thin-layer-chromatographic-tlc-separations-bioassays-plant-extracts

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

United States Department of Agriculture

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (Deltap) was only -25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZDeltapH) and the electrical membrane potential (DeltaPsi) increased. The Deltap was -125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but DeltaPsi increased to -70 mV. Protein synthesis inhibitors also significantly increased DeltaPsi. Non-growing, arginine-energized cells had a DeltaPsi of -80 mV at pH 6.7 or pH 4.7, but DeltaPsi was not detected if the F1F0 ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and DeltaPsi and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither DeltaPsi nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large Deltap to grow, and Deltap appears to serve as a mechanism of ATP dissipation or energy spilling.

The ability of acidic pH, growth inhibitors, and glucose to increase the proton motive force and energy spilling of amino acid-fermenting Clostridium sporogenes MD1 cultures.

Fermentation acids inhibited the growth and ammonia production of the amino-acid-fermenting bacterium Clostridium sporogenes MD1, but only when the pH was acidic. Such inhibition was traditionally explained by the ability of fermentation acids to act as uncouplers and decrease protonmotive force (Deltap), but C. sporogenes MD1 grows even if the Deltap is very low. Cell suspensions incubated with additional sodium chloride produced ammonia as rapidly at pH 5.0 as at pH 7.0, but cells incubated with additional sodium lactate were sensitive to even small decreases in extracellular pH. Similar results were obtained if the sodium lactate was replaced by sodium acetate or propionate. When extracellular pH declined, DeltapH increased even if sodium lactate was present. The cells accumulated intracellular lactate anion when the pH was acidic, and intracellular glutamate declined. Because amino acid deamination is linked to a transamination reaction involving glutamate dehydrogenase, the decrease in ammonia production could be explained by the decrease in intracellular glutamate. This latter hypothesis was consistent with the observation that extracellular glutamate addition restored amino acid deamination even though glutamate alone did not allow for the generation of ammonia.

Fermentation acids inhibit amino acid deamination by Clostridium sporogenes MD1 via a mechanism involving a decline in intracellular glutamate rather than protonmotive force.

At pH values >5.5, Clostridium sporogenes MD1 accumulated potassium even though it had little protonmotive force, but an ATPase inhibitor (N, N'- dicyclohexylcarbodiimide) prevented this uptake. The results suggested that potassium transport was ATP-driven, and a protonophore (3, 3', 4', 5 - tetrachlorosalicylanilide) did not eliminate uptake. However, potassium uptake could also be driven by an artificial pH gradient, and in this case the protonophore acted as an inhibitor. These latter results indicated that the cells also had a protonmotive force-driven transporter. When the pH <5.1, the cells could not retain potassium, rapid efflux was observed, and intracellular volume collapsed.

Effect of acidic pH on the ability of Clostridium sporogenes MD1 to take up and retain intracellular potassium.

Fresh plant materials can be fermented and preserved as silage for cattle, but clostridia that deaminate amino acids increase pH. If the pH of the silage rises, spoilage microorganisms proliferate, and undesirable products accumulate. Rod-shaped, anaerobic bacteria with spores were isolated from fresh alfalfa, fresh corn, and silages. Strain MD1 had the highest specific activity of amino acid deamination, and it was most closely related to Clostridium botulinum A and B. However, because strain MD1 did not produce a toxin, it was classified as Clostridium sporogenes. Washed cell suspensions of C. sporogenes MD1 had specific activities as great as 690 nmol ammonia mg protein(-1) min(-1), and this rate did not decrease until the pH was less than 4.5. Batch cultures of C. sporogenes MD1 did not initiate growth if the initial pH was less than 5.0, but continuous cultures (0.1 h(-1) dilution rate) persisted until the pH in the culture vessel was 4.6. When C. sporogenes MD1 was co-cultured with a bacteriocin-producing Streptococcus bovis HC5, ammonia production was greatly reduced. The ability of S. bovis HC5 to inhibit strain MD1 was pH-dependent. When the pH was 5.5 or less, strain MD1 could no longer be detected. These latter results support the idea that bacteriocin-producing bacteria may be used to improve silage quality.

