Occurrence of Helicobacter pylori DNA in the coastal environment of southern Italy (Straits of Messina)

AIMS: The occurrence of Helicobacter pylori in the coastal zone of the Straits of Messina (Italy) as free-living and associated with plankton was studied. METHODS AND RESULTS: Monthly sampling of seawater and plankton was carried out from April 2002 to March, 2003. All environmental samples analysed by cultural method, did not show the presence of H. pylori. The DNA extracted from all environmental samples was tested by PCR by using primers for H. pylori 16S rRNA, ureA and cagA. 16S rRNA PCR yielded amplified products of 522-bp in 15 of 36 (41.7%) of the environmental samples. By using the ureA primers to amplify the urea signal sequences, the predicted PCR products of 491-bp were obtained from eight (22.2%) of 36 environmental samples. PCR with cagA primers yielded amplified products of 349-bp in DNA extracted of seven of 36 (19.4%) of the environmental samples. When 16S rRNA, ureA and cagA amplified gene sequences were aligned with H. pylori 26695 and J99 genome sequences, we obtained a percentage of alignment over 90%. CONCLUSIONS: The detection of H. pylori genes in marine samples allows us to consider the marine environment a possible reservoir for this pathogenic bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct detection of H. pylori genes may be relevant in order to consider the marine environment as significant reservoir for this bacterium.     

Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients

AIMS: To characterize the molecular fingerprinting of Helicobacter pylori population isolated in duodenal ulcer patients treated with triple therapy. METHODS AND RESULTS: Gastric biopsy specimens from corpus and antrum, were cultured for H. pylori isolation. Helicobacter pylori eradication was evaluated after 4 and 16 weeks. DNAs of all isolates were characterized by random amplified polymorphic DNA typing and cagA gene was also detected. After the therapy, five patients harboured the microorganism at 4 weeks and two of them remained H. pylori positive at 16 weeks. The analysis of DNA fingerprinting of strains isolated from antrum and corpus of patients susceptible to treatment, showed similar patterns. Instead, when the therapy was not effective, strains isolated from sequential biopsies from initial and after 4 and 16 weeks, showed distinct fingerprintings and retained the cagA status, over time. CONCLUSIONS: The drugs used for therapy could exercise an effect in genotypical rearrangement among H. pylori cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The variableness among H. pylori strains represents a way to challenge environmental stress.     

Phylogenetic analysis of Leptospira strains of pathogenic serovars using 23S rDNA gene sequences.

The 23S ribosomal DNAs were amplified from 11 strains of Leptospira interrogans sensu lato by polymerase chain reaction (PCR) and sequenced. The PCR products of about 290-bp DNA fragments indicated more than 97% sequence similarity to each other. The phylogenetic tree based on the 23S ribosomal DNAs obtained in this study revealed that 11 strains of L. interrogans examined composed a cluster distinct to that of L. weilii and L. borgpetersenii, confirming that these strains were similar to strain Moulton of L. interrogans serovar canicola in 23S rDNA sequence.     

PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA.

PCR primers specific to the 16S ribosomal DNA (rDNA) of magnetic cocci were designed and used to amplify DNA from magnetically isolated magnetic cocci. The PCR products were subcloned by ligation into plasmid vector pCRII, and five clones containing approximately 270-bp fragments of amplified DNA were sequenced. The specific primers were also used to detect magnetic coccus 16S rDNA in environmental samples. Magnetic coccus 16S rDNA was amplified from the water column above sediment kept in an anoxic environment in the laboratory, but little was amplified from a water column kept in an oxic environment. These results suggest that magnetic cocci in the water column in an anoxic environment had migrated there from the sediment as a response to the microoxic or anoxic conditions, rather than having been present previously in a nonmagnetic form and having become magnetic due to these conditions. The specific primers were also used to detect magnetic cocci in aquatic sediment. DNA was extracted from sediment by direct lysis and purified for use as a PCR template by electrophoresis on an agarose-polyvinylpyrrolidone gel. 16S rDNA was then amplified and subcloned, and two clones were sequenced. The clones were screened for chimeric DNA by comparing sections of each with the GenBank database.     

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.     

Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species

AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA. METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized. We tested 11 type cultures of Saccharomyces, 27 different cultures of S. cerevisiae and four other ascomycetic genera. CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S. cerevisiae identification. HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.     

Evidence for S. cerevisiae fermentation in ancient wine

Saccharomyces cerevisiae is the principal yeast used in modern fermentation processes, including winemaking, breadmaking, and brewing. From residue present inside one of the earliest known wine jars from Egypt, we have extracted, amplified, and sequenced ribosomal DNA from S. cerevisiae. These results indicate that this organism was probably responsible for wine fermentation by at least 3150 B.C. This inference has major implications for the evolution of bread and beer yeasts, since it suggests that S. cerevisiae yeast, which occurs naturally on the surface bloom of grapes, was also used as an inoculum to ferment cereal products.     

