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The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.

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Recently, a unique bacterium, Eubacterium coprostanoligenes ATCC 51222, that reduces cholesterol to coprostanol was isolated. Because coprostanol is absorbed poorly, it was hypothesized that oral administration of Eu. coprostanoligenes might decrease cholesterol concentration in blood because the micro-organisms will decrease the absorption of endogenous and dietary cholesterol by conversion to coprostanol. To test the hypothesis, three adult New Zealand White rabbits received 4 ml of Eu. coprostanoligenes suspension (ca 2 x 10(7) cells ml-1) daily per os for 10 d; three other adult New Zealand White rabbits received the same dosage of boiled bacterial suspension. Plasma cholesterol concentration of experimental rabbits (183.3 +/- 11.0 mg dl-1, mean +/- S.E.) was significantly lower (P < 0.001) than that of controls (248.8 +/- 12.3 mg dl-1, mean +/- S.E.). The coprostanol-to-cholesterol ratios in contents of digestive tracts of experimental rabbits were greater than those of controls. The data indicate that oral administration of Eu. coprostanoligenes caused a significant hypocholesterolemic effect in rabbits and that this effect can be explained by the conversion of cholesterol to coprostanol in the intestine.

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A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen. The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.

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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.

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Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.

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Over the past 6 years, a revised classification of the streptococci and enterococci, based primarily on molecular techniques such as 16S rRNA sequencing and DNA-DNA hybridization, emerged. However, little attention was placed on routine physiological tests that could be used in food and clinical laboratories to differentiate between species of a new genus, Enterococcus, and fecal Streptococcus spp. The purpose of this study was to devise a convenient and reliable system to identify enterococci and fecal streptococci by using conventional procedures. Fifty-nine strains of 13 Enterococcus spp., including the type strains and many strains used by previous investigators, were characterized by using conventional tube tests, the API Rapid Strep system, and MicroScan Pos ID panels. Results were compared with each other and with previously published results. A comparison of conventional tube tests versus published tube test results yielded 17 discrepancies. Although not all tests were done with each of the three systems, 28 discrepancies between results obtained with the API system and those obtained with conventional tube tests were found. There were 24 discrepancies between results obtained with the MicroScan Pos ID panel and those obtained with conventional tube tests. There were 12 discrepancies between the results with the API Rapid Strep system and those with the MicroScan Pos ID panels. We devised flow charts of key tests that might be used to identify cultures without resorting to nucleic acid analysis and other labor- and equipment-intensive analyses.

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A simple well-plate technique was utilized to determine the effect of various metals on the growth of microorganisms in media containing different polyphosphates. Aspergillus flavus and four gram-positive bacteria were completely inhibited by media containing 1% of various alkaline polyphosphates, whereas four gram-negative bacteria were not. Significant differences were observed between the type of polyphosphate added, the type of metal added, and the species of gram-positive bacterium inhibited. The addition of Mg2+ stimulated growth of A. flavus and Bacillus cereus in the presence of tetrasodium pyrophosphate, whereas Mn2+ permitted growth of A. flavus and Staphylococcus aureus in the presence of sodium hexametaphosphate. Iron supplementation allowed the growth of S. aureus and Listeria monocytogenes on media containing 1 % tetrasodium pyrophosphate. A method for determining the amount of calcium and magnesium in water was modified to detect free Mg2+ by replacing EDTA with phosphate. The addition of free Mg2+, but not Mg2+ chelated by tetrasodium pyrophosphate, permitted the growth of B. cereus on a medium containing tetrasodium pyrophosphate. It is speculated that polyphosphates specifically inhibited A. flavus and gram-positive bacteria by removing essential metals from cation-binding sites located within their cell walls.

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Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.

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Simple enzymatic assays to detect heat-labile enterotoxins whose modes of action are similar to that of cholera toxin were evaluated. The assays are performed by using an artificial substrate, diethylamino benzylidine-aminoguanidine, which is an ADP-ribose acceptor. The product, formed in the presence of NAD+, can be quantitated by spectrofluorometric, spectrophotometric, or high-performance liquid chromatographic (HPLC) methods. As little as 25 ng (spectrofluorometry) or 125 ng (spectrophotometry or HPLC) of cholera toxin can be detected in an assay volume of 250 microliters. The detection limit for heat-labile enterotoxin by either the spectrophotometric or HPLC methods was 125 ng/250 microliters. Because the results are quantitative, the enzymatic methods can be used for medium development, determination of factors that influence toxin production, and other applications that heretofore could be accomplished only with difficulty. The enzymatic methods add a new dimension to the assay of toxins that ribosylate arginine residues of proteins. Sensitivities of the assays might be improved by developing better synthetic substrates, and applications could be broadened by the development of artificial substrates containing other functional groups.

