scholarly journals Blood bacterial DNA, intestinal adenoma and colorectal cancer

2021 ◽  
Author(s):  
Massimiliano Mutignani ◽  
Roberto Penagini ◽  
Giorgio Gargari ◽  
Simone Guglielmetti ◽  
Marcello Cintolo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Healthy Subjects ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Gene Copies ◽  
Intestinal Adenoma

Objective We aimed to investigate the relation of blood bacterial DNA load and profiling with intestinal adenoma (IA) and colorectal cancer (CRC) patients. Design We performed 16S rRNA gene analysis of blood from 100 incident histologically confirmed CRC cases, 100 IA and 100 healthy subjects, matched to cases by centre, sex and age. Bacterial load was analysed using multiple conditional logistic regression. Differences in terms of abundance of bacteria between groups were estimated through analysis based on negative binomial distribution normalization. Random Forest was applied to predict the group assignment. Results We found an overrepresentation of blood 16S rRNA gene copies in colon cancer as compared to tumor-free controls (IA and healthy subjects). The odds ratio of colon cancer for the highest versus the lowest three quintiles of gene copies was 2.62. (95% confidence interval=1.22-5.65). No difference was found for rectal cancer and IA. For high 16S rRNA, community diversity was higher in colon cancers than controls. CRC cases had an enrichment of Peptostreptococcaceae and Acetobacteriaceae and a reduced abundance of Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae. Identified variables predicted CRC from control and IA patients with an accuracy of 0.70. Conclusion Colon cancer patients had a higher DNA bacterial load and a different bacterial profiling as compared to healthy subjects, IA and rectal cancers, indicating a higher passage of bacteria from gastrointestinal tract to bloodstream. Further studies are needed to confirm this result and exploit it to conceive new non-invasive techniques for an early diagnosis of CRC.

scholarly journals Blood Bacterial DNA Load and Profiling Differ in Colorectal Cancer Patients Compared to Tumor-Free Controls

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6363
Author(s):  
Massimiliano Mutignani ◽  
Roberto Penagini ◽  
Giorgio Gargari ◽  
Simone Guglielmetti ◽  
Marcello Cintolo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Negative Binomial ◽  
Conditional Logistic Regression ◽  
Community Diversity ◽  
Rrna Gene ◽  
Barrier Dysfunction ◽  
Gene Copies

Inflammation and immunity are linked to intestinal adenoma (IA) and colorectal cancer (CRC) development. The gut microbiota is associated with CRC risk. Epithelial barrier dysfunction can occur, possibly leading to increased intestinal permeability in CRC patients. We conducted a case-control study including 100 incident histologically confirmed CRC cases, and 100 IA and 100 healthy subjects, matched to cases by center, sex and age. We performed 16S rRNA gene analysis of blood and applied conditional logistic regression. Further analyses were based on negative binomial distribution normalization and Random Forest algorithm. We found an overrepresentation of blood 16S rRNA gene copies in colon cancer as compared to tumor-free controls. For high levels of gene copies, community diversity was higher in colon cancer cases than controls. Bacterial taxa and operational taxonomic unit abundances were different between groups and were able to predict CRC with an accuracy of 0.70. Our data support the hypothesis of a higher passage of bacteria from gastrointestinal tract to bloodstream in colon cancer. This result can be applied on non-invasive diagnostic tests for colon cancer control.

scholarly journals Bacterial DNAemia is associated with serum zonulin levels in older subjects

2020 ◽  
Author(s):  
Giorgio Gargari ◽  
Valentina Taverniti ◽  
Cristian Del Bo’ ◽  
Stefano Bernardi ◽  
Cristina Andres-Lacueva ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Serum Levels ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Gene Copies ◽  
Older Subjects ◽  
Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

scholarly journals Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2080-2091 ◽  
Author(s):  
Anne-Laure Michon ◽  
Fabien Aujoulat ◽  
Laurent Roudière ◽  
Olivier Soulier ◽  
Isabelle Zorgniotti ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Diversity ◽  
Natural Populations ◽  
Variable Region ◽  
Housekeeping Gene ◽  
Single Copy ◽  
Population Based ◽  
Rrna Gene ◽  
Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

scholarly journals Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Vladimir Lazarevic ◽  
Nadia Gaïa ◽  
Myriam Girard ◽  
Jacques Schrenzel
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Rrna Gene

scholarly journals Six species of nontuberculous mycobacteria carry non-identical 16S rRNA gene copies

2018 ◽  
Vol 155 ◽  
pp. 34-36
Author(s):  
Keita Takeda ◽  
Kinuyo Chikamatsu ◽  
Yuriko Igarashi ◽  
Yuta Morishige ◽  
Yoshiro Murase ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nontuberculous Mycobacteria ◽  
Rrna Gene ◽  
Gene Copies

scholarly journals Fecal Short-Chain Fatty Acids Are Not Predictive of Colonic Tumor Status and Cannot Be Predicted Based on Bacterial Community Structure

