Developments in understanding and applying prebiotics in research and practice-an ISAPP conference paper.

AIMS: The concept of using specific dietary components to selectively modulate the gut microbiota to confer a health benefit, defined as prebiotics, originated in 1995. In 2018, a group of scientists met at the International Scientific Association for Probiotics and Prebiotics annual meeting in Singapore to discuss advances in the prebiotic field, focussing on issues affecting functionality, research methodology and geographical differences. METHODS AND RESULTS: The discussion ranged from examining scientific literature supporting the efficacy of established prebiotics, to the prospects for establishing health benefits associated with novel compounds, isolated from different sources. CONCLUSIONS: While many promising candidate prebiotics from across the globe have been highlighted in preliminary research, there are a limited number with both demonstrated mechanism of action and defined health benefits as required to meet the prebiotic definition. Prebiotics are part of a food industry with increasing market sales, yet there are great disparities in regulations in different countries. Identification and commercialization of new prebiotics with unique health benefits means that regulation must improve and remain up-to-date so as not to risk stifling research with potential health benefits for humans and other animals. SIGNIFICANCE AND IMPACT OF STUDY: This summary of the workshop discussions indicates potential avenues for expanding the range of prebiotic substrates, delivery methods to enhance health benefits for the end consumer and guidance to better elucidate their activities in human studies.

Impact of a very low-energy diet on the fecal microbiota of obese individuals.

PURPOSE: Study how the dietary intake affects the fecal microbiota of a group of obese individuals after a 6-week very low-energy diet (VLED) and thereafter during a follow-up period of 5, 8, and 12 months. Additionally, we compared two different methods, fluorescent in situ hybridization (FISH) and real-time PCR (qPCR), for the quantification of fecal samples. METHODS: Sixteen subjects participated in a 12-month dietary intervention which consisted of a VLED high in protein and low in carbohydrates followed by a personalized diet plan, combined with exercise and lifestyle counseling. Fecal samples were analyzed using qPCR, FISH, and denaturing gradient gel electrophoresis. RESULTS: The VLED affected the fecal microbiota, in particular bifidobacteria that decreased approximately two logs compared with the baseline numbers. The change in numbers of the bacterial groups studied followed the dietary intake and not the weight variations during the 12-month intervention. Methanogens were detected in 56% of the participants at every sampling point, regardless of the dietary intake. Moreover, although absolute numbers of comparable bacterial groups were similar between FISH and qPCR measurements, relative proportions were higher according to FISH results. CONCLUSIONS: Changes in the fecal microbial numbers of obese individuals were primarily affected by the dietary intake rather than weight changes.

Gut microbiota-derived propionate reduces cancer cell proliferation in the liver.

BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro. RESULTS: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines. CONCLUSION: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Functional dysbiosis within the gut microbiota of patients with constipated-irritable bowel syndrome.

BACKGROUND: The role of the gut microbiota in patho-physiology of irritable bowel syndrome (IBS) is suggested by several studies. However, standard cultural and molecular methods used to date have not revealed specific and consistent IBS-related groups of microbes. AIM: To explore the constipated-IBS (C-IBS) gut microbiota using a function-based approach. METHODS: The faecal microbiota from 14 C-IBS women and 12 sex-match healthy subjects were examined through a combined strictly anaerobic cultural evaluation of functional groups of microbes and fluorescent in situ hybridisation (16S rDNA gene targeting probes) to quantify main groups of bacteria. Starch fermentation by C-IBS and healthy faecal samples was evaluated in vitro. RESULTS: In C-IBS, the numbers of lactate-producing and lactate-utilising bacteria and the number of H(2) -consuming populations, methanogens and reductive acetogens, were at least 10-fold lower (P < 0.05) compared with control subjects. Concomitantly, the number of lactate- and H(2) -utilising sulphate-reducing population was 10 to 100 fold increased in C-IBS compared with healthy subjects. The butyrate-producing Roseburia - E. rectale group was in lower number (0.01 < P < 0.05) in C-IBS than in control. C-IBS faecal microbiota produced more sulphides and H(2) and less butyrate from starch fermentation than healthy ones. CONCLUSIONS: A major functional dysbiosis was observed in constipated-irritable bowel syndrome gut microbiota, reflecting altered intestinal fermentation. Sulphate-reducing population increased in the gut of C-IBS and were accompanied by alterations in other microbial groups. This could be responsible for changes in the metabolic output and enhancement in toxic sulphide production which could in turn influence gut physiology and contribute to IBS pathogenesis.

