Marcadores moleculares de tipo inserción en bacterias de las familias Moraxellaceae y Helicobacteraceae (phylum Proteobacteria) / Molecular markers of insertion type in bacterias of the Moraxellaceae and Helicobacteraceae (phylum Proteobacteria) families

Se efectuó un estudio con el empleo de la metodología de Gupta, en el Departamento de Biología y Geografía de la Universidad de Oriente en Santiago de Cuba, para determinar marcadores moleculares de tipo inserción en secuencias de las proteínas ADN polimerasa I y ADN polimerasa III (subunidad alfa), obtenidas de bases de datos internacionales y posteriormente alineadas con el programa ClustalX2. Las familias Moraxellaceae y Helicobacteraceae han sido ampliamente estudiadas, porque comprenden agentes patógenos causantes de numerosas enfermedades en humanos, pero pocas investigaciones han estado dirigidas a la identificación de las características moleculares que puedan distinguir a sus miembros de otros grupos de bacterias; de manera que los presentes resultados constituyen un aporte al conocimiento de la genética y la bioquímica de estas familias y proveen herramientas moleculares para la clasificación taxonómica y el diagnóstico de especies patógenas(AU)

A study with the use of Gupta methodology was carried out in the Biology and Geography Department of Oriente University in Santiago de Cuba, to determine molecular markers of insertion type in sequences of the DNA polimerase I proteins and DNA polimerase III (alpha subunity), obtained from international databases and later on aligned with the ClustalX2 program. The Moraxellaceae and Helicobacteraceae families have been broadly studied, because they comprise pathogen agents that cause numerous diseases in humans, but few investigations have been directed to the identification of the molecular characteristics that can distinguish their members from other groups of bacterias; so these results constitute a contribution to the knowledge of genetics and biochemistry of these families and provide molecular tools for the taxonomic classification and the diagnosis of pathogen species(AU)

Marcadores moleculares de tipo inserción en bacterias de las familias Moraxellaceae y Helicobacteraceae (phylum Proteobacteria) / Molecular markers of insertion type in bacterias of the Moraxellaceae and Helicobacteraceae (phylum Proteobacteria) families

Se efectuó un estudio con el empleo de la metodología de Gupta, en el Departamento de Biología y Geografía de la Universidad de Oriente en Santiago de Cuba, para determinar marcadores moleculares de tipo inserción en secuencias de las proteínas ADN polimerasa I y ADN polimerasa III (subunidad alfa), obtenidas de bases de datos internacionales y posteriormente alineadas con el programa ClustalX2. Las familias Moraxellaceae y Helicobacteraceae han sido ampliamente estudiadas, porque comprenden agentes patógenos causantes de numerosas enfermedades en humanos, pero pocas investigaciones han estado dirigidas a la identificación de las características moleculares que puedan distinguir a sus miembros de otros grupos de bacterias; de manera que los presentes resultados constituyen un aporte al conocimiento de la genética y la bioquímica de estas familias y proveen herramientas moleculares para la clasificación taxonómica y el diagnóstico de especies patógenas

A study with the use of Gupta methodology was carried out in the Biology and Geography Department of Oriente University in Santiago de Cuba, to determine molecular markers of insertion type in sequences of the DNA polimerase I proteins and DNA polimerase III (alpha subunity), obtained from international databases and later on aligned with the ClustalX2 program. The Moraxellaceae and Helicobacteraceae families have been broadly studied, because they comprise pathogen agents that cause numerous diseases in humans, but few investigations have been directed to the identification of the molecular characteristics that can distinguish their members from other groups of bacterias; so these results constitute a contribution to the knowledge of genetics and biochemistry of these families and provide molecular tools for the taxonomic classification and the diagnosis of pathogen species

Helicobacter pullorum induces nitric oxide release in murine macrophages that promotes phagocytosis and killing.

Helicobacter pullorum is an avian enterohepatic species that, more recently, has also been found as a naturally acquired infection in mice and rats, and isolated from patients with gastrointestinal and hepatobiliary diseases. In this work, the interaction between H. pullorum and murine macrophages was examined. Firstly, the impact of nitric oxide, which is an antimicrobial produced by mammalian macrophages, on H. pullorum 6350-92 viability and morphology was studied by colony-forming assays and light microscopy, respectively. Exposure to nitric oxide lowered H. pullorum viability, in a growth-phase-dependent manner, and decreased the mean cell size. However, the number of coccoid forms remained low, contrasting with what has been observed for other Helicobacter species. Confocal microscopy showed that H. pullorum is internalized by murine macrophages, triggering nitric oxide production that promotes phagocytosis and killing of the pathogen. Interaction between H. pullorum and macrophages stimulated secretion of pro-inflammatory cytokines, such as TNF-α, IL-1ß, IL-6 and MIP-2. These results show that H. pullorum is able to infect mammalian murine cells triggering an inflammatory response.

