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The Gram-negative bacterium

Helicobacter pylori is a common component of the human stomach microbiota, possibly dating back to the speciation of Homo sapiens. A history of pathogen evolution in allopatry has led to the development of genetically distinct H. pylori subpopulations, associated with different human populations, and more recent admixture among H. pylori subpopulations can provide information about human migrations. However, little is known about the degree to which some H. pylori genes are conserved in the face of admixture, potentially indicating host adaptation, or how virulence genes spread among different populations. We analyzed H. pylori genomes from 14 countries in the Americas, strains from the Iberian Peninsula, and public genomes from Europe, Africa, and Asia, to investigate how admixture varies across different regions and gene families. Whole-genome analyses of 723 H. pylori strains from around the world showed evidence of frequent admixture in the American strains with a complex mosaic of contributions from H. pylori populations originating in the Americas as well as other continents. Despite the complex admixture, distinctive genomic fingerprints were identified for each region, revealing novel American H. pylori subpopulations. A pan-genome Fst analysis showed that variation in virulence genes had the strongest fixation in America, compared with non-American populations, and that much of the variation constituted non-synonymous substitutions in functional domains. Network analyses suggest that these virulence genes have followed unique evolutionary paths in the American populations, spreading into different genetic backgrounds, potentially contributing to the high risk of gastric cancer in the region., This work was supported by “Consejo Nacional de Ciencia y Tecnología” (CONACYT; no. 576951) grant to ZYM-R, and by Magnus Bergvalls Foundation and Swedish Society for Medical Research (SSMF) to KT. SKS and BP are funded by the Medical Research Council (MR/L015080/1). Supported in part by the US National Cancer Institute National Cancer Institute to DRM (P01 CA028842, R01 CA190612, K07 CA125588, P30 CA068485). We are very grateful to Daniel Falush (Unit of Statistical Genetics of Bacteria, Institut Pasteur of Shanghai) for his critical input during the analysis of the presented data. We acknowledge the Aklavik H. pylori Project Planning Committee (Northwest Territories, Canada) for providing input on our presentation of this analysis.

Erratum in: Nat Commun. 2023 Mar 20;14(1):1539. doi: 10.1038/s41467-023-37302-5. Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori fromEurope and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks bymigration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck. This work was supported by Sequencing Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (221S0002, 18KK0266, 19H03473, 21H00346 and 22H02871) to Y.Y. F.F.V. is financed by FCT through Assistant Researcher grant CEECIND/03023/2017 and a project grant PTDC/BTM-TEC/3238/ 2020. I.K. studentship was funded by the National Strategic Reference Framework Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020, project No. MIS5002486) and sequencing of strains was supported by the InfeNeutra Project (NSRF 2007-2013, project no. MIS450598) of the Ministry of Culture and Edu- cation, Greece. K.T. and the sequencing of KI isolates was supported by Erik Philip-Sörensen Foundation grant G2016-08, and Swedish Society for Medical research (SSMF). All primary bioinformatics and parts of the comparative genomics were performed on resources provided by Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under projects snic2018-8-24 and uppstore2017270. Work by S.S. was supported by the German Research Foundation (DFG, project number 158 989 968–SFB 900/A1) and by the Bavarian Ministry of Sci- ence and the Arts in the framework of the Bavarian Research Network “New Strategies Against Multi-Resistant Pathogens by Means of Digital Networking—bayresq.net”. D.F. was supported by Shanghai Municipal Science and Technology Major Project No. 2019SHZDZX02. info:eu-repo/semantics/publishedVersion

