Impact of the Probiotic Organism Megasphaera elsdenii on Escherichia coli O157:H7 Prevalence in Finishing Cattle.

Feedlot cattle commonly shed the foodborne pathogen Escherichia coli O157:H7 in their feces. Megasphaera elsdenii (ME), a lactic acid-utilizing bacterium, is commonly administered to cattle to avoid lactate accumulation in the rumen and to control ruminal acidosis. The impact of administering ME on foodborne pathogen prevalence, specifically E. coli O157:H7, has not been explored. The purpose of this study was to quantify E. coli O157:H7 prevalence in finishing cattle administered ME. Cattle (n = 448) were assigned to treatments in a randomized complete block design with repeated measurements over two sampling periods. Treatments were arranged as a 2 × 2 factorial containing: ruminally protected lysine (RPL; included for a complementary study) fed at 0% or 0.45% of diet dry matter; with or without ME. Freeze-dried ME was administered as an oral drench (1 × 1010 CFU/steer on day one) and then top dressed onto basal diets (1 × 107 CFU/steer) daily thereafter. Rectoanal mucosal swabs (RAMS) were obtained from animals before harvest to determine the E. coli O157:H7 prevalence. The inclusion of RPL (P = 0.2136) and ME (P = 0.5012) did not impact E. coli O157:H7 prevalence, and RPL was not included in any significant interactions (P > 0.05). A significant interaction was observed between ME and sampling period (P = 0.0323), indicating that the effect of ME on E. coli O157:H7 prevalence varied over the sampling period. A diet containing ME reduced the odds of E. coli O157:H7 prevalence by 50% during sampling period 1 (8.0% and 14.7% for cattle with and without ME, respectively) and increased the odds by 23% during sampling period 2 (10.8% and 8.9% for cattle with and without ME, respectively). Administering ME in cattle diets did not impact E. coli O157:H7 in feedlot cattle. This is the first study to investigate the use of ME as a preharvest food safety intervention in cattle, and additional research is necessary to determine the efficacy.

Characterization and heterologous expression of plasmalogen synthase MeHAD from Megasphaera elsdenii.

Plasmalogens (Pls) are vinyl-ether bond-containing glycerophospholipids or glycosyl diradyl glycerols, and are of great importance in the physiological functions and stability of cell membrane. Here, we identified and characterized that the plasmalogen synthase MeHAD from anaerobic Megasphaera elsdenii was responsible for vinyl-ether bond formation. Different from the 2-hydroxyacyl-CoA dehydratase (HAD) family plasmalogen synthase PlsA-PlsR which are encoded by two genes in Clostridium perfringens, the HAD homolog (MeHAD) encoded by a single gene MELS_0169 was found in M. elsdenii. By heterologous expression of the MeHAD gene into a nonplasmalogen-producing Escherichia coli strain, the expressed MeHAD was found to be located in the cell membrane region. Plasmalogens were detected in the recombinant strain using GC-MS and LC-MS, demonstrating that MeHAD was the key enzyme for plasmalogen synthesis. Moreover, the synthesized plasmalogens could enhance the oxidative stress-resistance and osmotic pressure-resistance of the recombinant strain, probably due to the ROS scavenging and decreased membrane permeability by the plasmalogens, respectively. The four-cysteine (Cys125, Cys164, Cys445 and Cys484) site-mutant of MeHAD, which were predicted binding to the [4Fe-4S] cluster, was unable to synthesize plasmalogens, indicating that the cysteines are important for the catalytic activity of MeHAD. Our results revealed the single gene encoded plasmalogen synthase in M. elsdenii and established a recombinant E. coli strain with plasmalogen production potential.

Rapid-reaction kinetics of the butyryl-CoA dehydrogenase component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii.

We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB-bcd) from Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•- rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB-bcd.

Comparison of the effect of Saccharomyces cerevisiae-Megasphaera elsdenii and buffer on growth performance, digestibility, ruminal histomorphometry, and carcass characteristics of fattening lambs in high concentrate diet.

