Impact of Helicobacter pylori infection on fluid duodenal microbial community structure and microbial metabolic pathways

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16 S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16 S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection changed the relative mean abundance of three phyla (Proteobacteria, Actinobacteria, and TM7) and ten genera (Neisseria, Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

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Impact of Helicobacter Pylori Infection on Duodenal Microbial Community Structure and Microbial Metabolic Pathways

10.21203/rs.3.rs-166718/v2 ◽

2021 ◽

Author(s):

Tadashi Maeda ◽

Hiroaki Zai ◽

Yuto Fukui ◽

Yoshifumi Kato ◽

Eri Kumade ◽

...

Keyword(s):

Helicobacter Pylori ◽

16S Rrna ◽

Metabolic Pathways ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Operational Taxonomic Units ◽

H Pylori ◽

The Impact

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection increased the relative mean abundance of Proteobacteria and Neisseria and decreased the abundance of the two other phyla (Actinobacteria and TM7) and nine genera (Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

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Impact of Helicobacter Pylori Infection on Duodenal Microbial Community Structure and Microbial Metabolic Pathways

10.21203/rs.3.rs-166718/v1 ◽

2021 ◽

Author(s):

Tadashi Maeda ◽

Hiroaki Zai ◽

Yuto Fukui ◽

Yoshifumi Kato ◽

Eri Kumade ◽

...

Keyword(s):

Helicobacter Pylori ◽

Metabolic Pathways ◽

Acid Metabolism ◽

Ketone Bodies ◽

Ether Lipid ◽

Rrna Gene ◽

Related Factors ◽

Positive Group ◽

H Pylori ◽

The Impact

Abstract BackgroundRecent reports suggest that Helicobacter pylori infection may be related to the onset of certain diseases. However, the H. pylori-related factors that play a role in the etiology of these diseases have not been fully elucidated. This study aimed to elucidate the impact of H. pylori infection on the structure of commensal duodenal microbiota and their biofunctions.MethodsForty-seven (20 male, 27 female) subjects who underwent gastric cancer screening were enrolled. Duodenal fluid samples were aspirated from the descending duodenum and analyzed by 16S rRNA gene sequencing.ResultsThirteen subjects were positive for H. pylori, while thirty-four were negative. We observed 1404 bacterial operational taxonomic units from 23 phyla and 253 genera. In the H. pylori-positive group, we observed higher abundances of Proteobacteria and lower abundances of Actinobacteria and TM7 than that in the H. pylori-negative group. The abundances of 10 genera differed significantly between the H. pylori-positive and -negative groups. Microbiota features in the H. pylori-positive group was significantly influenced by 12 taxa primarily belonging to Gammaproteobacteria. Microbial functional annotation collated using the Kyoto Encyclopedia of Genes and Genomes Orthology database showed that 12 microbial metabolic pathways (Synthesis and degradation of ketone bodies, Tryptophan metabolism, N-glycan biosynthesis, Ether lipid metabolism, Lipoarabinomannan (LAM) biosynthesis, Linoleic acid metabolism, alpha-Linolenic acid metabolism, Biotin metabolism, Carotenoid biosynthesis, Phenylpropanoid biosynthesis, Biosynthesis of siderophore group nonribosomal peptides, Biosynthesis of terpenoids and steroids) were significantly affected by H. pylori infection.ConclusionsH. pylori infection disrupted the normal bacterial communities in the duodenum and changed the biofunctions of the commensal microbiota, primarily by upregulating specific metabolic pathways. This alteration may be related to the onset mechanisms of the diseases suspected of being related to H. pylori infection.

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1223. Impact of Helicobacter pylori Infection on Duodenal Microbial Community Structures and Microbial Metabolic Pathways

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1408 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S632-S632

Author(s):

Tadashi Maeda ◽

hiroaki zai ◽

Yuto Fukui ◽

yoshihumi katou ◽

eri kumade ◽

...

