DPO multiplex PCR as an alternative to culture and susceptibility testing to detect Helicobacter pylori and its resistance to clarithromycin

<p>Abstract</p> <p>Background</p> <p>Macrolide resistance in <it>Helicobacter pylori </it>is the major risk factor for treatment failure when using a proton pump inhibitor-clarithromycin containing therapy. Macrolide resistance is due to a few mutations on the 23S ribomosal subunit encoded by the 23S rRNA gene. The present study aimed at investigating the performance of the dual priming oligonucleotide (DPO)-PCR kit named Seeplex^®ClaR-<it>H. pylori </it>ACE detection designed to detect <it>H. pylori </it>and two types of point mutations causing clarithromycin resistance in <it>H. pylori</it>.</p> <p>Methods</p> <p>The performance of Seeplex^®ClaR-<it>H. pylori </it>ACE detection was evaluated on 127 gastric biopsies in comparison to conventional bacterial culture followed by the determination of susceptibility to clarithromycin by E-test, as well as by an in-house real-time PCR using a fluorescence resonance energy transfer (FRET) technology.</p> <p>Results</p> <p>Considering culture as the reference test, the sensitivity of DPO-PCR and real-time FRET-PCR was 97.7% and 100% while specificity was 83.1% and 80.7%, respectively. However, both PCR were concordant in detecting 14 <it>H. pylori </it>positive cases which were negative by culture. Globally, E-test and DPO-PCR were concordant with regard to clarithromycin susceptibility in 95.3% of the cases (41/43), while real-time FRET-PCR and DPO-PCR were concordant in 95% (57/60).</p> <p>Conclusion</p> <p>The DPO-PCR is an interesting tool to detect <it>H. pylori </it>on gastric biopsies and to study its susceptibility to clarithromycin in laboratories that cannot perform real-time PCR assays.</p

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200&nbsp;kilobases (kb), including a genome of 735&nbsp;kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems

Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathoge

Clades of huge phages from across Earth's ecosystems

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems

Human Bone Marrow-Derived Stem Cells Acquire Epithelial Characteristics through Fusion with Gastrointestinal Epithelial Cells

Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into a variety of cell types and are a potential source for epithelial tissue repair. Several studies have demonstrated their ability to repopulate the gastrointestinal tract (GIT) in bone marrow transplanted patients or in animal models of gastrointestinal carcinogenesis where they were the source of epithelial cancers. However, mechanism of MSC epithelial differentiation still remains unclear and controversial with trans-differentiation or fusion events being evoked. This study aimed to investigate the ability of MSC to acquire epithelial characteristics in the particular context of the gastrointestinal epithelium and to evaluate the role of cell fusion in this process. In vitro coculture experiments were performed with three gastrointestinal epithelial cell lines and MSC originating from two patients. After an 8 day coculture, MSC expressed epithelial markers. Use of a semi-permeable insert did not reproduce this effect, suggesting importance of cell contacts. Tagged cells coculture or FISH on gender-mismatched cells revealed clearly that epithelial differentiation resulted from cellular fusion events, while expression of mesenchymal markers on fused cells decreased over time. In vivo cell xenograft in immunodeficient mice confirmed fusion of MSC with gastrointestinal epithelial cells and self-renewal abilities of these fused cells. In conclusion, our results indicate that fusion could be the predominant mechanism by which human MSC may acquire epithelial characteristics when in close contact with epithelial cells from gastrointestinal origin . These results could contribute to a better understanding of the cellular and molecular mechanisms allowing MSC engraftment into the GIT epithelium

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Soil carbon dioxide emissions controlled by an extracellular oxidative metabolism identifiable by its isotope signature

Soil heterotrophic respiration is a major determinant of the carbon (C) cycle and its interactions with climate. Given the complexity of the respiratory machinery, it is traditionally considered that oxidation of organic C into carbon dioxide (CO2) strictly results from intracellular metabolic processes. Here we show that C mineralization can operate in soils deprived of all observable cellular forms. Moreover, the process responsible for CO2 emissions in sterilized soils induced a strong C isotope fractionation (up to 50 ‰) incompatible with respiration of cellular origin. The supply of 13C glucose in sterilized soil led to the release of 13CO2 suggesting the presence of respiratory-like metabolism (glycolysis, decarboxylation reaction, chain of electron transfer) carried out by soil-stabilized enzymes, and by soil mineral and metal catalysts. These findings indicate that CO2 emissions from soils can have two origins: (1) from the well-known respiration of soil heterotrophic microorganisms and (2) from an extracellular oxidative metabolism (EXOMET) or, at least, catabolism. These two metabolisms should be considered separately when studying effects of environmental factors on the C cycle because the likelihood is that they do not obey the same laws and they respond differently to abiotic factors

Study of Helicobacter pullorum proinflammatory properties on human epithelial cells in vitro

International audienceBackground and aims: Helicobacter pullorum is an enterohepatic Helicobacter species of avian origin detected in patients with acute diarrhoea and inflammatory bowel disease. The aim of the present study was to determine whether H pullorum exerts a direct effect on human intestinal epithelial cells in vitro and to characterise the bacterial mechanisms and the signalling pathways involved. Materials and methods: The proinflammatory properties of H pullorum from human and avian origins were measured on human gastric (AGS) and intestinal (CaCo-2 and HT-29) epithelial cell lines after co-culture with different H pullorum strains, and the extent of nuclear factor-κB (NF-κB) involvement was determined. Results: All of the H pullorum strains tested stimulated interleukin 8 (IL8) secretion by the three cell lines. Similar results were obtained with heat-killed H pullorum. Incubation of cells with filtered H pullorum culture supernatants did not stimulate IL8 secretion. The same observation was made when bacterial adherence was inhibited by Transwell inserts. H pullorum induced NF-κB activation and rapid nuclear translocation as demonstrated by immunofluorescent staining and cellular fractionation. NF-κB involvement was confirmed by using the specific inhibitor SN50 and small interfering RNA (siRNA) which abolished H pullorum-induced IL8 production. Conclusions: H pullorum strains stimulate IL8 secretion by human gastric and intestinal epithelial cell lines. This effect requires bacterial adherence and probably lipopolysaccharides, and is mediated by NF-κB signalling. The present study strengthens the argument that H pullorum is a potent human pathogen and highlights its putative role in acute and chronic digestive diseases such as inflammatory bowel disease