Inhibition of Shigella flexneri by the Normal Intestinal Flora II. Mechanisms of Inhibition by Coliform Organisms

Of 15 strains of coliform bacteria, all isolated from human feces, 14 inhibited the growth of Shigella flexneri in mixed culture. In every case, when inhibition occurred, exponential growth of Shigella was interrupted in the mixed culture and the organisms entered into either a stationary or a death phase. None of the test coliform strains produced colicines active against Shigella. An analysis of mixed-culture environments at the time Shigella inhibition occurred revealed that the inhibition was not due to nutrient depletion nor to the development of adverse pH or oxidation-reduction potentials in themselves. In mixed cultures, the coliform strains produced formic and acetic acids in concentrations that inhibited Shigella growth. With one exception, the coliform strains also greatly reduced the culture medium. In average concentrations produced, the formic and acetic acids exerted a bactericidal effect on Shigella under the reduced conditions found in mixed cultures. The acids were only moderately toxic for the coliform strains under the same conditions. Results indicate that volatile acid production and concomitant reduction of the medium are the mechanisms by which coliform bacteria inhibit Shigella growth in mixed cultures.     

Inhibition of a glucose-limited sequencing fed-batch culture of Salmonella enterica serovar enteritidis by volatile fatty acids representative of the ceca of broiler chickens

The effects of concentrations of volatile fatty acids on an anaerobic, glucose-limited, and pH-controlled growing culture of Salmonella enterica serovar Enteritidis were studied. Suddenly increasing volatile fatty acids to the concentrations representative of the ceca of 15-day-old broiler chickens caused washout of serovar Enteritidis. In contrast, a sudden increase to the volatile fatty acid concentrations representative of the ceca of younger broiler chickens caused a reduction in the biomass but not washout. Gradually increasing volatile fatty acids caused a gradual decrease in the biomass of serovar Enteritidis. We conclude that the concentrations of volatile fatty acids present in the ceca of broilers with a mature microflora can cause washout of serovar Enteritidis in an in vitro system mimicking cecal ecophysiology.     

Inhibition of methanogenesis and its reversal during biogas formation from cattle manure

The composition of volatile fatty acids in the biogas digester based on cattle manure as substrate and stabilised at 25°C showed that it contained 87–88% branched chain fatty acids, comprising of isobutyric and isovaleric acids, in comparison to 38 % observed in the digester operating at 35°C. Mixed cellulolytic cultures equilibrated at 25°C (C-25) and 35‡C (C-35) showed similar properties, but rates of hydrolysis were three times higher than that observed in a standard biogas digester. The proportion of isobutyric and isovaleric were drastically reduced when C-25 was grown with glucose or filter paper as substrates. The volatile fatty acids recovered from C-25 (at 25°C) inhibited growth of methanogens on acetate, whereas that from C-35 was not inhibitory. The inhibitory effects were due to the branched chain fatty acids and were observed with isobutyric acid at concentrations as low as 50 ppm. Addition of another micro-organismRhodotorula selected for growth on isobutyric completely reversed this inhibition. Results indicate that the aceticlastic methanogens are very sensitive to inhibition by branched chain fatty acids and reduction in methane formation in biogas digester at lower temperature may be due to this effect.     

Inhibition of a Glucose-Limited Sequencing Fed-Batch Culture of Salmonella enterica Serovar Enteritidis by Volatile Fatty Acids Representative of the Ceca of Broiler Chickens

The effects of concentrations of volatile fatty acids on an anaerobic, glucose-limited, and pH-controlled growing culture of Salmonella enterica serovar Enteritidis were studied. Suddenly increasing volatile fatty acids to the concentrations representative of the ceca of 15-day-old broiler chickens caused washout of serovar Enteritidis. In contrast, a sudden increase to the volatile fatty acid concentrations representative of the ceca of younger broiler chickens caused a reduction in the biomass but not washout. Gradually increasing volatile fatty acids caused a gradual decrease in the biomass of serovar Enteritidis. We conclude that the concentrations of volatile fatty acids present in the ceca of broilers with a mature microflora can cause washout of serovar Enteritidis in an in vitro system mimicking cecal ecophysiology.     

