scholarly journals Identification and Removal of Potential Contaminants in 16S rRNA Gene Sequence Datasets from Low Microbial Biomass Samples: An Example from the Mosquito Gut

2020 ◽  
Author(s):  
Sebastián Diaz ◽  
Juan Sebastián Escobar ◽  
Frank William Avila
Keyword(s):  
Microbial Biomass ◽  
16S Rrna ◽  
Gut Microbiota ◽  
Relative Abundance ◽  
Vector Competence ◽  
Negative Control ◽  
Rrna Gene ◽  
Physiological Processes ◽  
Physiological Relevance ◽  
The Impact

Abstract Background: The bacterial gut microbiota of the female mosquito influences numerous physiological processes, including vector competence. As a low-microbial-biomass ecosystem, mosquito gut tissue is prone to contamination from the laboratory environment and from reagents commonly used to dissect and/or isolate DNA from gut tissue. In this report, we analyze five 16S rRNA datasets, including new data obtained by us, to gain insight into the impact of potential contaminating sequences on the composition, diversity, and structure of the mosquito gut microbial community. Results: We present a clustering-free approach that, based on the relative abundance of amplicon sequence variants (ASVs) in gut and negative control samples , allowed for the identification of candidate contaminating sequences. Some of these sequences belong to bacterial taxa previously identified as common contaminants in metagenomic studies; they have also been identified as part of the mosquito core gut microbiota, with putative physiological relevance for the host. By using different relative abundance cutoffs, we show that contaminating sequences have a significant impact on gut microbiota diversity and structure.Conclusions: The approach presented here allows the identification and removal of purported contaminating sequences in datasets obtained from low-microbial biomass samples. While it was exemplified with the analysis of gut microbiota from mosquitos, it can easily extend to other datasets dealing with similar technical artifacts.

scholarly journals Different analysis strategies of 16S rRNA gene data from rodent studies generate contrasting views of gut bacterial communities associated with diet, health and obesity

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10372
Author(s):  
Jose F. Garcia-Mazcorro ◽  
Jorge R. Kawas ◽  
Cuauhtemoc Licona Cassani ◽  
Susanne Mertens-Talcott ◽  
Giuliana Noratto
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Future Research ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Analysis Strategies ◽  
The Impact

Background One of the main functions of diet is to nurture the gut microbiota and this relationship affects the health of the host. However, different analysis strategies can generate different views on the relative abundance of each microbial taxon, which can affect our conclusions about the significance of diet to gut health in lean and obese subjects. Here we explored the impact of using different analysis strategies to study the gut microbiota in a context of diet, health and obesity. Methods Over 15 million 16S rRNA gene sequences from published studies involving dietary interventions in obese laboratory rodents were analyzed. Three strategies were used to assign the 16S sequences to Operational Taxonomic Units (OTUs) based on the GreenGenes reference OTU sequence files clustered at 97% and 99% similarity. Results Different strategies to select OTUs influenced the relative abundance of all bacterial taxa, but the magnitude of this phenomenon showed a strong study effect. Different taxa showed up to 20% difference in relative abundance within the same study, depending on the analysis strategy. Very few OTUs were shared among the samples. ANOSIM test on unweighted UniFrac distances showed that study, sequencing technique, animal model, and dietary treatment (in that order) were the most important factors explaining the differences in bacterial communities. Except for obesity status, the contribution of diet and other factors to explain the variability in bacterial communities was lower when using weighted UniFrac distances. Predicted functional profile and high-level phenotypes of the microbiota showed that each study was associated with unique features and patterns. Conclusions The results confirm previous findings showing a strong study effect on gut microbial composition and raise concerns about the impact of analysis strategies on the membership and composition of the gut microbiota. This study may be helpful to guide future research aiming to investigate the relationship between diet, health, and the gut microbiota.

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

Impact of a probiotic, inulin, or their combination on the piglets’ microbiota at different intestinal locations

2015 ◽  
Vol 6 (4) ◽  
pp. 473-483 ◽  
Author(s):  
V.A. Sattler ◽  
K. Bayer ◽  
G. Schatzmayr ◽  
A.G. Haslberger ◽  
V. Klose
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Real Time ◽  
Relative Abundance ◽  
Real Time Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Feed Additives ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

scholarly journals Effect of Bifidobacterium thermophilum RBL67 and fructo-oligosaccharides on the gut microbiota in Göttingen minipigs

