Detection of colorectal-cancer-associated bacterial taxa in fecal samples using next-generation sequencing and 19 newly established qPCR assays.

We have previously identified increased levels of distinct bacterial taxa within mucosal biopsies from colorectal cancer (CRC) patients. Following prior research, the aim of this study was to investigate the detection of the same CRC-associated bacteria in fecal samples and to evaluate the suitability of fecal samples as a non-invasive material for the detection of CRC-associated bacteria. Next-generation sequencing (NGS) of the 16S ribosomal RNA (rRNA) V4 region was performed to evaluate the detection of the CRC-associated bacteria in the fecal microbiota of cancer patients, patients with adenomatous polyp and healthy controls. Furthermore, 19 novel species-specific quantitative PCR (qPCR) assays were established to detect the CRC-associated bacteria. Approximately, 75% of the bacterial taxa identified in biopsies were reflected in fecal samples. NGS failed to detect low-abundance CRC-associated taxa in fecal samples, whereas qPCR exhibited high sensitivity and specificity in identifying all targeted taxa. Comparison of fecal microbial composition between the different patient groups showed enrichment of Fusobacterium nucleatum, Parvimonas micra, and Gemella morbillorum in cancer patients. Our findings suggest that low-abundance mucosa-associated bacteria can be detected in fecal samples using sensitive qPCR assays.

Protocol for chromatin immunoprecipitation of histone modifications in frozen adipose tissue.

Chromatin immunoprecipitation (ChIP) combined with sequencing has revolutionized our understanding of gene regulation; however, its application to frozen adipose tissue presents unique challenges due to the high levels of lipid content. Here, we present a protocol for ChIP of histone modifications in human frozen adipose tissue. We describe steps for tissue preparation, chromatin isolation, sonication, pre-clearing of chromatin, and immunoprecipitation. We then detail procedures for elution, crosslink reversal, chromatin purification, quality control, and library synthesis.

Adipose Tissue Insulin Resistance in South Asian and Nordic Women after Gestational Diabetes Mellitus.

South Asians (SAs) have a higher risk of developing type 2 diabetes (T2D) than white Europeans, especially following gestational diabetes mellitus (GDM). Despite similar blood glucose levels post-GDM, SAs exhibit more insulin resistance (IR) than Nordics, though the underlying mechanisms are unclear. This study aimed to assess markers of adipose tissue (AT) IR and liver fat in SA and Nordic women post-GDM. A total of 179 SA and 108 Nordic women in Norway underwent oral glucose tolerance tests 1-3 years post-GDM. We measured metabolic markers and calculated the AT IR index and non-alcoholic fatty liver disease liver fat (NAFLD-LFS) scores. Results showed that normoglycaemic SAs had less non-esterified fatty acid (NEFA) suppression during the test, resembling prediabetes/T2D responses, and higher levels of plasma fetuin-A, CRP, and IL-6 but lower adiponectin, indicating AT inflammation. Furthermore, normoglycaemic SAs had higher NAFLD-LFS scores, lower insulin clearance, and higher peripheral insulin than Nordics, indicating increased AT IR, inflammation, and liver fat in SAs. Higher liver fat markers significantly contributed to the ethnic disparities in glucose metabolism, suggesting a key area for intervention to reduce T2D risk post-GDM in SAs.

Microbial dynamics with CRC progression: a study of the mucosal microbiota at multiple sites in cancers, adenomatous polyps, and healthy controls.

