Enhanced meat chicken productivity in response to the probiotic Bacillus amyloliquefaciens H57 is associated with the enrichment of microbial amino acid and vitamin biosynthesis pathways.

AIMS: Sub-therapeutic use of antibiotics as a growth promoter in animal diets has either been banned or voluntarily withdrawn from use in many countries to help curb the emergence of antibiotic-resistant pathogens. Probiotics may be an alternative to antibiotics as a growth promoter. We investigated the effects of a novel probiotic strain, Bacillus amyloliquefaciens H57 (H57) on the performance and microbiome-associated metabolic potential. METHODS AND RESULTS: Broiler chickens were fed either sorghum- or wheat-based diets supplemented with the probiotic H57. The growth rate, feed intake, and feed conversion in supplemented birds were compared with those in non-supplemented control. Caecal microbial metabolic functions were studied with shotgun metagenomic sequencing. H57 supplementation significantly increased the growth rate and daily feed intake of meat chickens relative to the non-supplemented controls without any effect on feed conversion ratio. In addition, relative to the non-supplemented controls, gene-centric metagenomics revealed that H57 significantly altered the functional capacity of the caecal microbiome, with amino acid and vitamin synthesis pathways being positively associated with H57 supplementation. CONCLUSIONS: Bacillus amyloliquefaciens H57 improves the performance of meat chickens or broilers and significantly modifies the functional potential of their caecal microbiomes, with enhanced potential capacity for amino acid and vitamin biosynthesis.

Sporulation capability and amylosome conservation among diverse human colonic and rumen isolates of the keystone starch-degrader Ruminococcus bromii.

Ruminococcus bromii is a dominant member of the human colonic microbiota that plays a 'keystone' role in degrading dietary resistant starch. Recent evidence from one strain has uncovered a unique cell surface 'amylosome' complex that organizes starch-degrading enzymes. New genome analysis presented here reveals further features of this complex and shows remarkable conservation of amylosome components between human colonic strains from three different continents and a R. bromii strain from the rumen of Australian cattle. These R. bromii strains encode a narrow spectrum of carbohydrate active enzymes (CAZymes) that reflect extreme specialization in starch utilization. Starch hydrolysis products are taken up mainly as oligosaccharides, with only one strain able to grow on glucose. The human strains, but not the rumen strain, also possess transporters that allow growth on galactose and fructose. R. bromii strains possess a full complement of sporulation and spore germination genes and we demonstrate the ability to form spores that survive exposure to air. Spore formation is likely to be a critical factor in the ecology of this nutritionally highly specialized bacterium, which was previously regarded as 'non-sporing', helping to explain its widespread occurrence in the gut microbiota through the ability to transmit between hosts.

Identification of Acid Hydrolysis Metabolites of the Pimelea Toxin Simplexin for Targeted UPLC-MS/MS Analysis.

Pimelea poisoning of cattle is a unique Australian toxic condition caused by the daphnane orthoester simplexin present in native Pimelea pasture plants. Rumen microorganisms have been proposed to metabolise simplexin by enzymatic reactions, likely at the orthoester and epoxide moieties of simplexin, but a metabolic pathway has not been confirmed. This study aimed to investigate this metabolic pathway through the analysis of putative simplexin metabolites. Purified simplexin was hydrolysed with aqueous hydrochloric acid and sulfuric acid to produce target metabolites for UPLC-MS/MS analysis of fermentation fluid samples, bacterial isolate samples, and other biological samples. UPLC-MS/MS analysis identified predicted hydrolysed products from both acid hydrolysis procedures with MS breakdown of these putative products sharing high-resolution accurate mass (HRAM) fragmentation ions with simplexin. However, targeted UPLC-MS/MS analysis of the biological samples failed to detect the H2SO4 degradation products, suggesting that the rumen microorganisms were unable to produce similar simplexin degradation products at detectable levels, or that metabolites, once formed, were further metabolised. Overall, in vitro acid hydrolysis was able to hydrolyse simplexin at the orthoester and epoxide functionalities, but targeted UPLC-MS/MS analysis of biological samples did not detect any of the identified simplexin hydrolysis products.

Microbial events in the hindgut during carbohydrate-induced equine laminitis.

Equine laminitis is the most serious foot disease of the horse, often resulting in death or euthanasia. Laminitis has long been recognized as an affliction of horses, as has the association of this condition with the ingestion of carbohydrates. Research into the pathophysiology of this condition has been facilitated by the development of reliable models for experimentally inducing laminitis, and DNA-based techniques for profiling complex microbiomes have dramatically increased the knowledge of the microbiology of this disease. Recent studies have provided substantial evidence showing equine hindgut streptococcal species to be the most likely causative agent. Although these studies are not definitive, they provide the foundations for future work to determine the source of laminitis trigger factors and their mechanisms of action.

Toxin Degradation by Rumen Microorganisms: A Review.

