Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.     

Modified Multiplex PCR Methods for Comprehensive Detection of Pectinatus and Beer-Spoilage Cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

Non-contiguous finished genome sequence and description of Megasphaera massiliensis sp. nov.

Megasphaera massiliensis strain NP3^T sp. nov. is the type strain of Megasphaera massiliensis sp. nov., a new species within the genus Megasphaera. This strain, whose genome is described here, was isolated from the fecal flora of an HIV-infected patient. M. massiliensis is a Gram-negative, obligate anaerobic coccobacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,661,757 bp long genome (1 chromosome but no plasmid) contains 2,577 protein-coding and 61 RNA genes, including 5 rRNA genes.     

Sequence of the gene encoding the 16S rRNA of the beer spoilage organismMegasphaera cerevisiae

The 16S ribosomal RNA gene from the beer-spoilage organism,Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position ofM. cerevisiae as a sister taxon ofMegasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.     

一株乳酸利用、丁酸产生菌的分离与鉴定及代谢特性的初步研究

利用改进型Hungate技术从猪粪中分离到一株乳酸利用、丁酸产生双重功能菌株LB01。常规生化检测表明菌株LB01为革兰氏阳性、严格厌氧菌,能利用葡萄糖、果糖、麦芽糖和乳酸等碳源,并产生大量的气体;16S rRNA序列比对表明其与GenBank中的Megasphaera hominis与Uncultured rumen bacterium 3c3d-18的同源性最高,同源性高达99%。菌株LB01可以利用乳酸,并将其主要转化为丁酸和丙酸,在有葡萄糖的情况下,菌株LB01尚能够利用乙酸并生成丁酸。与乳杆菌K9共培养时,菌株LB01有效地利用了乳杆菌K9代谢过程中产生的乳酸,减缓了由于乳酸积累而造成的pH值下降,并且将乳酸转化为丁酸和丙酸。这些代谢特征表明菌株LB01是一株具有潜在应用价值的肠道益生菌,它能够利用乳酸和乙酸(补充额外能量),能有效地防止乳酸和乙酸的积累,同时生成包括丁酸在内有益的短链脂肪酸,调控后肠道pH,营造着微酸的环境。