Accumulating evidence has related the gut microbiota to colorectal cancer (CRC). Fusobacterium nucleatum has repeatedly been linked to colorectal tumorigenesis. The aim of this study was to investigate microbial composition in different sampling sites, in order to profile the microbial dynamics with CRC progression. Further, we characterized the tumor-associated F. nucleatum subspecies. Here, we conducted Illumina Miseq next-generation sequencing of the 16S rRNA V4 region in biopsy samples, to investigate microbiota alterations in cancer patients, patients with adenomatous polyp, and healthy controls in Norway. Further, Fusobacterium positive tumor biopsies were subjected to MinION nanopore sequencing of Fusobacterium-specific amplicons to characterize the Fusobacterium species and subspecies. We found enrichment of oral biofilm-associated bacteria, Fusobacterium, Gemella, Parvimonas, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Selenomonas, Porphyromonas, and Prevotella in cancer patients compared to adenomatous polyp patients and control patients. Higher abundance of amplicon sequence variants (ASVs) classified as Phascolarctobacterium, Bacteroides vulgatus, Bacteroides plebeius, Bacteroides eggerthii, Tyzzerella, Desulfovibrio, Frisingicoccus, Eubacterium coprostanoligenes group, and Lachnospiraceae were identified in cancer and adenomatous polyp patients compared to healthy controls. F. nucleatum ssp. animalis was the dominating subspecies. F. nucleatum ssp. nucleatum, F. nucleatum ssp. vincentii, Fusobacterium pseudoperiodonticum, Fusobacterium necrophorum, and Fusobacterium gonidiaformans were identified in five samples. Several biofilm-associated bacteria were enriched at multiple sites in cancer patients. Another group of bacteria was enriched in both cancer and polyps, suggesting that they may have a role in polyp development and possibly early stages of CRC.

Mucosal Gene Transcript Signatures in Treatment Naïve Inflammatory Bowel Disease: A Comparative Analysis of Disease to Symptomatic and Healthy Controls in the European IBD-Character Cohort.

BACKGROUND: Studies of the mucosal transcriptomic landscape have given new insight into the pathogenesis of inflammatory bowel disease (IBD). Recently, the predictive biomarker potential of gene expression signatures has been explored. To further investigate the mucosal gene expression in IBD, we recruited a cohort of treatment naïve patients and compared them to both symptomatic and healthy controls. METHODS: Altogether, 323 subjects were included: Crohn's disease (N = 75), ulcerative colitis (N = 87) and IBD unclassified (N = 3). Additionally, there were two control groups: symptomatic controls (N = 131) and healthy controls (N = 27). Mucosal biopsies were collected during ileocolonoscopy and gene expression in inflamed and non-inflamed mucosa was explored. Gene expression profiling was performed using Agilent G3 Human Gene Expression 860K v3 One-Color microarray. We recorded information about treatment escalation to anti-TNF agents or surgery, and anti-TNF response, to explore predictive opportunities of the mucosal transcriptome. RESULTS: Gene expression profiles in symptomatic controls in whom IBD had been excluded resembled that of IBD patients and diverged from that of healthy controls. In non-inflamed Crohn's disease and ulcerative colitis, gene set enrichment analysis revealed dysregulation of pathways involved in basic cellular biological processes. Mitochondria-associated pathways were dysregulated both in non-inflamed and inflamed Crohn's disease and ulcerative colitis (>2.6 normalized enrichment scores <-1.8). Gene expression signatures of Crohn's disease and ulcerative colitis did not predict time for treatment escalation (p = 0.175). No significant association was found between gene expression signatures and anti-TNF response. CONCLUSION: Non-inflamed samples are probably superior to inflamed samples when exploring gene expression signatures in IBD and might reveal underlying mechanisms central for disease initiation. The gene expression signatures of the control groups were related to if they were symptomatic or not, which may have important implications for future study designs.

Associations of Serum Uromodulin and Urinary Epidermal Growth Factor with Measured Glomerular Filtration Rate and Interstitial Fibrosis in Kidney Transplantation.

