Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro.

A mixed culture chemostat system was used to distinguish between the effects of carbohydrate availability per se and the low pH generated from carbohydrate metabolism on the proportions of bacteria within microbial communities. Nine oral bacteria were grown at pH 7 and pulsed with glucose on ten consecutive days. In one chemostat, the pH was maintained automatically at 7 throughout the experimental period, while in the other, pH control was discontinued for six hours after each pulse. Glucose pulses at neutral pH had little effect on the composition of the microflora. Only the proportions of A. viscosus and V. dispar increased; L. casei and S. mutans remained at low levels (0.2% and 1.0%, respectively). Acetate and propionate were low. In contrast, when pH was allowed to fall after each glucose pulse, the composition of the microflora altered dramatically. The amounts of L. casei and S. mutans increased both as a proportion of the total count and in absolute numbers, as did V. dispar, whereas the amounts of the other Gram-negative organisms (B. intermedius, F. nucleatum, and N. subflava) and S. sanguis were considerably reduced. Lactate formed a major portion of the metabolic end-products. Successive glucose pulses resulted in both amplified changes in the microflora and a steadily greater rate and final extent of acid production. This is in agreement with the reported shifts in the oral microflora in vivo in response to frequent carbohydrate intake. Analysis of the data strongly suggests that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, is responsible for the widely reported shifts in composition and metabolism of the oral microflora in vivo.

Factors affecting the in vitro production of volatile fatty acids by mixed bacterial populations from the bovine rumen.

Factors affecting in vitro ruminal bacterial VFA production were examined. Treatments consisted of high and low initial pH (6.7, 5.7), osmolality (600, 400 mOsm) and concentrations of acetic (40, 0 mM) and propionic acids (20, 0 mM). Response variables measured included the production of acetic, propionic and total VFA, total gas and methane. Initial pH affected (P less than .05) most variables either independently or in combination with one or more of the other factors. Acetic acid production was reduced 40% (P = .03) when initial acetic acid concentrations were 40 mM compared with 0 mM. Also, acetic acid production was less (P less than .01) at low initial pH (5.7) than at high initial pH (6.7). Propionic acid production was greater (P = .05) at high vs low initial acetic acid concentrations. Propionic acid production was greater in response to low vs high initial osmolality, although the magnitude of this difference depended on initial pH (interaction P = .02). Total production of VFA was greater (P less than .01) at high than at low initial pH; however, at low initial pH, no difference (P greater than .05) was observed due to initial osmolality, whereas at high pH, production was greater (interaction P = .04) for low than for high initial osmolality. The diminished production of total VFA at pH 5.7 occurred primarily due to reduced acetic acid production, although increased production of propionic and butyric acids was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of enzyme induction on metabolite formation of bis(2-methoxyethyl) ether in the rat.

The effect of enzyme induction on the metabolism of the reproductive toxicant bis (2-methoxyethyl) ether (diglyme) was studied in male Sprague-Dawley rats. Rats were given either daily doses of diglyme at 5.1 mmol/kg body wt. by gavage or 0.1% (w/v) phenobarbital (PB) in the drinking water for 22 consecutive days. In one study, a significant reduction in the hexobarbital sleeping time was determined for rats pretreated with diglyme or PB in comparison with that determined for naive rats. In a second study, naive and pretreated rats given single oral doses of 14C-diglyme at 5.1 mmol/kg body wt. showed similar urinary 14C excretion patterns. Urinary metabolites were separated and quantified by hplc to evaluate the influence of pretreatment with either diglyme or PB on the 14C-diglyme urinary metabolite profile. The amount of (2-methoxyethoxy) acetic acid, the principal metabolite, was similar for rats given no pretreatment and for rats pretreated with either diglyme or PB. However, both pretreatments resulted in significant increases in the formation of methoxyacetic acid, a recognized reproductive toxicant.

Metabolism of metronidazole and antipyrine in isolated rat hepatocytes. Influence of sex and enzyme induction and inhibition.

The metabolism of metronidazole and antipyrine was investigated in freshly isolated hepatocytes from 7 male and 6 female control Wistar rats, 8 males and 5 females pretreated with phenobarbital (PB) and 3 males pretreated with 3-methylcholanthrene (MC). Pretreatment with PB increased the intrinsic clearance (CLi = Vmax/Km) of metronidazole to its acetic acid (MAA) and hydroxy metabolite (HM) 7- and 2.8-fold in the males and 3.2- and 3.0-fold in the females, whereas MC treatment increased the values 9- and 10-fold, respectively (P less than 0.05). The CLi of metronidazole to HM and its glucuronide conjugate was higher in the control and PB treated male than in the corresponding female groups, whereas the rank order was reversed for sulphate formation (P less than 0.05). SKF 525A was a more potent inhibitor of MAA formation than of HM formation, except in the PB treated male group. Pretreatment with MC increased the inhibitory potency of alpha-naphthoflavone and antipyrine toward MAA and HM formation. In male rats PB treatment increased the CLi of antipyrine to 3-hydroxymethyl-(HMAP), nor-(NORAP) and 4-hydroxyantipyrine (OHAP) 2.5-, 2.1- and 4.5-fold, respectively (P less than 0.05). Pretreatment with MC in male and with PB in female rats had no significant effect on antipyrine metabolism. SKF 525A was a more potent inhibitor of HMAP and OHAP formation than of NORAP formation. Treatment with MC increased the inhibitory potency of alpha-naphthoflavone toward the formation of all antipyrine metabolites. Metronidazole increased the formation rate of HMAP, but inhibited the formation of NORAP and OHAP, particularly the latter. The results suggest that the formation of MAA, HM, HMAP, NORAP and OHAP from metronidazole and antipyrine is catalyzed by different cytochrome P-450 isozymes, which may be supplemented or substituted by PB or MC induced species. The involved P-450 isozymes have more or less overlapping substrate and product specificity. Metronidazole appears to be a sensitive probe for detection and identification of PB and MC type induction.