Keyword(s):

Helicobacter Pylori ◽

Metabolic Pathways ◽

Amplicon Sequencing ◽

Helicobacter Pylori Infection ◽

Rrna Gene ◽

Negative Group ◽

Positive Group ◽

Principal Factors ◽

Gastric Cancer Screening ◽

H Pylori

Abstract Background Recent reports suggest that Helicobacter pylori infection causes extragastric diseases. However, the onset mechanisms of these diseases have not been fully elucidated, and the factors involved in the onset of these extragastric diseases remain obscure. Methods Forty-seven (20 male, 27 female) subjects who underwent gastric cancer screening were enrolled. Aspirated duodenal fluid samples were collected from the descending duodenum. Samples were analyzed by 16S rRNA gene amplicon sequencing to investigate whether the duodenal microbiota and microbial biofunctions were affected by H. pylori infection. Results Thirteen subjects were H. pylori positive while 34 were negative. We observed 1404 bacterial operational taxonomic units from 23 phyla and 253 genera. In the H. pylori positive group, we observed higher abundance of Proteobacteria and lower abundance of Actinobacteria and TM7 than in the H. pylori negative group. The abundance of 10 genera differed significantly between the H. pylori positive and negative groups. Aspects of microbiota in the H. pylori positive group were significantly influenced by 12 taxa primarily belonging to Gammaproteobacteria, compared with those in the H. pylori negative group. Microbial functional annotation collated using the Kyoto Encyclopedia of Genes and Genomes Orthology database showed that 12 microbial metabolic pathways were significantly affected by H. pylori infection. Conclusion 1. pylori infection disrupted the normal bacterial communities in the duodenum and changed aspects of the commensal microbial functions primarily by upregulating the metabolic pathways. This may be one of the principal factors in the development of extragastric diseases. Disclosures All Authors: No reported disclosures

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Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.8.3166-3170.2005 ◽

2005 ◽

Vol 49 (8) ◽

pp. 3166-3170 ◽

Cited By ~ 27

Author(s):

Erik Glocker ◽

Marco Berning ◽

Monique M. Gerrits ◽

Johannes G. Kusters ◽

Manfred Kist

Keyword(s):

Helicobacter Pylori ◽

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

Tetracycline Resistance ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Screening ◽

H Pylori

ABSTRACT The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.

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Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2621 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2621-2628 ◽

Cited By ~ 141

Author(s):

D E Taylor ◽

Z Ge ◽

D Purych ◽

T Lo ◽

K Hiratsuka

Keyword(s):

Helicobacter Pylori ◽

Natural Transformation ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Donor Strain ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

23S Rrna Genes ◽

H Pylori

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.

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An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities

Scientific Reports ◽

10.1038/s41598-021-89881-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Denise M. O’Sullivan ◽

Ronan M. Doyle ◽

Sasithon Temisak ◽

Nicholas Redshaw ◽

Alexandra S. Whale ◽

...

Keyword(s):

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Microbiome Analysis ◽

The Impact

AbstractDespite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

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Increased Production of Matrix Metalloproteinases in Helicobacter pylori Infection that Stimulates Gastric Cancer Stem Cells

International Journal of Veterinary Science ◽

10.37422/ijvs/20.043 ◽

2020 ◽

Keyword(s):