Inhibition by fatty acids during fermentation of pre-treated waste activated sludge

Fermentation of waste activated sludge produces volatile fatty acids (VFAs), which can be used as the carbon sources for numerous biological processes. However, product inhibition can limit extent of fermentation to VFAs. In this study, product inhibition during fermentation of waste activated sludge pre-treated by a thermal hydrolysis process (THP-WAS) was investigated. Product inhibition was confirmed as spiking reactors with high levels of a mix of VFAs prevented fermentation taking place. Various inhibition models were trialled and it was found that a threshold model (based on thermodynamics) provided the best fit between model and data. This is the first time that threshold type inhibition has been shown for a mixed substrate, mixed population system. Batch fermentations carried out with THP-WAS of different dilutions were used to evaluate the impact of different organic loadings. The threshold VFA concentration for the systems studied was determined to be 17±1gCOD(VFA)L(-1). Inhibition was shown to be due to the presence of a combination of VFAs containing 2-6 carbon atoms each. When evaluated individually, by spiking individual VFAs, all VFAs except for acetate had the same impact at this threshold; acetate being approximately 50% as inhibitory as the other organic acids (COD basis). Based on this, a weighted model could be proposed to better represent the data. Strategies to improve overall yield could be increased production of acetate, or dilution to below the inhibitory level.     

生物强化污泥厌氧发酵产酸研究进展*

污泥厌氧发酵生产挥发性脂肪酸相较产甲烷,是更具应用价值的污泥稳定途径及资源化利用方式,得到国内外学者的普遍重视。考虑到产酸量低和产酸过程的不稳定性是限制污泥发酵产酸的主要问题,采用生物强化方法实现挥发性脂肪酸的大量积累,与物理和化学方法相比,具有成本低、无二次污染等优点。根据生物强化制剂的类型,归纳了微生物纯培养物、微生物混合培养物及生物酶强化对污泥厌氧发酵产酸的影响,并在此基础上对生物强化技术控制污泥定向产酸、调控奇偶数碳比率等方面的应用进行讨论。此外,分析了影响挥发性脂肪酸产量和组分的因素,如pH、温度、底物、水力停留时间和污泥龄等。最后对生物强化技术的发展方向进行了展望,以期为深入探究污泥资源化利用提供借鉴。     

Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth

It is known that volatile fatty acids can inhibit growth of species of the family Enterobacteriaceae in vitro. However, whether these volatile fatty acids affect bacterial populations in the ceca of chickens is unknown. Therefore, a study was conducted to investigate if changes in volatile fatty acids in ceca of broiler chickens during growth affect bacterial populations. Results showed that members of the Enterobacteriaceae and enterococci are present in large numbers in 3-day-old broilers and start to decrease when broilers grow older. Lactobacilli are present in large numbers as well in 3-day-old broilers, but they remain stable during the growth of broilers. Acetate, butyrate, and propionate increase from undetectable levels in 1-day-old broilers to high concentrations in 15-day-old broilers, after which they stabilize. Significant negative correlations could be calculated between numbers of Enterobacteriaceae and concentrations of undissociated acetate, propionate, and butyrate. Furthermore, pure cultures of Enterobacteriaceae isolated from the ceca were grown in the presence of volatile fatty acids. Growth rates and maximal optical density decreased when these strains grew in the presence of increasing volatile fatty acid concentrations. It is concluded that volatile fatty acids are responsible for the reduction in numbers of Enterobacteriaceae in the ceca of broiler chickens during growth.     

Inhibition of swarming motility of Pseudomonas aeruginosa by branched-chain fatty acids.

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Inhibition of calcineurin by polyunsaturated lipids

From earlier studies on calcineurin, the presence of multiple double bonds in putative inhibitors was hypothesized as critical features for effective inhibition. Polyunsaturated fatty acids were tested as inhibitors of calcineurin and found to inhibit the phosphatase activity of calcineurin although effective inhibition was observed only in the absence of calmodulin. Calmodulin and fatty acids seemed to compete for the enzyme with the activation curve of calmodulin shifted approximately 100-fold in the presence of 50 microM eicosa-11Z,14Z-dienoic acid (20:2, n-6) or 50 microM eicosa-8Z,11Z,14Z-trienoic acid (20:3, n-6). Leukotriene B4 and derivatives also were screened as inhibitors. The most effective inhibition was caused by the 6-trans,12-epi-leukotriene B4 with an IC50 of 16.4 microM for the inhibition of calcineurin with pNPP as the substrate. Lipoxins A4 and B4 likewise caused inhibition in the presence of calmodulin with an IC50 of 42.7 microM for lipoxin B4. There was no protection by calmodulin, as found with the inhibition by the fatty acids. These data support the hypothesis that effective inhibition is bolstered by the presence of conjugated double bonds in the inhibitor. Consideration of cis- and trans-orientation of the double bonds suggests that presentation of the delocalized electron density is also a factor in effective inhibition of calcineurin.     