Understanding the effects of diet on bacterial metabolism in the large intestine.

Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations.

AIMS: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli. METHODS AND RESULTS: Through the adoption of a novel combination of multiple breakpoint selection, phenotypic characterization and the application of a polymerase chain reaction based gene identification system it proved possible to monitor the influence of antibiotic exposure on resistance gene possession. Using tetracycline as a case study a clear hierarchy was revealed between tet genes, strongly influenced by host antimicrobial exposure history. CONCLUSIONS: The antimicrobial exposure regime under which an animal is produced affects both the identity and magnitude of resistance gene possession of a selected bacterial population within its enteric microflora. Among the ramifications associated with such resistance gene selection is the degree of resistance conferred and the carriage of linked resistance determinants. This selection is applied by exposure to antibiotic concentrations well below recognized minimum inhibitory tetracycline concentration breakpoints widely adopted to characterize bacterial 'susceptibility'. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the ability of minimal antibiotic exposure to select for the continued persistence of resistance genes within the enteric microflora. It is clearly demonstrated that different antimicrobial regimes select for different resistance genes, the implications of which are discussed.

The role of conjugative transposons in spreading antibiotic resistance between bacteria that inhabit the gastrointestinal tract.

There is huge potential for genetic exchange to occur within the dense, diverse anaerobic microbial population inhabiting the gastrointestinal tract (GIT) of humans and animals. However, the incidence of conjugative transposons (CTns) and the antibiotic resistance genes they carry has not been well studied among this population. Since any incoming bacteria, including pathogens, can access this reservoir of genes, this oversight would appear to be an important one. Recent evidence has shown that anaerobic bacteria native to the rumen or hindgut harbour both novel antibiotic resistance genes and novel conjugative transposons. These CTns, and previously characterized CTns, can be transferred to a wide range of commensal bacteria under laboratory and in vivo conditions. The main evidence that gene transfer occurs widely in vivo between GIT bacteria, and between GIT bacteria and pathogenic bacteria, is that identical resistance genes are present in diverse bacterial species from different hosts.

Novel tetracycline resistance gene, tet(32), in the Clostridium-related human colonic anaerobe K10 and its transmission in vitro to the rumen anaerobe Butyrivibrio fibrisolvens.

A novel tetracycline resistance gene, designated tet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W). tet(32) was transmissible in vitro to the rumen anaerobe Butyrivibrio fibrisolvens 2221(R). The predicted gene product of tet(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain.

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Occurrence of the new tetracycline resistance gene tet(W) in bacteria from the human gut.

Members of our group recently identified a new tetracycline resistance gene, tet(W), in three genera of rumen obligate anaerobes. Here, we show that tet(W) is also present in bacteria isolated from human feces. The tet(W) genes found in human Fusobacterium prausnitzii and Bifidobacterium longum isolates were more than 99.9% identical to those from a rumen isolate of Butyrivibrio fibrisolvens.

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations.

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria.

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

Natural genetic transformation in the rumen bacterium Streptococcus bovis JB1.

