Potential involvement of several nitroreductases in metronidazole resistance in Helicobacter pylori

Susceptibility of Helicobacter pylori to the antibiotic metronidazole has been attributed to the activity of an oxygen-insensitive NADPH-dependent nitroreductase (RdxA), with resistance to this antimicrobial arising from null mutations in rdxA. To obtain a better understanding of the factors involved in resistance, nitroreductase and metronidazole reduction activities were investigated in matched pairs of clinical and laboratory-derived sensitive and resistant H. pylori strains. Significant differences in enzyme activities were observed between sensitive and resistant strains, suggesting that metronidazole susceptibility in H. pylori was associated with more than one enzyme activity. To establish the mutations occurring in rdxA, the genes from seventeen bacterial strains, including matched pairs were sequenced. To assess whether metronidazole was responsible for inducing random mutations in this gene, the complete nucleotide sequence of gene hp0630, encoding an NAD(P)H-quinone reductase which also has NADPH-dependent nitroreductase activity, was determined in the same strains. All resistant strains showed nonsense, missense, or frameshift mutations randomly throughout rdxA. In contrast, no mutations were observed in hp0630. The results confirmed the presence of rdxA null mutations in resistant strains and suggested that other factors involved in the metabolism of metronidazole contributed to the resistant phenotype.     

Evolutionary genomics of pathogenic bacteria

Complete genome sequences are now available for multiple strains of several bacterial pathogens and comparative analysis of these sequences is providing important insights into the evolution of bacterial virulence. Recently, DNA microarray analysis of many strains of several pathogenic species has contributed to our understanding of bacterial diversity, evolution and pathogenesis. Comparative genomics has shown that pathogens such as Escherichia coli, Helicobacter pylori and Staphylococcus aureus contain extensive variation in gene content whereas Mycobacterium tuberculosis nucleotide divergence is very limited. Overall, these approaches are proving to be a powerful means of exploring bacterial diversity, and are providing an important framework for the analysis of the evolution of pathogenesis and the development of novel antimicrobial agents.     

Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.     

comH, a novel gene essential for natural transformation of Helicobacter pylori

Helicobacter pylori is naturally competent for transformation, but the DNA uptake system of this bacterium is only partially characterized, and nothing is known about the regulation of competence in H. pylori. To identify other components involved in transformation or competence regulation in this species, we screened a mutant library for competence-deficient mutants. This resulted in the identification of a novel, Helicobacter-specific competence gene (comH) whose function is essential for transformation of H. pylori with chromosomal DNA fragments as well as with plasmids. Complementation of comH mutants in trans completely restored competence. Unlike other transformation genes of H. pylori, comH does not belong to a known family of orthologous genes. Moreover, no significant homologs of comH were identified in currently available databases of bacterial genome sequences. The comH gene codes for a protein with an N-terminal leader sequence and is present in both highly competent and less-efficient transforming H. pylori strains. A comH homolog was found in Helicobacter acinonychis but not in Helicobacter felis and Helicobacter mustelae.     

Microbial genome sequencing and pathogenesis

The year 1997 saw the publication of the complete nucleotide sequence of Helicobacter pylori and Escherichia coli. It is conceivable that the complete nucleotide sequence for all the major human bacterial pathogens will be available by the end of the century. Database alignments have been used to ascribe the putative functions of open reading frames in the sequenced isolates and to define the differences between bacterial species at the nucleotide level. The most striking finding from all genome projects has been the high proportion of open reading frames that have no known function. Experimental data demonstrating the utility of the genome sequencing projects are only just beginning to emerge.     

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.     

Serum- and animal tissue-free medium for transport and growth of Helicobacter pylori

The important human gastric pathogen Helicobacter pylori is the subject of many studies, and as a consequence it is frequently being transported between national and international laboratories. Unfortunately, common bacterial growth and transport media contain serum- and animal tissue-derived materials, which carry the risk of spreading infectious diseases. We have therefore developed a growth and transport medium for H. pylori, designated 'Serum- and Animal Tissue-Free Medium' (SATFM), which does not contain serum- or animal tissue-derived components. SATFM supported growth of H. pylori isolates to similar levels as obtained with serum-supplemented Brucella medium, and SATFM with 0.5% agar supported transport and storage of H. pylori strains, as 4/4 reference strains and 11/11 clinical isolates survived for at least 3 days at room temperature in SATFM, with some strains (2/15) even surviving for up to 7 days. In conclusion, SATFM can be used both as transport and growth medium for H. pylori. The formulation of SATFM may allow its use in international transport of H. pylori, and may also allow certified use in immunization studies requiring growth of H. pylori and other bacterial pathogens.     

