Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids.

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 ß-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.

Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks.

The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization.IMPORTANCE Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products.

Manipulating the gut microbiota to maintain health and treat disease.

BACKGROUND: The intestinal microbiota composition varies between healthy and diseased individuals for numerous diseases. Although any cause or effect relationship between the alterations in the gut microbiota and disease is not always clear, targeting the intestinal microbiota might offer new possibilities for prevention and/or treatment of disease. OBJECTIVE: Here we review some examples of manipulating the intestinal microbiota by prebiotics, probiotics, and fecal microbial transplants. RESULTS: Prebiotics are best known for their ability to increase the number of bifidobacteria. However, specific prebiotics could potentially also stimulate other species they can also stimulate other species associated with health, like Akkermansia muciniphila, Ruminococcus bromii, the Roseburia/Enterococcus rectale group, and Faecalibacterium prausnitzii. Probiotics have beneficial health effects for different diseases and digestive symptoms. These effects can be due to the direct effect of the probiotic bacterium or its products itself, as well as effects of the probiotic on the resident microbiota. Probiotics can influence the microbiota composition as well as the activity of the resident microbiota. Fecal microbial transplants are a drastic intervention in the gut microbiota, aiming for total replacement of one microbiota by another. With numerous successful studies related to antibiotic-associated diarrhea and Clostridium difficile infection, the potential of fecal microbial transplants to treat other diseases like inflammatory bowel disease, irritable bowel syndrome, and metabolic and cardiovascular disorders is under investigation. CONCLUSIONS: Improved knowledge on the specific role of gut microbiota in prevention and treatment of disease will help more targeted manipulation of the intestinal microbiota. Further studies are necessary to see the (long term) effects for health of these interventions.

Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a ß-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the ß-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans ß-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Biotics and bacterial function: impact on gut and host health.

The human gut microbiota, the vast community of microbes inhabiting the gastrointestinal tract, plays a pivotal role in maintaining health. Bacteria are the most abundant organism, and the composition of bacterial communities is strongly influenced by diet. Gut bacteria can degrade complex dietary carbohydrates to produce bioactive compounds such as short-chain fatty acids. Such products influence health, by acting on systemic metabolism, or by virtue of anti-inflammatory or anti-carcinogenic properties. The composition of gut bacteria can be altered through overgrowth of enteropathogens (e.g., Campylobacter, Salmonella spp.), leading to dysbiosis of the gut ecosystem, with some species thriving under the altered conditions whereas others decline. Various "biotics" strategies, including prebiotics, probiotics, synbiotics, and postbiotics, contribute to re-establishing balance within the gut microbial ecosystem conferring health benefits. Prebiotics enhance growth of beneficial members of the resident microbial community and can thus prevent pathogen growth by competitive exclusion. Specific probiotics can actively inhibit the growth of pathogens, either through the production of bacteriocins or simply by reducing the gastrointestinal pH making conditions less favorable for pathogen growth. This review discusses the importance of a balanced gut ecosystem, and strategies to maintain it that contribute to human health.

The influence of diet on the gut microbiota.

Diet is a major factor driving the composition and metabolism of the colonic microbiota. The amount, type and balance of the main dietary macronutrients (carbohydrates, proteins and fats) have a great impact on the large intestinal microbiota. The human colon contains a dense population of bacterial cells that outnumber host cells 10-fold. Bacteroidetes, Firmicutes and Actinobacteria are the three major phyla that inhabit the human large intestine and these bacteria possess a fascinating array of enzymes that can degrade complex dietary substrates. Certain colonic bacteria are able to metabolise a remarkable variety of substrates whilst other species carry out more specialised activities, including primary degradation of plant cell walls. Microbial metabolism of dietary carbohydrates results mainly in the formation of short chain fatty acids and gases. The major bacterial fermentation products are acetate, propionate and butyrate; and the production of these tends to lower the colonic pH. These weak acids influence the microbial composition and directly affect host health, with butyrate the preferred energy source for the colonocytes. Certain bacterial species in the colon survive by cross-feeding, using either the breakdown products of complex carbohydrate degradation or fermentation products such as lactic acid for growth. Microbial protein metabolism results in additional fermentation products, some of which are potentially harmful to host health. The current 'omic era promises rapid progress towards understanding how diet can be used to modulate the composition and metabolism of the gut microbiota, allowing researchers to provide informed advice, that should improve long-term health status.

