Showing papers by "Martin J. Blaser published in 2003"

Traces of human migrations in Helicobacter pylori populations

Abstract: Helicobacter pylori, a chronic gastric pathogen of human beings, can be divided into seven populations and subpopulations with distinct geographical distributions. These modern populations derive their gene pools from ancestral populations that arose in Africa, Central Asia, and East Asia. Subsequent spread can be attributed to human migratory fluxes such as the prehistoric colonization of Polynesia and the Americas, the neolithic introduction of farming to Europe, the Bantu expansion within Africa, and the slave trade.

Population attributable risks of esophageal and gastric cancers.

Abstract: Background Several risk factors have been identified for esophageal adenocarcinoma, gastric cardia adenocarcinoma, esophageal squamous cell carcinoma, and noncardia gastric adenocarcinoma, but no study has comprehensively examined their contributions to the cancer burden in the general population. Herein, we estimate the population attributable risks (PARs) for various risk factors observed in a multicenter population-based case-control study. Methods We calculated PARs by using 293 patients with esophageal adenocarcinoma, 261 with gastric cardia adenocarcinoma, 221 with esophageal squamous cell carcinoma, 368 with noncardia gastric adenocarcinoma, and 695 control subjects. We included smoking for all four tumor types and Helicobacter pylori infection for noncardia gastric adenocarcinoma as established causal risk factors as well as several other factors for which causality is under evaluation. Results Ever smoking, body mass index above the lowest quartile, history of gastroesophageal reflux, and low fruit and vegetable consumption accounted for 39.7% (95% confidence interval [CI] = 25.6% to 55.8%), 41.1% (95% CI = 23.8% to 60.9%), 29.7% (95% CI = 19.5% to 42.3%), and 15.3% (95% CI = 5.8% to 34.6%) of esophageal adenocarcinomas, respectively, with a combined PAR of 78.7% (95% CI = 66.5% to 87.3%). Ever smoking and body mass index above the lowest quartile were responsible for 45.2% (95% CI = 31.3% to 59.9%) and 19.2% (95% CI = 4.9% to 52.0%) of gastric cardia adenocarcinomas, respectively, with a combined PAR of 56.2% (95% CI = 38.1% to 72.8%). Ever smoking, alcohol consumption, and low fruit and vegetable consumption accounted for 56.9% (95% CI = 36.6% to 75.1%), 72.4% (95% CI = 53.3% to 85.8%), and 28.7% (95% CI = 11.1% to 56.5%) of esophageal squamous cell carcinomas, respectively, with a combined PAR of 89.4% (95% CI = 79.1% to 95.0%). Ever smoking, history of gastric ulcers, nitrite intake above the lowest quartile, and H. pylori infection were responsible for 18.3% (95% CI = 6.5% to 41.8%), 9.7% (95% CI = 5.4% to 16.8%), 40.7% (95% CI = 23.4% to 60.7%), and 10.4% (95% CI = 0.3% to 79.6%) of noncardia gastric adenocarcinomas, respectively, with a combined PAR of 59.0% (95% CI = 16.2% to 91.4%). Conclusion In this population, a few known risk factors account for a majority of esophageal and gastric cancers. These results suggest that the incidence of these cancers may be decreased by reducing the prevalence of smoking, gastroesophageal reflux, and being overweight and by increasing the consumption of fruits and vegetables.

Evolutionary Implications of Microbial Genome Tetranucleotide Frequency Biases

Abstract: We compared nucleotide usage pattern conservation for related prokaryotes by examining the representation of DNA tetranucleotide combinations in 27 representative microbial genomes. For each of the organisms studied, tetranucleotide usage departures from expectations (TUD) were shared between related organisms using both Markov chain analysis and a zero-order Markov method. Individual strains, multiple chromosomes, plasmids, and bacteriophages share TUDs within a species. TUDs varied between coding and noncoding DNA. Grouping prokaryotes based on TUD profiles resulted in relationships with important differences from those based on 16S rRNA phylogenies, which may reflect unequal rates of evolution of nucleotide usage patterns following divergence of particular organisms from a common ancestor. By both symmetrical tree distance and likelihood analysis, phylogenetic trees based on TUD profiles demonstrate a level of congruence with 16S rRNA trees similar to that of both RpoA and RecA trees. Congruence of these trees indicates that there exists phylogenetic signal in TUD patterns, most prominent in coding region DNA. Because relationships demonstrated in TUD-based analyses utilize whole genomes, they should be considered complementary to phylogenies based on single genetic elements, such as 16S rRNA.

