Acid-stable capsid structure of Helicobacter pylori bacteriophage KHP30 by single-particle cryoelectron microscopy.

The acid-stable capsid structures of Helicobacter pylori phages KHP30 and KHP40 are solved at 2.7 and 3.0 Å resolutions by cryoelectron microscopy, respectively. The capsids have icosahedral T = 9 symmetry and consist of each 540 copies of 2 structural proteins, a major capsid protein, and a cement protein. The major capsid proteins form 12 pentagonal capsomeres occupying icosahedral vertexes and 80 hexagonal capsomeres located at icosahedral faces and edges. The major capsid protein has a unique protruding loop extending to the neighboring subunit that stabilizes hexagonal capsomeres. Furthermore, the capsid is decorated with trimeric cement proteins with a jelly roll motif. The cement protein trimer sits on the quasi-three-fold axis formed by three major capsid protein capsomeres, thereby enhancing the particle stability by connecting these capsomeres. Sequence and structure comparisons between the related Helicobacter pylori phages suggest a possible mechanism of phage adaptation to the human gastric environment.

Infección por Helicobacter pylori y factores asociados en adultos con sospecha clínica de úlcera duodenal / Helicobacter pylori infection and associated factors in adults with a clinical suspicion of duodenal ulcer

RESUMEN Introducción: la infección por Helicobacter pylori es la enfermedad bacteriana crónica que afecta con mayor prevalencia al ser humano. Objetivo: identificar la frecuencia de infección por Helicobacter pylori y su relación con variables consideradas factores de riesgo de esta infección. Materiales y métodos: estudio de corte transversal realizado en el Policlínico Docente Camilo Cienfuegos, del municipio Habana del Este, durante el año 2018, en un universo de 42 pacientes con 18 años y más de edad, con sospecha clínica y hallazgo endoscópico de úlcera duodenal e informe del resultado de estudio histológico para el diagnóstico de la infección. Se confeccionó una planilla de recolección de datos que incluyó variables como hacinamiento, agua de consumo, lugar de nacimiento, estancia en una institución, contacto con animales y antecedentes familiares. Se determinó relación entre variables con la prueba de chi cuadrado (c2) con significación estadística ɒ = 0,05, y se identificaron variables cuyos coeficientes fueron significativamente diferentes de 0 (p < 0,05). La fuerza de asociación se determinó mediante odds ratio. Resultados: la prevalencia fue de 59,5 %. Se encontró asociación estadística y constituyeron factores de riesgo de infección por Helicobacter pylori, el hacinamiento (c2 = 4,37; OR = 3,89), el agua de consumo (c2 = 4,92; OR = 3,43), el contacto con animales (c2 = 7,41; OR = 6,17) y los antecedentes familiares (c2 = 13,18; OR = 13). Conclusiones: el estudio permitió determinar la prevalencia de infección por Helicobacter pylori y las principales variables asociadas, coincidiendo con otros estudios revisados que tratan el tema (AU).

ABSTRACT Introduction: the infection by Helicobacter pylori is the chronic bacterial disease that affects the human being with greater prevalence. Objective: to identify the frequency of the infection by Helicobacter pylori and its relationship with variables considered risk factors for this infection. Materials and methods: a cross-sectional study was carried out in the teaching Polyclinic Camilo Cienfuegos, municipality Habana del Este, during 2018. In a universe of 42 patients aged 18 years and over, with clinical suspicion and endoscopic diagnosis of duodenal ulcer and histological study report for the diagnosis of the infection. A data collection form was made, which included variables such as: overcrowding, consumption water, place of birth, staying in an institution, contact with animals, and family history. The relationship within variables was found using the chi-square test (c2) with statistical significance ɒ = 0.05, and there were identified variables significantly different from 0 (p < 0.05). The association strength was determined through odds ratio. Results: the prevalence was 59.5%. Statistical association was found and overcrowding (c2 = 4.37, OR = 3.89), consumption water (c2 = 4.92; OR = 3.43), contact with animals (c2 = 7.41, OR = 6.17) and family history (c2 = 13.18, OR = 13) were found risk factors for Helicobacter pylori infection. Conclusions: the study allowed to determine the prevalence of Helicobacter pylori infection and the main associated variables, coinciding with other reviewed studies dealing with the subject (AU).

Bacteriophage Tail-Spike Proteins Enable Detection of Pseudaminic-Acid-Coated Pathogenic Bacteria and Guide the Development of Antiglycan Antibodies with Cross-Species Antibacterial Activity.

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 (Ab-54149) to form a glycoconjugate for preparing anti-Ab-54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab-54149 than an antibody purified from anti- Ab-54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides (e.g., Helicobacter pylori and Enterobacter cloacae), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti-Ab-54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae, indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Clinical factors associated with initial Helicobacter pylori eradication therapy: a retrospective study in China.

