Biomarker Characterization and Prediction of Virulence and Antibiotic Resistance from Helicobacter pylori Next Generation Sequencing Data.

The Gram-negative bacterium Helicobacter pylori colonizes c.a. 50% of human stomachs worldwide and is the major risk factor for gastric adenocarcinoma. Its high genetic variability makes it difficult to identify biomarkers of early stages of infection that can reliably predict its outcome. Moreover, the increasing antibiotic resistance found in H. pylori defies therapy, constituting a major human health problem. Here, we review H. pylori virulence factors and genes involved in antibiotic resistance, as well as the technologies currently used for their detection. Furthermore, we show that next generation sequencing may lead to faster characterization of virulence factors and prediction of the antibiotic resistance profile, thus contributing to personalized treatment and management of H. pylori-associated infections. With this new approach, more and permanent data will be generated at a lower cost, opening the future to new applications for H. pylori biomarker identification and antibiotic resistance prediction.

Glutaredoxin: Discovery, redox defense and much more.

Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.

An American lineage of Helicobacter pylori prophages found in Colombia.

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.

A 500-year tale of co-evolution, adaptation, and virulence: Helicobacter pylori in the Americas.

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.

Bacteriophages of Helicobacter pylori.

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.

Rapid Characterization of Virulence Determinants in Helicobacter pylori Isolated from Non-Atrophic Gastritis Patients by Next-Generation Sequencing.

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 < 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.

Prevalence, antibiotic resistance, and MLST typing of Helicobacter pylori in Algiers, Algeria.

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.

The expression of Helicobacter pylori tfs plasticity zone cluster is regulated by pH and adherence, and its composition is associated with differential gastric IL-8 secretion.

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.

Genomic structure and insertion sites of Helicobacter pylori prophages from various geographical origins.

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.

Helicobacter pylori infection - recent developments in diagnosis.

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.

Overview of the phytomedicine approaches against Helicobacter pylori.

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.

Alternative therapies for Helicobacter pylori: probiotics and phytomedicine.

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.

Genome sequencing reveals a phage in Helicobacter pylori.

UNLABELLED: 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.

Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration.

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.

Genomic methylation: a tool for typing Helicobacter pylori isolates.

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.