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Marc A. Sze ◽  
Begüm D. Topçuoğlu ◽  
Nicholas A. Lesniak ◽  
Mack T. Ruffin ◽  
Patrick D. Schloss
Keyword(s):  
Colorectal Cancer ◽  
Fatty Acids ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Data ◽  
Short Chain Fatty Acids ◽  
Tumor Burden ◽  
Short Chain ◽  
Rrna Gene ◽  
Chain Fatty Acids

ABSTRACT Colonic bacterial populations are thought to have a role in the development of colorectal cancer with some protecting against inflammation and others exacerbating inflammation. Short-chain fatty acids (SCFAs) have been shown to have anti-inflammatory properties and are produced in large quantities by colonic bacteria that produce SCFAs by fermenting fiber. We assessed whether there was an association between fecal SCFA concentrations and the presence of colonic adenomas or carcinomas in a cohort of individuals using 16S rRNA gene and metagenomic shotgun sequence data. We measured the fecal concentrations of acetate, propionate, and butyrate within the cohort and found that there were no significant associations between SCFA concentration and tumor status. When we incorporated these concentrations into random forest classification models trained to differentiate between people with healthy colons and those with adenomas or carcinomas, we found that they did not significantly improve the ability of 16S rRNA gene or metagenomic gene sequence-based models to classify individuals. Finally, we generated random forest regression models trained to predict the concentration of each SCFA based on 16S rRNA gene or metagenomic gene sequence data from the same samples. These models performed poorly and were able to explain at most 14% of the observed variation in the SCFA concentrations. These results support the broader epidemiological data that questions the value of fiber consumption for reducing the risks of colorectal cancer. Although other bacterial metabolites may serve as biomarkers to detect adenomas or carcinomas, fecal SCFA concentrations have limited predictive power. IMPORTANCE Considering that colorectal cancer is the third leading cancer-related cause of death within the United States, it is important to detect colorectal tumors early and to prevent the formation of tumors. Short-chain fatty acids (SCFAs) are often used as a surrogate for measuring gut health and for being anticarcinogenic because of their anti-inflammatory properties. We evaluated the fecal SCFA concentrations of a cohort of individuals with different colonic tumor burdens who were previously analyzed to identify microbiome-based biomarkers of tumors. We were unable to find an association between SCFA concentration and tumor burden or use SCFAs to improve our microbiome-based models of classifying people based on their tumor status. Furthermore, we were unable to find an association between the fecal community structure and SCFA concentrations. Our results indicate that the association between fecal SCFAs, the gut microbiome, and tumor burden is weak.

scholarly journals Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

1999 ◽  
Vol 37 (10) ◽  
pp. 3281-3290 ◽  
Author(s):  
Michael M. Tunney ◽  
Sheila Patrick ◽  
Martin D. Curran ◽  
Gordon Ramage ◽  
Donna Hanna ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Immunofluorescence Microscopy ◽  
Joint Infection ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Prosthetic Joint ◽  
Culture Positive ◽  
Bacterial 16S Rrna Gene

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific forPropionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

scholarly journals Microbiota in Exhaled Breath Condensate and the Lung

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Laura Glendinning ◽  
Steven Wright ◽  
Peter Tennant ◽  
Andrew C. Gill ◽  
David Collie ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Exhaled Breath Condensate ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
Exhaled Breath ◽  
Bacterial Dna ◽  
Less Invasive ◽  
Colistimethate Sodium ◽  
Breath Condensate

ABSTRACT The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection. We also sought to assess whether aerosolized antibiotic treatment would influence the lung microbiota and whether this change could be detected in EBC. EBC was collected from 6 conscious sheep and then from the same anesthetized sheep during mechanical ventilation. Following the latter EBC collection, PSB samples were collected from separate sites within each sheep lung. On the subsequent day, each sheep was then treated with nebulized colistimethate sodium. Two days after nebulization, EBC and PSB samples were again collected. Bacterial DNA was quantified using 16S rRNA gene quantitative PCR. The V2-V3 region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina MiSeq. Quality control and operational taxonomic unit (OTU) clustering were performed with mothur. The EBC samples contained significantly less bacterial DNA than the PSB samples. The EBC samples from anesthetized animals clustered separately by their bacterial community compositions in comparison to the PSB samples, and 37 bacterial OTUs were identified as differentially abundant between the two sample types. Despite only low concentrations of colistin being detected in bronchoalveolar lavage fluid, PSB samples were found to differ by their bacterial compositions before and after colistimethate sodium treatment. Our findings indicate that microbiota in EBC samples and PSB samples are not equivalent. IMPORTANCE Sampling of the lung microbiota usually necessitates performing bronchoscopic procedures that involve a hospital visit for human participants and the use of trained staff. The inconvenience and perceived discomfort of participating in this kind of research may deter healthy volunteers and may not be a safe option for patients with advanced lung disease. This study set out to evaluate a less invasive method for collecting lung microbiota samples by comparing samples taken via protected specimen brushings (PSB) to those taken via exhaled breath condensate (EBC) collection. We found that there was less bacterial DNA in EBC samples compared with that in PSB samples and that there were differences between the bacterial communities in the two sample types. We conclude that while EBC and PSB samples do not produce equivalent microbiota samples, the study of the EBC microbiota may still be of interest.

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