This study aimed to investigate the effect of rumen pH-adjusting additives in the high-concentrated diet on functional traits, nutrient digestion, some meat parameters, and histomorphometry, and rumen histopathology. Twenty-four Arabia male lambs with 3 to 4 months old and initial body weight of 23.9 ± 3.15 kg were used in a completely randomized design with three treatments and eight replicates. The study was 77 days, including 14 days of the adaptation period and 63 days of the record taking and sampling period. The experimental treatments consisted of a control diet, control diet + sodium bicarbonate buffer, control diet + Megasphaera elsdenii, and Saccharomyces cerevisiae (bacterial-yeast). Rumen fluid was taken by stomach tube at 3 h after morning feeding to measure pH. The lambs were weighed every 3 weeks during the period, and the body weight changes, average daily gain, and total weight gain were measured, and the feed conversion ratio was calculated. At the end of the experiment, the lambs were slaughtered, and the longissimus dorsi muscle was prepared to determine the meat parameters. For histological studies, the abdominal rumen sac was sampled. There were no differences among treatments in dry matter intake (DMI), daily weight gain (ADG), and feed conversion ratio (P > 0.05). Propionate concentration was higher in the bacteria-yeast treatment than other treatments (P < 0.05). Protein digestibility was higher in control and bacteria-yeast treatments than buffer treatment (P < 0.05). The percentage of meat protein, carcass weight, and dressing percentage in bacterial-yeast treatment was higher than other treatments (P < 0.05). Rumen wall thickness in the buffer and bacterial-yeast receiving treatments was greater than the control treatment and was significant in the buffer treatment compared to the control treatment (P < 0.05). The thickness of rumen epithelial tissue in the buffer and bacterial-yeast recipient treatments was less than the control treatment (P < 0.05). Rumen papillae thickness was higher in the control treatment than other treatments (P < 0.05). Hydropic degeneration and parakeratosis were less in pH-regulating treatments than in control. The results showed that the use of Megasphaera elsdenii could be an effective way to modulate the ruminal fermentation conditions of lambs fed with high concentrate diets. In addition, to increaseing dressing percentage and meat protein, it can also reduce tissue damage and improve ruminal tissue structure.

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge.

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1-8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9-11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, D-, L-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3-N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3-N concentration was reduced to approximately 2 mg/dL as the challenge progressed.

Ambient pH regulates lactate catabolism pathway of the ruminal Megasphaera elsdenii BE2-2083 and Selenomonas ruminantium HD4.

AIMS: To explore the impact of ambient pH on lactate catabolism by Megasphaera elsdenii BE2-2083 and Selenomonas ruminantium HD4 in both pure culture and in binary mixed culture. METHODS AND RESULTS: The growth rate, substrate consumption, product formation, enzymatic activity and gene expression of M. elsdenii and S. ruminantium at various pHs were examined. Furthermore, the metabolism of lactate catabolism pathways for M. elsdenii and S. ruminantium in the co-culture system was investigated by chasing the conversion of sodium L-[3-13 C]-lactate in nuclear magnetic resonance. In the pure culture systems, ambient pH had significant effects on the growth of M. elsdenii, whereas S. ruminantium was less sensitive to pH changes. In addition, lactate metabolic genes and activities of key enzymes were affected by ambient pH in M. elsdenii and S. ruminantium. In the co-culture system, low ambient pH reduced the contribution lactate catabolism by M. elsdenii. CONCLUSION: M. elsdenii BE2-2083 and S. ruminantium HD4 lactate degradation affected by ambient pH. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the regulatory mechanisms of lactate decomposing bacteria in lactate catabolism under the condition of subacute ruminal acidosis.

Biohydrogen production by mixed culture of Megasphaera elsdenii with lactic acid bacteria as Lactate-driven dark fermentation.

