Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

Identification of the mechanism for dehalorespiration of monofluoroacetate in the phylum Synergistota.

OBJECTIVE: Monofluoroacetate (MFA) is a potent toxin that blocks ATP production via the Krebs cycle and causes acute toxicity in ruminants consuming MFA-containing plants. The rumen bacterium, Cloacibacillus porcorum strain MFA1 belongs to the phylum Synergistota and can produce fluoride and acetate from MFA as the end-products of dehalorespiration. The aim of this study was to identify the genomic basis for the metabolism of MFA by this bacterium. METHODS: A draft genome sequence for C. porcorum strain MFA1 was assembled and quantitative transcriptomic analysis was performed thus highlighting a candidate operon encoding four proteins that are responsible for the carbon-fluorine bond cleavage. Comparative genome analysis of this operon was undertaken with three other species of closely related Synergistota bacteria. RESULTS: Two of the genes in this operon are related to the substrate-binding components of the glycine reductase protein B (GrdB) complex. Glycine shares a similar structure to MFA suggesting a role for these proteins in binding MFA. The remaining two genes in the operon, an antiporter family protein and an oxidoreductase belonging to the radical S-adenosyl methionine superfamily, are hypothesised to transport and activate the GrdB-like protein respectively. Similar operons were identified in a small number of other Synergistota bacteria including type strains of Cloacibacillus porcorum, C. evryensis, and Pyramidobacter piscolens, suggesting lateral transfer of the operon as these genera belong to separate families. We confirmed that all three species can degrade MFA, however, substrate degradation in P. piscolens was notably reduced compared to Cloacibacillus isolates possibly reflecting the loss of the oxidoreductase and antiporter in the P. piscolens operon. CONCLUSION: Identification of this unusual anaerobic fluoroacetate metabolism extends the known substrates for dehalorespiration and indicates the potential for substrate plasticity in amino acid-reducing enzymes to include xenobiotics.

Butyrylated starch increases colonic butyrate concentration but has limited effects on immunity in healthy physically active individuals.

BACKGROUND: Butyrate delivery to the large bowel may positively modulate commensal microbiota and enhance immunity. OBJECTIVE: To determine the effects of increasing large bowel butyrate concentration through ingestion of butyrylated high amylose maize starch (HAMSB) on faecal biochemistry and microbiota, and markers of immunity in healthy active individuals. DESIGN: Male and female volunteers were assigned randomly to consume either two doses of 20 g HAMSB (n = 23; age 37.9 +/- 7.8 y; mean +/- SD) or a low amylose maize starch (LAMS) (n = 18; age 36.9 = 9.5 y) twice daily for 28 days. Samples were collected on days 0, 10 and 28 for assessment of faecal bacterial groups, faecal biochemistry, serum cytokines and salivary antimicrobial proteins. RESULTS: HAMSB led to relative increases in faecal free (45%; 12-86%; mean; 90% confidence interval; P = 0.02), bound (950%; 563-1564%; P < 0.01) and total butyrate (260%; 174-373%; P < 0.01) and faecal propionate (41%; 12-77%; P = 0.02) from day 0 to day 28 compared to LAMS. HAMSB was also associated with a relative 1.6-fold (1.2- to 2.0-fold; P < 0.01) and 2.5-fold (1.4- to 4.4-fold; P = 0.01) increase in plasma IL-10 and TNF-alpha but did not alter other indices of immunity. There were relative greater increases in faecal P. distasonis (81-fold (28- to 237-fold; P < 0.01) and F. prausnitzii (5.1-fold (2.1- to 12-fold; P < 0.01) in the HAMSB group. CONCLUSIONS: HAMSB supplementation in healthy active individuals promotes the growth of bacteria that may improve bowel health and has only limited effects on plasma cytokines.

Detection of changes in regional colonic fermentation in response to supplementing a low FODMAP diet with dietary fibres by hydrogen concentrations, but not by luminal pH.

BACKGROUND: Carbohydrate fermentation plays a pivotal role in maintaining colonic health with excessive proximal and deficient distal fermentation being detrimental. AIMS: To utilise telemetric gas- and pH-sensing capsule technologies for defining patterns of regional fermentation following dietary manipulations, alongside conventional techniques of measuring fermentation. METHODS: In a double-blind crossover trial, 20 patients with irritable bowel syndrome were fed low FODMAP diets that included no extra fibre (total fibre content 24 g/day), or additional poorly fermented fibre, alone (33 g/day) or with fermentable fibre (45 g/day) for 2 weeks. Plasma and faecal biochemistry, luminal profiles defined by tandem gas- and pH-sensing capsules, and faecal microbiota were assessed. RESULTS: Plasma short-chain fatty acid (SCFA) concentrations (µmol/L) were median (IQR) 121 (100-222) with fibre combination compared with 66 (44-120) with poorly fermented fibre alone (p = 0.028) and 74 (55-125) control (p = 0.069), but no differences in faecal content were observed. Luminal hydrogen concentrations (%), but not pH, were higher in distal colon (mean 4.9 [95% CI: 2.2-7.5]) with fibre combination compared with 1.8 (0.8-2.8) with poorly fermented fibre alone (p = 0.003) and 1.9 (0.7-3.1) control (p = 0.003). Relative abundances of saccharolytic fermentative bacteria were generally higher in association with supplementation with the fibre combination. CONCLUSIONS: A modest increase in fermentable plus poorly fermented fibres had minor effects on faecal measures of fermentation, despite increases in plasma SCFA and abundance of fermentative bacteria, but the gas-sensing capsule, not pH-sensing capsule, detected the anticipated propagation of fermentation distally in the colon. The gas-sensing capsule technology provides unique insights into localisation of colonic fermentation. TRIAL REGISTRATION: ACTRN12619000691145.