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Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.

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Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PPi-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PPi-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P2). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P2), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PPi-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.

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Separation of fertirelin acetate (FA) from process impurities, potential degradation products and related peptides including luteinizing hormone releasing hormone has been achieved by reversed-phase high-performance liquid chromatography (HPLC). A number of chromatographic conditions (column type, mobile phase composition, isocratic/gradient elution) and detection systems have been utilized to examine the bulk drug and formulation of FA. Examples of separations designed for potency and impurity determinations are described. Complete recovery of FA is obtained with an isocratic HPLC system. An external standard method is used to determine potency with a precision of less than 1% R.S.D. A gradient HPLC system is used to determine impurities with a precision of ca. 5-10% R.S.D. at the 1-2% impurity level. As little as ca. 0.1% (area%) of related peptides are detected at 214 nm.

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Six strains of Oxalobacter formigenes (anaerobic oxalate-degrading bacteria) were examined for their ability to colonize the gastrointestinal tracts of adult laboratory rats. These rats did not harbor O. formigenes. Strain OxCR6, isolated from the cecal contents of a laboratory rat that was naturally colonized by oxalate-degrading bacteria, colonized the ceca and colons of adult rats fed a diet that contained 4.5% sodium oxalate. Five days after rats were inoculated intragastrically with 10(9) viable cells of strain OxCR6, oxalate degradation rates in cecal and colonic contents increased by 19 and 40 times, respectively. Viable counts of strain OxCR6 from these rats averaged 10(8)/g (dry weight) of cecal contents. Strain OxCR6 was not detected in the cecal contents of inoculated rats fed diets that contained less than 3.0% sodium oxalate. Strains of O. formigenes isolated from the cecal contents of swine, guinea pigs, and wild rats and from human feces also colonized the ceca of laboratory rats; a ruminal strain failed to colonize the rat cecum.

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Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.

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Rates of oxalate degradation by mixed bacterial populations in cecal contents from wild rats ranged from 2.5 to 20.6 mumol/g (dry weight) per h. The oxalate-degrading activity in cecal contents from three strains of laboratory rats (Long-Evans, Wistar, and Sprague-Dawley) from four commercial breeders was generally lower, ranging from 1.8 to 3.5 mumol/g (dry weight) of cecal contents per h. This activity did not increase when diets were supplemented with oxalate. When Sprague-Dawley rats from a fifth commercial breeder were fed an oxalate diet, rates of oxalate degradation in cecal contents increased from 2.0 to 23.1 mumol/g (dry weight) per h. Obligately anaerobic, oxalate-degrading bacteria, similar to ruminal strains of Oxalobacter formigenes, were isolated from the latter group of laboratory rats and from wild rats. Viable counts of these bacteria were as high as 10(8)/g (dry weight) of cecal contents, which was less than 0.1% of the total viable population. This report presents the first evidence for the presence of anaerobic oxalate-degrading bacteria in the cecal contents of rats and represents the first direct measurement of the concentration of these bacteria in the large bowel of monogastric animals. We propose that methods used for the maintenance of most commercial rat colonies often preclude the intestinal colonization of laboratory rats with anaerobic oxalate-degrading bacteria.

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Mixed populations of rumen bacteria or Clostridium hastiforme (a rumen isolate) catalyzed the oxidation of vitamin D3 to 5(E)-19-nor-10-keto-vitamin D3. The reaction depended upon small amounts of O2 (less than 0.1% dissolved O2); when O2 was available, supernatant obtained from heat-killed mixed cultures also produced 5(E)-19-nor-10-keto-vitamin D3. Results obtained by ultrafiltration indicated that at least two heat-stable factors of bacterial origin were involved. Lower rates of the same oxidation were observed when O2 was introduced to solutions containing vitamins D3 and L-cysteine. Oxygen radicals are known to be produced in such solutions and the involvement of such radicals in the D3 oxidation is probable since production in cysteine solutions was inhibited by superoxide dismutase and catalase.

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Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification.

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Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

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Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide possessed beta-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported.

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