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

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

Regulation of the HpyII restriction–modification system of Helicobacter pylori by gene deletion and horizontal reconstitution

Helicobacter pylori, Gram-negative, curved bacteria colonizing the human stomach, possess strain-specific complements of functional restriction-modification (R-M) systems. Restriction-modification systems have been identified in most bacterial species studied and are believed to have evolved to protect the host genome from invasion by foreign DNA. The large number of R-Ms homologous to those in other bacterial species and their strain-specificity suggest that H. pylori may have horizontally acquired these genes. A type IIs restriction-modification system, hpyIIRM, was active in two out of the six H. pylori strains studied. We demonstrate now that in most strains lacking M.HpyII function, there is complete absence of the R-M system. Direct DNA repeats of 80 bp flanking the hpyIIRM system allow its deletion, resulting in an "empty-site" genotype. We show that strains possessing this empty-site genotype and strains with a full but inactive hpyIIRM can reacquire the hpyIIRM cassette and functional activity through natural transformation by DNA from the parental R-M+ strain. Identical isolates divergent for the presence of an active HpyII R-M pose different restriction barriers to transformation by foreign DNA. That H. pylori can lose HpyII R-M function through deletion or mutation, and can horizontally reacquire the hpyIIRM cassette, is, in composite, a novel mechanism for R-M regulation, supporting the general hypothesis that H. pylori populations use mutation and transformation to regulate gene function.     

利用相似致病菌中保守的致病菌特有基因预测新的毒力相关基因

目的:在致病机制相似的致病菌中寻找保守的致病菌特有基因,预测新的毒力相关基因。方法:首先选取致病机制相似的致病菌EHEC与EPEC,利用本实验室构建的包含115 152条致病菌特有基因片段的数据库进行本地Blast,得到致病菌特有基因,对致病菌特有基因在相似致病菌中的保守性进行分析,得到新的可能的毒力相关基因。结果:在6株EHEC菌中找到95条保守的致病菌特有基因,其中大部分为已知的毒力相关基因,还有许多可能的毒力相关基因;在9株相似致病菌(EHEC、EPEC)中找到10条保守的致病菌特有的蛋白基因,其中9条为已知的致病相关基因,1条为可能的致病相关基因。结论:应用本方法可以发现新的毒力基因,为后续对致病菌致病机制的实验研究奠定了基础。     

The evaluation of vacA gene alleles frequency in Helicobacter pylori strains in children and adults in Podlaskie region

The frequency of Helicobacter pylori infection in population can depend on the organism resistance, genetic condition, and bacterial strains virulence. A vacA gene, of mosaic structure, which encodes vacuolating cytotoxin is one of the known genes of H. pylori. The existence of several different genotypes of s and m regions enables the formation of numerous combinations of vacA gene genome. The studies on vacA genotype revealed that the frequency of occurrence of H. pylori containing s1 or s2, as well as m1 and m2 alleles varies in different parts of the world. The aim of the studies performed in the group of children and adults was to evaluate the prevalence of particular vacA gene alleles distribution in the population of the Podlasie province. The allele s1, which occurred in 84.3% of the examined group (86.8% in children and 81.3% in adults), turned out to be the most frequently observed of the signal encoding region. Statistically significant differences in s1 and s2 alleles distribution in relation to a dwelling place were not detected. The allele m2 (42.1% in children and 59% in adults) was the allele of midregion, most frequently occurring in our studies. The allele m2 was observed more often in H. pylori strains in the inhabitants from the urban areas (data statistically significant).     

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.     