Long-term persistence of resistant Enterococcus species after antibiotics To eradicate Helicobacter pylori

Abstract: A common Helicobacter pylori regimen (clarithromycin, metronidazole, and omeprazole) selects for highly resistant enterococci that can persist in the gastrointestinal tract for at least 3 years.

Extensive repetitive DNA facilitates prokaryotic genome plasticity

Abstract: Prokaryotic genomes are substantially diverse, even when from closely related species, with the resulting phenotypic diversity representing a repertoire of adaptations to specific constraints. Within the microbial population, genome content may not be fixed, as changing selective forces favor particular phenotypes; however, organisms well adapted to particular niches may have evolved mechanisms to facilitate such plasticity. The highly diverse Helicobacter pylori is a model for studying genome plasticity in the colonization of individual hosts. For H. pylori, neither point mutation, nor intergenic recombination requiring the presence of multiple colonizing strains, is sufficient to fully explain the observed diversity. Here we demonstrate that H. pylori has extensive, nonrandomly distributed repetitive chromosomal sequences, and that recombination between identical repeats contributes to the variation within individual hosts. That H. pylori is representative of prokaryotes, especially those with smaller (<2 megabases) genomes, that have similarly extensive direct repeats, suggests that recombination between such direct DNA repeats is a widely conserved mechanism to promote genome diversification.

Use of an Open-Reading Frame–Specific Campylobacter jejuni DNA Microarray as a New Genotyping Tool for Studying Epidemiologically Related Isolates

Abstract: Findings from use of an open-reading frame-specific Campylobacter jejuni DNA microarray to investigate genetic diversity among clinical isolates associated with 5 independent clusters of infection were compared with data from random amplified polymeric DNA (RAPD) and Penner serotyping analyses. The DNA microarray provides a highly specific epidemiological typing tool for analysis of C. jejuni isolates and reveals both divergent and highly conserved gene classes among isolates.

Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system component.

Abstract: DNA rearrangement permits bacteria to regulate gene content and expression. In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system. Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes. In individual hosts, H. pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition. Using repetitive DNA, H. pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion.

Transient and Persistent Helicobacter pylori Colonization in Native American Children

Abstract: Helicobacter pylori is chiefly acquired in childhood, but the exact timing of acquisition is not well understood. The main goal of this study was to assess H. pylori acquisition in a pediatric population. We studied two cohorts of Native American children: a birth cohort of 50 children and 58 older children (mean age, 53 months). We measured serum immunoglobulin G (IgG), IgM, and IgA antibodies to H. pylori whole-cell antigen and IgG antibodies to CagA. Among 44 birth cohort children monitored for more than 12 months, 24 (54.5%) had seroconversions, 7 (15.9%) were transient, and 17 (38.6%) were persistent. Among the older children, 49 (84.5%) of the 58 children were monitored for 1 year; 34 (69.4%) had H. pylori antibodies at study entry. During the next year, 7 (20.6%) children seroreverted, and of 15 initially negative children, 5 (33.3%) seroconverted. In both groups, evaluation of CagA antibodies increased the sensitivity of H. pylori detection. Serum pepsinogen I (PGI) levels in H. pylori-negative children rose significantly until age 6 months and remained constant for the next 19 months. At the time of H. pylori seroconversion, PGI peaked to levels significantly higher than in the never-seroconverted (P = 0.02) and the pre-seroconverted (P = 0.03) children, but then declined to levels paralleling those of H. pylori-negative children. Thus, H. pylori acquisition, accompanied by a transient PGI increase, was frequent in this population, especially in the second and third years of life, but often was brief.