Establishing genetic manipulation for novel strains of human gut bacteria.

Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as Bifidobacterium, Bacteroides and Roseburia. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota.

Evaluation of novel assays to assess the influence of different iron sources on the growth of Clostridium difficile.

The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 µM DPP) and iron-limited (75 µM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO(4), FeCl(3), Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.

Comparison of microbial signatures between paired faecal and rectal biopsy samples from healthy volunteers using next-generation sequencing and culturomics.

BACKGROUND: Faecal samples are frequently used to characterise the gut microbiota in health and disease, yet there is considerable debate about how representative faecal bacterial profiles are of the overall gut community. A particular concern is whether bacterial populations associated with the gut mucosa are properly represented in faecal samples, since these communities are considered critical in the aetiology of gastrointestinal diseases. In this study we compared the profiles of the faecal and mucosal microbiota from ten healthy volunteers using bacterial culturing (culturomics) and next-generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene. Paired fresh rectal biopsies and faecal samples were processed under stringent anaerobic conditions to maintain the viability of the bacteria. Four different sample types were analysed: faecal (F), faecal homogenised (FHg), biopsy tissue (B) and biopsy wash (BW) samples.  RESULTS: There were no significant statistical differences in either bacterial richness or diversity between biopsy washes (BW) and faecal (F) or faecal homogenised (FHg) samples. Principal coordinates analysis of a Bray-Curtis distance matrix generated from sequence variant tables did not show distinct clustering between these samples (PERMANOVA; p = 0.972) but showed strong clustering of samples from individual donors. However, the rectal biopsy tissue (B) samples had a significantly altered bacterial signature with greater abundance of Proteobacteria and Acidobacteria compared to faecal (F) and faecal homogenised (FHg) samples. A total of 528 bacteria encompassing 92 distinct bacterial species were isolated and cultured from a subset of six volunteer samples (biopsy washes and faeces). This included isolation of 22 novel bacterial species. There was significant similarity between the bacterial species grown in anaerobic culture and those identified by 16S rRNA gene sequencing (Spearman correlation; rho = 0.548, p = 0.001). CONCLUSION: This study showed that the bacterial profiles of paired faecal and rectal biopsy wash samples were very similar, validating the use of faecal samples as a convenient surrogate for rectal biopsy-associated microbiota. Anaerobic bacterial culture results showed similar taxonomic patterns to the amplicon sequence analysis disproving the dogma that culture-based methods do not reflect findings of molecular assessments of gut bacterial composition. Video abstract.

Changes in the gut microbiota of Nigerian infants within the first year of life.

The composition of the gut microbiota plays an important role in maintaining the balance between health and disease. However, there is considerably less information on the composition of the gut microbiota of non-Western communities. This study was designed to investigate the evolution in the gut microbiota in a cohort of Nigerian infants within the first year of life. Faecal samples were obtained monthly from 28 infants from birth for one year. The infants had been born by a mix of natural birth and caesarean section and were either breast-fed or mixed fed. Sequencing of the V1-V2 region of the 16S rRNA gene was used to characterise the microbiota. Short chain fatty acids and lactate present in each faecal sample were identified by gas chromatography. Microbial differences were observed between the vaginal and caesarean section delivered infants in samples collected within 7 days of life, although these differences were not observed in later samples. Exclusively breastfed infants had predominance of Ruminococcus gnavus, Collinsella, and Sutterella species. Different Bifidobacterium species dominated breast-fed compared to mixed fed infants. Clostridium, Enterococcus, Roseburia, and Coprococcus species were observed once the infants commenced weaning. Butyrate was first detected when weaning started between months 4-6 in the majority of the infants while total short chain fatty acid concentrations increased, and acetate and lactate remained high following the introduction of solid foods. The observed taxonomic differences in the gut microbiota between Nigerian infants, as well as butyrate production during weaning, were strongly influenced by diet, and not by birthing method. Introduction of local/solid foods encouraged the colonisation and evolution of specific marker organisms associated with carbohydrate metabolism.