Conservation and microdiversity of the phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries

Phospholipase activity is important in bacterial pathogenicity and could contribute to the pathogenic role of Helicobacter pylori by degradation of the gastric mucus, and in maintaining long-term colonisation. Our aim was to determine the degree of variation in the phospholipase A gene (pldA) of H. pylori from different geographic locations, and to investigate links between pldA genotype and clinical disease severity, as well as with variation in cagA status and vacA genotypes. PCR-restriction fragment length polymorphism (RFLP) analysis with MboI and HaeIII was used to study 124 isolates from 10 countries that included the two genome-sequenced strains (26695 and J99), as well as Tx30a and NCTC 11637 (type strain). The 925-bp pldA fragment was amplified with a frequency of 90%. The presence of pldA was confirmed in the other strains using an alternative forward primer. Isolates were distinguished by PCR-RFLP analysis with 10 MboI and four HaeIII restriction patterns that combined to give 25 distinct pldA RFLP types. The pldA M2H2 strain genotype was most common (20%) in the UK but similar strains came from several other countries. Microdiversity was evident in pldA sequences of strains representing different RFLP types, and five M2H2 strains each had a distinct pldA sequence type. Intragenic variation was independent of gastric disease severity as well as strain cagA status and vacA genotype, with the exception of eight geographically diverse strains all with the pldA M4H3/cagA+/vacA s1m1 genotype predominantly from peptic ulcer patients. The study indicated a spectrum of genotypic variants and was supportive of a pldA function in H. pylori colonisation and persistence rather than in chronicity of infection.     

Functional organization and insertion specificity of IS607, a chimeric element of Helicobacter pylori

A search by subtractive hybridization for sequences present in only certain strains of Helicobacter pylori led to the discovery of a 2-kb transposable element to be called IS607, which further PCR and hybridization tests indicated was present in about one-fifth of H. pylori strains worldwide. IS607 contained two open reading frames (ORFs) of possibly different phylogenetic origin. One ORF (orfB) exhibited protein-level homology to one of two putative transposase genes found in several other chimeric elements including IS605 (also of H. pylori) and IS1535 (of Mycobacterium tuberculosis). The second IS607 gene (orfA) was unrelated to the second gene of IS605 and might possibly be chimeric itself: it exhibited protein-level homology to merR bacterial regulatory genes in the first approximately 50 codons and homology to the second gene of IS1535 (annotated as "resolvase," apparently due to a weak short recombinase motif) in the remaining three-fourths of its length. IS607 was found to transpose in Escherichia coli, and analyses of sequences of IS607-target DNA junctions in H. pylori and E. coli indicated that it inserted either next to or between adjacent GG nucleotides, and generated either a 2-bp or a 0-bp target sequence duplication, respectively. Mutational tests showed that its transposition in E. coli required orfA but not orfB, suggesting that OrfA protein may represent a new, previously unrecognized, family of bacterial transposases.     

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.     

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。     

Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India

The genotypes of 78 strains of Helicobacter pylori from Calcutta, India (55 from ulcer patients and 23 from more-benign infections), were studied, with a focus on putative virulence genes and neutral DNA markers that were likely to be phylogenetically informative. PCR tests indicated that 80 to 90% of Calcutta strains carried the cag pathogenicity island (PAI) and potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA), independent of disease status. This was higher than in the West (where cag PAI(+) vacAs1 genotypes are disease associated) but lower than in east Asia. The iceA2 gene was weakly disease associated in Calcutta, whereas in the West the alternative but unrelated iceA1 gene at the same locus is weakly disease associated. DNA sequence motifs of vacAm1 (middle region) alleles formed a cluster that was distinct from those of east Asia and the West, whereas the cagA sequences of Calcutta and Western strains were closely related. An internal deletion found in 20% of Calcutta iceA1 genes was not seen in any of approximately 200 strains studied from other geographic regions and thus seemed to be unique to this H. pylori population. Two mobile DNAs that were rare in east Asian strains were also common in Calcutta. About 90% of Calcutta strains were metronidazole resistant. These findings support the idea that H. pylori gene pools differ regionally and emphasize the potential importance of studies of Indian and other non-Western H. pylori populations in developing a global understanding of this gastric pathogen and associated disease.     

Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)]

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.