Detection of a luxS-Signaling Molecule in Bacillus anthracis

Abstract: Quorum-sensing regulation of density-dependent genes has been described for numerous bacterial species. The partially annotated genome sequence of Bacillus anthracis contains an open reading frame (BA5047) predicted to encode an ortholog of luxS, required for synthesis of the quorum-sensing signaling molecule autoinducer-2 (AI-2). To determine whether B. anthracis produces AI-2, the Vibrio harveyi luminescence bioassay was used. Cell-free conditioned media from vaccine (Sterne) strain 34F2 induced luminescence in V. harveyi reporter strain BB170, indicating its production of AI-2. Cloned BA5047, expressed in Escherichia coli DH5α cells, restored AI-2 activity to these cells. To evaluate whether BA5047 is essential for AI-2 synthesis, it was deleted through allelic exchange with marker rescue; the resulting mutant had no functional luxS activity and had reduced growth in vitro. In the wild-type strain, AI-2 activity was greatest during the exponential phase of growth. In total, these data indicate that BA5047 is a functional luxS ortholog in B. anthracis necessary for growth-phase-specific AI-2 expression. Thus, B. anthracis may utilize extracellular signaling molecules to regulate density-dependent gene expression.

Natural Variation in Populations of Persistently Colonizing Bacteria Affect Human Host Cell Phenotype

Abstract: The highly diverse bacterium Helicobacter pylori, which persistently colonizes the human stomach, provides models to study the role of genome plasticity in host adaptation. Within H. pylori populations from 2 colonized individuals, intragenomic recombination between cagA DNA repeat sequences leads to deletion or duplication of tyrosine phosphorylation sites in the CagA protein, which is injected by a type IV secretion system into host cells. Experimental coculture of gastric epithelial cells with the strains containing these naturally occurring CagA phosphorylation site variants induced markedly divergent host cell morphologic responses. Mutants were constructed in which a phosphorylation site was either added or deleted in the expressed CagA protein; coculture studies confirmed that the naturally occurring differences in CagA phosphorylation are responsible for the observed phenotypic variation. These findings indicate that within an individual host, intragenomic recombination between H. pylori repetitive DNA produces strain variants differing in their signals to host cells.

Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus

Abstract: Surface layer proteins (SLPs) are essential for induction of abortion by Campylobacter fetus subsp. fetus in experimentally challenged ewes. These proteins are encoded by multiple sap genes and vary in size and antigenicity. The role of SLP antigenic variation during experimental ovine infection was investigated. Following subcutaneous challenge, the SLPs were highly antigenic, and antibodies were detected in serum, milk, bile, and urine. Fecal anti-SLP antibodies were detected only in animals challenged orally. Ewes challenged with wild-type strain 23D with variable SLPs developed detectable circulating anti-SLP immunoglobulin G (IgG) antibodies by 2 weeks postchallenge. In contrast, ewes challenged with mutants of 23D that had fixed expression of a single SLP developed antibodies within 1 week postchallenge, suggesting that antigenic variation in SLPs may delay the host antibody response. Although not statistically significant, the data from challenge experiments in which vaccinated ewes were used suggested that SLP-expressing vaccines could protect animals from abortion and that this effect was independent of the SLP expressed, indicating involvement of conserved epitopes in the SLP. The conserved 184-amino-acid N-terminal region of the SLP, identified from previously published sequences, was epitope mapped with rabbit anti-SLP antisera by using overlapping synthetic 20-mer peptides. Two putative epitopes were identified at amino acids 81 to 110 and 141 to 160. Amino acids 81 to 100 also bound serum IgG antibodies from experimentally challenged sheep. Conserved antigenic regions of the SLP that induce protective immune responses may enable development of synthetic vaccine candidates for C. fetus subsp. fetus-associated ovine abortion.