INTRODUCTION: Noninvasive biomarkers that reflect tubular health and allow early recognition of accelerated graft fibrosis development are warranted. Serum uromodulin (sUmod) and urinary epidermal growth factor (uEGF) originate from kidney tubules and may reflect functional nephron mass. The aim of this study was to investigate the associations between sUmod and uEGF with measured glomerular filtration rate (mGFR) and kidney allograft interstitial fibrosis percentage (IF%) score. METHODS: sUmod and uEGF measurements, mGFR by iohexol-clearance and kidney allograft biopsies were obtained from kidney transplant recipients (KTRs) included in the Omega-3 fatty acids in Renal Transplantation (ORENTRA) trial at 8 weeks (baseline) and at 1 year after transplantation (end of study). Associations were analyzed with univariable and multivariable linear regression. RESULTS: Ninety patients at baseline and 48 patients at end of study had complete study variable assessments. uEGF normalized to urinary creatinine (uEGF/Cr) was associated with mGFR both at baseline (standardized ß-coefficient [Std. ß-coeff] = 0.457 [p = <0.001]) and at end of study (Std. ß-coeff = 0.637 [p = <0.001]). sUmod was only associated with mGFR at end of study (Std. ß-coeff = 0.443 [p = 0.002]). uEGF/Cr, sUmod, and mGFR were associated with graft IF% score both at baseline (Std. ß-coeff = -0.349 [p = 0.001], -0.274 [p = 0.009] and -0.289 [p = 0.006], respectively) and at end of study (Std. ß-coeff = -0.365 [p = 0.011], -0.347 [p = 0.016] and -0.405 [p = 0.004], respectively). The results remained largely unchanged in multivariable analysis. CONCLUSION: uEGF/Cr and sUmod were associated with mGFR and graft IF% score. Our results indicate a possible role of uEGF/Cr and sUmod in the follow-up of KTRs.

Marine n-3 Polyunsaturated Fatty Acids and Cellular Senescence Markers in Incident Kidney Transplant Recipients: The Omega-3 Fatty Acids in Renal Transplantation (ORENTRA) Randomized Clinical Trial.

RATIONALE & OBJECTIVE: Deterioration of kidney graft function is associated with accelerated cellular senescence. Marine n-3 polyunsaturated fatty acids (PUFAs) have favorable properties that may counteract cellular senescence development and damage caused by the senescence-associated secretory phenotype (SASP) secretome. Our objective was to investigate the potential effects of marine n-3 PUFA supplementation on the SASP secretome in kidney transplant recipients. STUDY DESIGN: Exploratory substudy of the Omega-3 Fatty Acids in Renal Transplantation trial. SETTING & PARTICIPANTS: Adult kidney transplant recipients with a functional kidney graft (defined as having an estimated glomerular filtration rate of >30 mL/min/1.73 m2) 8 weeks after engraftment were included in this study conducted in Norway. ANALYTICAL APPROACH: The intervention consisted of 2.6 g of a marine n-3 PUFA or olive oil (placebo) daily for 44 weeks. The outcome was a predefined panel of SASP components in the plasma and urine. RESULTS: A total of 132 patients were enrolled in the Omega-3 Fatty Acids in Renal Transplantation trial, and 66 patients were allocated to receive either the study drug or placebo. The intervention with the marine n-3 PUFA was associated with reduced plasma levels of granulocyte colony-stimulating factor, interleukin 1α, macrophage inflammatory protein 1α, matrix metalloproteinase (MMP)-1, and MMP-13 compared with the intervention in the control group. LIMITATIONS: Post hoc analysis. CONCLUSIONS: The results suggest that marine n-3 PUFA supplementation has mitigating effects on the plasma SASP components granulocyte colony-stimulating factor, interleukin 1α, macrophage inflammatory protein 1α, MMP-1, and MMP-13 in kidney transplant recipients. Future studies with kidney transplant recipients in maintenance phase, combined with an evaluation of cellular senescence markers in kidney transplant biopsies, are needed to further elucidate the potential antisenescent effect of marine n-3 PUFAs. This trial is registered as NCT01744067.

Fecal microbiota profiles in treatment-naïve pediatric inflammatory bowel disease - associations with disease phenotype, treatment, and outcome.