Numerous attempts have been made to upscale biohydrogen production via dark fermentation (DF); however, the Achilles' heel of DF, i.e., lactic acid bacteria (LAB) contamination and overgrowth, hinders such upscaling. Key microbes are needed to develop a lactate-driven DF system that can serve as a lactate fermentation platform. In this study, the utility of Megasphaera elsdenii and LAB co-culturing in lactate-driven DF was evaluated. When inoculated simultaneously with LAB or after LAB culture, M. elsdenii achieved a stable hydrogen yield of 0.95-1.49 H2-mol/mol-glucose, approximately half that obtained in pure M. elsdenii cultures. Hydrogen production was maintained even at an initial M. elsdenii-to-LAB cell ratio of one-millionth or less. Moreover, M. elsdenii produced hydrogen via lactate-driven DF from unusable sugars such as xylose or cellobiose. Thus, M. elsdenii could be a Game changer instrumental in unlocking the full potential of DF.

Spectral deconvolution of redox species in the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii. A flavin-dependent bifurcating enzyme.

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

Influence of three microbial feed additives of Megasphaera elsdenii, Saccharomyces cerevisiae and Lactobacillus sp. on ruminal methane and carbon dioxide production, and biofermentation kinetics.

AIMS: This study was performed to investigate the effects of Megasphaera elsdenii (Me), Saccharomyces cerevisiae (SC) and lactic acid bacteria (FP-Lactobacillus fermentum plus Lactobacillus plantarum) alone or in combination on biogas production and ruminal biofermentation parameter in a heterofermenter system. METHODS AND RESULTS: Eight treatments were evaluated; (i) control (without additive; CON); (ii) Me; (iii) SC; (iv) FP; (v) Me plus SC (MSC); (vi) Me plus FP (MFP); (vii) SC plus FP (SCFP) and (viii) Me plus SC plus FP (MSCFP). Doses of FP, Me and SC were 1·5 × 108 (CFU per ml), 1·5 × 108 (CFU per ml) and 1·4 × 107 (CFU 0·002-1  g), respectively. Biogas production in all time increased (P < 0·05) by MSCFP than CON additive. The proportional methane (CH4 ) decreased (P < 0·05) in MSCFP and FP, while carbon dioxide (CO2 ) was decreased (P < 0·05) by SC compared MSCFP and MSC. The proportional CO2 decreased (P < 0·05) by MSCFP and FP additive. The mean concentration of NH3 -N was not affected by treatments. Concentration of total volatile fatty acids and the percent of acetate and propionate was not affected by treatments. The highest (P < 0·05) percent of butyrate and valerate were observed in MSCFP additive. The experiment showed that microbial additives of FP, SCFP and MSCFP reduced proportional CH4 and CO2 . CONCLUSIONS: Microbial additives of MFP and MSCFP had a sustainable positive efficiency on pH and volatile fatty acids and mitigate CH4 and CO2 . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of microbial additives control on the ruminal pH (MFP) and improve VFA such as butyrate (MSC, MSCFP) and valerate (MSCFP) and reduce the greenhouse gases production showed a reduced risk of ruminal acidosis.

A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.

Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals.

Vaginal dysbiosis associated-bacteria Megasphaera elsdenii and Prevotella timonensis induce immune activation via dendritic cells.

Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria leads to bacterial vaginosis (BV) which is associated with increased inflammation in the genital mucosa. Moreover, BV increases susceptibility to sexual transmitted infections (STIs) and is associated with adverse pregnancy outcomes. It remains unclear how specific vaginal aerobic and anaerobic bacteria affect health and disease. We selected different vaginal bacteria ranging from true commensals to species associated with dysbiosis and investigated their effects on activation of dendritic cells (DCs). Commensal Lactobacilli crispatus did not induce DC maturation nor led to production of pro-inflammatory cytokines. In contrast, BV-associated bacteria Megasphaera elsdenii and Prevotella timonensis induced DC maturation and increased levels of pro-inflammatory cytokines. Notably, DCs stimulated with Prevotella timonensis suppressed Th2 responses and induced Th1 skewing, typically associated with preterm birth. In contrast, Lactobacillus crispatus and Megasphaera elsdenii did not affect Th cell polarization. These results strongly indicate that the interaction of vaginal bacteria with mucosal DCs determines mucosal inflammation and we have identified the anaerobic bacterium Prevotella timonensis as a strong inducer of inflammatory responses. Specifically targeting these inflammation-inducing bacteria might be a therapeutic strategy to prevent BV and associated risks in STI susceptibility and preterm birth.

