Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.     

Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Effect of heterotrophic ruminal bacteria on xylan metabolism by the anaerobic fungus Piromyces communis

Six rumen bacteria were cocultured with the rumen fungus Piromyces communis and the effects on xylanolysis determined. The rate and extent of xylan utilization was enhanced in cocultures with Prevotella ruminicola or Succinivibrio dextrinosolvens. The positive effects of Suc. dextrinosolvens and Prev. ruminicola on xylanolysis by P. communis correlated with effective cross-feeding by the bacteria on arabinose and xylose released from xylan. Xylanolysis was not enhanced in cocultures of P. communis with Streptococcus bovis, Veillonella parvula or Ruminococcus flavefaciens. A comparison of fermentation product profiles and of extracellular enzyme activities showed that whereas saccharolytic species and Butyrivibrio fibrisolvens were dominant in cocultures, P. communis dominated in the culture with R. flavefaciens. Extracellular xylanase and β-xylosidase activities were not increased by cocultivation of P. communis with any of the heterotrophic bacteria.     

Real-time PCR法检测日粮中添加小黑麦干酒糟对肉牛瘤胃Prevotella属菌数量的影响

通过在肉牛日粮中添加不同比例的小黑麦干酒糟及其可溶物(TDDGS),运用Real-time PCR方法检测在添加TDDGS后对3种瘤胃普雷沃菌(Prevotella ruminicola、Prevotella brevis和Prevotella bryantii)数量的影响。结果表明,TDDGS组(20%、25%和30%TDDGS)与对照组(CG)相比,瘤胃中P.ruminicola和P.brevis菌数量均有升高,且在20%TDDGS组的数量分别显著升高47倍(P0.05)和794倍(P0.05),而P.bryantii菌数量却有所降低,并且在20%TDDGS组的数量显著降低5倍(P0.05);另一方面,TDDGS组间相比,除了P.ruminicola菌数量在20%和30%TDDGS组间存在显著差异,其余TDDGS组间的3种菌数量差异均不显著。结论是在肉牛日粮中添加20%TDDGS对3种瘤胃普雷沃菌数量都产生了显著影响,3种菌在TDDGS组间的数量变化差异不明显。     

Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.     

Activity and properties of fumarate reductase in ruminal bacteria

Fumarate-reducing bacteria were sought from the main ruminal bacteria. Fibrobacter succinogenes, Selenomonas ruminantium subsp. ruminantium, Selenomonas ruminantium subsp. lactilytica, and Veillonella parvula reduced fumarate by using H(2) as an electron donor. Ruminococcus albus, Prevotella ruminicola, and Anaerovibrio lipolytica consumed fumarate, although they did not oxidize H(2). Of these bacteria, V. parvula, two strains of Selenomonas, and F. succinogenes had a high capacity to reduce fumarate. In all the fumarate-reducing bacteria examined, fumarate reductase existed in the membrane fraction. Based on the activity per cell mass and the affinity of fumarate reductase to fumarate, these bacteria were divided into two groups, which corresponded to the capacity to use H(2): A group of bacteria with higher activity and affinity were able to use H(2) as an electron donor for fumarate reduction. The bacteria in this group should gain an advantage over the bacteria in another group in fumarate reduction in the rumen. Cellulose digestion by R. albus was improved by fumarate reduction by S. lactilytica as a result of an increased growth of R. albus, which may have been caused by the fact that S. lactilytica immediately consumed H(2) produced by R. albus. Thus fumarate reduction may play an important role in keeping a low partial pressure of H(2) in the rumen.     

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.     

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.     

