scholarly journals Microbiome Analysis from Paired Mucosal and Fecal Samples of a Colorectal Cancer Biobank

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3702
Author(s):  
Ulrich Wirth ◽  
Debora Garzetti ◽  
Lara M. Jochum ◽  
Stefanie Spriewald ◽  
Florian Kühn ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Graphical Representation ◽  
Association Studies ◽  
Normal Mucosa ◽  
Rrna Gene ◽  
Storage Duration ◽  
Microbiome Analysis ◽  
Α Diversity ◽  
And Storage ◽  
Microbiome Profiling

The role of gut microbiota in colorectal cancer is subject to extensive research. Before usage of biorepositories for microbiome studies, it is crucial to evaluate technical feasibility of microbiome profiling from various biospecimens. The aim of this study was to assess the feasibility of DNA-extraction and microbiome profiling of samples from different sample sites, tissue sites and storage duration of a colorectal cancer biobank. Mucosa samples, mucosal scrapings and feces as well as different tissue sites (tumor, normal mucosa) were analyzed. 16S rRNA gene-based microbiome profiling with taxonomic assignment was performed on the Illumina MiSeq (Illumina, San Diego, USA) platform from stored snap frozen samples. For statistical analysis, α- and β-diversity measures, PCoA, permutational multivariate analysis of variance and graphical representation were performed. Microbiome analysis could be successfully performed in most of the samples (overall 93.3%) with sufficient numbers of high-quality reads. There were no differences between sample sites, while in some measures significant differences were found between tumor and normal mucosa (α-diversity, Shannon/Simpson Indices p = 0.028/0.027, respectively). Samples stored for up to eight years were used and storage conditions had no significant influence on the results. Tumor and tissue samples of a biobank stored long term can be successfully used for microbiome analysis. As large sample sizes are needed for association studies to evaluate microbial impact on tumorigenesis or progression of colorectal cancer, an already established biorepository may be a useful alternative to prospective clinical studies.

scholarly journals Tumour Microbiome-Based Subtypes of Colorectal Cancer Correlate with Clinical Variables

2020 ◽  
Author(s):  
Barbora Zwinsová ◽  
Vyacheslav Petrov ◽  
Martina Hrivňáková ◽  
Stanislav Smatana ◽  
Lenka Micenková ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gut Microbiome ◽  
Primary Tumour ◽  
Distant Metastases ◽  
Normal Mucosa ◽  
Tnm Staging ◽  
Response To Treatment ◽  
Clinical Variable ◽  
Rrna Gene ◽  
Clinical Variables

Abstract BackgroundLong-term dysbiosis of the gut microbiome has a significant impact on the development, progression and the aggressiveness of colorectal cancer (CRC) and may explain part of the observed heterogeneity of the disease from phenotypic, prognostic and response to treatment perspectives. Although the shifts in gut microbiome in the normal-adenoma-carcinoma sequence have been described, the landscape of microbiome within CRC and its associations with clinical variables remain under-explored. ResultsWe performed 16S rRNA gene sequencing of paired tumour tissue, adjacent visually-normal mucosa and stool swabs of N=186 patients with stage 0-IV CRC to describe the tumour microbiome and its association with clinical variable and to derive tumour microbial subtypes.We identified new genera never previously associated with CRC tumour mucosa (Flavonifractor, Haemophilus, Howardella, Pseudomonas, Sutterella, Treponema 2) or CRC (Actinobacillus, Aggregatibacter, Bergeyella, Phocaeiola, Defluviitaleaceae UCG-011, Massilia, Tyzzerella 4). The bacteria residing on tumour-mucosa were dominated by genera belonging to (potential) oral pathogens. Based on tumour microbial profiles, we stratified CRC patients into three subtypes. The subtypes were significantly associated with prognostic factors such as tumor grade, primary tumour sidedness and TNM staging, with one subtype enriched in tumours with poor prognosis. Further, we inspected the associations of microbiome with clinical variables in a subtype-agnostic setting. The primary tumour-associated clinical variables predominantly correlated with tumour mucosal microbiome, while the presence of local and distant metastases was mostly associated with the stool microbiome.ConclusionsUnderstanding the interactions of the bacteria residing on tumour mucosa within different CRC tumour microbiome subtypes will help to better understand the underlying biological background of the heterogeneity of this disease. Indeed, the tumour microbiome is a possible source of additional integrative markers of CRC patients’ survival and prognosis. We found that CRC microbiome is strongly correlated with clinical variables, but these associations are dependent on the microbial environment (tumour mucosa, normal mucosa, stool). Our study thus identifies limitations of the usage of microbiome composition as marker of CRC progression, suggesting the need of combining several sampling sites (e.g. stool and tumour swabs).