Animal feeds may contain exogenous compounds that can induce toxicity when ruminants ingest them. These toxins are secondary metabolites originating from various sources including plants, bacteria, algae and fungi. Animal feed toxins are responsible for various animal poisonings which negatively impact the livestock industry. Poisoning is more frequently reported in newly exposed, naïve ruminants while 'experienced' ruminants are observed to better tolerate toxin-contaminated feed. Ruminants can possess detoxification ability through rumen microorganisms with the rumen microbiome able to adapt to utilise toxic secondary metabolites. The ability of rumen microorganisms to metabolise these toxins has been used as a basis for the development of preventative probiotics to confer resistance against the poisoning to naïve ruminants. In this review, detoxification of various toxins, which include plant toxins, cyanobacteria toxins and plant-associated fungal mycotoxins, by rumen microorganisms is discussed. The review will include clinical studies of the animal poisoning caused by these toxins, the toxin mechanism of action, toxin degradation by rumen microorganisms, reported and hypothesised detoxification mechanisms and identified toxin metabolites with their toxicity compared to their parent toxin. This review highlights the commercial potential of rumen inoculum derived probiotics as viable means of improving ruminant health and production.

Purified plant cell walls with adsorbed polyphenols alter porcine faecal bacterial communities during in vitro fermentation.

A substantial fraction of ingested polyphenols accumulate in the large intestine (LI), attached to undigested plant cell walls (PCW) (dietary fibre). Yet, whether these PCW-bound polyphenols alter the structure and function of the resident microbiota remains unclear. This study characterised bacterial populations during the in vitro fermentation of three standard polyphenols: ferulic acid (FER), (±)-catechin (CAT), and cyanidin-3-glucoside (CYAN), adsorbed individually or in combination to apple cell walls (ACW). During fermentation with porcine faeces, samples were collected at regular time-points (up to 72 hours) for bacterial 16S rRNA gene amplicon sequencing and fermentation end-product analyses (short-chain fatty acids and ammonium). The metabolic end-products differed to only a small extent between substrates, though significantly for propionate (P < 0.0001). Significant differences in microbial populations were noted between substrates tested (P < 0.0001). The presence of cyanidin-3-glucoside resulted in the most significant differences between bacterial communities during fermentation of the ACW substrate. Key microbes identified to be associated with the ACW with adsorbed polyphenols as well as individual polyphenols were: Phascolarctobacterium with ACW + FER and FER, the Lachnospiraceae family with ACW + CYAN, Parabacteroides with ACW + CYAN and CYAN, Collinsella and Coprococcus with ACW + CAT, and the Clostridiales order with ACW + CAT and CAT. This study has demonstrated the use of a simplified model to indicate any microbial effects of polyphenols associated with dietary fibre in whole fruits. This work has shown that individual polyphenols, or those adsorbed to PCW, have potentially very different effects on the gut bacteria. Future work could examine further polyphenols associated with a range of fresh fruits.

Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria.

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

Near complete genome sequence of the animal feed probiotic, Bacillus amyloliquefaciens H57.

Bacillus amyloliquefaciens H57 is a bacterium isolated from lucerne for its ability to prevent feed spoilage. Further interest developed when ruminants fed with H57-inoculated hay showed increased weight gain and nitrogen retention relative to controls, suggesting a probiotic effect. The near complete genome of H57 is ~3.96 Mb comprising 16 contigs. Within the genome there are 3,836 protein coding genes, an estimated sixteen rRNA genes and 69 tRNA genes. H57 has the potential to synthesise four different lipopeptides and four polyketide compounds, which are known antimicrobials. This antimicrobial capacity may facilitate the observed probiotic effect.

Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing.

Kangaroos ferment forage material in an enlarged forestomach analogous to the rumen, but in contrast to ruminants, they produce little or no methane. The objective of this study was to identify the dominant organisms and pathways involved in hydrogenotrophy in the kangaroo forestomach, with the broader aim of understanding how these processes are able to predominate over methanogenesis. Stable isotope analysis of fermentation end products and RNA stable isotope probing (RNA-SIP) were used to investigate the organisms and biochemical pathways involved in the metabolism of hydrogen and carbon dioxide in the kangaroo forestomach. Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos. In in vitro fermentations, the microbial community of the kangaroo foregut produced very little methane, but produced a significantly greater proportion of acetate derived from carbon dioxide than the microbial community of the bovine rumen. A bacterial operational taxonomic unit closely related to the known reductive acetogen Blautia coccoides was found to be associated with carbon dioxide and hydrogen metabolism in the kangaroo foregut. Other bacterial taxa including members of the genera Prevotella, Oscillibacter and Streptococcus that have not previously been reported as containing hydrogenotrophic organisms were also significantly associated with metabolism of hydrogen and carbon dioxide in the kangaroo forestomach.

Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

Microbial ecology of the equine hindgut during oligofructose-induced laminitis.

Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3-7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.

Naturally occurring DNA transfer system associated with membrane vesicles in cellulolytic Ruminococcus spp. of ruminal origin.

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.

Ecology of uncultivated Oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches.

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Family of shuttle vectors for ruminal Bacteroides.

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 10(5) transformants/microg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11-0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0.53% per generation.