Promoter analysis of Helicobacter pylori genes with enhanced expression at low pH

Abstract: Summary To identify Helicobacter pylori genes with expression that is enhanced under low pH conditions, we used subtractive hybridization methodology. We identified 28 acid-induced genes, of which 18 have known or putative functions. Six pairs of genes were co-transcribed. Primer extension analysis identified single or multiple transcriptional start points (tsp) for 14 of the 22 loci. Sequence analysis of the −10 regions upstream of the tsps revealed consensus motifs for multiple RNA polymerase sigma factors present in H. pylori (σ80, σ54 and σ28). No sequences resembling the −35 Escherichia coli consensus sequence (TTGACA) were present upstream of any of the genes. Both increased gene transcription and decreased mRNA decay contribute to the observed increase in H. pylori transcript abundance at acid pH. These studies document the complex response of H. pylori to environmental pH changes, and provide insight into mechanisms used for intragastric survival.

Probiotics and Prebiotics: Where Are We Going?

Abstract: “Probiotics and prebiotics have become part of the lexicon of food technologists,” writes Gerald W. Tannock, editor of the provocative new book, Probiotics and Prebiotics: Where Are We Going? Probiotics were defined by Fuller in 1989, as “live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance.” The concept of probiotics is not new, however. Approximately 100 years ago, Elie Metchnikoff, the father of immunology, investigated intestinal microbes as causative agents in aging, a process he called “autointoxication.” He believed that lactic acid–producing bacteria (such as those found in yogurt) would suppress the growth of more proteolytic, autointoxicating bacteria. Prebiotics have been defined as “nondigestible food ingredients (usually carbohydrates) that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon.” Potential prebiotics have included bifidobacteria, indigenous microbes epidemiologically associated with long life and other healthful conditions. The concept of probiotics has been developing over recent decades, and the use of prebiotics extends this idea. Although the ideas are intriguing, the central theories to be tested and the tools necessary to test them have been lacking. This book contributes substantially to addressing these difficulties. The opening chapter, by Tannock, is rich in ideas and sets the appropriate tone for the rest of the book. The other nine chapters, by 20 other authors from seven countries, address both hypotheses and specifics in state-of-the-art reviews. The central issue in this field is the following: how can the metabolic activities of the bacterial population in the colon be manipulated to promote health? Rigorous scientific exploration of this question has been limited by two factors: the colonic biota (flora) is vast and also largely undefined. In consequence, many studies, fueled by commercial self-interest, have lacked the stringency necessary for true scientific advancement. Accordingly, a substantial portion of this book discusses the methods, current or being developed, that will help address these deficiencies. Improved methods hold the promise of better defining which bacteria are present, distinguishing how much the biota varies from person to person, and measuring how well persons respond to probiotics and prebiotics. Another important issue is exploring the relationship of the microbial biota and the host, especially the healthy host; such microecologic studies are critical to understanding potential microbial contributions to disease. Again, development of standard methods would permit these assessments; without extensive cataloging, we cannot establish the baseline. A third and related issue is defining conditions that might be ameliorated by probiotic or prebiotic therapies. The authors provide a long list of such diseases, including colon cancer, inflammatory bowel disease, and some less obvious candidates such as osteoporosis and atopic diseases (for example, asthma). Some researchers have hypothesized that these latter diseases result from a childhood deprived of specific pathogens, the “hygiene hypothesis.” Use of probiotics and prebiotics has been advanced as one solution to that problem. In summary, Probiotics and Prebiotics is an important book, from which I have learned much. One deficiency, however, is the book’s remarkable absence of discussion about the role of these agents in the selection of particular bacterial populations. The focus of the book, and of the field, is largely on metabolism, but any of the anticipated therapies, especially prebiotics, will select for particular bacterial species or phenotypes. A greater focus on the biology of selection in this milieu would have been helpful. Nevertheless, this limitation does not substantially reduce the great utility of this volume to those interested in ecology, microbiology, medicine, or nutrition. This book explores a field that is out of the mainstream of human biology and medicine but deserves to be more central. For a field often marked by hyperbole because of commercially based conflicts of interest, this book is appropriately subdued and scientifically balanced. The editor and authors should be credited for their scholarly approach.