Higher total faecal short-chain fatty acid concentrations correlate with increasing proportions of butyrate and decreasing proportions of branched-chain fatty acids across multiple human studies.

Metabolites produced by microbial fermentation in the human intestine, especially short-chain fatty acids (SCFAs), are known to play important roles in colonic and systemic health. Our aim here was to advance our understanding of how and why their concentrations and proportions vary between individuals. We have analysed faecal concentrations of microbial fermentation acids from 10 human volunteer studies, involving 163 subjects, conducted at the Rowett Institute, Aberdeen, UK over a 7-year period. In baseline samples, the % butyrate was significantly higher, whilst % iso-butyrate and % iso-valerate were significantly lower, with increasing total SCFA concentration. The decreasing proportions of iso-butyrate and iso-valerate, derived from amino acid fermentation, suggest that fibre intake was mainly responsible for increased SCFA concentrations. We propose that the increase in % butyrate among faecal SCFA is largely driven by a decrease in colonic pH resulting from higher SCFA concentrations. Consistent with this, both total SCFA and % butyrate increased significantly with decreasing pH across five studies for which faecal pH measurements were available. Colonic pH influences butyrate production through altering the stoichiometry of butyrate formation by butyrate-producing species, resulting in increased acetate uptake and butyrate formation, and facilitating increased relative abundance of butyrate-producing species (notably Roseburia and Eubacterium rectale).

Nutritional influences on the gut microbiota and the consequences for gastrointestinal health.

The human colonic microbiota degrades dietary substrates that are indigestible in the upper GIT (gastrointestinal tract), releasing bacterial metabolites, some of which are important for gut health. Advances in molecular biology techniques have facilitated detailed analyses of the composition of the bacterial community resident in the lower GIT. Such analyses have indicated that more than 500 different bacterial species colonize an individual, and that, although there is much functional consistency in the resident bacterial groups, there is considerable inter-individual variation at the species/strain level. The bacterial community develops during early childhood until it reaches an adult-like composition. Whereas colonization and host factors influence the species composition, dietary factors also have an important impact, with specific bacterial groups changing in response to specific dietary interventions. Since bacterial species have different metabolic activities, specific diets have various consequences for health, dependent on the effect exerted on the bacterial population.

Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics.

In May 2019, the International Scientific Association for Probiotics and Prebiotics (ISAPP) convened a panel of nutritionists, physiologists and microbiologists to review the definition and scope of synbiotics. The panel updated the definition of a synbiotic to "a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host". The panel concluded that defining synbiotics as simply a mixture of probiotics and prebiotics could suppress the innovation of synbiotics that are designed to function cooperatively. Requiring that each component must meet the evidence and dose requirements for probiotics and prebiotics individually could also present an obstacle. Rather, the panel clarified that a complementary synbiotic, which has not been designed so that its component parts function cooperatively, must be composed of a probiotic plus a prebiotic, whereas a synergistic synbiotic does not need to be so. A synergistic synbiotic is a synbiotic for which the substrate is designed to be selectively utilized by the co-administered microorganisms. This Consensus Statement further explores the levels of evidence (existing and required), safety, effects upon targets and implications for stakeholders of the synbiotic concept.

Daily Fermented Whey Consumption Alters the Fecal Short-Chain Fatty Acid Profile in Healthy Adults.