Impact of feedstocks and downstream processing technologies on the economics of caproic acid production in fermentation by Megasphaera elsdenii T81.

Caproic acid (CA) was produced by Megasphaera elsdenii T81 with Jerusalem artichoke tubers (JA) as a feedstock. More CA was produced under the medium with the acid hydrolysate of JA than the comparative medium with a carbon composition similar to that of JA. CA was produced up to 13.0 g/L and 0.52 g/L/h with extractive fermentation using a mixed solvent of alamine 336 in oleyl alcohol at 37 °C. The JA cost to produce 1 ton of CA is only 505 USD, which is much lower than that required for purchasing sucrose (860 USD) in CA production. As a result of the analysis performed using SuperPro Designer, including the cost of distillation to obtain pure CA, the estimated production cost for CA from dry JA is 1869 USD/ton CA at the production scale of 2000 ton/year, which is lower than the current market price for petroleum-derived CA (~2500 USD/ton).

Medium chain carboxylic acids production from waste biomass: Current advances and perspectives.

Alternative chemicals to diverse fossil-fuel-based products is urgently needed to mitigate the adverse impacts of fossil fuel depletion on human development. To this end, researchers have focused on the production of biochemical from readily available and affordable waste biomass. This is consistent with current guidelines for sustainable development and provides great advantages related to economy and environment. The search for suitable biochemical products is in progress worldwide. Therefore, this review recommends a biochemical (i.e., medium chain carboxylic acids (MCCAs)) utilizing an emerging biotechnological production platform called the chain elongation (CE) process. This work covers comprehensive introduction of the CE mechanism, functional microbes, available feedstock types and corresponding utilization strategies, major methods to enhance the performance of MCCAs production, and the challenges that need to be addressed for practical application. This work is expected to provide a thorough understanding of the CE technology, to guide and inspire researchers to solve existing problems in depth, and motivate large-scale MCCAs production.

Efficacy of Megasphaera elsdenii inoculation in subacute ruminal acidosis in cattle.

Two consecutive experiments were carried out to determine efficacy of Megasphaera elsdenii inoculation in alleviation of subacute ruminal acidosis (SARA). In the first experiment, SARA was induced by feeding corn- and wheat-based diets (20%, 40%, 60% and 80% of TMR, DM basis) in six ruminally cannulated heifers. Continuous pH was obtained using data loggers embedded in rumen. In corn (80%)- and wheat (60%)-based diets ruminal pH ranged from 5.2 to 5.6 for 7.77 and 5.93 hr. In the second experiment (5 day), M. elsdenii (200 ml; 2.4 x 1010 cfu/ml) was inoculated during the first two days. During the SARA induction period, M. elsdenii and S. bovis in rumen liquor were more abundant in wheat-based feeding (7.97 and 8.77) than in corn-based feeding (7.06 and 7.95 per ml, log basis; p < 0.0001 for both). M. elsdenii inoculation increased total volatile fatty acids (VFA) concentration when corn-based diet was fed, whereas it decreased total VFA concentration when wheat-based diet was fed (p < 0.004). There was a decrease in the propionic acid proportion (24.04%-19.08%; p < 0.002), whereas no alteration in lactate and ammonia concentrations was observed. M. elsdenii inoculation increased protozoa count (from 5.39 to 5.55 per ml, log basis; p < 0.009) and decreased S. bovis count (from 9.18 to 7.95 per ml, log basis; p < 0.0001). The results suggest that M. elsdenii inoculation may help prevent SARA depending on dietary grain through altering rumen flora as reflected by a decrease in S. bovis count and an increase in protozoa count.

Rational design of thiolase substrate specificity for metabolic engineering applications.

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse ß-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.

Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O2 Resistance and Catalytic Bias?

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

Valerate production by Megasphaera elsdenii isolated from pig feces.