2015 ◽  
Vol 114 (5) ◽  
pp. 746-755 ◽  
Author(s):  
Sabine A. Tanner ◽  
Christophe Lacroix ◽  
Christophe Del’Homme ◽  
Christoph Jans ◽  
Annina Zihler Berner ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Skim Milk ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Suitable Model ◽  
Cross Sectional ◽  
Bifidobacterium Thermophilum ◽  
The Impact

Modulating the gut microbiota via dietary interventions is a common strategy to enhance the natural defence mechanisms of the host. Several in vitro studies have highlighted the probiotic potential of Bifidobacterium thermophilum RBL67 (RBL67) selected for its anti-Salmonella effects. The present study aimed to investigate the impact of RBL67 alone and combined with fructo-oligosaccharides (FOS) on the gut microbiota of Göttingen minipigs. Minipigs were fed a basal diet supplemented with 8 g/d probiotic powder (1×109 CFU/g in skim milk matrix) (probiotic diet (PRO)), 8 g/d probiotic powder plus 8 g/d FOS (synbiotic diet (SYN)) or 8 g/d skim milk powder (control), following a cross-sectional study design. Faecal and caecal microbiota compositions were analysed with pyrosequencing of 16S rRNA genes and quantitative PCR. Metabolic activity in the caecum and colon was measured by HPLC. 16S rRNA gene amplicon sequencing revealed that minipig faeces show close similarity to pig microbiota. During the treatments and at the time of killing of animals, RBL67 was consistently detected in faeces, caecum and colon at numbers of 105–106 16S rRNA copies/g content after feeding PRO and SYN diets. At the time of killing of animals, significantly higher Bifidobacterium numbers in the caecum and colon of SYN-fed minipigs were measured compared with PRO. Our data indicate that the Göttingen minipig may be a suitable model for gut microbiota research in pigs. Data from this first in vivo study of RBL67 colonisation suggest that the combination with FOS may represent a valuable symbiotic strategy to increase probiotic bacteria levels and survival in gastrointestinal tracts for feed and food applications.

scholarly journals 16S rRNA Sequencing Analysis of the Gut Microbiota in Broiler Chickens Prophylactically Administered with Antimicrobial Agents

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 146 ◽  
Author(s):  
Matteo Cuccato ◽  
Selene Rubiola ◽  
Diana Giannuzzi ◽  
Elena Grego ◽  
Paola Pregel ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Broiler Chickens ◽  
Antimicrobial Agents ◽  
Alpha Diversity ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Poultry Production ◽  
Growth Promoters ◽  
The Impact

In poultry production, gut microbiota (GM) plays a pivotal role and influences different host functions related to the efficiency of production performances. Antimicrobial (AM) use is one of the main factors affecting GM composition and functions. Although several studies have focused their attention on the role of AMs as growth promoters in the modulation of GM in broilers, the consequences of higher AM concentrations administered during prophylactic treatments need to be better elucidated. For this purpose, 16S rRNA gene sequencing was performed to evaluate the impact of different prophylactic AM protocols on the composition and diversity of the broiler GM. Diversity analysis has shown that AM treatment significantly affects alpha diversity in ileum and beta diversity in both ileum and caecum. In ileal samples, the Enterobacteriaceae family has been shown to be particularly affected by AM treatments. AMs have been demonstrated to affect GM composition in broiler. These findings indicate that withdrawal periods were not enough for the restoral of the original GM. Further studies are needed for a better elucidation of the negative effects caused by an altered GM in broilers.

scholarly journals Monosodium Glutamate Induces Changes in Hepatic and Renal Metabolic Profiles and Gut Microbiome of Wistar Rats

Nutrients ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1865
Author(s):  
Kanokwan Nahok ◽  
Jutarop Phetcharaburanin ◽  
Jia V. Li ◽  
Atit Silsirivanit ◽  
Raynoo Thanan ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Gut Microbiome ◽  
Wistar Rats ◽  
Monosodium Glutamate ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Male Wistar Rats ◽  
The Impact

The short- and long-term consumption of monosodium glutamate (MSG) increases urinary pH but the effects on the metabolic pathways in the liver, kidney and the gut microbiota remain unknown. To address this issue, we investigated adult male Wistar rats allocated to receive drinking water with or without 1 g% MSG for 2 weeks (n = 10, each). We performed a Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolomic study of the jejunum, liver, and kidneys, while faecal samples were collected for bacterial DNA extraction to investigate the gut microbiota using 16S rRNA gene sequencing. We observed significant changes in the liver of MSG-treated rats compared to controls in the levels of glucose, pyridoxine, leucine, isoleucine, valine, alanine, kynurenate, and nicotinamide. Among kidney metabolites, the level of trimethylamine (TMA) was increased, and pyridoxine was decreased after MSG-treatment. Sequencing of the 16S rRNA gene revealed that MSG-treated rats had increased Firmicutes, the gut bacteria associated with TMA metabolism, along with decreased Bifidobacterium species. Our data support the impact of MSG consumption on liver and kidney metabolism. Based on the gut microbiome changes, we speculate that TMA and its metabolites such as trimethylamine-N-oxide (TMAO) may be mediators of the effects of MSG on the kidney health.