The effect of pH and a bacteriocin (bovicin HC5) on Clostridium sporogenes MD1, a bacterium that has the ability to degrade amino acids in ensiled plant materials.

Metabolic inhibition of Clostridium thermocellum, when grown in a high solids environment, was investigated by comparing submerged fermentation (SmF), solid-substrate cultivation (SSC) and solid-substrate cultivation with media replacement by periodic flushing (FSSC). Cellulose conversion extent and end-product concentrations were measured over time. SmF converted approximately 65% of the cellulose in 240 h (10 days), whereas SSC converted <8% in the same period. FSSC converted approximately 25% and 47% of initial substrate after 240 h; 45% and 71% of initial substrate after 25 days, with media replacement every 24 and 12h, respectively. The SSC experienced higher initial production rates for all fermentation products, but could not sustain production rates. When acetate concentrations reached a critical point, the acetate decreased the intracellular volume of C. thermocellum cell suspensions at pH values similar to those observed in SSC. Acids produced by fermentation exacerbated the already unfavorable osmotic condition of SSC, resulting in metabolic inhibition. Consistent with this finding, approximately constant amounts of ethanol, acetate and lactate were produced during each flush of the FSSC. Flushed solid-substrate cultivation maintained favorable growth conditions for C. thermocellum even up to 25 days, allowing more total product to be formed than in the other cultivation methods.

Investigation of the metabolic inhibition observed in solid-substrate cultivation of Clostridium thermocellum on cellulose.

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H(2)) are also produced. The effect of hydrogenase inhibitors (H(2), carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N(2) to H(2), acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H(2) or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD(+) from NADH by H(2), lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H(2) production for the conversion of synthetic gases to chemicals.

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate.

Red clover (Trifolium pratense) is rich in phenolic compounds. Both the crude phenolic extract and biochanin A, an isoflavonoid component of the extract, suppress growth of Clostridium sticklandii, a bovine, Gram-positive, ruminal hyper-ammonia producing bacterium (HAB). The purpose of this study was to determine if other components of red clover extract contributed to its anti-HAB activity. Extracts of the Kenland cultivar of red clover, collected 0 h and 24 h after cutting, were separated by normal-phase thin-layer chromatography (TLC) in either ethyl acetate-hexanes (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by either overlaying the plates with agar seeded with C. sticklandii, or setting the excised bands face-down onto plates of bacteria-seeded agar. Biochanin A inhibited C. sticklandii growth on TLC plates when as little as 8 nmol was present in the extract. An antimicrobial band, seen in a previous bioassay, was not found, suggesting that this second compound may be more labile than biochanin A.

Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium.

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.

Article	Year
The ability of acidic pH, growth inhibitors, and glucose to increase the proton motive force and energy spilling of amino acid-fermenting Clostridium sporogenes MD1 cultures. Archives of microbiology\| PubMed ID: 15891933	2005
Fermentation acids inhibit amino acid deamination by Clostridium sporogenes MD1 via a mechanism involving a decline in intracellular glutamate rather than protonmotive force. Microbiology (Reading, England)\| PubMed ID: 16946257	2006
Effect of acidic pH on the ability of Clostridium sporogenes MD1 to take up and retain intracellular potassium. FEMS microbiology letters\| PubMed ID: 17233675	2007
The effect of pH and a bacteriocin (bovicin HC5) on Clostridium sporogenes MD1, a bacterium that has the ability to degrade amino acids in ensiled plant materials. FEMS microbiology ecology\| PubMed ID: 19712336	2004
Investigation of the metabolic inhibition observed in solid-substrate cultivation of Clostridium thermocellum on cellulose. Bioresource technology\| PubMed ID: 20362436	2010
Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate. Applied microbiology and biotechnology\| PubMed ID: 22218768	2012
Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium. Natural product communications\| PubMed ID: 23413564	2012
Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: responses to antibiotic challenge. Veterinary microbiology\| PubMed ID: 23769300	2013

Michael D. Flythe

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