The Regional Specificity of Mucosa-Associated Microbiota in Patients with Distal Colorectal Cancer

Digestion ◽  
2021 ◽  
pp. 1-9
Author(s):  
Yoshihiko Kono ◽  
Ryo Inoue ◽  
Takumi Teratani ◽  
Mineyuki Tojo ◽  
Yuko Kumagai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fusobacterium Nucleatum ◽  
Amplicon Sequencing ◽  
Normal Mucosa ◽  
Rrna Gene ◽  
Normal Tissues ◽  
Streptococcus Anginosus ◽  
Significant Difference ◽  
Regional Specificity ◽  
Tissue Specimens

<b><i>Background/Aims:</i></b> Recent studies have demonstrated that the populations of several microbes are significantly increased in fecal samples from patients with colorectal cancer (CRC), suggesting their involvement in the development of CRC. The aim of this study was to identify microbes which are increased in distal CRCs and to identify the specific location of microbes increased in mucosal tissue around the tumor. <b><i>Methods:</i></b> Tissue specimens were collected from surgical resections of 28 distal CRCs. Five samples were collected from each specimen (location A: tumor, B: adjacent normal mucosa, C: normal mucosa 1 cm proximal to the tumor, D: normal mucosa 3 cm proximally, and E: normal mucosa 6 cm proximally). The microbiota in the sample were analyzed using 16S rRNA gene amplicon sequencing and the relative abundance (RA) of microbiota compared among the 5 locations. <b><i>Results:</i></b> At the genus level, the RA of <i>Fusobacterium</i> and <i>Streptococcus</i> at location A was the highest among the 5 locations, significantly different from that in location E. The dominant species of each genus was <i>Fusobacterium nucleatum</i> and <i>Streptococcus anginosus.</i> The RAs of these species gradually decreased from locations B to E with a statistically significant difference in <i>F. nucleatum</i>. The genus <i>Peptostreptococcus</i> also showed a similar trend, and the RA of <i>Peptostreptococcus stomatis</i> in location A was significantly associated with depth of tumor invasion and tumor size. <b><i>Conclusion:</i></b> Although the clinical relevance is not clear yet, these results suggest that <i>F. nucleatum, S. anginosus</i>, and <i>P. stomatis</i> can spread to the adjacent normal tissues and may change the surrounding microenvironment to support the progression of CRC.

scholarly journals Association between colorectal cancer and Fusobacterium nucleatum and Bacteroides fragilis bacteria in Iranian patients: a preliminary study

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Aref Shariati ◽  
Shabnam Razavi ◽  
Ehsanollah Ghaznavi-Rad ◽  
Behnaz Jahanbin ◽  
Abolfazl Akbari ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Relative Abundance ◽  
Normal Tissue ◽  
Bacteroides Fragilis ◽  
Fusobacterium Nucleatum ◽  
Normal Mucosa ◽  
Clinicopathologic Characteristics ◽  
Tissue Samples ◽  
Adjacent Normal Mucosa ◽  
Iranian Patients

Abstract Background and aim Recent studies have proposed that commensal bacteria might be involved in the development and progression of gastrointestinal disorders such as colorectal cancer (CRC). Therefore, in this study, the relative abundance of Fusobacterium nucleatum, Bacteroides fragilis, Streptococcus bovis/gallolyticus, and Enteropathogenic Escherichia coli (EPEC) in CRC tissues, and their association with clinicopathologic characteristics of CRC was investigated in Iranian patients. Moreover, the role of these bacteria in the CRC-associated mutations including PIK3CA, KRAS, and BRAF was studied. Method To these ends, the noted bacteria were quantified in paired tumors and normal tissue specimens of 30 CRC patients, by TaqMan quantitative Real-Time Polymerase Chain Reaction (qPCR). Next, possible correlations between clinicopathologic factors and mutations in PIK3CA, KRAS, and BRAF genes were analyzed. Results In studied samples, B. fragilis was the most abundant bacteria that was detected in 66 and 60% of paired tumor and normal samples, respectively. Furthermore, 15% of the B. fragilis-positive patients were infected with Enterotoxigenic B. fragilis (ETBF) in both adenocarcinoma and matched adjacent normal samples. F. nucleatum was also identified in 23% of tumors and 13% of adjacent normal tissue samples. Moreover, the relative abundance of these bacteria determined by 2-ΔCT was significantly higher in CRC samples than in adjacent normal mucosa (p < 0.05). On the other hand, our findings indicated that S. gallolyticus and EPEC, compared to adjacent normal mucosa, were not prevalent in CRC tissues. Finally, our results revealed a correlation between F. nucleatum-positive patients and the KRAS mutation (p = 0.02), while analyses did not show any association between bacteria and mutation in PIK3CA and BRAF genes. Conclusion The present study is the first report on the analysis of different bacteria in CRC tissue samples of Iranian patients. Our findings revealed that F. nucleatum and B. fragilis might be linked to CRC. However, any link between gut microbiome dysbiosis and CRC remains unknown.