PURPOSE: Imbalance in the microbiota, dysbiosis, has been identified in inflammatory bowel disease (IBD). We explored the fecal microbiota in pediatric patients with treatment-naïve IBD, non-IBD patients with gastrointestinal symptoms and healthy children, its relation to IBD subgroups, and treatment outcomes. PATIENTS AND METHODS: Fecal samples were collected from 235 children below 18 years of age. Eighty children had Crohn's disease (CD), 27 ulcerative colitis (UC), 3 IBD unclassified, 50 were non-IBD symptomatic patients, and 75 were healthy. The bacterial abundance of 54 predefined DNA markers was measured with a 16S rRNA DNA-based test using GA-Map™ technology at diagnosis and after therapy in IBD patients. RESULTS: Bacterial abundance was similarly reduced in IBD and non-IBD patients in 51 of 54 markers compared to healthy patients (P<0.001). Only Prevotella was more abundant in patients (P<0.01). IBD patients with ileocolitis or total colitis had more Ruminococcus gnavus (P=0.02) than patients with colonic CD or left-sided UC. CD patients with upper gastrointestinal manifestations had higher Veillonella abundance (P<0.01). IBD patients (58%) who received biologic therapy had lower baseline Firmicutes and Mycoplasma hominis abundance (P<0.01) than conventionally treated. High Proteobacteria abundance was associated with stricturing/penetrating CD, surgery (P<0.01), and nonmucosal healing (P<0.03). Low Faecalibacterium prausnitzii abundance was associated with prior antibiotic therapy (P=0.001), surgery (P=0.02), and nonmucosal healing (P<0.03). After therapy, IBD patients had unchanged dysbiosis. CONCLUSION: Fecal microbiota profiles differentiated IBD and non-IBD symptomatic children from healthy children, but displayed similar dysbiosis in IBD and non-IBD symptomatic patients. Pretreatment fecal microbiota profiles may be of prognostic value and aid in treatment individualization in pediatric IBD as severe dysbiosis was associated with an extensive, complicated phenotype, biologic therapy, and nonmucosal healing. The dysbiosis persisted after therapy, regardless of treatments and mucosal healing.

Cervical microbiota in women with cervical intra-epithelial neoplasia, prior to and after local excisional treatment, a Norwegian cohort study.

BACKGROUND: Local treatment for cervical intraepithelial neoplasia (CIN) by Loop Electrosurgical Excision Procedure (LEEP) has been correlated with reproductive morbidity, while the cervicovaginal microbiota is also known to affect the risk of preterm delivery. CIN and treatment by LEEP might change the cervical microbiota. The main aim of this study was to describe the cervical microbiota before and after LEEP and assess its associaton with cone depth and HPV persistence. Further, we aimed to compare the microbiota to references with normal cervical cytology. METHODS: Between 2005 and 2007, we prospectively identified 89 women planned for LEEP in a Norwegian hospital and recruited 100 references with a normal cervical cytology. Endocervical swabs were collected prior to treatment and at six (n = 77) and 12 months (n = 72) post LEEP for bacterial culture and PCR, and post LEEP for DNA testing for human papillomavirus (HPV). We compared the cervical microbiota composition before and after treatment and between women planned for LEEP vs references. RESULTS: There was a reduction in the number of non-Lactobacillus bacterial species six and 12 months after LEEP compared to before treatment and a tendency towards a concomitant increase in Lactobacillus. No association between the detection of cervical bacteria, HPV persistence or cone depth was found. Women planned for LEEP carried significantly more Bacteroides spp., Gardnerella vaginalis, Mycoplasma hominis and Ureaplasma parvum as well as a greater number of bacterial species than the references. CONCLUSIONS: Local excisional treatment appears to alter the cervical microbiota towards a less diverse microbiota. Women with CIN have a more diverse cervical microbiota compared to women with normal cervical cytology.

The prevalence and transcriptional activity of the mucosal microbiota of ulcerative colitis patients.

Active microbes likely have larger impact on gut health status compared to inactive or dormant microbes. We investigate the composition of active and total mucosal microbiota of treatment-naïve ulcerative colitis (UC) patients to determine the microbial picture at the start-up phase of disease, using both a 16S rRNA transcript and gene amplicon sequencing. DNA and RNA were isolated from the same mucosal colonic biopsies. Our aim was to identify active microbial members of the microbiota in early stages of disease and reveal which members are present, but do not act as major players. We demonstrated differences in active and total microbiota of UC patients when comparing inflamed to non-inflamed tissue. Several taxa, among them the Proteobacteria phyla and families therein, revealed lower transcriptional activity despite a high presence. The Bifidobacteriaceae family of the Actinobacteria phylum showed lower abundance in the active microbiota, although no difference in presence was detected. The most abundant microbiota members of the inflamed tissue in UC patients were not the most active. Knowledge of active members of microbiota in UC patients could enhance our understanding of disease etiology. The active microbial community composition did not deviate from the total when comparing UC patients to non-IBD controls.