Isolation and identification of sodium fluoroacetate degrading bacteria from caprine rumen in Brazil.

The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100 µL in mineral medium added with 20 mmol L(-1) sodium fluoroacetate (SF), incubated at 39°C in an orbital shaker. Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08) and Ancylobacter dichloromethanicus (ECPB09). These bacteria degraded sodium fluoroacetate, releasing 20 mmol L(-1) of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L(-1) of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.

Defluorination of sodium fluoroacetate by bacteria from soil and plants in Brazil.

The aim of this work was to isolate and identify bacteria able to degrade sodium fluoroacetate from soil and plant samples collected in areas where the fluoroacetate-containing plants Mascagnia rigida and Palicourea aenofusca are found. The samples were cultivated in mineral medium added with 20 mmol L(-1) sodium fluoroacetate. Seven isolates were identified by 16S rRNA gene sequencing as Paenibacillus sp. (ECPB01), Burkholderia sp. (ECPB02), Cupriavidus sp. (ECPB03), Staphylococcus sp. (ECPB04), Ancylobacter sp. (ECPB05), Ralstonia sp. (ECPB06), and Stenotrophomonas sp. (ECPB07). All seven isolates degraded sodium-fluoroacetate-containing in the medium, reaching defluorination rate of fluoride ion of 20 mmol L(-1). Six of them are reported for the first time as able to degrade sodium fluoroacetate (SF). In the future, some of these microorganisms can be used to establish in the rumen an engineered bacterial population able to degrade sodium fluoroacetate and protect ruminants from the poisoning by this compound.

Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.

OBJECTIVES: Mucosa-associated bacteria are increased in inflammatory bowel disease (IBD), which suggests the possibility of an increased source of digestible endogenous mucus substrate. We hypothesized that mucolytic bacteria are increased in IBD, providing increased substrate to sustain nonmucolytic mucosa-associated bacteria. METHODS: Mucolytic bacteria were characterized by the ability to degrade human secretory mucin (MUC2) in pure and mixed anaerobic cultures. Real-time PCR was used to enumerate mucosa-associated mucolytic bacteria in 46 IBD and 20 control patients. Bacterial mucolytic activity was tested in vitro using purified human MUC2. RESULTS: We confirm increased total mucosa-associated bacteria 16S rRNA gene in macroscopically and histologically normal intestinal epithelium of both Crohn's disease (CD) (mean 1.9-fold) and ulcerative colitis (UC) (mean 1.3-fold). We found a disproportionate increase in some mucolytic bacteria. Mean Ruminococcus gnavus were increased >4-fold and Ruminococcus torques ∼100-fold in macroscopically and histologically normal intestinal epithelium of both CD and UC. The most abundantly detected mucolytic bacterium in controls, Akkermansia muciniphila, was reduced many fold in CD and in UC. Coculture of A. muciniphila with MUC2 as the sole carbon source led to reduction in its abundance while it augmented growth of other bacteria. CONCLUSIONS: Mucolytic bacteria are present in healthy humans, where they are an integral part of the mucosa-associated bacterial consortium. The disproportionate increase in R. gnavus and R. torques could explain increased total mucosa-associated bacteria in IBD.

An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction.

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.

Genetic Markers Are Associated with the Ruminal Microbiome and Metabolome in Grain and Sugar Challenged Dairy Heifers.

Dairy heifers were subjected to a non-life-threatening challenge designed to induce ruminal acidosis by feeding grain and sugar. Large among animal variation in clinical signs of acidosis, rumen metabolite concentrations, and the rumen microbiome occurred. This exploratory study investigates sources of the variation by examining associations between the genome, metabolome, and microbiome, albeit with a limited population. The broader objective is to provide a rationale for a larger field study to identify markers for susceptibility to ruminal acidosis. Initially, heifers (n = 40) allocated to five feed additive groups were fed 20-days pre-challenge with a total mixed ration and additives. Fructose (0.1% of bodyweight/day) was added for the last 10 days pre-challenge. On day 21 heifers were challenged with 1.0% of bodyweight dry matter wheat + 0.2% of bodyweight fructose + additives. Rumen samples were collected via stomach tube weekly (day 0, 7, and 14) and at five times over 3.6 h after challenge and analyzed for pH and volatile fatty acid, ammonia, D-, and L-lactate concentrations. Relative abundance of bacteria and archaea were determined using Illumina MiSeq. Genotyping was undertaken using a 150K Illumina SNPchip. Genome-wide association was performed for metabolite and microbiome measures (n = 33). Few genome associations occurred with rumen pH, concentration of acetate, propionate, total volatile fatty acids, or ammonia, or the relative abundance of the Firmicutes, Bacteroidetes, and Spirochaetes phyla. Metabolites and microbial phyla that had markers associated and quantitative trait loci (QTL) were: acetate to propionate ratio (A:P), D-, L-, and total lactate, butyrate, acidosis eigenvalue, Actinobacteria, Chloroflexi, Euryarchaeota, Fibrobacteres, Planctomycetes, Proteobacteria, and Tenericutes. A putative genomic region overlapped for Actinobacteria, Euryarchaeota, and Fibrobacteres and covered the region that codes for matrix extracellular phosphoglycoprotein (MEPE). Other overlapping regions were: (1) Chloroflexi, Tenericutes, and A:P, (2) L- and total lactate and Actinobacteria, and (3) Actinobacteria, Euryarchaeota, Fibrobacteres, and A:P. Genome-wide associations with the metabolome and microbiome occurred despite the small population, suggesting that markers for ruminal acidosis susceptibility exist. The findings may explain some of the variation in metabolomic and microbial data and provide a rationale for a larger study with a population that has variation in acidosis.