All genomes mutate but the consequences of the resulting deleterious mutational load are poorly understood. Helicobacter pylori lives in the human stomach, has a higher mutation rate than most bacteria and has accompanied anatomically modern humans in migrations including the out-of-Africa expansion more than 50,000 years ago. H. pylori from East Asia have accumulated at least 500 more non-synonymous mutations than African strains, which we propose is due to reduced efficacy of selection during the out-of-Africa bottleneck. H. pylori from Europe and the Middle East trace a substantially higher fraction of ancestry from modern African populations than the humans that carry them, which we find is due to at least three separate admixture events. African ancestry is elevated at positions in the genome where non-synonymous mutations are at high frequency in Asia. We propose that this is due to replacement of deleterious mutations that accumulated during the bottleneck, with the high overall African ancestry proportion due to clonal expansion of strains of African origin. We use simulations to show that a Muller9s ratchet like effect can lead to long-term segregation of deleterious mutations within bacterial populations after a bottleneck, despite high rates of homologous recombination, but that population fitness can be restored by migration of small numbers of bacteria from non-bottlenecked populations. Our results demonstrate that population bottlenecks can have long-term genomic and demographic consequences, even in species with enormous population sizes.

Here, we present the draft genome sequences of 29 Colombian Helicobacter pylori strains. These strains were isolated in Bogotá, Colombia, from patients diagnosed with chronic gastritis. The genomic characterization of these strains will provide more information on the genetic composition of H. pylori strains from Colombia., We thank the entities that financially supported the development of this work. A.B.M. is a recipient of a scholarship from the Centro de Estudios Interdisciplinarios Básicos y Aplicados (CEIBA) Foundation, Colombia; and A.B.M., C.A., J.S., and A.A.T.-R. are recipients of a project grant (120380763025/2018) from MinCiencias, Colombia. The work is partially supported by Research Vice-Rectory, Pontificia Universidad Javeriana (PPTA_7676) and F.P.I.T. BanRepCultural (project 3956). F.F.V. is financed by national funds from the Fundação para a Ciência e a Tecnologia (FCT) through an assistant researcher grant (CEECIND/03023/2017), a project grant (PTDC/BTM-SAL/28978/2017), and projects UIDB/04138/2020 and UIDP/04138/2020; these funds partially supported this work. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Background Helicobacter pylori is a human gastric carcinogen that is highly prevalent in Latin American. The prophages of H. pylori show a structured population and contribute to the diversity of this bacterium. However, H. pylori prophages present in American strains have not been described to date. In this study, we identified, characterized, and present the phylogenetic analysis of the prophages present in Colombian H. pylori strains. Methods To characterize Colombian H. pylori strains and their prophages, a Multilocus Sequences Typing (MLST) and a Prophage Sequences Typing (PST), using the integrase and holin genes, were performed. Furthermore, five Colombian H. pylori had their full genome sequenced, and six Colombian H.pylori retrieved from databases, allowing to determine the prophage complete genome and insertion site. Results The integrase gene frequency was 12.6% (27/213), while both integrase and holin genes were present in 4.2% (9/213) of the samples analyzed. The PST analysis showed that Colombian prophages belong to different populations, including hpSWEurope, hpNEurope, hpAfrica1, and a new population, named hpColombia. The MLST analysis classified most of the Colombia strains in the hpEurope population. Conclusions The new H. pylori prophage population revealed that Colombian prophages follow a unique evolutionary trajectory, contributing to bacterial diversity. The global H. pylori prophage phylogeny highlighted five phylogenetic groups, one more than previously reported. After the arrival of Europeans, the Colombian H. pylori bacteria and their prophages formed an independent evolutionary line to adapt to the new environment and new human hosts.

The bacterium Helicobacter pylori colonize the stomach in approximately half of the world’s population. Infection with this bacterium is associated with gastritis, peptic ulcer, adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Besides being a pathogen with worldwide prevalence, H. pylori show increasingly high antibiotic resistance rates, making the development of new therapeutic strategies against this bacterium challenging. Furthermore, H. pylori is a genetically diverse bacterium, which may be influenced by the presence of mobile genomic elements, including prophages. In this review, we analyze these issues and summarize various reports and findings related to phages and H. pylori, discussing the relationship between the presence of these elements and the genomic diversity, virulence, and fitness of this bacterium. We also analyze the state of the knowledge on the potential utility of bacteriophages as a therapeutic strategy for H. pylori., This work was supported by the following sources: AM is a recipient of a scholarship from the Centro de Estudios Interdisciplinarios Básicos y Aplicados (CEIBA Foundation), Colombia. AM, JS, and AT are recipients of a project grant (120380763025/2018) from the Departamento Administrativo de Ciencia, Tecnología e Innovación de Colombia (Colciencias) and a project grant (PPTA_7676) from Research Vice-Rectory, Pontificia Universidad Javeriana. FV is the recipient of a project grant (PTDC/BTM-SAL/28978/2017) from the Fundação para a Ciência e a Tecnologia (FCT).

