Reducing bias in microbiome research: Comparing methods from sample collection to sequencing

BackgroundMicrobiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially available mock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples.MethodsHuman fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls.ResultsSamples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs.ConclusionOur study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecal microbiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation

Reconstructing functional networks in the human intestinal tract using synthetic microbiomes : Systems Biology • Nanobiotechnology

The human intestinal tract harbors one of the most densely populated and open microbial ecosystems. The application of multi-omics approaches has provided insight into a wide array of complex interactions between the various groups of mainly anaerobic colonic microbes as well as the host-microbe dialogue. Integration of multi-omits techniques in cultivation based experiments that vary in complexity from monocultures to synthetic microbial communities identified key metabolic players in the trophic interactions as well as their ecological dynamics. A synergy between these approaches will be of utmost importance to reconstruct the functional interaction networks at the ecosystem level within the human intestinal microbiome. The improved understanding of microbiome functioning at ecosystem level will further aid in developing better predictive models and design of effective microbiome modulation strategies for health benefits.Peer reviewe

Intestinal microbiome landscaping : insight in community assemblage and implications for microbial modulation strategies

High individuality, large complexity and limited understanding of the mechanisms underlying human intestinal microbiome function remain the major challenges for designing beneficial modulation strategies. Exemplified by the analysis of intestinal bacteria in a thousand Western adults, we discuss key concepts of the human intestinal microbiome landscape, i.e. the compositional and functional 'core', the presence of community types and the existence of alternative stable states. Genomic investigation of core taxa revealed functional redundancy, which is expected to stabilize the ecosystem, as well as taxa with specialized functions that have the potential to shape the microbiome landscape. The contrast between Prevotella-and Bacteroides-dominated systems has been well described. However, less known is the effect of not so abundant bacteria, for example, Dialister spp. that have been proposed to exhibit distinct bistable dynamics. Studies employing time-series analysis have highlighted the dynamical variation in the microbiome landscape with and without the effect of defined perturbations, such as the use of antibiotics or dietary changes. We incorporate ecosystem-level observations of the human intestinal microbiota and its keystone species to suggest avenues for designing microbiome modulation strategies to improve host health.Peer reviewe

Flux, Impact, and Fate of Halogenated Xenobiotic Compounds in the Gut

Humans and their associated microbiomes are exposed to numerous xenobiotics through drugs, dietary components, personal care products as well as environmental chemicals. Most of the reciprocal interactions between the microbiota and xenobiotics, such as halogenated compounds, occur within the human gut harboring diverse and dense microbial communities. Here, we provide an overview of the flux of halogenated compounds in the environment, and diverse exposure routes of human microbiota to these compounds. Subsequently, we review the impact of halogenated compounds in perturbing the structure and function of gut microbiota and host cells. In turn, cultivation-dependent and metagenomic surveys of dehalogenating genes revealed the potential of the gut microbiota to chemically alter halogenated xenobiotics and impact their fate. Finally, we provide an outlook for future research to draw attention and attract interest to study the bidirectional impact of halogenated and other xenobiotic compounds and the gut microbiota.Peer reviewe

Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity

BACKGROUND: The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool. FINDINGS: The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation. CONCLUSIONS: Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact

Inter-species Metabolic Interactions in an In-vitro Minimal Human Gut Microbiome of Core Bacteria

Knowledge of the functional roles and interspecies interactions are crucial for improving our understanding of the human intestinal microbiome in health and disease. However, the complexity of the human intestinal microbiome and technical challenges in investigating it pose major challenges. In this proof-of-concept study, we rationally designed, assembled and experimentally tested a synthetic Diet-based Minimal Microbiome (Db-MM) consisting of ten core intestinal bacterial species that together are capable of efficiently converting dietary fibres into short chain fatty acids (SCFAs). Despite their genomic potential for metabolic competition, all ten bacteria coexisted during growth on a mixture of dietary fibres, including pectin, inulin, xylan, cellobiose and starch. By integrated analyses of metabolite production, community composition and metatranscriptomics-based gene expression data, we identified interspecies metabolic interactions leading to production of key SCFAs such as butyrate and propionate. While public goods, such as sugars liberated from colonic fibres, are harvested by non-degraders, some species thrive by cross-feeding on energetically challenging substrates, including the butyrogenic conversion of acetate and lactate. Using a reductionist approach in an in-vitro system combined with functional measurements, our study provides key insights into the complex interspecies metabolic interactions between core intestinal bacterial species.Peer reviewe

Dynamic metabolic interactions and trophic roles of human gut microbes identified using a minimal microbiome exhibiting ecological properties

