Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods.
Materials and methods:  Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH).
Results:  In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis /flexispira taxon 8, H. cinaedi , and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH.
Conclusions:  This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.     

胆型螺旋杆菌(Helicobacter bilis)的分离、鉴定与序列分析

鉴定分离到的微需氧菌为螺旋杆菌,并对该菌进行分型。小鼠皮下或肌肉注射地塞米松使其免疫抑制,取小鼠肠内容物培养,对分离到的细菌,经油镜,电镜观察,然后提取细菌DNA,用根据螺旋杆菌（Helicobacter sp.)rRNA保守区设计的引物P7/P8进行扩增,并对扩增产物分别用MboI,HhaI,XspI内切酶酶切,酶切产物用10%PAGE分析。再用根据螺旋杆菌胆型（H.bilis)rRNA设计特异引物P7/Pb扩增,将扩增产物测序分析。最后。将该细菌在Scid小鼠上作动物感染。细菌在油镜下呈鸟翼状,电镜下观察到双极鞭毛。无周质纤毛。引物P7/P8扩增出374bp的特异带,此片段能分别被MboI,HhaI,Zsp内切酶酶切,引物P7/Pb扩增出364bp的条带,测得的DNA序列中存在MboI,HhaI,Xsp内切酶酶切。引物P7/Pb扩增出364bp的条带,测得的DNA序列中存在MboI,HhaI,XspI的内切位点,与文献中H.bilis序列比较,同源性为97.5%。动物感染试验符合Koch准则。分离到的细菌确为胆型螺旋杆菌。     

Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections

Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals.
Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus , and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns.
Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H . bilis , in 91% for H. hepaticus, and in 82% for H. ganmani . The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium .
Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.     

Analysis of polymorphism of 18S rRNA gene in Wuchereria bancrofti microfilariae

The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes Ssp I, Msp I and Hha I showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any difference in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf isolates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.     

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.     

Evaluation of a molecular identification scheme based on 23S rRNA gene polymorphisms for differentiating canine and feline gastric Helicobacter spp

A scheme for the rapid identification of Helicobacter spp. using restriction fragment length polymorphism digestion profiles of PCR amplified 23S rRNA genes is described. The efficacy of this scheme for speciation of the closely related gastric species H. felis, H. bizzozeronii and H. salomonis was evaluated. It was difficult to distinguish between some RFLP profiles obtained and often, more than one profile was seen with each species examined. Some evidence was found that the 23S rRNA gene copies of these species may not be identical. Moreover, the identification scheme was ineffective in discriminating these species from each other, although they could be differentiated, as a group, from other Helicobacter spp. The results indicate that this scheme should be carefully evaluated with a number of isolates if it is to be applied to additional, highly related Helicobacter spp.     

A novel enterohepatic Helicobacter species 'Helicobacter mastomyrinus' isolated from the liver and intestine of rodents

BACKGROUND: A number of novel Helicobacter species have been isolated from both animals and humans. Many of these helicobacters colonize the lower gastrointestinal tract and hepatobiliary tract and are associated with diseases. METHODS: A spiral-shaped bacterium, with bipolar single-sheathed flagella, was isolated from the liver and cecum of mastomys (the African rodent, Mastomys natalenis), from the feces and ceca of normal mice, and also from the cecum of a mouse with proctitis. 16S ribosomal RNA gene sequence analysis, restriction fragment length polymorphism (RFLP) and fluorophore-enhanced repetitive element polymerase chain reaction (FERP or rep-PCR) analysis were used to classify the organism. RESULTS: The bacterium grew at 37 and 42 degrees C under microaerobic conditions, rapidly hydrolyzed urea, and was catalase and oxidase positive. It did not reduce nitrate to nitrite, and was resistant to cephalothin and nalidixic acid. Like many other enterohepatic Helicobacter species, this organism expressed cytolethal distending toxin and causes cell distention. CONCLUSIONS: The organism was classified as a novel Helicobacter species for which we propose the name 'Helicobacter mastomyrinus'. Although 'H. mastomyrinus', like Helicobacter hepaticus and Helicobacter bilis, colonizes the liver of rodents, the pathogenic potential of this novel helicobacter is unknown.     

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various restriction enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave species-specific RFLP profiles on simplified agarose gel electrophoresis. 16S rDNA gragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pair of methanogen-specific primers and cloned directly by the T-A cloning technique. The cloned 16S rDNAs from recombinants were reamplified by PCR, and RFLP pattern analysis was performed following digestion with HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol can be completed within one day, provided that sufficient amounts of test cells are available, and has great promise as a simple and rapid technique for identification of methanogens. A combined method consisting of PCR amplification, direc cloning with T vectors, and RFLP analysis of 16S rDNA is also useful for rapid estimation of the mixed population structure of methanogens without the need for cultivation and isolation.     