For a long time Helicobacter pylori infections have been treated using the macrolide antibiotic, clarithromycin. Clarithromycin resistance is increasing worldwide and is the most common cause of H. pylori treatment failure. Here we review the mechanisms of antibiotic resistance to clarithromycin, detailing the individual and combinations of point mutations found in the 23S rRNA gene associated with resistance. Additionally, we consider the methods used to detect clarithromycin resistance, emphasizing the use of high-throughput next-generation sequencing methods, which were applied to 17 newly sequenced pairs of H. pylori strains isolated from the antrum and corpus of a recent colonized paediatric population. This set of isolates was composed of six pairs of resistant strains whose phenotype was associated with two point mutations found in the 23S rRNA gene: A2142C and A2143G. Other point mutations were found simultaneously in the same gene, but, according to our results, it is unlikely that they contribute to resistance. Further, among susceptible isolates, genomic variations compatible with mutations previously associated with clarithromycin resistance were detected. Exposure to clarithromycin may select low-frequency variants, resulting in a progressive increase in the resistance rate due to selection pressure., F. F. V. is the recipient of a project grant (PTDC/BTM-SAL/28978/2017) from the Fundação para a Ciência e a Tecnologia (FCT), which supported this work. J. V.’s research group was financed by New England Biolabs, Inc. (USA).

Bacteriophages are the most prevalent biological entities impacting on the ecosystem and are characterized by their extensive diversity. However, there are two aspects of phages that have remained largely unexplored: genetic flux by recombination between phage populations and characterization of specific phages in terms of the pan-genome. Here, we examined the recombination and pan-genome in Helicobacter pylori prophages at both the genome and gene level. In the genome-level analysis, we applied, for the first time, chromosome painting and fineSTRUCTURE algorithms to a phage species, and showed novel trends in inter-population genetic flux. Notably, hpEastAsia is a phage population that imported a higher proportion of DNA fragments from other phages, whereas the hpSWEurope phages showed weaker signatures of inter-population recombination, suggesting genetic isolation. The gene-level analysis showed that, after parameter tuning of the prokaryote pan-genome analysis program, H. pylori phages have a pan-genome consisting of 75 genes and a soft-core genome of 10 genes, which includes genes involved in the lytic and lysogenic life cycles. Quantitative analysis of recombination events of the soft-core genes showed no substantial variation in the intensity of recombination across the genes, but rather equally frequent recombination among housekeeping genes that were previously reported to be less prone to recombination. The signature of frequent recombination appears to reflect the host–phage evolutionary arms race, either by contributing to escape from bacterial immunity or by protecting the host by producing defective phages.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs, babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P &lt, 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

The study of the gastric pathogen Helicobacter pylori brought us interesting data on the history of mankind. Based on multi-locus sequence typing, it was possible to trace the migration of Homo sapiens all around the world, and to infer the time when he went Out of Africa. Beside these phylogeographic aspects, paleomicrobiology gave us important information on life in the Neolithic period, following the discovery of Ötzi, the Iceman, who was living in the Tyrolean Alps 5200 years ago, and from whom a Helicobacter pylori genome was sequenced. This review presents the data accumulated in these different fields.