Fluoroacetate in plants - a review of its distribution, toxicity to livestock and microbial detoxification.

Fluoroacetate producing plants grow worldwide and it is believed they produce this toxic compound as a defence mechanism against grazing by herbivores. Ingestion by livestock often results in fatal poisonings, which causes significant economic problems to commercial farmers in many countries such as Australia, Brazil and South Africa. Several approaches have been adopted to protect livestock from the toxicity with limited success including fencing, toxic plant eradication and agents that bind the toxin. Genetically modified bacteria capable of degrading fluoroacetate have been able to protect ruminants from fluoroacetate toxicity under experimental conditions but concerns over the release of these microbes into the environment have prevented the application of this technology. Recently, a native bacterium from an Australian bovine rumen was isolated which can degrade fluoroacetate. This bacterium, strain MFA1, which belongs to the Synergistetes phylum degrades fluoroacetate to fluoride ions and acetate. The discovery and isolation of this bacterium provides a new opportunity to detoxify fluoroacetate in the rumen. This review focuses on fluoroacetate toxicity in ruminant livestock, the mechanism of fluoroacetate toxicity, tolerance of some animals to fluoroaceate, previous attempts to mitigate toxicity, aerobic and anaerobic microbial degradation of fluoroacetate, and future directions to overcome fluoroacetate toxicity.

Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids.

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091(T), strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091(T) genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091(T) genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these 'low-methane-emitting' herbivores.

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes.

Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but the degradation of fluorinated compounds under similar conditions has rarely been described. Here, we report on the isolation of a bovine rumen bacterium that metabolizes fluoroacetate under anaerobic conditions, the mode of degradation and its presence in gut ecosystems. The bacterium was identified using 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. Growth was stimulated by amino acids with greater quantities of amino acids metabolized in the presence of fluoroacetate, but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and H(2) were end products of amino acid metabolism. Acetate was the primary end product of fluoroacetate dehalogenation, and the amount produced correlated with the stoichiometric release of fluoride which was confirmed using fluorine nuclear magnetic resonance ((19) F NMR) spectroscopy. Hydrogen and formate produced in situ were consumed during dehalogenation. The growth characteristics of strain MFA1 indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Nested 16S rRNA gene-specific PCR assays detected the bacterium at low numbers in the gut of several herbivore species.

Dysbiosis of fecal microbiota in Crohn's disease patients as revealed by a custom phylogenetic microarray.

BACKGROUND: A custom phylogenetic microarray composed of small subunit ribosomal RNA probes, representing ≈500 bacterial species from the human and animal gut, was developed and evaluated for analysis of gut microbial diversity using fecal samples from healthy subjects and Crohn's disease (CD) patients. METHODS: Oligonucleotide probes (≈40 mer) used on the microarray were selected from published articles or designed with the "GoArray" microarray probe design program using selected bacterial 16S rRNA sequences. Fecal 16S rDNA from individual samples of six healthy subjects and six CD patients were used as template to generate fluorescently labeled cRNA that was hybridized to the microarray. Differences revealed by the microarray in relative abundance of microbial populations between healthy and diseased patients were verified using quantitative real-time polymerase chain reaction (PCR) with species-specific primer sets. RESULTS: The microarray analyses showed that Eubacterium rectale, Bacteroides fragilis group, B. vulgatus, Ruminococcus albus, R. callidus, R. bromii, and Faecalibacterium prausnitzii were 5-10-fold more abundant in the healthy subjects than in the CD patients, while Enterococcus sp., Clostridium difficile, Escherichia coli, Shigella flexneri, and Listeria sp. were more abundant in the CD group. CONCLUSIONS: The microarray detected differences in abundance of bacterial populations within the phylum Firmicutes that had been reported previously for the same samples based on phylogenetic analysis of metagenomic clone libraries. In addition, the microarray showed that Enterococcus sp. was in higher abundance in the CD patients. This microarray should be another useful tool to examine the diversity and abundance of human intestinal microbiota.