Natural transformation of Streptococcus bovis JB1 was demonstrated after development of competence in normal culture medium. Transformation efficiencies were not significantly increased when heat-inactivated horse serum was added to the medium before growth. This is the first time that a resident rumen bacterial species has been shown to be naturally transformable. Transformation allowed the acquisition of plasmids or integration of sequences into the chromosome. No transformation was observed in the presence of undiluted autoclaved or filter-sterilised ovine rumen fluid or filter-sterilised ovine saliva, suggesting that transformation in the ruminant digestive tract is a rare event, although transformation was observed in the presence of 1% and 0.5% filter-sterilised rumen fluid. The use of natural transformation of S. bovis should facilitate further molecular biological studies on this species.

Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva.

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria.

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens. Donor strains were postulated to carry two TcR genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet(O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible TcR gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet(W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet(W) genes with almost identical sequences are also shown to be present in TcR strains of B. fibrisolvens from Australian sheep and in TcR strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus. This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet(W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus.

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences.

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

High-frequency transfer of a naturally occurring chromosomal tetracycline resistance element in the ruminal anaerobe Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens strains resistant to tetracycline were isolated from the bovine rumen. Two of three Tcr B. fibrisolvens tested were able to donate tetracycline resistance at frequencies ranging from 10(-7) to 10(-1) per donor cell in anaerobic filter matings to a rifampin-resistant mutant of the type strain of B.fibrisolvens, 2221R. The recipient strain 2221R exhibited rapid autoaggregation, which might be a factor in the high transfer rates observed. Tcr transconjugants of B. fibrisolvens 2221R were also capable of further transferring tetracycline resistance to a fusidic acid-resistant mutant, 2221F. Comparison of genomic DNAs by pulsed-field gel electrophoresis demonstrated altered band profiles in transconjugants, consistent with the acquisition of a large mobile chromosomal element. The transferable elements from the two B. fibrisolvens donors 1.23 and 1.230 (TnB123 and TnB1230, respectively) showed the same preferred insertion site in the B. fibrisolvens 2221R chromosome and are likely to be similar, or identical, elements. Hybridization experiments showed no close relationship between TnB1230 and int-xis regions from Tn916 or Tn5253. Although DNA from the B. fibrisolvens donor strains hybridized with probes carrying tet(M) or tet(O) sequences, transconjugants were found to have acquired a distinct band that hybridized only weakly with these probes, suggesting that a second, distantly related Tcr determinant had been transferred.

Transfer of plasmids between strains of Escherichia coli under rumen conditions.

Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10(-6) per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol l-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol l-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic but environment.

Sequence analysis and gene content of potato mop-top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses.

The complete sequence of the 2315 nucleotides in RNA 3 of potato mop-top furovirus (PMTV) isolate T was obtained by analysis of cDNA clones and by direct RNA sequencing. The sequence contains an open reading frame for the coat protein (20K) terminated by an amber codon, followed by an in-phase coding region for an additional 47K. PMTV therefore resembles soil-borne wheat mosaic (SBWMV) and beet necrotic yellow vein (BNYVV) viruses (two other fungus-transmitted viruses with rod-shaped particles) in having a coat protein-readthrough product. Comparison of the 3' untranslated regions of PMTV RNA 2 and RNA 3 reveals a long conserved block of 150 nucleotides, which contains two repeated sequences and has the potential to form consecutive pseudoknot structures. PMTV RNA 3 ends a few nucleotides downstream of this conserved block, but RNA extends for a further 140 nucleotides, which can potentially form a 3'-terminal tRNA-like structure similar to those in the RNA species of SBWMV, tymoviruses, and some tobamoviruses. PMTV coat protein has amino acid sequence identities of 30 and 28% with SBWMV and BNYVV coat proteins, respectively, and apparent structural similarities with tobacco mosaic virus coat protein. The coat protein readthrough domains of PMTV, SBWMV, and BNYVV have shared residues throughout their length but no extended sequences are conserved. The presence in RNA 3 of coding sequences for only the coat protein and readthrough domain distinguishes PMTV from SBWMV and BNYVV, both of which have them in RNA 2 along with one or more other genes. Comparison of the genomes of PMTV, BNYVV, and SBWMV shows that furoviruses exhibit considerable heterogeneity in genome organization.