Gut microbiota influences many aspects of host health including immune, metabolic, and gut health. We examined the effect of a fermented whey concentrate (FWC) drink rich in L-(+)-Lactic acid, consumed daily, in 18 healthy men (n = 5) and women (n = 13) in free-living conditions. Objective: The aims of this 6-weeks pilot trial were to (i) identify changes in the gut microbiota composition and fecal short chain fatty acid (SCFA) profile, and (ii) to monitor changes in glucose homeostasis. Results: Total fecal SCFA (mM) concentration remained constant throughout the intervention. Proportionally, there was a significant change in the composition of different SCFAs compared to baseline. Acetate levels were significantly reduced (-6.5%; p < 0.01), coupled to a significant increase in the relative amounts of propionate (+2.2%; p < 0.01) and butyrate (+4.2%; p < 0.01), respectively. No changes in the relative abundance of any specific bacteria were detected. No significant changes were observed in glucose homeostasis in response to an oral glucose tolerance test. Conclusion: Daily consumption of a fermented whey product led to significant changes in fecal SCFA metabolite profile, indicating some potential prebiotic activity. These changes did not result in any detectable differences in microbiota composition. Post-hoc analysis indicated that baseline microbiota composition might be indicative of participants likely to see changes in SCFA levels. However, due to the lack of a control group these findings would need to be verified in a rigorously controlled trial. Future work is also required to identify the biological mechanisms underlying the observed changes in microbiota activity and to explore if these processes can be harnessed to favorably impact host health. Clinical Trial Registration: www.clinicaltrials.gov, identifier NCT03615339; retrospectively registered on 03/08/2018.

Tetracycline resistome of the organic pig gut.

The occurrence of genes conferring resistance to tetracyclines in the organic pig gut was assessed through the metagenomic approach. Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known tet(C), tet(W), and tet(40) genes, as well as novel genes encoding resistance to the tetracyclines minocycline and doxycycline. The latter are different from the known tet genes and are homologous to genes encoding UDP-glucose 4-epimerases, with the domain structure characteristic for these enzymes. The majority of the resistance genes were associated with putative mobile genetic elements. The sequence of a novel 9.7-kb plasmid carrying tet(W) and tet(40) was also identified. Conserved flanking regions identified around the tet(W) and tet(40) genes in our metagenomic library may play a role in genetic exchange of these genes. This is the first report describing the occurrence of tet(40) outside the human intestine. The maintenance of antibiotic resistance genes in apparently antibiotic-free animals is probably due to their presence on mobile genetic elements, the fitness cost of which for the cell is ameliorated during the previous antibiotic selection.

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

Transfer of conjugative elements from rumen and human Firmicutes bacteria to Roseburia inulinivorans.

Studies on Firmicutes bacteria from the gut are hampered by a lack of gene transfer systems. Here the human colonic anaerobe Roseburia inulinivorans A2-194 was shown to be a transfer recipient for two conjugative transposons, Tn1545 from Eubacterium cellulosolvens and TnK10 from Clostridium saccharolyticum K10.

Assessment of metabolic diversity within the intestinal microbiota from healthy humans using combined molecular and cultural approaches.

The human gut harbours a wide range of bacterial communities that play key roles in supplying nutrients and energy to the host through anaerobic fermentation of dietary components and host secretions. This fermentative process involves different functional groups of microorganisms linked in a trophic chain. Although the diversity of the intestinal microbiota has been studied extensively using molecular techniques, the functional aspects of this biodiversity remain mostly unexplored. The aim of the present work was to enumerate the principal metabolic groups of microorganisms involved in the fermentative process in the gut of healthy humans. These functional groups of microorganisms were quantified by a cultural approach, while the taxonomic composition of the microbiota was assessed by in situ hybridization on the same faecal samples. The functional groups of microorganisms that predominated in the gut were the polysaccharide-degrading populations involved in the breakdown of the most readily available exogenous and endogenous substrates and the predominant butyrate-producing species. Most of the functional groups of microorganisms studied appeared to be present at rather similar levels in all healthy volunteers, suggesting that optimal numbers of these various bacterial groups are crucial for efficient gut fermentation, as well as for host nutrition and health. Significant interindividual differences were, however, confirmed with respect to the numbers of methanogenic archaea, filter paper-degrading and acetogenic bacteria and the products formed by lactate-utilizing bacteria.