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Effects of an Aspergillus oryzae fermentation extract and other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii.

The objective of this study was to determine the effects of an Aspergillus oryzae fermentation extract (Amaferm) as well as other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii B159. Addition of Amaferm or a filter-sterilized Amaferm filtrate stimulated L-lactate uptake by both M. elsdenii and the ruminal selenomonad strain H18. Growth of M. elsdenii in medium that contained DL-lactate (2 g/L), Trypticase, and yeast extract was only slightly stimulated by the addition of 5% (vol/vol) Amaferm filtrate after 24 h. However, growth of M. elsdenii in a similar medium lacking Trypticase and yeast extract was increased over twofold by the addition of either 2 or 5% (vol/vol) Amaferm filtrate. These results suggest that Amaferm provides growth factors (i.e., amino acids, B vitamins) to support growth of M. elsdenii on lactate. There was no inhibition of L-lactate uptake when lactate-grown cells of M. elsdenii were incubated with excess (10 mM) glucose, sucrose, or maltose. In addition, when cells were grown on glucose, sucrose, or maltose rather than lactate there was little difference in L-lactate uptake, suggesting that L-lactate transport in M. elsdenii is not subject to catabolite repression by these soluble sugars. Both K+ and Na+ had little effect on L-lactate uptake. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, whereas pH values of 5.0 and 4.0 increased uptake. In addition, L-lactate uptake was inhibited between 34 and 61% by protonophores. These results suggest that protons may be involved in the uptake of L-lactate by M. elsdenii B159.

Animal Feed↗

Preventing in vitro lactate accumulation in ruminal fermentations by inoculation with Megasphaera elsdenii.

In vitro fermentations containing a mixed culture of ruminal bacteria (ruminal fluid from a hay-fed steer), buffer, and primarily rapidly degradable substrates (starch, glucose, cellulose, cellobiose, and trypticase) were inoculated with an overnight culture of Megasphaera elsdenii B159. Triplicate flasks were either uninoculated or inoculated to obtain a final concentration of 8.7 x 10(5) and 8.7 x 10(6) colony forming units of M. elsdenii per milliliter of culture fluid. Inoculation with M. elsdenii prevented an accumulation of lactic acid and excessive drop in pH. Lactate peaked at more than 40 mM in untreated cultures. In cultures inoculated with a low dose of M. elsdenii, lactate concentration peaked at approximately 25 mM at 5 h of fermentation but decreased rapidly to less than 5 mM by 7 h of fermentation. With the addition of the high dose of M. elsdenii, lactate was never greater than 2 mM (P < .05) throughout fermentation. Cultures treated with M. elsdenii had greater amounts (P < .05) of isobutyrate, butyrate, isovalerate, and valerate than untreated cultures. After 24 h of fermentation, one-half of the culture fluid was transferred to an equal volume of fresh buffer with substrate but was not inoculated with further quantities of M. elsdenii. Six hours after transfer, cultures that had been originally treated with M. elsdenii had lower (P < .05) amounts of lactate than untreated cultures. Inoculation with M. elsdenii has potential to prevent lactate accumulation in diets containing readily fermentable carbohydrates.

Animals↗

Characterization of endotoxin from the rumen bacterium Megasphaera elsdenii.

Phenol-water extraction of Megasphaera elsdenii, a predominant gram-negative coccus in rumens of cattle fed high-grain diets, yielded material that exhibited typical characteristics of endotoxin. The extract was lethal to mice and to chicken embryos, caused biphasic fever in rabbits, leukopenia in mice, and local and generalized Shwartzman reactions; and induced tolerance to the lethal effect of the endotoxin in mice. The material contained carbohydrate, protein, lipid, phosphorus, and 2-keto-3-deoxyoctonate, but no nucleic acid. The beta-hydroxymyristic acid was absent. Results imply that M elsdenii endotoxin has many biological and chemical characteristics common to enterobacterial endotoxin. However, the median lethal doses in mice and in chicken embryos, and minimal dose required to elicit a local Shwartzman reaction, indicate that M elsdenii endotoxin's potency is low, which may explain why the large gram-negative bacterial population in the rumen of cattle is generally innocuous.

Animals↗

A pulse-radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism.

Pulse-radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is -(480 +/- 30) mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2 approximately equal to 10(5)S-1) the intermediate state decays to the stable semiquinone form of flavodoxin. The delta G of the conformational change at pH 8 is about 29 kJ mol -1 (0.3 eV). This means that the upper limit for the pK of N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mol -1 (0.45 eV). The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion-controlled with a forward rate constant of (7 +/- 1) X 10(8) M -1S -1 (pH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 +/- 5) mV. A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two-electron process, the midpoint potential of which should be located between -440 mV less than Em (Fld/FldH-) less than -290 mV. The exact value will depend on the delta G of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two-electron donor.

Electron Transport↗

[Megasphaera elsdenii--occurrence in 2,255 fecal samples of humans, chimpanzees and mice].

In human gnotobiotic studies the number of cfu/g feces expresses the interspecies-specific relations within this biotop. In our study we tried to culture the resident anaerobe microflora diluting 1 g feces to 10(-6) - 10(-9), and using the cystein-blood-agar (Beerens mod. Werner) together with the dextrose-water-blue agar. M. elsdenii appear as big, smooth and yellow colonies, which may be recognised at "first sight" diagnosis. In a retrospective study of 2255 fecal samples we compared the isolation-rate of M. elsdenii in the range of greater than or equal to 10(7)/g feces from healthy people to that of persons with gastrointestinal troubles. M. elsdenii has not been isolated from fecal samples of healthy people but in 12% from those of adults and 7.4% from children, both suffering from gastrointestinal disorders. So we consider that M. elsdenii in the range of greater than or equal to 10(7)/g feces does not belong to the resident anaerobe flora of the intestine. Further studies have to establish wether its normal range may be 10(3)-10(4)/g feces like that of C. perfringens. Comparing the isolation-rate from patients with colon-carcinoma to that of patients with dermatological affections, we found a relation of 8% to 20%. The biochemical activities of our isolates were according to the Anaerobe Laboratory Manual. Additional tests e.g. API-Zym and others make us assume an activity inner the metabolism of fatty-acids and amino acids e.g. inner the aspartat and pyruvat-family.

Adult↗

Cell envelope morphology of rumen bacteria.

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.

Adaptation, Biological↗

Obligately anaerobic bacterial species isolated from foot-rot lesions in goats.

Lesions showing clinical signs of foot-rot from 120 goats were cultured on six selective media during October 1987 to November 1988. A total of 582 strictly anaerobic microorganisms belonging to 50 different species was isolated and identified. The anaerobes most frequently isolated belonged to the following genera: Bacteroides (80%), Peptostreptococcus (63.6%), Megasphaera (40%), Fusobacterium (29.2%), Clostridium (22.5%), Propionibacterium (12.5%), Eubacterium (11.7%) and Leptotrichia (10.8%). Percentages for Acidaminococcus, Peptococcus, Tissierella, Wolinella and Veillonella were below 10%. The following species were identified in 10% or more of cases: Peptostreptococcus anaerobius (61.7%), Bacteroides buccae (51.7%), Bacteroides nodosus (42.5%), Megasphaera elsdenii (40%), Bacteroides ruminicola subsp. brevis (22.5%), Fusobacterium necrophorum (19.2%), Leptotrichia buccalis (11.7%) and Clostridium perfringens (10%). Lower percentages were obtained for the remaining species. The efficiency and selectivity of the six culture media used for isolation are discussed.

Animals↗