacteria having differences in pathogenic factors associated with ulcers.
Materials and Methods. The cagA gene of H. pylori was used to test for mixed infection because it is present in only some strains, and its presence has been associated with ulcers. Polymerase chain reaction (PCR) assays for the cagA gene were applied to H. pylori culture isolates and endoscopic gastric aspirates. Individual bacterial clones were tested for genetic similarity by random primer amplification and restriction endonuclease digestion of urease gene PCR products.
Results. The majority of H. pylori -positive patients had strongly cagA -positive culture isolates and endoscopic samples (62.5% and 69.6%, respectively). However, many of these patients had evidence of mixed infection with cagA negative and cagA positive strais in cultures isolates and endoscopic samples (25% and 17.4%, respectively). Mixed infection was found to be due to genetically unrelated strains in two patients in whom genetic analysis was performed.
Conclusion. Mixed infection with differences in substrain pathogenic factors might occur in H. pylori infection and might contribute to differences in clinical outcome.     

新疆有毒焮麻内生菌的分离及AP-PCR与ERIC-PCR分析

目的分离焮麻的内生菌,探讨ERIC-PCR引物在植物内生细菌分型中的优化及其与AP-PCR的结合在植物内生菌分型中的应用价值。方法通过改进的碾碎法对焮麻的内生菌进行分离,依据形态学与生理生化特性同时对比AP-PCR和ERIC-PCR图谱,对所分离菌进行分类鉴定。结果所筛选的12株内生细菌,2株为奈氏菌属,1株为葡萄球菌属,1株为链球菌属,3株为芽胞杆菌属,2株为短杆菌属,3株为肠杆菌属。优化引物后的ERIC-PCR条带稳定、丰富、清晰。结论ERIC-PCR与AP-PCR结合使用分型效果明显增强。     

Comparison of five molecular subtyping methods for differentiation of Salmonella Kentucky isolates in Tunisia

Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16 %) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1 %) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.     

First evidence for a restriction-modification system in Leptospira sp

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.