Megasphaera elsdenii is able to produce several short-chain fatty acids (SCFAs), such as acetate, propionate, butyrate, and valerate. These SCFAs serve as an energy source for host animals and play an important role in gut health. In this study, M. elsdenii was isolated from pig feces that had been collected from two farms located in distinct areas of Japan. These M. elsdenii isolates were genotyped, and 7 representative strains were selected. When these 7 strains and M. elsdenii JCM 1772T were cultured with lactate for 24 h, all 7 strains produced valerate as a predominant SCFA. Therefore, the valerate-producing M. elsdenii inhabits a wide area of Japan. In contrast, M. elsdenii JCM 1772T produced acetate, propionate, butyrate, and valerate at similar levels. When the Y2 strain, one of the 7 representative strains, was cultured without lactate, low levels of valerate accumulated. In contrast, in a time course of lactate fermentation by the Y2 strain, lactate was rapidly consumed, and acetate and propionate were produced after 6 h of incubation. Thereafter, acetate and propionate were consumed from 6 to 12 h after the start of the incubation, and valerate and butyrate were produced. In most of the previously described M. elsdenii strains, valerate was not a predominant SCFA. Therefore, the M. elsdenii Y2 strain showed an unique metabolism in which valerate was produced as a primary end product of lactate fermentation.

Lifespan Extension of Caenorhabditis elegans by Butyricicoccus pullicaecorum and Megasphaera elsdenii with Probiotic Potential.

Butyricicoccus pullicaecorum and Megasphaera elsdenii inhabit the human intestine and have probiotic potential. The aim of this study was to evaluate the effects of B. pullicaecorum and M. elsdenii on the lifespan of Caenorhabditis elegans. They significantly (P < 0.05) extended the lifespan of C. elegans compared with Escherichia coli OP50, a standard food for the worm. Analysis of age-related biomarkers such as lipofuscin, body size, and locomotory activity showed that they retarded aging. They all failed to extend the lifespan of daf-12 or dbl-1 loss-of-function C. elegans mutants compared with E. coli OP50-fed worms. However, the increase in lifespan was observed in daf-16, jnk-1, pmk-1, and skn-1 mutants. Moreover, they increased the resistance of C. elegans to a human pathogen, Salmonella typhimurium. In conclusion, B. pullicaecorum and M. elsdenii extend the lifespan of C. elegans via the transforming growth factor-beta (TGF-ß) pathway associated with anti-inflammatory processes in the innate immune system.

Short communication: Does early-life administration of a Megasphaera elsdenii probiotic affect long-term establishment of the organism in the rumen and alter rumen metabolism in the dairy calf?

Megasphaera elsdenii is a bacterial species of the rumen that can utilize lactate to produce butyrate, a key volatile fatty acid often implicated in driving calf rumen development. Because lactate is abundant in the rumen of young calves, administration of M. elsdenii to increase butyrate production and thus promote calf rumen development is an appealing possibility. The main objective of this study was to determine whether M. elsdenii administration to calves via oral drench at 14 d of age affected its long-term establishment at 70 d postadministration. Ruminal volatile fatty acid and lactate profiles and blood glucose and ß-hydroxybutyrate concentrations were also examined to determine potential influence on rumen metabolism. Six neonatal Holstein heifer calves were blocked on d 1 by body weight (41.3 ± 1.8 kg) and total serum protein (5.23 ± 0.16 g/dL) and assigned to either the M. elsdenii (n = 3) or control (n = 3) treatment groups. On d 14, calves in the M. elsdenii group orally received 25 mL of a commercially available M. elsdenii suspension, whereas calves in the control group received 25 mL of the same product that had been autoclaved. Rumen contents and blood samples were collected weekly from each animal until 84 d of age. The oral administration of M. elsdenii at 14 d did not increase the abundance of M. elsdenii 70 d postdosing, alter rumen fermentation, or change blood metabolites associated with butyrate. These results suggest that a single administration of the M. elsdenii probiotic may not affect the rumen establishment of the organism.

Preferential isolation of Megasphaera elsdenii from pig feces.

Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60-100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9-99.2% identity). These three strains were approximately 1.5 µm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.