Carbon Dioxide Requirement of Various Species of Rumen Bacteria

The carbon dioxide requirement of 32 strains of rumen bacteria, representing 11 different species, was studied in detail. Increasing concentrations of CO(2) were added as NaHCO(3) to a specially prepared CO(2)-free medium which was tubed and inoculated under nitrogen. Prior depletion of CO(2) in the inoculum was found to affect the level of requirement; however, the complexity and buffering capacity of the medium did not appear to be involved. An absolute requirement for CO(2) was observed for eight strains of Bacteroides ruminicola, three strains of Bacteroides succinogenes, four strains of Ruminococcus flavefaciens, two strains of Lachnospira multiparus, one strain of Succinimonas amylolytica, and two strains of Butyrivibrio fibrisolvens. Inconsistent growth responses were obtained in CO(2)-free media with one strain each of B. fibrisolvens, Ruminococcus albus, and Selenomonas ruminantium. Growth of six additional strains of B. fibrisolvens, and single strains of Eubacterium ruminantium and Succinivibrio dextrinosolvens was markedly increased or stimulated by increasing concentrations of CO(2). Peptostreptococcus elsdenii B159 was the only organism tested which appeared to have no requirement, either absolute or partial, for CO(2). Higher concentrations of CO(2) were required for the initiation of growth, as well as for optimal growth, by those species which produce succinic acid as one of their primary end products.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

Influence of the rumen anaerobic fungus Neocallimastix frontalis on the proteolytic activity of a defined mixture of rumen bacteria growing on a solid substrate

A grass + fishmeal ruminant feed was incubated for 7 d in a mineral salts medium with the non-proteolytic rumen bacteria Bacteroides succinogenes, Ruminococcus flavefaciens, Megasphaera elsdenii and proteolytic strains of Bacteroides ruminicola, Selenomonas ruminantium and Streptococcus bovis in the presence and absence of the anaerobic fungus Neocallitnastix frontalis . The fungus increased the dry matter digestion from 65·0 to 69·4%, and more than doubled the proteolytic activity of the culture filtrate. However, a greater difference was observed with the solid material, where the proteolytic activity increased from 0·71 to 6·89 mg ¹⁴C-casein hydrolysed/g/h, due mainly to EDTA-sensitive fungal protease.     

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

Xylanolysis by cocultures of the rumen fungus Neocallimastix frontalis and ruminal bacteria

The effect of fibrolytic and saccharolytic rumen bacteria on xylanolysis by the rumen fungus Neocallimastix frontalis has been investigated. In cocultivations N. frontalis interacted synergistically with Bacteroides ruminicola, Succinivibrio dextrin-osolvens and Selenomonas ruminantium during xylan utilization. Xylan utilization decreased in cocultures containing Lachnospira multiparus or Streptococcus bovis. Ruminococcus flavefaciens appeared to inhibit fungal growth.     

Adhesion of cellulolytic ruminal bacteria to barley straw

Adhesion of the cellulolytic ruminal bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes to barley straw was measured by incubating bacterial suspensions with hammer-milled straw for 30 min, filtering the mixtures through sintered glass filters, and measuring the optical densities of the filtrates. Maximum adhesion of both species occurred at pH 6.0 and during mid- to late-exponential phase. Adhesion was saturable at 33 and 23 mg (dry weight) g of straw for R. flavefaciens and F. succinogenes, respectively. Methyl cellulose and carboxymethyl cellulose inhibited adhesion by 24 to 33%. Competition between species was determined by measuring characteristic cell-associated enzyme activities in filtrates of mixtures incubated with straw; p-nitrophenyl-beta-d-lactopyranoside hydrolysis was used as a marker for F. succinogenes, while either beta-xylosidase or carboxymethyl cellulase was used for R. flavefaciens, depending on the other species present. R. flavefaciens had no influence on F. succinogenes adhesion, and F. succinogenes had only a minor (<20%) effect on R. flavefaciens adhesion. The noncellulolytic ruminal bacteria Bacteroides ruminicola and Selenomonas ruminantium had no influence on adhesion of either cellulolytic species, although these organisms also adhered to the straw. We concluded that R. flavefaciens and F. succinogenes have separate, specific adhesion sites on barley straw that are not obscured by competition with non-cellulolytic species.     

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.     