scholarly journals Salmon Gut Microbiota Correlates With Disease Infection Status: Potential for Monitoring Health in Farmed Animals  

2020 ◽  
Author(s):  
Davide Bozzi ◽  
Jacob A. Rasmussen ◽  
Christian Carøe ◽  
Harald Sveier ◽  
Kristian Nordøy ◽  
...  
Keyword(s):  
Health Status ◽  
Infectious Diseases ◽  
Gut Microbiota ◽  
Relative Abundance ◽  
Rrna Gene ◽  
Host Immune System ◽  
Positive Correlation ◽  
Growth Performances ◽  
Probiotic Treatment ◽  
The Impact

Abstract Background: Infectious diseases cause significant production losses in aquaculture every year. Given the pivotal role played by the gut microbiota in regulating the host immune system, health and physiology, interest has risen in the possibility of controlling the fish health status by modulating the gut microbiota. An altered gut microbiota is often associated with a disease status. However, few studies have examined the association between disease severity and degree of gut dysbiosis, especially when the gut is not the site of the primary infection. Moreover, there is a lack of knowledge on the impact of formalin, a commonly used disinfectant in aquaculture, on the gut microbiome. Here we investigate, through 16S rRNA gene metabarcoding, changes in the distal gut microbiota composition of a captive-reared cohort of Atlantic salmon (Salmo salar L.), in consequence of an external bacterial skin infection and the subsequent formalin treatment.Results: We show that the distal gut of diseased salmon presented a different composition from that of healthy individuals, with an increased relative abundance of strains regarded as opportunistic in sick fish. Conversely, healthy salmon were dominated by a new, yet undescribed, Mycoplasma genus. We also observed a positive correlation between fish weight and Mycoplasma sp. relative abundance, potentially indicating a beneficial effect for its host. Moreover, we observed that the treatment with formalin, while suited to resolve the external infection, was unable to recover the gut microbiota characteristic of healthy fish prior to treatment, potentially compromising the subsequent health status and growth performances of all treated fish.Conclusions: We conclude that infectious diseases have the potential of affecting the host gut microbiota at different body sites and that treatment optimization procedures should account for that. The formalin treatment is not an optimal solution from a holistic perspective, we suggest its coupling with a probiotic treatment aimed at re-establishing the original community. Lastly, we have observed a positive correlation of Mycoplasma sp. with salmon health and growth performances and, while inviting further research on this microorganism, we also encourage further investigations towards its potential utilization as a biomarker for monitoring health in salmon and potentially other farmed fish species.

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

2020 ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas CA Hitch ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
Amanda E Ramer-Tait ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Data Processing ◽  
Relative Abundance ◽  
Large Scale ◽  
Peat Soil ◽  
Rrna Gene ◽  
The Impact

Abstract Background: 16S rRNA gene amplicon sequencing is a very popular approach for studying microbiomes. However, varying standards exist for sample and data processing and some basic concepts such as the occurrence of spurious sequences have not been investigated in a comprehensive manner, which was done in the present study. Methods: Using defined communities of bacteria in vitro and in vivo , we searched for sequences not matching the expected species ( i.e. , spurious taxa) and determine a threshold of occurrence relevant for adequate data analysis. The origin of spurious taxa was then investigated via large-scale amplicon queries. We also assessed the impact of varying sequence filtering stringency on diversity readouts in human fecal and peat soil communities. Results: 16S rRNA gene amplicon data processing based on Operational Taxonomic Units (OTUs) clustering and singleton removal, a commonly used approach that discards any taxa represented by only one sequence across all samples, delivered approx. 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa on average. This spurious fraction of taxa was lower based on amplicon sequence variants (ASVs) analysis but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was identified as a threshold below which the analysis of spurious taxa can be prevented to a large extent. Most spurious taxa (approx. 70%) detected in simplified communities occurred in samples multiplexed in the same sequencing run and were present in only one of ten runs. Use of the 0.25% relative abundance threshold decreased the coefficient of variations calculated on richness in the same six human fecal samples across seven sequencing runs by 38% compared with singleton filtering. The output of beta -diversity analyses of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparing samples. Importantly, major findings were confirmed by using data generated in a second sequencing facility. Conclusions: Handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. A threshold of relative abundance of 0.25% is more appropriate than singleton removal, although study-specific analysis strategies are mandatory. We propose the concept of effective richness, which will help comparing results across studies.

