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.

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.

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.

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.

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.

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.

Towards standards for human fecal sample processing in metagenomic studies.

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

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.

16S sequencing and functional analysis of the fecal microbiome during treatment of newly diagnosed pediatric inflammatory bowel disease.

The human microbiome is of considerable interest to pediatric inflammatory bowel disease (PIBD) researchers with 1 potential mechanism for disease development being aberrant immune handling of the intestinal bacteria. This study analyses the fecal microbiome through treatment in newly diagnosed PIBD patients and compares to cohabiting siblings where possible. Patients were recruited on clinical suspicion of PIBD before diagnosis. Treatment-naïve fecal samples were collected, with further samples at 2 and 6 weeks into treatment. Samples underwent 16S ribosomal ribonucleic acid (RNA) gene sequencing and short-chain fatty acids (SCFAs) analysis, results were analyzed using quantitative-insights-into-microbial-ecology. Six PIBD patients were included in the cohort: 4 Crohn disease (CD), 1 ulcerative colitis (UC), 1 inflammatory bowel disease (IBD) unclassified, and median age 12.6 (range 10-15.1 years); 3 patients had an unaffected healthy sibling recruited. Microbial diversity (observed species/Chao1/Shannon diversity) was reduced in treatment-naïve patients compared to siblings and patients in remission. Principal coordinate analysis using Bray-Curtis dissimilarity and UniFrac revealed microbial shifts in CD over the treatment course. In treatment-naïve PIBD, there was reduction in functional ability for amino acid metabolism and carbohydrate handling compared to controls (P = .038) and patients in remission (P = .027). Metabolic function returned to normal after remission was achieved. SCFA revealed consistent detection of lactate in treatment-naïve samples. This study adds in-depth 16S rRNA sequencing analysis on a small longitudinal cohort to the literature and includes sibling controls and patients with UC/IBD unclassified. It highlights the initial dysbiosis, reduced diversity, altered functional potential, and subsequent shifts in bacteria from diagnosis over time to remission.

First-Pass Meconium Samples from Healthy Term Vaginally-Delivered Neonates: An Analysis of the Microbiota.

BACKGROUND: Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility. METHODS: Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples. RESULTS: The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37-40 weeks gestation. 8/15 were male, mean birth weight was 3.4 kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2-41.8 x 10(4) cells/g with a mean of 15.4 x 10(4) cells/g. (The limit of detection for automated counting is 10(6) cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab. CONCLUSION: This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.

16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

BACKGROUND: Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning. RESULTS: The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers. CONCLUSIONS: This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

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.

Links between diet, gut microbiota composition and gut metabolism.

The gut microbiota and its metabolic products interact with the host in many different ways, influencing gut homoeostasis and health outcomes. The species composition of the gut microbiota has been shown to respond to dietary change, determined by competition for substrates and by tolerance of gut conditions. Meanwhile, the metabolic outputs of the microbiota, such as SCFA, are influenced both by the supply of dietary components and via diet-mediated changes in microbiota composition. There has been significant progress in identifying the phylogenetic distribution of pathways responsible for formation of particular metabolites among human colonic bacteria, based on combining cultural microbiology and sequence-based approaches. Formation of butyrate and propionate from hexose sugars, for example, can be ascribed to different bacterial groups, although propionate can be formed via alternative pathways from deoxy-sugars and from lactate by a few species. Lactate, which is produced by many gut bacteria in pure culture, can also be utilised by certain Firmicutes to form butyrate, and its consumption may be important for maintaining a stable community. Predicting the impact of diet upon such a complex and interactive system as the human gut microbiota not only requires more information on the component groups involved but, increasingly, the integration of such information through modelling approaches.

Can prebiotics and probiotics improve therapeutic outcomes for undernourished individuals?

It has become clear in recent years that the human intestinal microbiota plays an important role in maintaining health and thus is an attractive target for clinical interventions. Scientists and clinicians have become increasingly interested in assessing the ability of probiotics and prebiotics to enhance the nutritional status of malnourished children, pregnant women, the elderly, and individuals with non-communicable disease-associated malnutrition. A workshop was held by the International Scientific Association for Probiotics and Prebiotics (ISAPP), drawing on the knowledge of experts from industry, medicine, and academia, with the objective to assess the status of our understanding of the link between the microbiome and under-nutrition, specifically in relation to probiotic and prebiotic treatments for under-nourished individuals. These discussions led to four recommendations:   (1) The categories of malnourished individuals need to be differentiated To improve treatment outcomes, subjects should first be categorized based on the cause of malnutrition, additional health-concerns, differences in the gut microbiota, and sociological considerations. (2) Define a baseline "healthy" gut microbiota for each category Altered nutrient requirement (for example, in pregnancy and old age) and individual variation may change what constitutes a healthy gut microbiota for the individual. (3) Perform studies using model systems to test the effectiveness of potential probiotics and prebiotics against these specific categories These should illustrate how certain microbiota profiles can be altered, as members of different categories may respond differently to the same treatment. (4) Perform robust well-designed human studies with probiotics and/or prebiotics, with appropriate, defined primary outcomes and sample size These are critical to show efficacy and understand responder and non-responder outcomes. It is hoped that these recommendations will lead to new approaches that combat malnutrition. This report is the result of discussion during an expert workshop titled "How do the microbiota and probiotics and/or prebiotics influence poor nutritional status?" held during the 10th Meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) in Cork, Ireland from October 1-3, 2012. The complete list of workshop attendees is shown in Table 1.

Phylogenetic distribution of three pathways for propionate production within the human gut microbiota.

Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl-CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota.

Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro.

Dietary macronutrients affect the composition of the gut microbiota, and prebiotics are used to improve and maintain a healthy gut. The impact of prebiotics on dominant gut bacteria other than bifidobacteria, however, is under-researched. Here, we report carbohydrate utilisation patterns for representative butyrate-producing anaerobes, belonging to the Gram-positive Firmicutes families Lachnospiraceae and Ruminococcaceae, by comparison with selected Bacteroides and Bifidobacterium species. Growth assessments using anaerobic Hungate tubes and a new rapid microtitre plate assay were generally in good agreement. The Bacteroides strains tested showed some growth on basal medium with no added carbohydrates, utilising peptides in the growth medium. The butyrate-producing strains exhibited different growth profiles on the substrates, which included starch, inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS) and xylooligosaccharides (XOS). Eleven were able to grow on short-chain FOS, but this number decreased as the chain length of the fructan substrates increased. Long-chain inulin was utilised by Roseburia inulinivorans, but by none of the Bifidobacterium species examined here. XOS was a more selective growth substrate than FOS, with only six of the 11 Firmicutes strains able to use XOS for growth. These results illustrate the selectivity of different prebiotics and help to explain why some are butyrogenic.