Milton J. Allison

Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract

This report describes a new group of anaerobic bacteria that degrade oxalic acid. The new genus and species, Oxalobacter formigenes, are inhabitants of the rumen and also of the large bowel of man and other animals where their actions in destruction of oxalic acid may be of considerable importance to the host. Isolates from the rumen of a sheep, the cecum of a pig, and from human feces were all similar Gram-negative, obligately anaerobic rods, but differences between isolates in cellular fatty acid composition and in serologic reaction were noted. Measurements made with type strain OxB indicated that 1 mol of protons was consumed per mol of oxalate degraded to produce approximately 1 mol of CO2 and 0.9 mol of formate. Substances that replaced oxalate as a growth substrate were not found.

Synergistes jonesii, gen. nov., sp.nov.: A Rumen Bacterium That Degrades Toxic Pyridinediols

Summary This study was conducted to identify and characterize rumen bacteria that are able to degrade the toxic compound, 3-hydroxy-4(lH)-pyridone (3,4 DHP), that is produced in the rumen from mimosine. Mimosine is a non-protein amino acid that is found in leaves and seeds of Leucaena leucocephala , a leguminous tree used as a forage crop for ruminants in the tropics, and degradation of 3,4 DHP by ruminal microbes is critical for protection of animals from leucaena toxicity. Microbes with this capacity are, however, not ubiquitous and microbial populations in the rumens of animals in some parts of the world are unable to metabolize 3,4 DHP. Four strains of obligately anaerobic, gram-negative, rod-shaped bacteria that degrade 3,4 DHP were isolated from rumen contents from a goat in Hawaii. The isolates do not ferment carbohydrates, but are able to use both 3,4 DHP and its isomer, 3-hydroxy-2(lH)-pyridone (2,3 DHP), as well as arginine and histidine as substrates for growth. Comparisons of the 16S rRNA sequence from one of these isolates with sequences from a widely diverse group of bacteria agree with other information indicating that these isolates do not fit into any existing taxon. Thus, we are hereby proposing a new genus and species designation, Synergistes jonesii , for these organisms.

Oxalobacter formigenes and Its Potential Role in Human Health

ABSTRACT Oxalate degradation by the anaerobic bacterium Oxalobacter formigenes is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because Oxalobacter is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of O. formigenes. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts. O. formigenes strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of O. formigenes bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of O. formigenes by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.

Biosynthesis of branched-chain amino acids from branched-chain fatty acids by rumen bacteria.

When R. flavefaciens was cultured in a medium containing C14O2 and unlabeled isobutyrate, isovalerate, and 2-methylbutyrate, the carboxyl carbons of valine, leucine, and isoleucine were labeled. Carbon dioxide or a derivative of CO2 was apparently involved in carboxylation reactions with these fatty acids to produce the carbon skeletons of the branched-chain amino acids. Formate carbon was incorported into various cellular components, but was not used in branched-chain amino acid biosynthesis. Radioactivity from acetate-2-C14 was incorporated into several amino acids, but leucine was not appreciably labeled. This suggests that the pathway for leucine biosynthesis that has been demonstrated in a number of other microorganisms does not function in R. flavefaciens. When whole rumen contents were incubated for 2 hr. in the presence of isovalerate-1-C14, most of the radioactivity in the “bacterial fraction” recovered from the fermentation was in leucine. With isobutyrate-1-C14, most of the radioactivity in the “bacterial fraction” was in valine. Although the rates of the reactions are not yet known, it is suggested that in the rumen a significant quantity of microbial branchedchain amino acids may be synthesized from branched-chain fatty acids.

Denitrobacterium detoxificans gen. nov., sp. nov., a ruminal bacterium that respires on nitrocompounds

A new group of anaerobic, Gram-positive, high G + C (56-60 mol%) bacteria was isolated from the bovine rumen. Of four strains characterized, all were non-motile and none produced spores. The isolates did not produce indole or H2S and did not hydrolyse gelatin. Cells of each strain exhibited similar rod-shaped morphology (0.5-1.0 x 1.0-1.5 microns) although bulbous ends were sometimes present. None of the four strains were able to grow via oxidation of a variety of potentially fermentable substrates but rather obtained energy for growth via anaerobic respiration processes, oxidizing hydrogen, formate or lactate for reduction of various oxidized nitrogen compounds. Trimethylamine oxide and DMSO were also used as electron acceptor. All four strains shared greater than 99% 16S rRNA gene sequence identity. The closest match found between the 16S rRNA gene sequence of all four strains, NPOH1T, NPOH2, NPOH3 and MAJ1, to sequences available in GenBank was that of Coriobacterium glomerans (86% sequence similarity), a phenotypically dissimilar anaerobe within the class Actinobacteria. To accommodate these bacteria the creation of a new genus and species, Denitrobacterium detoxificans, for placement within the family Coriobacteriaceae is proposed. The type strain, NPOH1T (ATCC 700546T), grew equally well over a narrow range of incubation temperatures tested (32-39 degrees C).