scholarly journals Age-Related Differences in the Gut Microbiome of Rhesus Macaques

2020 ◽  
Vol 75 (7) ◽  
pp. 1293-1298 ◽  
Author(s):  
Julie Adriansjach ◽  
Scott T Baum ◽  
Elliot J Lefkowitz ◽  
William J Van Der Pol ◽  
Thomas W Buford ◽  
...  
Keyword(s):  
Sex Differences ◽  
Gut Microbiota ◽  
Relative Abundance ◽  
Rhesus Macaques ◽  
Alpha Diversity ◽  
Multiple Comparisons ◽  
Rrna Gene ◽  
Alpha And Beta Diversity ◽  
Age Related ◽  
The Impact

Abstract Aging is a multifactorial process characterized by progressive changes in gut physiology and the intestinal mucosal immune system. These changes, along with alterations in lifestyle, diet, nutrition, inflammation and immune function alter both composition and stability of the gut microbiota. Given the impact of environmental influences on the gut microbiota, animal models are particularly useful in this field. To understand the relationship between the gut microbiota and aging in nonhuman primates, we collected fecal samples from 20 male and 20 female rhesus macaques (Macaca mulatta), across the natural macaque age range, for 16S rRNA gene analyses. Operational taxonomic units were then grouped together to summarize taxon abundance at different hierarchical levels of classification and alpha- and beta-diversity were calculated. There were no age or sex differences in alpha diversity. At the phylum level, relative abundance of Proteobacteria and Firmicutes and Firmicutes to Bacteriodetes ratio were different between age groups though significance disappeared after correction for multiple comparisons. At the class level, relative abundance of Firmicutes_Bacilli decreased and Proteobacteria_Alphaproteobacteria and Proteobacteria_Betaproteobacteria increased with each successively older group. Only differences in Firmicutes_Bacilli remained significant after correction for multiple comparisons. No sex differences were identified in relative abundances after correction for multiple comparisons. Our results are not surprising given the known impact of environmental factors on the gut microbiota.

scholarly journals Characterization of the gut microbiota in the Anjak (Schizocypris altidorsalis), a native fish species of Iran

2019 ◽  
Author(s):  
Mahdi Ghanbari ◽  
Mansooreh Jami ◽  
Hadi Shahraki ◽  
Konrad J. Domig
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Fish Species ◽  
River System ◽  
Rrna Gene ◽  
Physiological Relevance ◽  
Commercially Important ◽  
Quality Filtering ◽  
First Time

AbstractThe antiquity, diversity and dietary variations among fish species present an exceptional opportunity for understanding the variety and nature of symbioses in vertebrate gut microbial communities. In this study for the first time the composition of the gut bacterial communities in the anjak was surveyed by 16S rRNA gene 454-pyrosequencing. Anjak (Schizocypris altidorsalis), is a native benthopelagic and commercially important fish species in Hamun Lake and Hirmand River system in Sistan basin, Iran. Thirteen 16S rRNA PCR libraries were generated, representing 13 samples of Anjak gut microbiota. After quality filtering, a total of 137 978 read pairs remained from the V3–V6 16S rRNA gene regions, each measuring 680 bp, and were assigned to bacterial OTU’s at a minimum sequence homology of 97%, resulted in total 608 OTUs. The results provided evidence of the presence of a very diverse microbial community present in this fish. The majority of sequences belonged to members of the Firmicutes; however, members of the Proteobacteria, Tenericutes, Bacteroidetes and Cyanobacteria were also well represented in the anjak gut microbiota. The most abundant classes recorded in the samples were uncultured CK-1C4-19 class of Tenericutes, Flavobacteriia, α-Proteobacteria, Erysipelotrichi, Synechococcophycideae, γ-Proteobacteria, and Clostridia. The presence of many different types of bacteria in the anjak is not unexpected since it has been shown that eukaryotes with a detritivorous and planktivorous diet have a higher microbial diversity compared to omnivorous and herbivorous fish species. Many of these microbes might be of high physiological relevance for snow trout and may play key roles in the functioning of a healthy gut in this fish.

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