Outer membrane phospholipase A's roles in Helicobacter pylori acid adaptation.

BACKGROUND: The pH of the human gastric mucosa varies around 2.5 so that only bacteria with strong acidic stress tolerance can colonize it. The ulcer causing Helicobacter pylori thrives in the gastric mucosa. We analyse the roles of the key outer membrane protein OMPLA in its roles in acid tolerance. RESULTS: The homology model of Helicobacter pylori outer membrane phospholipase A (OMPLA) reveals a twelve stranded ß-barrel with a pore that allows molecules to pass with a diameter up to 4 Å. Structure based multiple sequence alignments revealed the functional roles of many amino acids, and led to the suggestion that OMPLA has multiple functions. Besides its role as phospholipase it lets urea enter and ammonium exit the periplasm. Combined with an extensive literature study, our work leads to a comprehensive model for H. pylori's acid tolerance. This model is based on the conversion of urea into ammonium, and it includes multiple roles for OMPLA and involves two hitherto little studied membrane channels in the OMPLA operon. CONCLUSION: The three-dimensional model of OMPLA predicts a transmembrane pore that can aid H. pylori's acid tolerance through urea influx and ammonium efflux. After urea passes through OMPLA into the periplasm, it passes through the pH-gated inner membrane channel UreI into the cytoplasm where urease hydrolyses it into NH3 and CO2. Most of the NH3 becomes NH4+ that is likely to need an inner membrane channel to reach the periplasm. Two genes that are co-regulated with OMPLA in gastric Helicobacter operons could aid this transport. The NH4+ that might leave the cell through the OMPLA pore has been implicated in H. pylor's pathogenesis.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

BACKGROUND: Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. RESULTS: A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. CONCLUSIONS: The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

In Silico Structure and Sequence Analysis of Bacterial Porins and Specific Diffusion Channels for Hydrophilic Molecules: Conservation, Multimericity and Multifunctionality.

Diffusion channels are involved in the selective uptake of nutrients and form the largest outer membrane protein (OMP) family in Gram-negative bacteria. Differences in pore size and amino acid composition contribute to the specificity. Structure-based multiple sequence alignments shed light on the structure-function relations for all eight subclasses. Entropy-variability analysis results are correlated to known structural and functional aspects, such as structural integrity, multimericity, specificity and biological niche adaptation. The high mutation rate in their surface-exposed loops is likely an important mechanism for host immune system evasion. Multiple sequence alignments for each subclass revealed conserved residue positions that are involved in substrate recognition and specificity. An analysis of monomeric protein channels revealed particular sequence patterns of amino acids that were observed in other classes at multimeric interfaces. This adds to the emerging evidence that all members of the family exist in a multimeric state. Our findings are important for understanding the role of members of this family in a wide range of bacterial processes, including bacterial food uptake, survival and adaptation mechanisms.

Up-regulation of CLDN1 in gastric cancer is correlated with reduced survival.

BACKGROUND: The genetic changes in gastric adenocarcinoma are extremely complex and reliable tumor markers have not yet been identified. There are also remarkable geographical differences in the distribution of this disease. Our aim was to identify the most differentially regulated genes in 20 gastric adenocarcinomas from a Norwegian selection, compared to matched normal mucosa, and we have related our findings to prognosis, survival and chronic Helicobacter pylori infection. METHODS: Biopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 20 patients immediately following surgical resection of the tumor. Whole genome, cDNA microarray analysis was performed on the RNA isolated from the sample pairs to compare the gene expression profiles between the tumor against matched mucosa. The samples were microscopically examined to classify gastritis. The presence of H. pylori was examined using microscopy and immunohistochemistry. RESULTS: 130 genes showed differential regulation above a predefined cut-off level. Interleukin-8 (IL-8) and Claudin-1 (CLDN1) were the most consistently up-regulated genes in the tumors. Very high CLDN1 expression in the tumor was identified as an independent and significant predictor gene of reduced post-operative survival. There were distinctly different expression profiles between the tumor group and the control mucosa group, and the histological subsets of mixed type, diffuse type and intestinal type cancer demonstrated further sub-clustering. Up-regulated genes were mapped to cell-adhesion, collagen-related processes and angiogenesis, whereas normal intestinal functions such as digestion and excretion were associated with down-regulated genes. We relate the current findings to our previous study on the gene response of gastric epithelial cells to H. pylori infection. CONCLUSIONS: CLDN1 was highly up-regulated in gastric cancer, and CLDN1 expression was independently associated with a poor post-operative prognosis, and may have important prognostic value. IL-8 and CLDN1 may represent central links between the gene response seen in acute H. pylori infection of gastric epithelial cells, and ultimately gastric cancer.