Morphologic, genetic, and biochemical characterization of Helicobacter magdeburgensis, a novel species isolated from the intestine of laboratory mice.

BACKGROUND: The presence of enterohepatic Helicobacter species (EHS) is commonly noted in mouse colonies. These infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. The aim of this study was to isolate and characterize a putative novel EHS that has previously been detected by PCR screening of specific-pathogen-free mice. MATERIALS AND METHODS: Biochemical analysis of enzyme activities (API campy), morphologic investigation (Gram-staining and electron microscopy) and genetic analyses (16SrRNA and 23SrRNA analyses, DNA fingerprinting, restriction fragment polymorphisms, and pulsed-field gel electrophoresis) were used to characterize isolated EHS. Genomic DNA fragments were sequenced to develop a species-specific PCR detection assay. RESULTS: Scanning electron microscopy revealed the presence of spiral-shaped EHS, which varied in length (2.5-6 µm) and contained single monopolar or single bipolar sheathed flagella. The bacteria were grown under anaerobic conditions, preferably on agar plates containing serum or blood. The 16SrRNA, genetic, and biochemical analyses indicated the identification of a novel EHS species, named Helicobacter magdeburgensis. We also examined the genome content using pulsed-field gel electrophoresis. Based on the pattern produced by two restriction enzymes, BamIII and KspI, the genome size was determined to be about 1.7-1.8 Mbp. CONCLUSION: We isolated and characterized a novel EHS species, H. magdeburgensis, morphologically, biochemically, and genetically. These results are important for future studies on the prevalence and pathophysiologic relevance of such infections. Our PCR assay can be used to detect and discriminate H. magdeburgensis from other Helicobacter species.

Helicobacter equorum sp. nov., a urease-negative Helicobacter species isolated from horse faeces.

Gram-negative, curved, motile bacteria (strains EqF1T and EqF2) were isolated from faecal samples from two clinically healthy horses. Both strains possessed a single, monopolar, sheathed flagellum and were urease-negative. The novel strains grew at 37 degrees C under microaerobic conditions and were positive for oxidase, catalase and alkaline phosphatase activities. The isolates reduced nitrate to nitrite, but gamma-glutamyl transpeptidase activity was not detected. The novel isolates did not grow at 42 degrees C or on media containing 1 % glycine. They were resistant to cephalotin and nalidixic acid and susceptible to metronidazole. Analysis of the 16S and 23S rRNA gene sequences of the two novel strains identified them as representing a single species within the genus Helicobacter. In terms of 16S rRNA gene sequence similarity, Helicobacter pullorum and Helicobacter canadensis were the most closely related species (98 % similarity). 23S rRNA gene sequence analysis also classified strains EqF1T and EqF2 within the enterohepatic division of the genus Helicobacter, but only 94 % similarity was detected with H. pullorum and H. canadensis, which are helicobacters with unsheathed flagella. The most closely related species in terms of 23S rRNA gene sequence similarity was Helicobacter canis (95 %). Numerical analysis of whole-cell protein extracts by SDS-PAGE was performed and the novel isolates were clearly differentiated from H. pullorum, H. canadensis, H. canis and other species of the genus Helicobacter. This finding was also confirmed by sequence analysis of the hsp60 gene. On the basis of these genetic, biochemical and protein data, the isolates are classified as representing a novel species, for which the name Helicobacter equorum sp. nov. is proposed (type strain EqF1T=LMG 23362T=CCUG 52199T).

Helicobacter cynogastricus sp. nov., isolated from the canine gastric mucosa.

A Gram-negative, microaerophilic helical rod, isolated from the gastric mucosa of a dog and designated strain JKM4(T), was subjected to a polyphasic taxonomic study. The tightly coiled organism, measuring 10-18 mum long and up to 1 mum wide, was motile by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril running along the external side of the helix. Strain JKM4(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 30 and 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed > 97 % similarity to Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, three species previously isolated from the canine gastric mucosa. Protein profiling of strain JKM4(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin and from Helicobacter canis. Additionally, the urease gene sequence of strain JKM4(T) was different from those of urease genes of H. felis, H. bizzozeronii, H. salomonis and "Candidatus Helicobacter heilmannii". It is thus proposed that strain JKM4(T) (=LMG 23188(T)) represents a novel species within this genus, Helicobacter cynogastricus sp. nov.