The suicide phenomenon in motile aeromonads.

Certain strains of motile Aeromonas species, including all those of Aeromonas caviae examined, were shown to be suicidal. When they were grown in the presence of glucose at both 30 and 37 degrees C, there was rapid die-off of the organisms after 12 h of incubation, and viable cells generally could not be recovered after 24 h. It was shown that this phenomenon was due to the production of relatively high levels of acetic acid by these strains, even during growth under highly aerobic conditions, and to the greater susceptibility of these strains to acetic acid-mediated death. Suicide did not occur when the pH was maintained above 6.5 or in the presence of high concentration of Pi. These observations were consistent with our inability to isolate suicidal Aeromonas spp. from acidic lakes in New England and with their recovery from alkaline waters in Israel and from sewage. Suicidal aeromonads appear to be better adapted than the nonsuicidal biotypes to anaerobic growth in low-nutrient environments.

Concentrations of volatile fatty acids and acetate production rates in the forestomachs of grazing camels.

1. Concentration profiles of volatile fatty acids (VFA), fluid volumes and turnover rates, and acetate production rates were measured in two different seasons in the forestomachs of four fistulated dromedary camels grazing in the Kenyan thornbush savannah. 2. VFA profiles and average concentrations were similar under both feeding conditions but, due to a smaller fluid turnover, VFA outflow to lower gastric sections in the dry season was reduced by almost 50%. 3. The mean acetate production rate fell from 2234 mmol/hr in the green season to 816 mmol/hr in the dry season, i.e. by approximately 64%.

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii.

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.

Short-chain fatty acid production from mono- and disaccharides in a fecal incubation system: implications for colonic fermentation of dietary fiber in humans.

An in vitro fecal incubation system was used to demonstrate how lactose, lactulose and monosaccharides (mainly constituents of dietary fiber) influence short-chain fatty acid production in colon. Short-chain fatty acids were formed from all mono- and disaccharides tested (except L-glucose): D-glucose, D-galactose, D-fructose, D-mannose, L-rhamnose, D-sorbitol, D-arabinose, D-xylose, D-ribose, D-galacturonate, D-glucuronate, lactose and lactulose. All saccharides increased acetate formation; propionate production was increased from rhamnose, arabinose, xylose, ribose, galacturonic and glucuronic acid, whereas the synthesis of butyrate was elevated in assays incubated with sorbitol, galacturonic and glucuronic acid, and to a lesser degree ribose. Isobutyrate, valerate, isovalerate and hexanoate were produced in increased amounts in assays incubated with albumin, but in fact decreased in many incubations with saccharides. It is speculated that saccharide fermentation always results in formation of acetate, and that the relative production of acetate, propionate and butyrate is related to the monosaccharide composition of dietary fiber available for colonic bacteria. However, the production of isobutyrate, valerate, isovalerate and hexanoate is probably not due to saccharide fermentation, but is rather of polypeptide origin.

Effect of xylitol-containing carbohydrate mixtures on acid and ammonia production in suspensions of salivary sediment.

pH changes and the production of lactic acid, acetic acid and ammonia were studied in suspensions of salivary sediment supplemented with mixtures of xylitol and other carbohydrate sweeteners. The only mixtures which increased the pH values of the suspensions were those containing xylitol alone or mixtures of xylitol and sorbitol. Mixtures of xylitol and Lycasin 80/55 caused a relatively small pH reduction. Xylitol was not able to inhibit the acid production from the easily fermented glucose, fructose and Lycasin 05/60. The levels of lactic acid, determined in the incubation mixtures, directly reflected these pH changes. The levels of acetic acid and ammonia were, however, relatively similar in all incubation mixtures. The results suggest that the inhibitory effects of xylitol on acid production of oral flora should be retained, provided that xylitol is used either alone or in mixtures with slowly fermentable carbohydrates, such as sorbitol and Lycasin 80/55.

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle.

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.

The effect of arginine on in-vitro acid production by the oral bacterium Streptococcus mutans 10449 in various concentrations of glucose.

Acid production was measured both with constant pH and with the pH being allowed to fall. Under both conditions 10 mM arginine reduced it. There was also evidence that arginine reduced the rate of uptake of glucose by the bacterium. Thus arginine may affect the rate of glucose transport or catabolism by Strep. mutans 10449, and has more than a simple buffering effect on these cultures.