The eradication rate of Helicobacter pylori (H. pylori) has been decreasing every year, mainly due to the increase in antibiotic resistance. In fact, many other factors may affect H. pylori eradication. To analyze the clinical factors affecting the initial eradication therapy in Chinese patients with H. pylori infection. We conducted a retrospective study on 264 outpatients who were diagnosed with H. pylori-associated chronic gastritis and peptic ulcer disease between January and December 2015 at a large tertiary hospital in China. The patients were divided into three groups: ECA, RCA, and RCM (R: 20 mg rabeprazole, E: 40 mg esomeprazole, C: 0.5 g clarithromycin, A: 1.0 g amoxicillin and M: 0.4 g metronidazole). The patients were treated for 14 days and followed up for 1 year. The 14C-urea breath test (14C-UBT) was performed 4 weeks after the completion of the eradication therapy. The eradication rate was higher in ≥ 40-year-old patients than in < 40-year-old-patients (85.7% vs. 54.7%, p = 0.002). Multivariate analyses revealed only age ≥ 40 years to be significantly associated with a high H. pylori eradication rate [odds ratio (OR) 4.58, p = 0.003]. The H. pylori eradication rate in patients with duodenal ulcers was significantly higher than that in patients with gastric ulcers (79% vs. 60%, p = 0.012). Age could be a predictor of successful H. pylori eradication. Patients with duodenal ulcers had a higher H. pylori eradication rate than those with other lesions.

Analysis of genetic recombination and the pan-genome of a highly recombinogenic bacteriophage species.

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.

The Story of Helicobacter pylori: Depicting Human Migrations from the Phylogeography.

Helicobacter pylori is a spiral-shaped Gram-negative bacterium, which has infected more than half of the human population. Besides its colonisation capability, the genetic diversity of H. pylori is exceptionally well structured and belongs to several distinct genetic populations, depicting various prehistorical human migration events. The evolutionary relationship of H. pylori with its host had been started at least ~100,000 years ago. In addition, the discovery of the ancient H. pylori genome from a European Copper Age glacier mummy, "The Iceman", gave the idea that the second out of Africa migration resulted in the recombinant population of hpEurope at least about 5300 years ago. The advancement of next-generation genome sequencing discovered the prophage of H. pylori and could discriminate the big population of hpEurope into two different subpopulations. In addition, the implementation of the chromopainter/fineSTRUCTURE algorithm to the whole genome analysis of H. pylori provides a finer resolution population genetics of H. pylori; therefore it could also depict the recent migrations within the past 500 years after colonial expansion. This discovery shows that the genetic recombination of H. pylori strains is far more dynamic compared to its human host, but still maintains the similarity to its host, suggesting that H. pylori is a handy tool to reconstruct the human migration both in the past and the recent.

Relating Phage Genomes to Helicobacter pylori Population Structure: General Steps Using Whole-Genome Sequencing Data.

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.

Polymorphisms in the Helicobacter pylori NY43 strain and its prophage-cured derivatives.

This study aimed to determine the characteristics of the Helicobacter pylori host NY43 strain and its prophage-cured derivative. H. pylori colonizing the human stomach cause many diseases. They show high genetic diversity, allowing the development of mutant strains that can form bacterial communities adapted to specific environmental conditions. Bacteriophage activities are associated with bacterial evolution, including pathogenicity development. Herein, we reported the complete genome sequence and genomic organization of two H. pylori prophages, KHP30 and KHP40; the effects of KHP30 on the behaviours of NY43 are not yet known. We showed that approximately 57 % prophage-cured derivatives spontaneously appeared in the exponential phase during liquid culture, and the biological characteristics of these derivatives differed from those of the host NY43. KHP30 reinfected the cured derivatives, and the curing ratio was influenced by culture conditions. KHP30 was shown to promote the development of a flexible H. pylori community with variable characteristics.

Isolation of Bacteriophages for Fastidious Bacteria.

One of the most important factors for successful bacteriophage therapy is, undoubtedly, the isolation of excellent therapeutic candidate bacteriophages. There are only a few reports about active bacteriophages in the fastidious bacteria Helicobacter pylori. In this chapter, we describe a method for isolating and purifying KHP30-like bacteriophages in H. pylori, which have lytic and pseudolysogenic life cycles.

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.

Screening of KHP30-like prophages among Japanese Helicobacter pylori strains, and genetic analysis of a defective KHP30-like prophage sequence integrated in the genome of the H. pylori strain NY40.

We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR, in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.