USA300 methicillin-resistant Staphylococcus aureus in Norway.

Moen AEF*, Tannaes TM, Leegaard TM. USA300 methicillin-resistant Staphylococcus aureus (MRSA) in Norway. Community-associated methicillin-resistant S. aureus USA300 is known for its ability to disseminate and colonize and the clone has been detected globally. We studied the most populated area in Norway in search for the strain and for signs of endemic establishment. Strain typing techniques such as spa-, SCCmec- and dru-typing, pulsed-field gel electrophoresis and detection of USA300 molecular markers, lukS/F-PV and arcA, were performed. USA300 has been present in the study area since 2003 and has increased in incidence in parallel with the total MRSA incidence. Most USA300 isolates were found in the community, but isolates were also detected in health care institutions.

In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA).

BACKGROUND: In the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) genes, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both intra- and inter-species evolutionary analyses. RESULTS: In this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found. CONCLUSIONS: Our findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.

Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells.

BACKGROUND: The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA⁺ and OMPLA⁻ H. pylori variants. RESULTS: Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment. CONCLUSION: In whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA⁺/⁻ did not affect the acute inflammatory response to H. pylori.

Temporal and spatial diversity of the tap water microbiota in a Norwegian hospital.

We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.

High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease.

Helicobacter pylori phospholipase A (OMPLA) degrades bacterial membrane phospholipids to lysophospholipids. High levels of lysophospholipids are associated with higher hemolytic activity, increased release of urease and vacA and better adherence to epithelial cells in vitro. The phospholipase A gene (pldA) displays phase variation due to a slippage in a homopolymeric tract. The aim of this study was to determine if the relative amount of lysophospholipids in the cell wall is associated with ulcer disease, and to further investigate the significance of pldA phase variation. H. pylori isolates of 40 patients were examined. The relative lysophospholipid content of each isolate was determined and the pldA gene was sequenced. The study indicated that H. pylori can regulate its OMPLA activity by phase variation in the pldA gene or by protein level regulation among phase variants in the pldA 'ON' status. We found a significant difference between the relative amount of lysophospholipids of the ulcer group and the non-ulcer group (p=0.022). When the lysophospholipid/phospholipid ratios were compared with outcome, the OR for ulcer disease was 9.0 (95% CI 1.6-49.4; p=0.014). Isolates with a high OMPLA activity are significantly associated with patients with ulcer disease.

Cholesteryl-6-O-acyl-alpha-D-glucopyranoside of Helicobacter pylori relate to relative lysophospholipid content.

The presence of cholesteryl glucosides and high levels of lysophospholipids are elements making the cell wall of Helicobacter pylori unique. In this study, we have investigated the relationship between lysophospholipid content and cholesteryl glucoside composition of variants of 6 clinical isolates. The samples were characterized by diverse outer membrane phospholipase A activity measured as lysophospholipid content of the cell wall. A pldA negative mutant was also included in the study. Thin-layer chromatography showed that cholesteryl glucosides were present in all samples. However, the distribution of cholesteryl-6-O-acyl-alpha-D-glucopyranoside, cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside varied according to lysophospholipid content. Cholesteryl-6-O-acyl-alpha-D-glucopyranoside was exclusively observed in the isolates/variants with an intact pldA and where a significant amount of lysophospholipids could be demonstrated. High lysophospholipid content destabilizes membranes. The balance between cholesteryl-6-O-acyl-alpha-D-glucopyranoside, cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside in H. pylori is probably important for the stability of the membrane when the lysophospholipid content varies.