Structure and genotypic plasticity of the Campylobacter fetus sap locus

Abstract: Summary The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild-type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553–2622 bp 5′ regions, with partial sharing of 5′ and 3′ non-coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP– strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter-specific polymerase chain reactions (PCRs) and sapA homologue-specific reverse transcription (RT)-PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5′ conserved regions can involve each of the eight sapA homologues, with frequencies from 10−1 to 10−3. Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10−1−10−2 frequency, allows the formation of new sap homologues. As divergent SLP C-termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.

Evolutionary History of hrgA, Which Replaces the Restriction Gene hpyIIIR in the hpyIII Locus of Helicobacter pylori

Abstract: A recently identified Helicobacter pylori gene, hrgA, was previously reported to be present in 70 (33%) of 208 strains examined (T. Ando, T. M. Wassenaar, R. M. Peek, R. A. Aras, A. I. Tschumi, L.-J. Van Doorn, K. Kusugami, and M. J. Blaser, Cancer Res. 62:2385-2389, 2002). Sequence analysis of nine such strains indicated that in each strain hrgA replaced hpyIIIR, which encodes a restriction endonuclease and which, together with the gene for its cognate methyltransferase, constitutes the hpyIII locus. As a consequence of either the hrgA insertion or independent mutations, hpyIIIM function was lost in 11 (5%) of the 208 strains examined, rendering chromosomal DNA sensitive to MboI digestion. The evolutionary history of the locus containing either hpyIII or hrgA was reconstructed. By homologous recombination involving flanking sequences, hrgA and hpyIIIR can replace one another in the hpyIII locus, and there is simultaneous replacement of several flanking genes. These findings, combined with the hpyIM/iceA2 locus discovered previously, suggest that the two most strongly conserved methylase genes of H. pylori, hpyIIIM and hpyIM, are both preceded by alternative genes that compete for presence at their loci. Type II restriction-modification (R-M) systems are comprised of paired enzymes, a restriction endonuclease that cleaves DNA within a specific 4- to 8-bp sequence and a methyltransferase that specifically methylates the DNA within the same sequence, protecting the sequence from cleavage (4, 5, 14). Helicobacter pylori, a gram-negative bacterium that colonizes the human stomach, affects the risk of getting upper gastrointestinal tract diseases, including gastric cancer (13). H. pylori strains are highly heterogeneous in terms of the number and nature of the R-M systems that they carry (2, 10, 11, 18, 19, 21, 23). During characterization of the hpyIIIR-hpyIIIM locus in Asian and Western strains, we found numerous strains with a novel gene that we designated hrgA in place of hpyIIIR (encoding an isoschizomer of Moraxella bovis MboI). The presence of hrgA appears to have predictive value for virulence in cagA-positive strains from Asia (3). Neither gene is essential, but since no strain that lacks or contains both genes has been identified thus far, it is hypothesized that there is selection for the presence of either gene. The work described here addressed the following questions. How conserved is hrgA? How did the hrgA-hpyIII locus evolve? And are hrgA and hpyIIIR functional, and can the two genes be exchanged by natural transformation? Mutants bearing antibiotic resistance cassettes were constructed to investigate exchange of the genes between strains by natural transformation. The results indicate that exchange of these genes is possible and may involve transfer of a DNA fragment containing substantial flanking sequences, thus increasing the potential for genomic plasticity in H. pylori. MATERIALS AND METHODS

Helicobacter pylori and TT virus prevalence in Japanese children.

Abstract: The major transmission route of Helicobacter pylori, oral-oral or fecal-oral, remains to be established. TT virus (TTV), a recently discovered microbe that is prevalent in healthy persons, is believed to be mainly transmitted by nonparenteral routes. The purpose of this study was to test the hypothesis that these two microorganisms have a common mode of transmission.

An email alert system for internal medicine physicians.

Abstract: This study evaluated the effectiveness of an email-based alerting system for internal medicine house staff and faculty in geographically dispersed locales. Responses to a test alert email message were used to quantify the rapidity by which physicians read the message, and to define subgroups in which this communication modality proved most successful. The results of this study are being used to improve our preparedness for emergencies.