Inhibition of hyaluronidases and chondroitinases by fatty acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C10:0 to C22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C14:1 to C24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis-trans form of oleic acid (C18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.     

Inhibition of Hyaluronidases and Chondroitinases by Fatty Acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C 10:0 to C 22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C 14:1 to C 24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis - trans form of oleic acid (C 18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C 20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C 24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.     

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.     

Inhibition of yeast phosphatidic-acid synthesis by free fatty acids

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl-CoA thioesters. When palmitoyl-CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1-Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl-CoA thioesters as acyl donor. These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1-Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.     

Inhibition of Ochromonas Respiration by Hypocholesteremic Compounds and its Annulment by Unsaturated Fatty Acids

SYNOPSIS. Eleven of 14 hypocholesteremic drugs inhibited aerobic respiration of the phytoflagellate Ochromonas danica. Lecithin and unsaturated fatty acids annulled the inhibition of respiration by triparanol. Saturated fatty acids, squalene, cholesterol and a mixture of amino acids, purines, and water-soluble vitamins were inactive. Unsaturated fatty acids only annul the triparanol inhibition if preincubated or added simultaneously with triparanol. Evidence is presented suggesting that triparanol and oleic acid form a complex or micelle; presumably in this form the triparanol no longer inhibits aerobic respiration.     

Inhibition of ion permeability control properties of acetylcholine receptor from Torpedo californica by long-chain fatty acids

The characteristics of fatty acid inhibition of acetylcholine receptor function were examined in membrane vesicles prepared from Torpedo californica electroplax. Inhibition of the carbamylcholine-induced increase in sodium ion permeability was correlated with the bulk melting point of exogenously incorporated fatty acids. Above its melting temperature, a fatty acid could inhibit the large increase in cation permeability normally elicited by agonist binding to receptor. Below its melting temperature, a fatty acid was ineffective. None of the fatty acids altered any of the ligand binding properties of the receptor. Inhibitory fatty acids did not induce changes in membrane fluidity, as determined by electron paramagnetic resonance using spin-labeled fatty acids. The spin-labeled fatty acids also acted as inhibitors, and the extent of inhibition depended largely on the position of the nitroxide group along the fatty acid chain. Addition of noninhibitory fatty acid to the vesicle membranes did not protect the receptor from inhibition by spin-labeled fatty acids. The effects of free fatty acids on acetylcholine receptor function are attributed to the disruptions of protein-lipid interactions.     

Inhibition of bacterial perchlorate reduction by zero-valent iron

Perchlorate was reduced by a mixed bacterial culture over a pH range of 7.0–8.9. Similar rates of perchlorate reduction were observed between pH 7.0 and 8.5, whereas significantly slower reduction occurred at pH 8.9. Addition of iron metal, Fe(0), to the mixed bacterial culture resulted in slower rates of perchlorate reduction. Negligible perchlorate reduction was observed under abiotic conditions with Fe(0) alone in a reduced anaerobic medium. The inhibition of perchlorate reduction observed in the presence of Fe(0) is in contrast to previous studies that have shown faster rates of contaminant reduction when bacteria and Fe(0) were combined compared to bacteria alone. The addition of Fe(0) resulted in a rise in pH, as well as precipitation of Fe minerals that appeared to encapsulate the bacterial cells. In experiments where pH was kept constant, the addition of Fe(0) still resulted in slower rates of perchlorate reduction suggesting that encapsulation of bacteria by Fe precipitates contributed to the inhibition of the bacterial activity independent of the effect of pH on bacteria. These results provide the first evidence linking accumulation of iron precipitates at the cell surface to inhibition of environmental contaminant degradation. Fe(0) was not a suitable amendment to stimulate perchlorate-degrading bacteria and the bacterial inhibition caused by precipitation of reduced Fe species may be important in other combined anaerobic bacterial–Fe(0) systems. Furthermore, the inhibition of bacterial activity by iron precipitation may have significant implications for the design of in situ bioremediation technologies for treatment of perchlorate plumes.     

Inhibition of topoisomerases by fatty acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).