Development of PCR assay to identify Pseudomonas fluorescens and its biotype

The paper provides a simple protocol that uses the polymerase chain reaction to amplify a specific portion of the 16S gene, allowing the recognition of Pseudomonas fluorescens from other group I Pseudomonas. The amplified DNA patterns of 16S rRNA and ITS1, from the restriction fragment length polymorphisms VspI, HaeIII and TaqI digestion, produced band patterns that distinguished the biotypes of Ps. fluorescens. In addition to distinguishing the biotypes C and 3 we used a phenotypical method for levan production.     

Helicobacter bilis-associated hepatitis in outbred mice

Although Helicobacter bilis infects mice worldwide, it is not known whether H. bilis causes enterohepatic disease in outbred Swiss Webster (SW) mice. Intestinal and liver specimens from four groups of 39 SW mice, five of which were treated with creatine in the drinking water, were obtained for culture for the presence of H. bilis and were analyzed as to whether infection status was associated with H. bilis seroconversion and/or hepatitis. Helicobacter bilis was isolated from the colon of all 27 mice of groups I-III, but only from the liver of one 12- to 13-month-old female mouse. Ten of 27 livers were H. bilis-positive based on polymerase chain reaction (PCR) analysis; 8 of 10 (80%) of the positive results were for older mice. Results of PCR analysis for H. bilis were negative, and H. bilis was not isolated from 12 control mice (group IV). Irrespective of treatment group or controls, severity of histologic lobular and periportal chronic inflammatory lesions in the liver of H. bilis-infected outbred mice ranged from minimally to moderately severe. Helicobacter bilis infection was associated with increased portal inflammation in group III mice, compared with age-matched, helicobacter-free, group IV mice (P < 0.03). A comparison of potential sex effects in group III mice indicated that H. bilis-infected female mice developed more severe portal inflammation than did H. bilis-infected male mice (P < 0.01). On the basis of results of an ELISA, 8 of 11, 6- to 8-month-old H. bilis-infected mice of group III seroconverted to H. bilis outer membrane antigen. Helicobacter bilis infection is associated with hepatitis in SW mice and can confound experimental results.     

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.     

16S rDNA_RFLP分析繁茂膜海绵可培养放线菌的多样性

海绵是迄今为止已知海洋天然产物的最大来源。由于海绵的底栖过滤性摄食和消化选择性等生理特性,其体内蕴藏了丰富的微生物种群。近几年来,发现越来越多的海绵微生物产生很强的生物活性物质,有些并被证明是海绵天然产物的真正生产者。对大连海域繁茂膜海绵中的放线菌进行分离,对于得到的菌株进行16SrDNA扩增和RFLP及测序分析。研究表明海绵中蕴含着丰富的放线菌资源。其中包含链霉菌(Streptomycetes),拟诺卡氏菌(Nocardiopsis),假诺卡氏菌(Pseudonocardia),诺卡氏菌(Nocardia),小单孢菌(Micromonospora),红球菌(Rhodococcus),异壁放线菌(Actinoalloteichus)等属。利用限制性内切酶HhaⅠ对16SrDNA进行的RFLP分析能够对海绵中放线菌的16SrDNA多样性进行有效的分析,结果达到属,部分到种的级别。揭示了繁茂膜海绵中蕴藏着丰富的放线菌资源。     

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes

The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.     

Use of the P167 recombinant antigen for serodiagnosis of Helicobacter bilis

Helicobacter bilis is widespread among research mouse colonies. Serodiagnosis of Helicobacter infections involves use of bacterial lysates or membrane antigen preparations that lack specificity, necessitating the need to identify a specific and sensitive antigen. A previously reported recombinant protein (P167) was evaluated for use as an H. bilis-specific antigen for serologic testing. Seventy-six mice naturally infected with Helicobacter spp. were identified from commercially bred or sentinel mice. Infection was confirmed and speciated by use of cecal specimen culture and fecal polymerase chain reaction (PCR) analysis, followed by restriction enzyme digest of the amplicon. Forty-one mice were determined to be monoinfected with H. bilis, 27 mice were determined to be monoinfected with H. hepaticus, and eight mice were infected with another species of Helicobacter. Serum was diluted 1:100 to evaluate the immunoreactivity to enzyme-linked immunosorbent assay preparations of H. bilis membrane extract and the immunodominant C and D fragments of the p167 gene. The sensitivity was greatest for the membrane extract preparation (76%), whereas sensitivity to the P167C and D recombinants was lower (62 and 51%, respectively). However, the specificity of the membrane extract preparation was low (87%), compared with the much improved specificity of the recombinant P167C and D fragments (96 and 96%, respectively). These findings suggest that the recombinant P167C and D fragments of the p167 gene product from H. bilis can be used as specific reagents in the serodiagnosis of H. bilis infection in mice.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

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.