Effect of sodium monensin and cinnamaldehyde on the growth and phenotypic characteristics of Prevotella bryantii and Prevotella ruminicola

Two representative strains of Gram-negative rumen bacteria from the genus Prevotella were used as model organisms in order to evaluate the effect of cinnamaldehyde (the secondary metabolite found in extracts of the Cinnamomum family) vs. sodium monensin on growth, cell size and cell protein production. Prevotella bryantii B(1)4 was found to be remarkably more resistant to the action of both compounds than Prevotella ruminicola 23. The approximate IC(50) concentrations of sodium monensin influenced the increase in cell size of both strains during growth, which was much more pronounced in the case of the B(1)4 strain. A similar effect was observed in strain B(1)4 when 1.438 mmol/L cinnamaldehyde was added to the growth medium, indicating a possible interference with cell division. The action of cinnamaldehyde on P. bryantii B(1)4 was concentration-dependent, in contrast to the effect observed on P. ruminicola 23.     

Design and use of 16S ribosomal DNA-directed primers in competitive PCRs to enumerate proteolytic bacteria in the rumen

Competitive Polymerase Chain Reaction primers were designed for Streptococcus, B. fibrisolvens, P. bryantii, Eubacterium sp., Prevotella, and a universal primer for the eubacteria. DNA was extracted from rumen contents collected from eight dairy cows fed four diets: adequate nitrogen, adequate nitrogen plus carbohydrate, low nitrogen, and low nitrogen plus carbohydrate. B. fibrisolvens was significantly higher on the adequate nitrogen plus carbohydrate and the low nitrogen plus carbohydrate diets compared with the other diets, while P. bryantii was significantly higher on the low nitrogen plus carbohydrate diet compared with the adequate nitrogen diet. The population of Eubacterium sp. was significantly lower on both the adequate nitrogen plus carbohydrate and low nitrogen plus carbohydrate diets. Streptococcus populations were significantly lower on the low nitrogen plus carbohydrate diet compared with all three other diets, whereas there were no significant differences in populations of Prevotella or total eubacteria on any of the diets.     

Antibiotic activity of an isocyanide metabolite of Trichoderma hamatum against rumen bacteria

A metabolite of Trichoderma hamatum, 3-(3-isocyanocyclopent-2-enylidene)propionic acid, was tested for its effects on growth of and carbohydrate metabolism in 11 strains of functionally important rumen bacteria. To standardize the biological activity of this unstable metabolite, a rapid, aerobic disc diffusion assay was developed using Escherichia coli ATCC 11775. In an anaerobic broth dilution assay using a medium lacking rumen fluid and containing a soluble carbohydrate, the minimum inhibitory concentration of the metabolite which completely inhibited growth of the rumen bacteria for 18 h at 39 degrees C was generally less than 10 micrograms X mL-1; however, the minimum inhibitory concentrations for Megasphaera elsdenii B159 and Streptococcus bovis Pe(1)8 were 10-25 and 25-64 micrograms X mL-1, respectively. In general, the Gram-negative strains were more sensitive than the Gram positive. The minimum inhibitory concentration for Bacteroides ruminicola 23 grown with glucose was 1 micrograms X mL-1; for B. ruminicola GA33 (glucose), B. succinogenes S85 (cellobiose), and Succinivibrio dextrinosolvens 24 (maltose), it was 2 microgram X mL-1. When added to a cellulose-containing rumen fluid medium, 1-4 micrograms X mL-1 of the metabolite delayed cellulose hydrolysis by B. succinogenes S85, Ruminococcus albus 7, and R. flavefaciens FD1 for up to 4 days, and 6-7 micrograms X mL-1 prevented hydrolysis for at least 1 month. In the presence of the metabolite, the proportion of acetate produced from soluble carbohydrate by the majority of strains increased, but with some strains net production of acetate decreased relative to production of other acidic fermentation products.(ABSTRACT TRUNCATED AT 250 WORDS)