ESR signal of the iron-sulfur center F(X) and its function in the homodimeric reaction center of Heliobacterium modesticaldum.

Electron transfer in the membranes and the type I reaction center (RC) core protein complex isolated from Heliobacterium modesticaldum was studied by optical and ESR spectroscopy. The RC is a homodimer of PshA proteins. In the isolated membranes, illumination at 14 K led to accumulation of a stable ESR signal of the reduced iron-sulfur center F(B)(-) in the presence of dithiothreitol, and an additional 20 min illumination at 230 K induced the spin-interacting F(A)(-)/F(B)(-) signal at 14 K. During illumination at 5 K in the presence of dithionite, we detected a new transient signal with the following values: g(z)= 2.040, g(y)= 1.911, and g(x)= 1.896. The signal decayed rapidly with a 10 ms time constant after the flash excitation at 5 K and was attributed to the F(X)(-)-type center, although the signal shape was more symmetrical than that of F(X)(-) in photosystem I. In the purified RC core protein, laser excitation induced the absorption change of a special pair, P800. The flash-induced P800(+) signal recovered with a fast 2-5 ms time constant below 150 K, suggesting charge recombination with F(X)(-). Partial destruction of the RC core protein complex by a brief exposure to air increased the level of the P800(+)A(0)(-) state that gave a lifetime (t(1/2)) of 100 ns at 77 K. The reactions of F(X) and quinone were discussed on the basis of the three-dimensional structural model of RC that predicts the conserved F(X)-binding site and the quinone-binding site, which is more hydrophilic than that in the photosystem I RC.

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica.

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.

Phylogenetic characterization of 'Candidatus Helicobacter bovis', a new gastric helicobacter in cattle.

Recently helicobacter-like organisms have been reported in the pyloric part of the abomasum of calves and adult cattle. Cultivation of these spiral bacteria has not been successful to date. In the present study, comparative 16S rDNA sequence analysis was used to determine the taxonomic position of these bacteria. Seven abomasal biopsies of adult cattle were sampled from different Belgian and Dutch farms. In all samples the presence of helicobacter-like organisms was demonstrated by biochemical, immunohistochemical and electron microscopical data. Bacterial 16S rDNA was amplified by PCR and sequences were determined either by direct or indirect sequence analysis. Pairwise comparisons revealed all sequences to be more than 99% homologous. Phylogenetic analysis placed the organism, corresponding to the reference sequence R2XA, within the genus Helicobacter. A diagnostic PCR assay was designed, differentiating all of the bovine 16S rDNA sequences from Helicobacter and Wolinella species. The low similarity level towards Helicobacter bilis (92.8%), its closest validly named neighbour, indicates that this novel taxon is indeed a novel Helicobacter species. An in situ hybridization procedure associated the bovine sequences to the helicobacter-like organisms in the abomasum. The name 'Candidatus Helicobacter bovis' is proposed for this new abomasal helicobacter from cattle.

Helicobacter heilmannii associated erosive gastritis.

The spiral bacteria, Helicobacter heilmannii (H. heilmannii), distinct from Helicobacter pylori (H. pylori), was found in the gastric mucosa of a 71-year-old man without clinical symptoms. The endoscopic examination revealed erosive gastritis. Rapid urease test from the antral specimen was positive, but both culture and immunohistological staining for H. pylori were negative. Touch smear cytology showed tightly spiral bacteria, which were consistent with H. heilmannii. At the second endoscopy after medication regimen for eradication of H. pylori, inflammation was decreased and the rapid urease test was negative. The second cytology showed no evidence of H. heilmannii. Anti-H. pylori therapy may be a useful medication for H. heilmannii.

Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii.

Cultures of Helicobacter pylori obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking. Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for H. pylori whereas bacteria from broth cultures had the morphologic appearance of Helicobacter heilmannii. Bacteria harvested from blood agar plates assumed an H. heilmannii-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of H. pylori when grown on blood agar plates. Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (H. pylori) was responsible for both morphologies.

Isolation of Helicobacter strains from wild bird and swine feces.

We report the first isolations of Helicobacter strains from wild birds and swine. Genus-specific oligonucleotide probes identified nine Cape Cod isolates from gull, tern, house sparrow, and pig feces as Helicobacter spp. and not Campylobacter spp. Antibiotic sensitivity and urease tests distinguished three phenotypes. Strains examined rapidly lost culturability under simulated natural conditions.