The review uses the Helicobacter pylori, the gastric bacterium that colonizes the human stomach, to address how to obtain information from bacterial genomes about prophage biology. In a time of continuous growing number of genomes available, this review provides tools to explore genomes for prophage presence, or other mobile genetic elements and virulence factors. The review starts by covering the genetic diversity of H. pylori and then moves to the biologic basis and the bioinformatics approaches used for studding the H. pylori phage biology from their genomes and how this is related with the bacterial population structure. Aspects concerning H. pylori prophage biology, evolution and phylogeography are discussed.

Background Helicobacter pylori infection is common in Algeria, but there are few data on the characterization of isolated strains. The aim of this study was to update data on the prevalence of H. pylori in patients submitted to endoscopy, antibiotic resistance, and phylogeography of H. pylori strains isolated in Algiers. Materials and Methods This is a prospective study carried out between November 2015 and August 2016. The culture of H. pylori was performed on antral and fundic gastric biopsies of adult patients from 3 hospitals. A real-time PCR using the fluorescence resonance energy transfer (FRET) principle for the detection of H. pylori followed by a melting curve analysis for the detection of mutations associated with resistance to clarithromycin was applied. Differentiation between antral and fundic isolates of the same patient was also determined by RAPD, and an MLST typing was performed for characterization of the phylogeographic group of H. pylori. Results By real-time PCR, the prevalence of H. pylori infection among the 147 patients included was 57%. Culture was positive in only 29% of the cases. Twenty-seven percent of patients had received H. pylori eradication treatment. The primary and secondary resistance rates to clarithromycin were 23% and 36%, respectively, and to metronidazole, 45% and 71%, respectively. Only one isolate was resistant to levofloxacin, and no resistance to amoxicillin, tetracycline, and rifampicin was detected. A double population was present in 14 patients. The MLST analysis classified the 42 H. pylori strains from 38 patients in 2 haplotypes: hpEurope (33) and hpNEAfrica (9). Conclusion The prevalence of H. pylori remains high in Algeria but appears to be decreasing in recent years. High resistance to clarithromycin requires increased monitoring of the evolution of antibiotic resistance and adaptation of eradication therapy.

As Helicobacter pylori infects half the world's population and displays an extensive intraspecies diversity, genomics is a powerful tool to understand evolution and disease, to identify factors that confer higher risk of severe sequelae, and to find new approaches for therapy both among bacterial and host targets. In line with these objectives, this review article summarizes the major findings in Helicobacter genomics in papers published between April 2016 and March 2017.

The human gastric colonizer Helicobacter pylori is useful to track human migrations given the agreement between the bacterium phylogeographic distribution and human migrations. As Portugal was an African and Brazilian colonizer for over 400 years, we hypothesized that Portuguese isolates were likely genetically closer with those from countries colonized by Portuguese in the past. We aimed to characterize the population structure of several Portuguese-speaking countries, including Portugal, Brazil, Angola, and Cape Verde.We included strains isolated in Portugal from Portuguese and from former Portuguese colonies. These strains were typed by multilocus sequence typing (MLST) for seven housekeeping genes. We also retrieved from Multi Locus Sequence Typing Web site additional housekeeping gene sequences, namely from Angola and Brazil.We provided evidence that strains from Portuguese belong to hpEurope and that the introgression of hpEurope in non-European countries that speak Portuguese is low, except for Brazil and Cape Verde, where hpEurope accounted for one quarter and one half of the population, respectively. We found genetic similarity for all strains from Portuguese-speaking countries that belong to hpEurope population. Moreover, these strains showed a predominance of ancestral Europe 2 (AE2) over ancestral Europe 1 (AE1), followed by ancestral Africa 1.H. pylori is a useful marker even for relative recent human migration events and may become rapidly differentiated from founder populations. H. pylori from Portuguese-speaking countries assigned to hpEurope appears to be a hybrid population resulting from the admixture of AE1, AE2 and ancestral hpAfrica1.