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

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.

scholarly journals Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zijian Chen ◽  
Zenghong Huang ◽  
Yanxin Luo ◽  
Qi Zou ◽  
Liangliang Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Expression Profiles ◽  
Methylation Status ◽  
Gene Promoter ◽  
Normal Mucosa ◽  
Suppressor Gene ◽  
Survival Outcome ◽  
Prognostic Models

Abstract Background Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). Methods An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). Results The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. Conclusions This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

scholarly journals A Combined Analysis of Gut and Skin Microbiota in Infants with Food Allergy and Atopic Dermatitis: A Pilot Study

Nutrients ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1682
Author(s):  
Ewa Łoś-Rycharska ◽  
Marcin Gołębiewski ◽  
Marcin Sikora ◽  
Tomasz Grzybowski ◽  
Marta Gorzkiewicz ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Food Allergy ◽  
Gut Microbiota ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Healthy Children ◽  
Skin Microbiota ◽  
Fecal Samples ◽  
Healthy Infants ◽  
Α Diversity

The gut microbiota in patients with food allergy, and the skin microbiota in atopic dermatitis patients differ from those of healthy people. We hypothesize that relationships may exist between gut and skin microbiota in patients with allergies. The aim of this study was to determine the possible relationship between gut and skin microbiota in patients with allergies, hence simultaneous analysis of the two compartments of microbiota was performed in infants with and without allergic symptoms. Fifty-nine infants with food allergy and/or atopic dermatitis and 28 healthy children were enrolled in the study. The skin and gut microbiota were evaluated using 16S rRNA gene amplicon sequencing. No significant differences in the α-diversity of dermal or fecal microbiota were observed between allergic and non-allergic infants; however, a significant relationship was found between bacterial community structure and allergy phenotypes, especially in the fecal samples. Certain clinical conditions were associated with characteristic bacterial taxa in the skin and gut microbiota. Positive correlations were found between skin and fecal samples in the abundance of Gemella among allergic infants, and Lactobacillus and Bacteroides among healthy infants. Although infants with allergies and healthy infants demonstrate microbiota with similar α-diversity, some differences in β-diversity and bacterial species abundance can be seen, which may depend on the phenotype of the allergy. For some organisms, their abundance in skin and feces samples may be correlated, and these correlations might serve as indicators of the host’s allergic state.

scholarly journals Pre-digest of unprotected DNA by Benzonase improves the representation of living skin bacteria and efficiently depletes host DNA

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yacine Amar ◽  
Ilias Lagkouvardos ◽  
Rafaela L. Silva ◽  
Oluwaseun Ayodeji Ishola ◽  
Bärbel U. Foesel ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Bacterial Load ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Skin Microbiome ◽  
Mock Community ◽  
Skin Bacteria ◽  
Α Diversity ◽  
Microbial Turnover ◽  
Microbial Dna

Abstract Background The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities. Results We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 105 CFU and that Benzonase digest is not sufficient to overcome this bias. Conclusions The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. Graphical abstract

Gut microbiome of the emerald ash borer, Agrilus planipennis Fairmaire, and its relationship with insect population density

2020 ◽  
Vol 96 (8) ◽  
Author(s):  
Judith Mogouong ◽  
Philippe Constant ◽  
Robert Lavallée ◽  
Claude Guertin
Keyword(s):  
Population Density ◽  
Gut Microbiome ◽  
Emerald Ash Borer ◽  
Agrilus Planipennis ◽  
Economic Damage ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Local Conditions ◽  
Β Diversity ◽  
Α Diversity

ABSTRACT The gut microbial communities of beetles play crucial roles in their adaptive capacities. Environmental factors such as temperature or nutrition naturally affect the insect microbiome, but a shift in local conditions like the population density on a host tree could also lead to changes in the microbiota. The emerald ash borer (EAB), Agrilus planipennis Fairmaire, is an exotic wood borer that causes environmental and economic damage to ash trees in North America. This study aimed to describe the taxonomic structure of the EAB gut microbiome and explore its potential relationship with borer population size. The number of EAB adults collected per tree through a 75 km transect from an epicenter allowed the creation of distinct classes of population density. The Gammaproteobacteria and Ascomycota predominated in bacterial and fungal communities respectively, as determined by sequencing of the bacterial 16S rRNA gene and the fungal internal transcribed spacer ITS2. Species richness and diversity of the bacterial community showed significant dependence on population density. Moreover, α-diversity and β-diversity analysis revealed some indicator amplicon sequence variants suggesting that the plasticity of the gut microbiome could be related to the EAB population density in host trees.

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