Gastric Cancer ◽

Stem Cells ◽

Helicobacter Pylori ◽

Cancer Stem Cells ◽

Histopathological Examination ◽

Physiological Saline ◽

Rrna Gene ◽

Peptic Ulcers ◽

Gastric Cancer Stem Cells ◽

H Pylori

Background and aim: Helicobacter pylori (H. pylori) is an incriminated pathogen causing diseases in both animals and humans and considered a zoonotic pathogen. H. pylori infection is considered a cause of gastric cancer, which rests a significant health care challenge. This study analyzes the expression pattern of matrix metalloprotein 2 (MMP-2) in patients with Helicobacter pylori-associated gastritis and the effect of H. pylori on gastric cancer stem cells, as well as study the role of helicon bacteriosis in dog in transmission of H. pylori infection to human. Materials and methods: Fifty-five of each sample (gastric biopsy, blood and stool) were collected from patients suffering from dyspepsia, chronic vomiting and perforated peptic ulcers and also from apparent healthy dogs. The investigation detected H. pylori by serological and histopathological examination. Biopsies were stored in physiological saline for identification of H. pylori by conventional time PCR. MMP-2 and Gastric cancer stem cells were then identified by immunohistochemistry. Results: Serological identification for H. pylori Antigen and Antibodies revealed (63% human, 50% dogs) and (87% human, 90% dogs) respectively were positive. Genotyping of H. pylori based on 16S rRNA gene showed 54.5% of human and 35% of dogs were positive. Immunohistochemistry revealed strong expression of CD44 in H. pylori- associated gastric cancer cases, MMP-2 expression was observed in all neoplastic lesions associated with H. pylori infection. Conclusion: H. pylori infection affects gastric mucosa and induces changes in gastric stem cells altering their differentiation and increased expression of MMP’s and CD44with a resultant potentiation of oncogenic alteration. In addition the up-regulation of both markers could be an instrumental to interpret the origination of gastric cancer.

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A Retrospective Experience of Helicobacter pylori Histology in a Large Sample of Subjects in Northern Italy

Life ◽

10.3390/life11070650 ◽

2021 ◽

Vol 11 (7) ◽

pp. 650

Author(s):

Davide Giuseppe Ribaldone ◽

Carlo Zurlo ◽

Sharmila Fagoonee ◽

Chiara Rosso ◽

Angelo Armandi ◽

...

Keyword(s):

Helicobacter Pylori ◽

Large Population ◽

Population Data ◽

Signet Ring Cell ◽

Current Data ◽

Low Grade ◽

Gastric Cardia Adenocarcinoma ◽

Significant Difference ◽

H Pylori ◽

The Impact

Updated data about the prevalence of Helicobacter pylori (H. pylori) and its correlation with histological results are scarce. The aim of our study was to provide current data on the impact of H. pylori in a third-level endoscopy service. We performed a large, retrospective study analyzing the results of all histological samples of gastroscopy from the year 2019. In total, 1512 subjects were included. The prevalence of H. pylori was 16.8%. A significant difference between the prevalence in subjects born in Italy and those from eastern Europe, south America, or Africa was found (p < 0.0001, p = 0.006, and p = 0.0006, respectively). An association was found between H. pylori and active superficial gastritis (p < 0.0001). Current H. pylori and/or a previous finding of H. pylori was related to antral atrophy (p < 0.0001). Fifteen patients had low-grade dysplasia. There were no statistically significant associations with current or past H. pylori infection. One patient presented gastric cardia adenocarcinoma with regular gastric mucosa. One patient, H. pylori positive, was diagnosed with gastric signet ring cell adenocarcinoma in a setting of diffuse atrophy, without metaplasia.. Our study provides updated, solid (biopsy diagnosis and large population) data on the prevalence of H. pylori infection in a representative region of southern Europe.

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Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11353-4 ◽

2021 ◽

Author(s):

Annemarie Siebert ◽

Katharina Hofmann ◽

Lena Staib ◽

Etienne V. Doll ◽

Siegfried Scherer ◽

...

Keyword(s):

Sample Preparation ◽

Dna Extraction ◽

Raw Milk ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Dna ◽

Microbiome Analysis ◽

Eukaryotic Dna ◽

Selective Lysis ◽

The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

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Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Sandra Reitmeier ◽

Thomas C. A. Hitch ◽

Nicole Treichel ◽

Nikolaos Fikas ◽

Bela Hausmann ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Diversity Analysis ◽

Careful Attention ◽

Operational Taxonomic Units ◽

Basic Concepts ◽

Gnotobiotic Mice ◽

Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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