Functional conservation and cell cycle localization of the Nhp2 core component of H + ACA snoRNPs in fission and budding yeasts.

We report the identification of a novel nucleolar protein from fission yeast, p17(nhp2), which is homologous to the recently identified Nhp2p core component of H+ACA snoRNPs in Saccharomyces cerevisiae. We show that the fission yeast p17(nhp2) localizes to the nucleolus in live S. cerevisiae or Schizosaccharomyces pombe cells and is functionally conserved since the fission yeast gene can complement a deletion of the NHP2 gene in budding yeast. Analysis of p17(nhp2) during the mitotic cell cycles of living fission and budding yeast cells shows that this protein, and by implication H+ACA snoRNPs, remains localized with nucleolar material during mitosis, although the gross organization of partitioning of p17(nhp2) during anaphase is different in a comparison of the two yeasts. During anaphase in S. pombe p17(nhp2) trails segregating chromatin, while in S. cerevisiae the protein segregates alongside bulk chromatin. The pattern of segregation comparing haploid and diploid S. cerevisiae cells suggests that p17(nhp2) is closely associated with the rDNA during nuclear division.     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

Prevalence of Helicobacter pylori pathogenicity-associated cagA and vacA genotypes among Pakistani dyspeptic patients

The cytotoxin-associated gene A ( cagA ), and the vacuolating cytotoxin gene A ( vacA ) products are considered the most important pathogenic determinants of Helicobacter pylori , a gram-negative bacterium causing gastrointestinal disorders such as duodenal ulcers, gastritis and mucosa-associated lymphoid tissue disease. A higher prevalence of H. pylori has been reported in various regions in the Pakistani population; however, no data are available about the virulence-associated genetic determinants. The objective of this study was to determine the prevalence of virulence-associated genes, cagA, vacA and particularly vacA allelic variants among dyspeptic patients from Pakistan. Gastric biopsy samples were obtained from 78 adult patients presenting dyspepsia symptoms. DNA was isolated and analyzed for the presence of H. pylori and its genotypes by PCR. Genus-specific PCR involving 16S rRNA gene revealed that 66 of the 78 patients were positive for H. pylori , an overall prevalence of 84.6% for this particular study. The most common vacA genotype was s1b/m2 (54.5%) followed by s1a/m1 (19.7%). cagA was positive in 24.2% of the cases and strongly associated with s1a/m1, vacA . The prevalence of virulent cagA , and vacA allelic form s1a/m1 was lower than that reported from neighboring countries.     

两株希木龙假丝酵母14α-去甲基化酶基因(ERG11)的克隆及其功能初步验证

为探讨希木龙假丝酵母(假丝酵母又称念珠菌)的耐药机制,首先克隆出两株希木龙念珠菌ERG11基因,初步验证其功能,从而为后续研究奠定基础。从美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)基因数据库中获取白念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌Erg11蛋白的保守序列,设计简并引物,聚合酶链反应(polymerase chain reaction,PCR)扩增获得希木龙念珠菌ERG11cDNA部分片段;用快速cDNA末端扩增法(rapid amplification of cDNA ends,RACE)分别扩增其5′和3′端,获得完整的ERG11编码序列(coding sequence,CDS);将CDS克隆到pYES2表达载体中,在尿嘧啶营养缺陷型酿酒酵母中过表达ERG11;用微量液基稀释法检测转化后的酿酒酵母对氟康唑的敏感性,初步验证其功能。结果显示,简并PCR扩增获得预期708bp片段,5′RACE和3′RACE分别获得385bp和1 336bp片段,经纯化、克隆、测序、比对分析,获得两株菌的ERG11CDS;比对其编码的蛋白,与其他念珠菌的Erg11蛋白高度同源;分别检测克隆了这两株希木龙念珠菌ERG11CDS表达载体的酿酒酵母对氟康唑的敏感性,发现过表达ERG11明显降低其对氟康唑的敏感性。结果提示,简并PCR联合RACE能准确有效地克隆出希木龙念珠菌ERG11基因,用pYES2酿酒酵母表达系统能初步验证其功能。     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

The size of cagA based on repeat sequence has the responsibility of the location of Helicobacter pylori in the gastric mucus and the degree of gastric mucosal inflammation