Dormant phages of Helicobacter pylori reveal distinct populations in Europe.

Prophages of Helicobacter pylori, a bacterium known to co-evolve in the stomach of its human host, were recently identified. However, their role in the diversity of H. pylori strains is unknown. We demonstrate here and for the first time that the diversity of the prophage genes offers the ability to distinguish between European populations, and that H. pylori prophages and their host bacteria share a complex evolutionary history. By comparing the phylogenetic trees of two prophage genes (integrase and holin) and the multilocus sequence typing (MLST)-based data obtained for seven housekeeping genes, we observed that the majority of the strains belong to the same phylogeographic group in both trees. Furthermore, we found that the Bayesian analysis of the population structure of the prophage genes identified two H. pylori European populations, hpNEurope and hpSWEurope, while the MLST sequences identified one European population, hpEurope. The population structure analysis of H. pylori prophages was even more discriminative than the traditional MLST-based method for the European population. Prophages are new players to be considered not only to show the diversity of H. pylori strains but also to more sharply define human populations.

Comparative genomics of a Helicobacter pylori isolate from a Chinese Yunnan Naxi ethnic aborigine suggests high genetic divergence and phage insertion.

Helicobacter pylori is a common pathogen correlated with several severe digestive diseases. It has been reported that isolates associated with different geographic areas, different diseases and different individuals might have variable genomic features. Here, we describe draft genomic sequences of H. pylori strains YN4-84 and YN1-91 isolated from patients with gastritis from the Naxi and Han populations of Yunnan, China, respectively. The draft sequences were compared to 45 other publically available genomes, and a total of 1059 core genes were identified. Genes involved in restriction modification systems, type four secretion system three (TFS3) and type four secretion system four (TFS4), were identified as highly divergent. Both YN4-84 and YN1-91 harbor intact cag pathogenicity island (cagPAI) and have EPIYA-A/B/D type at the carboxyl terminal of cagA. The vacA gene type is s1m2i1. Another major finding was a 32.5-kb prophage integrated in the YN4-84 genome. The prophage shares most of its genes (30/33) with Helicobacter pylori prophage KHP30. Moreover, a 1,886 bp transposable sequence (IS605) was found in the prophage. Our results imply that the Naxi ethnic minority isolate YN4-84 and Han isolate YN1-91 belong to the hspEAsia subgroup and have diverse genome structure. The genome has been extensively modified in several regions involved in horizontal DNA transfer. The important roles played by phages in the ecology and microevolution of H. pylori were further emphasized. The current data will provide valuable information regarding the H. pylori genome based on historic human migrations and population structure.

Cáncer gástrico en una zona con alto potencial para la cultura apícola y su aplicación en la prevención de las lesiones gástricas premalignas y malignas / Gastric cancer in an area of high potential for beekiping culture and its application in the prevention of premalignat and malignat gastric lesion

El cáncer gástrico es la principal causa de muerte por cáncer del estado Táchira - Venezuela. Con una particular elevada tasa de mortalidad en los estados andinos. Los esfuerzos realizados en la detección temprana basados en estudios radiológicos y endoscópicos no han permitido cubrir gran parte de la población. La identificación de lesiones pre malignas y cáncer asociadas a la infección por Helicobacter pylori han obligado al desarrollo de proyectos epidemiológicos de prevención primaria que tiendan a incidir sobre estilos de vida y factores dietéticos. La erradicación de H. pylori con el uso de terapia antimicrobiana convencional ha dado resultados controvertidos en diferentes latitudes, con los mismos esquemas terapéuticos. La capacidad antimicrobiana de diferentes tipos de propóleos ante el H. pylori, ya ha sido evaluada, siendo el principal responsable los flavonoides: pinocembrina, galangina y crisina. La alta prevalencia de H. pylori en la Aldea Potrero de las Casas, Táchira, Venezuela, aunado al potencial y cultura apícola de la zona promueven estudios de intervención con productos de la colmena que son accesibles y de bajo costo

Gastric Cancer is the leading cause of cancer death in Táchira State - Venezuela with a high mortality rate especially in the Andean States. Efforts at early gastric cancer detection based on radiological and endoscopic studies have failed to cover all the population. The identification of premalignant gastric lesion and gastric cancer associated with Helicobacter Pylori infection have led to the development of epidemiological primary prevention projects that led to influence on dietary and life style factors Helicobacter Pylori eradication using conventional therapy has been controversial results in differents latitudes with the same therapeutics regimens. The antimicrobial activity of different types of propolis against Helicobacter Pylori has been evaluated, the main responsibility are flavonoids,pinocembrin, galactin and chrysin. The high prevalence of Helicobacter Pylori in the village of Potrero de las Casas, Táchira Venezuela together with the potential of the area in the bee culture induce to think about to promote intervention studies with bee products that are accessible and low cost