Microbe-microbe interactions in the human gut are influenced by host-derived glycans and diet. The high complexity of the gut microbiome poses a major challenge for unraveling the metabolic interactions and trophic roles of key microbes. Synthetic minimal microbiomes provide a pragmatic approach to investigate their ecology including metabolic interactions. Here, we rationally designed a synthetic microbiome termed Mucin and Diet based Minimal Microbiome (MDb-MM) by taking into account known physiological features of 16 key bacteria. We combined 16S rRNA gene-based composition analysis, metabolite measurements and metatranscriptomics to investigate community dynamics, stability, inter-species metabolic interactions and their trophic roles. The 16 species co-existed in the in vitro gut ecosystems containing a mixture of complex substrates representing dietary fibers and mucin. The triplicate MDb-MM's followed the Taylor's power law and exhibited strikingly similar ecological and metabolic patterns. The MDb-MM exhibited resistance and resilience to temporal perturbations as evidenced by the abundance and metabolic end products. Microbe-specific temporal dynamics in transcriptional niche overlap and trophic interaction network explained the observed co-existence in a competitive minimal microbiome. Overall, the present study provides crucial insights into the co-existence, metabolic niches and trophic roles of key intestinal microbes in a highly dynamic and competitive in vitro ecosystem.Peer reviewe

Molecular ecology of the yet uncultured bacterial Ct85-cluster in the mammalian gut

In our previous studies on irritable bowel syndrome (IBS) –associated microbiota by molecular methods, we demonstrated that a particular 16S rRNA gene amplicon was more abundant in the feces of healthy subjects or mixed type IBS (IBS-M) –sufferers than in the feces of individuals with diarrhea-type IBS (IBS-D). In the current study, we demonstrated that this, so called Ct85-amplicon, consists of a cluster of very heterogeneous 16S rRNA gene sequences, and defined six 16S rRNA gene types, a to f, within this cluster, each representing a novel species-, genus- or family level taxon. We then designed specific PCR primers for these sequence types, mapped the distribution of the Ct85-cluster sequences and that of the newly defined sequence types in several animal species and compared the sequence types present in the feces of healthy individuals and IBS sufferers using two IBS study cohorts, Finnish and Dutch. Various Ct85-cluster sequence types were detected in the fecal samples of several companion and production animal species with remarkably differing prevalences and abundances. The Ct85 sequence type composition of swine closely resembled that of humans. One of the five types (d) shared between humans and swine was not present in any other animals tested, while one sequence type (b) was found only in human samples. In both IBS study cohorts, one type (e) was more prevalent in healthy individuals than in the IBS-M group. By revealing various sequence types in the widespread Ct85-cluster and their distribution, the results improve our understanding of these uncultured bacteria, which is essential for future efforts to cultivate representatives of the Ct85-cluster and reveal their roles in IBS.Peer reviewe

Unveiling the impact of STEC infecting phages on the colon microbiota using an in vitro fermentation model

(Bacterio)phages are considered safe for humans consumption, being regard as excellent biocontrol tools to prevent foodborne pathogens spread. Phages major advantage is their inherent specificity towards a bacterial species, yet some reports have shown phages ability to evolve to infect different hosts when transiting the gastrointestinal tract (GIT). And so, it is of extreme importance to understand the safety outcome of using phages as biocontrol agents in food, with particular interest in the ones that target species from Enterobacteriaceae family, commonly found in the human GIT microbiota. In this study, the impact of a phage infecting Shiga toxin-producing Escherichia coli (STEC), named E. coli phage vB_EcoS_Ace (Ace), towards the colon microbiota was investigated. An in vitro batch fermentation model was used, and the inoculum was the fecal material of three healthy donors. Fermentations metabolome was analyzed through GC and HPLC, and the concentration of both phage Ace and STEC strain were monitored along time (up to 24h). The interference with the gut microbiota composition and functional potential was assessed by shot gun metagenomics. We observed an increase in phage titre only when the host was present, suggesting that there was no other suitable host within the different microbiotas used. Also, the microbiotas composition did not alter when phage Ace was added. Nevertheless, the attenuated version of STEC strain did indeed create some perturbation in the microbiota, which led to different functional potential. This was corroborated by the differences observed for both gas and short chain fatty acid acids dynamics. The microbiotas individuality was an important factor for the observed perturbations. Moreover, phage Ace revealed to be a safe phage when intended to be used as a biocontrol agent for food products. Also, we concluded that the in vitro fermentation model is a reliable, easy, and nonexpensive safety screening methodology for phages.info:eu-repo/semantics/publishedVersio

An in vitro fermentation model to study the impact of bacteriophages targeting shiga toxin-encoding escherichia coli on the colonic microbiota

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae. Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.info:eu-repo/semantics/publishedVersio