Lachnospira pectinoschiza sp. nov., an Anaerobic Pectinophile from the Pig Intestine

Pectinophiles are bacteria that utilize pectin and only a few related compounds as substrates. Obligately anaerobic pectinophiles have been isolated from the intestinal tracts and gingivae of humans and from the rumina of cattle. We isolated three strains of pectinophilic bacteria from colonic contents of pigs but were unable to isolate pectinophiles from the rumen contents of four sheep, even when the animals were fed a high-pectin diet. The pectinophiles isolated from pigs were strictly anaerobic, motile, gram-positive rods (0.36 to 0.56 by 2.4 to 3.1 microns). Pectin, polygalacturonic acid, and gluconate were the only substrates that supported rapid growth. All three strains grew slowly on either lactose or cellobiose and fermented fructose after a lag of several days. Pectin was degraded by means of an extracellular pectin methylesterase and a Ca(2+)-dependent exopectate lyase. A comparison of the 16S rRNA sequences of these isolates with the 16S rRNA sequences of other gram-positive bacteria revealed a specific relationship with Lachnospira multipara (level of similarity, 94%). The Gram reaction, formation of spore-like structures, and the utilization of lactose and cellobiose differentiated the pig isolates from previously described pectinophiles. The pig isolates represent a previously undescribed species of the genus Lachnospira, for which we propose the name Lachnospira pectinoschiza.

Anaeroplasma abactoclasticum gen.nov., sp.nov.: an Obligately Anaerobic Mycoplasma from the Rumen

Obligately anaerobic, filterable microorganisms have been isolated from the rumens of cattle and sheep. On the basis of typical colonial appearance, lack of cell wall, filterability through a 450-nm membrane filter, absence of reversion to a bacterial form, and inhibition of growth by homologous antiserum, these organisms fit the description for organisms in the order Mycoplasmatales. They exhibit unique cultural, biochemical, and serological properties, and we have thus placed them in a new genus, Anaeroplasma. A. abactoclasticum is the type species of the new genus. Strain 6-1, a sterol-requiring strain, has been designated as the type strain of A. abactoclasticum and has been deposited in the American Type Culture Collection as ATCC 27879; a non-sterol-requiring strain, number 161, has been deposited as ATCC 27880. The relationship of Anaeroplasma abactoclasticum to Acholesplasma bactoclasticum, an anaerobic mycoplasma possessing extracellular proteolytic and bactoclastic enzymes, is discussed.

Amino acid utilization by the ruminal bacterium Synergistes jonesii strain 78-1

The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-14C)-arginine metabolism were CO2 acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-14C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.

ETHANOL ACCUMULATION IN THE RUMEN AFTER OVERFEEDING WITH READILY FERMENTABLE CARBOHYDRATE.

A neutral volatile substance in ruminal contents from sheep suffering from acute indigestion due to overfeeding has been identified as ethanol. Ethanol was consistently observed in ruminal material from both cattle and sheep after they had been fed large amounts of readily fermentable carbohydrate.

Transfer of Acholeplasma bactoclasticum Robinson and Hungate to the Genus Anaeroplasma (Anaeroplasma bactoclasticum [Robinson and Hungate] comb.nov.): Emended Description of the Species

An anaerobic bacteriolytic mycoplasma was isolated by Robinson and Hungate and designated Acholeplasma bactoclasticum sp.n. We found that this strain and four similar isolates from the ovine rumen have a requirement for cholesterol and thus do not belong in the genus Acholeplasma. The morphological, cultural, and biochemical properties of these organisms are similar to those of a group of non-bacteriolytic anaerobic mycoplasmas that do not fit well in genera previously established for mycoplasmas and thus have been placed in a newly described genus, Anaeroplasma. We propose that the organisms isolated by Robinson and Hungate be transferred to the new genus as Anaeroplasma bactoclasticum (Robinson and Hungate) comb. nov., of which ATCC 27112 is the type strain.