Diagnóstico de infección por Helicobacter pylori en cuerpo gástrico con magnificación endoscópica y "flexible spectral imaging colour enhancement" (FICE) / Diagnosis of Helicobacter pylori infection in gastric body with endoscopic magnification and flexible spectral imaging colour enhancement (FICE)

La endoscopia estándar no diagnostica infección por Helicobacter pylori. Con magnificación y "Flexible Spectral Imaging Colour Enhancement" (FICE) se observan patrones de mucosa gástrica que sugieren su presencia. Diagnosticar infección por Helicobacter pylori con magnificación endoscópica y "Flexible Spectral Imaging Colour Enhancement" (FICE). Previo consentimiento se incluyeron a los individuos con indicación electiva de endoscopia digestiva superior. Se realizó endoscopia digestiva superior con equipo Fujinon Inc. EG 590 ZW, y procesador EPX 4400. En ambas caras del cuerpo gástrico se realizó consecutivamente: a) alta resolución, b) magnificación, c) alta resolución, d)FICE, e)magnificación y f) biopsia en el antro y del patrón mas prevalente en cada cara del cuerpo evaluadas sin información del paciente. Todo el procedimiento se grabó, se fotografió y se guardó en JPEG en programa Power Point. Se evaluaron 60 áreas en 30 pacientes: 10 hombres y 20 mujeres con edades de 20-82 años y promedio 49,60 años. Solo magnificación y FICE identificaron los patrones de mucosa en cuerpo gástrico. En 37,03% se diagnosticó Helicobacter pylori con histología, 53,33% y 61,11% en patrón Z2 y Z3 respectivamente. La magnificación y FICE permiten identificar los patrones de mucosa gástrica que sugieren infección por Helicobacter pylori

Helicobacter pylori infection is not diagnosed with standard endoscopy. With high resolution and magnification patterns of gastric mucosa suggesting its presence are observed. Diagnose Helicobacter pylori infection with endoscopic magnification and "Flexible Spectral Imaging Colour Enhancement" (FICE). Individuals scheduled to undergo routine upper gastrointestinal endoscopy were enrolled. Upper gastrointestinal endoscopy was performed with Fujinon Inc. 590 EG ZW and EPX 4400 processor. Endoscopy was practiced on both sides of the gastric body consecutively with: a) high-resolution, b) magnification, c) high-resolution, d) FICE, e) magnification and g) biopsy of the antrum and the pattern more prevalent on each side of the body evaluated without patient information. The entire procedure was recorded, was photographed and was saved in JPEG in program Power Point. 60 Areas in 30 patients were evaluated: 10 men and 20 women with ages of 20-82 years and average 49.60. Only magnification and FICE identified patterns of mucosa in gastric body. Helicobacter pylori was diagnosed in 37.03% with histology and in pattern Z2 and Z3 in 53.33% and 61.11% respectively. The endoscopic magnification and Flexible Spectral Imaging Colour Enhancement (FICE) identify patterns of gastric mucosa suggesting Helicobacter pylori infection

Characterization of Helicobacter pylori bacteriophage KHP30.

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.

Complete genome sequences of two Helicobacter pylori bacteriophages isolated from Japanese patients.

Helicobacter pylori causes peptic ulcers and gastric cancer, which lead to significantly higher morbidity in Japan than elsewhere in the world. As bacteriophage (phage) and host bacteria coevolve, the study of H. pylori phages is important to extend understanding of the evolution and pathogenesis of H. pylori. Here we report two complete genome sequences of H. pylori phages KHP30 and KHP40, which were released spontaneously from the most pathogenic East Asian-type isolates from Japanese patients.

Genome, integration, and transduction of a novel temperate phage of Helicobacter pylori.

Helicobacter pylori is a common human pathogen that has been identified to be carcinogenic. This study isolated the temperate bacteriophage 1961P from the lysate of a clinical strain of H. pylori isolated in Taiwan. The bacteriophage has an icosahedral head and a short tail, typical of the Podoviridae family. Its double-stranded DNA genome is 26,836 bp long and has 33 open reading frames. Only 9 of the predicted proteins have homologs of known functions, while the remaining 24 are only similar to unknown proteins encoded by Helicobacter prophages and remnants. Analysis of sequences proximal to the phage-host junctions suggests that 1961P may integrate into the host chromosome via a mechanism similar to that of bacteriophage lambda. In addition, 1961P is capable of generalized transduction. To the best of our knowledge, this is the first report of the isolation, characterization, genome analysis, integration, and transduction of a Helicobacter pylori phage.