The aim of this study was to examine whether there is a relationship between cagA size of Japanese Helicobacter pylori strains and the location of these strains in the mucous layer, the degree of gastric inflammation and acid survival. Upper gastrointestinal endoscopies were done to 144 patients with dyspeptic symptom with informed consent, sera, biopsy specimens and H. pylori strains were obtained, and gastric histology and susceptibility to pH 3 of the strains were evaluated. To determine cagA size of Japanese strains using PCR, cagA of strain CPY3401 was sequenced. 74 H. pylori samples (72 cagA+) were obtained from the body and 56 samples (56 cagA +) obtained from the antrum. cagA size of 72 H. pylori strains from the body was mainly classified into 3 groups (short (48), middle (8), long (9), and others (7)) by PCR and all of that of 56 strains from the antrum except 2 was short. The size of cagA of isolated strains from the body is associated with enhanced gastritis, acid survival, and the location in the mucus. The long size cagA of which strain is acid sensitive, may be a strong selective pressure on strain that colonizes close to the host, which enhanced gastritis.     

应用ARDRA技术研究Frankia菌多样性

应用原核生物16SrDNA特异性引物rD1和fD1,对分自4个分类接种群的12株纯培养Frankia菌总DNA进行扩增,得到1条长约1500bp的扩增产物.选用2种内切酶HinfI,MspI对扩增产物进行酶切,得到稳定的酶切图谱.对图谱的分析结果表明,Frankia菌间存在极其丰富的遗传多样性.     

细菌性阴道病患者阴道分泌物16SrDNA序列分析

目的分析健康妇女及细菌性阴道病(Bacterial vaginosis,BV)患者阴道分泌物16S rDNA序列。方法提取20例健康妇女及40例BV患者阴道分泌物标本中的总DNA,针对细菌16S rDNA保守区设计通用引物进行PCR扩增、克隆、测序,将获得的16S rDNA序列与美国国立生物技术信息中心(NCBI)数据库中的发表序列进行比对,分析克隆群中细菌种类和比例。结果通过阴道分泌物16S rDNA序列分析,发现健康妇女阴道分泌物中以卷曲乳酸杆菌(Lactobacillus crispatus),惰性乳酸杆菌(Lactobacillus iners),加氏乳酸杆菌(Lactobacillus gasseri)为优势菌种,而BV患者阴道菌群种类繁多,以加德纳菌属(Gardnerella)和奇异菌属(Atopobium vaginae)克隆子占较大比例,仅4例患者可见卷曲乳酸杆菌(Lactobacillus crispatus),其他患者均未见有乳酸杆菌克隆子且奇异菌属阴道病患者甲硝唑治疗疗效较差。结论健康妇女和BV患者阴道分泌物菌群种类有较大区别,BV患者在治疗前进行16S rDNA序列分析检测有较大的临床意义。     

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.     

Differentiation between brewing and non-brewing yeasts using a combination of PCR and RFLP

In order to differentiate brewing from non-brewing yeasts, a specific polymerase chain reaction (PCR) which targeted the open reading frame of FLO1 was employed. Non-brewing yeasts include 'non-brewing Saccharomyces yeasts' and 'non-Saccharomyces yeasts'. The molecular sizes of the PCR products differed between brewing and non-brewing Saccharomyces yeasts. No FLO1 PCR products were obtained from non-Saccharomyces yeasts. Specific PCR, using oligonucleotide primers that targeted the region between the 5S and 26S rRNA genes, could be used to differentiate brewing yeasts from some non-brewing yeasts. These PCR products were digested with restriction enzymes, Scr FI and Msp I. Different restriction profiles were obtained from brewing and non-brewing yeasts which could not be differentiated using specific PCR of rDNA. These results suggest that it is possible to identify brewing from non-brewing yeasts using specific PCR of FLO1 and rDNA, and detection of restriction fragment polymorphism of rDNA.     

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

AIM: To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.     

A method for assessment of Helicobacter pylori genotype using stool specimens

Helicobacter pylori infection has been regarded as a major factor associated with the development of gastric diseases. The characterization of infected H. pylori in asymptomatic individuals is important for the prediction of the onset of such diseases. However, because of the difficulty in obtaining gastric biopsy samples, H. pylori in healthy subjects have not been studied sufficiently. Therefore, we tested a noninvasive method for the characterization of H. pylori using stool specimens. This method involved H. pylori antigen detection in stool specimens by immunochromatography; confirmation of H. pylori DNA by real-time PCR that involved the detection of its 16S rRNA gene in the DNA extracted from stool specimens; and nested PCR with genotype-specific primer pairs. A total of 80 samples obtained from asymptomatic subjects were assessed using this method. The results showed that the prevalence of H. pylori in asymptomatic Japanese individuals was 37.5%. The detection rate of the virulence factor gene cagA was 18.8%. Furthermore, all the detected cagA belonged to the highly virulent East-Asian type. These data suggest that the method used in this study is valuable for studying the molecular epidemiology of H. pylori infection in asymptomatic people.