Substrate stereochemistry of the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans.

The steric course of the decarboxylation of glutaconyl-CoA to crotonyl-CoA, catalysed by the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans, was elucidated using the sequence: chiral acetate----citrate----glutamate----glutaconyl-CoA----crotonyl-CoA ----chiral acetate. Since glutaconyl-CoA or glutaconate labeled at C-4 was subjected to rapid chemical or enzymatic exchanges, glutamate was fermented to acetate by growing cells of A. fermentans. The analysis of the final chiral acetates gave following deviations from 50% in the fumarase exchange: + 13.8% starting with (R)-acetate and - 13.9% starting with (S)-acetate. The results demonstrated a retention of configuration during the decarboxylation. Thus glutaconyl-CoA decarboxylase adds to the list of biotin enzymes in which exclusive retention of configuration was observed. Glutaconate CoA-transferase from A. fermentans catalysed a 3H exchange of [2,4,4-3H]glutaconate with water when acetyl-CoA was present. At low concentration of acetyl-CoA (20 microM) the exchange ceased after exactly one atom 3H was released into the water, at high concentrations (1 mM) the exchange proceeded further. The apparent Km of acetyl-CoA in the exchange (1.1 microM) was 150 times smaller than that of the complete CoA transfer. It was concluded that either a mixed anhydride, between a carboxyl group of the enzyme and [2,4,4-3H]glutaconate, or enzyme-bound glutaconyl-CoA was the exchanging species.

Analysis by gas liquid chromatography of production of volatile fatty acids by anaerobic bacteria grown on solid medium.

Volatile fatty acids produced in Robertson's cooked meat medium by a range of clinically relevant anaerobes were compared by gas liquid chromatography with those produced in blood agar. The same volatile fatty acid profiles were obtained in both media, although the concentration of acids was lower in blood agar. We conclude that detection of volatile fatty acids from a pure culture of an organism on solid medium is practicable and offers advantages over the conventional technique.

Importance of high pKA acids in cariogenic potential of plaque.

Through the use of computer simulation, it is shown that anions of high pKA acids (e.g., acetic, propionic, butyric), present in resting plaque fluid, act as effective buffers during the production of stronger acids (e.g., lactic) by oral micro-organisms. Using reported organic acid compositions for plaque, obtained from caries-resistant and caries-susceptible individuals, analyzed at various times following sucrose exposure, it is shown that the calculated pH values of the aqueous phase of plaque describe typical Stephan curves. Furthermore, it is shown that for higher acid anion contents in resting plaque, a higher degree of saturation of the aqueous plaque phase with respect to enamel is maintained during acid production, resulting in a lower demineralization potential of plaque. Enamel demineralization experiments conducted in vitro confirmed the significance of the results of the computer simulations, thus providing evidence for a correlation between the known metabolic activity of plaque (organic acid composition), physical chemistry of plaque, and caries susceptibility.

Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis.

The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups of acetyl-CoA, then equilibrate the carbonyl with CO in the solution and re-form acetyl-CoA. CoA is not necessary for the exchange and, in fact, inhibits the reaction. These studies support the view that CO dehydrogenase is the condensing enzyme that forms acetyl-CoA from its component parts. Carbon dioxide also exchanges with the C-1 of acetyl-CoA, but at a much lower rate than does CO. At 50 degrees C and pH 5.3, the optimal pH, the turnover number is 70 mol of CO exchanged per min/mol of enzyme. Low potential electron carriers are stimulatory. The Km app for stimulation by ferredoxin is 50-fold less than the value for flavodoxin. Neither ATP or Pi stimulate the exchange. The EPR spectrum of the CO-reacted enzyme is markedly changed by binding of CoA or acetyl-CoA. Arginine residues of the CO dehydrogenase appear to be involved in the active site, possibly by binding acetyl-CoA. Mersalyl acid, methyl iodide, 5,5-dithiobis-(2-nitrobenzoate), and sodium dithionite inhibit the exchange reaction. A scheme is presented to account for the role of CO dehydrogenase in the exchange reaction and in the synthesis of acetate.

Metabolism of propionate to acetate in the cockroach Periplaneta americana.

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.

In-vitro acid production by the oral bacterium Streptococcus mutans 10449 in various concentrations of glucose, fructose and sucrose.

At intermediate and high concentrations, the results with the sugars were similar, with lactic acid as the main end product. Over 4 h, the pH fell from approx. 7 to 4. At low monosaccharide concentrations (2 mM glucose, 2 and 5 mM fructose), after an initial pH drop and period of lactic-acid production, evidence of pH rise and lactic-acid consumption were noted. This did not happen when sucrose was added to the bacteria. There was evidence of a heterolactic-acid fermentation pattern at low-sugar concentrations, lactic, acetic and formic acids being produced in similar amounts. The results suggest that, when low-sugar concentrations are present in dental plaque, Strep. mutans is capable of consuming previously-formed lactic acid.