Helicobacter pylori genetic diversity is known to be influenced by mobile genomic elements. Here we focused on prophages, the least characterized mobile elements of H. pylori. We present the full genomic sequences, insertion sites and phylogenetic analysis of 28 prophages found in H. pylori isolates from patients of distinct disease types, ranging from gastritis to gastric cancer, and geographic origins, covering most continents. The genome sizes of these prophages range from 22.6-33.0 Kbp, consisting of 27-39 open reading frames. A 36.6% GC was found in prophages in contrast to 39% in H. pylori genome. Remarkably a conserved integration site was found in over 50% of the cases. Nearly 40% of the prophages harbored insertion sequences (IS) previously described in H. pylori. Tandem repeats were frequently found in the intergenic region between the prophage at the 3' end and the bacterial gene. Furthermore, prophage genomes present a robust phylogeographic pattern, revealing four distinct clusters: one African, one Asian and two European prophage populations. Evidence of recombination was detected within the genome of some prophages, resulting in genome mosaics composed by different populations, which may yield additional H. pylori phenotypes. This work was supported by the the Fundação para a Ciência e a Tecnologia (FCT) project grant PTDC/EBBEBI/119860/2010 and by the University of Malaya-Ministry of Education (UM-MoE) High Impact Research (HIR) Grant UM.C/HIR/MOHE/13/5 (h-50001-00-A000033). F.F.V. is recipient of a postdoctoral fellowship from FCT (SFRH/BPD/95125/2013). info:eu-repo/semantics/publishedVersion

Aims To understand whether the variability found in the proteome of Helicobacter pylori relates to the genomic methylation, virulence and associated gastric disease. Methods and Results We applied the Minimum-Common-Restriction-Modification (MCRM) algorithm to genomic methylation data of 30 Portuguese H. pylori strains, obtained by genome sensitivity to Type II restriction enzymes' digestion. All the generated dendrograms presented three clusters with no association with gastric disease. Comparative analysis of two-dimensional gel electrophoresis (2DE) maps obtained for total protein extracts of 10 of these strains, representative of the three main clusters, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (P

The development of high-throughput whole genome sequencing (WGS) technologies is changing the face of microbiology, facilitating the comparison of large numbers of genomes from different lineages of a same organism. Our aim was to review the main advances on Helicobacter pylori “omics” and to understand how this is improving our knowledge of the biology, diversity and pathogenesis of H. pylori. Since the first H. pylori isolate was sequenced in 1997, 510 genomes have been deposited in the NCBI archive, providing a basis for improved understanding of the epidemiology and evolution of this important pathogen. This review focuses on works published between April 2015 and March 2016. Helicobacter “omics” is already making an impact and is a growing research field. Ultimately these advances will be translated into a routine clinical laboratory setting in order to improve public health.

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit to the host. Probiotics are not part of the current treatment therapies prescribed for Helicobacter pylori eradication, but there are numerous studies, most of them using probiotics as adjuvants to therapy, showing a reduction of side effects in the greater majority of cases. The probiotics administered vary hugely in the composition of microorganisms used, as well as the duration and mode of administration, which renders the comparison difficult. However, the most used probiotics for H. pylori infection are composed of Lactobacillus, Bifidobacterium, Saccharomyces, and Streptococcus. The mode of action of these probiotics relies on competition for nutrients and for adhesion to cell receptors, antimicrobial activity, and modulation of the immune system and microbiota.

© The Author 2007. Published by Oxford University Press. All rights reserved., Motivation: The genomic methylation analysis is useful to type bacteria that have a high number of expressed type II methyltransferases. Methyltransferases are usually committed to Restriction and Modification (R-M) systems, in which the restriction endonuclease imposes high pressure on the expression of the cognate methyltransferase that hinder R-M system loss. Conventional cluster methods do not reflect this tendency. An algorithm was developed for dendrogram construction reflecting the propensity for conservation of R-M Type II systems. Results: The new algorithm was applied to 52 Helicobacter pylori strains from different geographical regions and compared with conventional clustering methods. The algorithm works by first grouping strains that share a common minimum set of R-M systems and gradually adds strains according to the number of the R-M systems acquired. Dendrograms revealed a cluster of African strains, which suggest that R-M systems are present in H.pylori genome since its human host migrates from Africa. Availability: The software files are available at http://www.ff.ul.pt/paginas/jvitor/Bioinformatics/MCRM_algorithm.zip

We present draft genome sequences of 10 Helicobacter pylori clinical strains isolated from children. This will be important for future studies of comparative genomics in order to better understand the virulence determinants underlying peptic ulcer disease. This work was supported by the FCT-PTDC/BIM-MEC/1051/2012 grant from the Fundação para a Ciência e a Tecnologia (FCT) (to M.O.). A.N. and F.F.V. are recipients of postdoctoral fellowships (SFRH/BPD/75295/2010 and SFRH/BPD/95125/2013, respectively) from FCT, and R.R. is a recipient of a fellowship (BRJ-DDI/2012) from the National Institute of Health.

© 2017 John Wiley & Sons Ltd., Background: Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. Methods: The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. Results: We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. Conclusions: A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains., This work was supported by the FCT-PTDC/BIM-MEC/1051/2012 grant from the Fundação para a Ciência e a Tecnologia (FCT) (to M.O.). B.S. and F.F.V. are recipients of postdoctoral fellowships (PTDC/BIM- MEC/1051/2012 and SFRH/BPD/95125/2013, respectively) from FCT, and R.R is recipient of a fellowship (BRJ-DDI/2012) from the National Institute of Health. WGS and capillary sequencing were performed at Unidade de Tecnologia e Inovação (Departamento de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal).

Considering the recommended indications for Helicobacter pylori (H. pylori) eradication therapy and the broad spectrum of available diagnostic methods, a reliable diagnosis is mandatory both before and after eradication therapy. Only highly accurate tests should be used in clinical practice, and the sensitivity and specificity of an adequate test should exceed 90%. The choice of tests should take into account clinical circumstances, the likelihood ratio of positive and negative tests, the cost-effectiveness of the testing strategy and the availability of the tests. This review concerns some of the most recent developments in diagnostic methods of H. pylori infection, namely the contribution of novel endoscopic evaluation methodologies for the diagnosis of H. pylori infection, such as magnifying endoscopy techniques and chromoendoscopy. In addition, the diagnostic contribution of histology and the urea breath test was explored recently in specific clinical settings and patient groups. Recent studies recommend enhancing the number of biopsy fragments for the rapid urease test. Bacterial culture from the gastric biopsy is the gold standard technique, and is recommended for antibiotic susceptibility test. Serology is used for initial screening and the stool antigen test is particularly used when the urea breath test is not available, while molecular methods have gained attention mostly for detecting antibiotic resistance.

Helicobacter pylori is a common human pathogen infecting about 30% of children and 60% of adults worldwide and is responsible for diseases such as gastritis, peptic ulcer and gastric cancer. Treatment against H. pylori is based on the use of antibiotics, but therapy failure can be higher than 20% and is essentially due to an increase in the prevalence of antibiotic-resistant bacteria, which has led to the search for alternative therapies. In this review, we discuss alternative therapies for H. pylori, mainly phytotherapy and probiotics. Probiotics are live organisms or produced substances that are orally administrated, usually in addition to conventional antibiotic therapy. They may modulate the human microbiota and promote health, prevent antibiotic side effects, stimulate the immune response and directly compete with pathogenic bacteria. Phytomedicine consists of the use of plant extracts as medicines or health-promoting agents, but in most cases the molecular mode of action of the active ingredients of these herbal extracts is unknown. Possible mechanisms include inhibition of H. pylori urease enzyme, disruption of bacterial cell membrane, and modulation of the host immune system. Other alternative therapies are also reviewed.

Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori., IMPORTANCE Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population, and while most of the infected individuals do not develop disease, H. pylori infection doubles the risk of developing gastric cancer. An abundance and diversity of viruses (phages) infect microbial populations in most environments and are important mediators of microbial diversity. Our finding of a 24.6-kb prophage integrated inside an H. pylori genome and the observation of circular integrase gene-containing DNA and phage-like particles inside cells upon UV treatment demonstrate that we have discovered a viable H. pylori phage. The additional finding of integrase genes in a large proportion of screened isolates of diverse geographic origins indicates that the prevalence of prophages may have been underestimated in H. pylori. Since phages are important drivers of microbial evolution, the discovery should be important for understanding and predicting genetic diversity in H. pylori.

Helicobacter pylori is a Gram-negative microaerophilic bacterium that has colonized the human gastric mucosa. This infection is very common and affects more than half of the human population. The prevalence is however unbalanced between rural developing areas (more than 80%) and urban developed areas (less than 40%). H. pylori is responsible for several pathologies, such as gastritis, peptic ulcer and gastric cancer but its transmission pathway is still not clear. The risk factors for H. pylori infection include poor social and economic development; poor hygienic practices; absence of hygienic drinking water; and unsanitary prepared food. There is evidence supporting a gastro-oral, oral-oral and faecal-oral transmission, but no predominant mechanism of transmission has been yet identified. Transmission may occur in a vertical mode (e.g. from parents to child) or in a horizontal mode (across individuals or from environmental contamination). In either case, the involvement of water and food cannot be excluded as vehicles or sources of infection. Indirect evidence of presence of H. pylori in water and food, namely the detection of its DNA and survival studies after artificial contamination of food and water has been described. This paper reviews data both favourable and against the role of water and food in the transmission of H. pylori, exploring their role as a potential transmission vehicle for person-to-person and food-chain transmission. The likelihood of the transmission pathway in developing rural and developed urban areas appears to be different. In developed areas, person-to-person transmission within families appears to be dominant, while in the rural developing areas the transmission pathway appears to be more complex. In this later case, the transmission by contaminated food, water, or via intensive contact between infants and non-parental caretakers may have a greater influence than within-family transmission.

Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations., This work was partially supported by New England Biolabs, Inc. (USA)

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction., This work was partially supported by New England Biolabs, Inc.

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

Helicobacter pylori is a human pathogen involved in gastritis and gastric cancer whose mode of transmission remains unknown. Association of H. pylori with humans is thought to date from remote antiquity and the bacterium has apparently evolved together with the human host. A few studies have shown the presence of H. pylori in aquatic environments, which might provide a route of transmission of the bacteria to humans. A recent study has also disclosed the association of the bacteria with Acantamoeba castellanii. Amoeba are known to harbor and promote the persistence of several human pathogens in the environment, representing a significant source of contamination in community and hospital acquired infections.

Helicobacter pylori (H. pylori) successfully colonizes the human stomach of the majority of the human population. This infection always causes chronic gastritis, but may evolve to serious outcomes, such as peptic ulcer, gastric carcinoma or mucosa-associated lymphoid tissue lymphoma. H. pylori first line therapy recommended by the Maastricht-4 Consensus Report comprises the use of two antibiotics and a proton-pomp inhibitor, but in some regions failure associated with this treatment is already undesirable high. Indeed, treatment failure is one of the major problems associated with H. pylori infection and is mainly associated with bacterial antibiotic resistance. In order to counteract this situation, some effort has been allocated during the last years in the investigation of therapeutic alternatives beyond antibiotics. These include vaccines, probiotics, photodynamic inactivation and phage therapy, which are briefly revisited in this review. A particular focus on phytomedicine, also described as herbal therapy and botanical therapy, which consists in the use of plant extracts for medicinal purposes, is specifically addressed, namely considering its history, category of performed studies, tested compounds, active principle and mode of action. The herbs already experienced are highly diverse and usually selected from products with a long history of employment against diseases associated with H. pylori infection from each country own folk medicine. The studies demonstrated that many phytomedicine products have an anti-H. pylori activity and gastroprotective action. Although the mechanism of action is far from being completely understood, current knowledge correlates the beneficial action of herbs with inhibition of essential H. pylori enzymes, modulation of the host immune system and with attenuation of inflammation.