Lipophilic 9,10-Dehydrofukinone Action on Pathogenic and Non-Pathogenic Bacterial Biofilms. Why Is This Main Volatile Metabolite in Senecio?

The effect of a natural sesquiterpene ketone, 9,10-dehydrofukinone (DHF), on pathogenic Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from chronic infectious processes, was the focus of the present study. Lipophilic DHF produced important antibacterial synergistic effects in association with ciprofloxacin (CPX) against two biofilm-forming strains of S. aureus HT1 (FIC=0.21) and P. aeruginosa HT5 (FIC=0.05). Hence, this mixture constitutes an excellent strategy to combat these biofilm-producing bacteria that overexpress drug efflux pumps as a resistance mechanism. Additionally, a substantial rise in beneficial Lactobacillus biofilm biomass was determined as a very significant finding of this association. Particularly, a non-pathogenic biofilm increment of 119 % was quantified when the mixture was added to a probiotic L. acidophilus ATCC SD-5212 culture. A surface activity enhanced in 71 % with respect to untreated L. acidophilus culture was also generated by the DHF and CPX association, and therefore, a glycoprotein synthesis induction mediated by the mixture is discussed. The results obtained could help in the development of new selective antibiotics. From an ecological standpoint, the present study strongly suggests that DHF is a polyfunctional organic molecule produced with a high yield in Senecio punae that exerts a positive impact on a non-pathogenic plant bacterium L. plantarum CE105.

Compounds from Lactobacillus plantarum culture supernatants with potential pro-healing and anti-pathogenic properties in skin chronic wounds.

CONTEXT: It is necessary to advance the field of alternative treatments for chronic wounds that are financially accessible to the least economically developed countries. Previously we demonstrated that topical applications of Lactobacillus plantarum culture supernatants (LAPS) on human-infected chronic wounds reduce the pathogenic bioburden, the amount of necrotic tissue, and the wound area, as well as promote debridement, granulation tissue, and wound healing. OBJECTIVE: To study LAPS chemically and biologically and to find potential molecules responsible for its pro-healing and anti-pathogenic properties in chronic wounds. MATERIALS AND METHODS: (1) Chemical analysis: extracts were subjected to a column chromatography and the fractions obtained were studied by GCMS. (2) Quantification: dl-lactic acid (commercial kit), phenolic compounds (Folin-Ciocalteu), H2O2 (micro-titration), and cations (flame photometry). (3) Biological analysis: autoinducers type 2 (AI-2) (Vibrio harveyi BB170 bioassay), DNAase activity (Agar DNAase), and Pseudomonas aeruginosa biofilm inhibition (crystal violet technique). RESULTS: According to its biological activity, the most significant molecules found by GCMS were the following: antimicrobials (mevalonolactone, 5-methyl-hydantoine, benzoic acid, etc.); surfactants (di-palmitin, distearin, and 1,5-monolinolein); anesthetics (barbituric acid derivatives), and AI-2 precursors (4,5-dihydroxy-2,3-pentanedione and 2-methyl-2,3,3,4-tetrahydroxytetrahydrofurane). Concentrations measured (µg/mL): DL-lactic acid (11.71 ± 1.53) and H2O2 (36 ± 2.0); phenolic compounds (485.2 ± 15.20); sodium (370 ± 17); potassium 920 ± 24); calcium (20 ± 4); and magnesium (15 ± 3). DNAase from LAPS had activity on genomic DNA from PMNs and P. aeruginosa. DISCUSSION AND CONCLUSION: The molecules and biological activities found in LAPS could explain the observed effects in human chronic wounds.

Microbiological culture broth designed from food waste.

The current trend of increasing air, water, and soil pollution is, in part, due to inadequate management of municipal solid waste (MSW). The relationship between public health and the collection, storage and improper disposal of solid waste has encouraged several studies and the results were attributed to the spread of over twenty human and animal diseases due to this interrelationship. The term "single cell protein" (SCP) refers to microbial biomass used as a dietary additive. It has high nutritional value because of its high content of vitamins, lipids, and proteins of biological quality (the presence of all essential amino acids) (Lal, 2005). The aim of this work was to design a culture media for microbiological assays and to produce SCP for animal feeding, using nutrients contained in organic waste. In order to compare the effectiveness of food waste (FW) and LAPTg media, different strains of Lactobacillus, Enterococcus, Staphylococcus, Shigella, Salmonella, Saccharomyces and Schizosaccharomyces were studied. In all cases, the growth obtained from FW and LAPTg culture media were not significantly different (p > 0.05). In addition, the growth of Saccharomyces cerevisiae was studied in order to produce SCP for animal feeding. Comparative experiments involving molasses broth, FW broth, and basal broth were carried out. The biomass yield calculated at 24 h from FW broth was 13% lower than from molasses broth. The FW broth provided a significantly lower biomass yield; however, it can be very useful in areas where molasses are not available. FW broth can be elaborated at low cost, in any populated region of the world because its ingredients are wastes generated by humans. It has great versatility, allowing the development of a wide variety of microorganisms, both Gram negative and Gram positive bacteria as well as yeasts. The production of safe protein additives, with high biological quality and low cost, is necessary due to the increasing global demand for food for humans and animals.

Wine composition plays an important role in the control of carcinogenic precursor formation by Lactobacillus hilgardii X1B.

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X1B. Glucose and L-malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors.

Citrus sinensis Essential Oils an Innovative Antioxidant and Antipathogenic Dual Strategy in Food Preservation against Spoliage Bacteria.

The present study evaluates the chemical compositions and antioxidant and antipathogenic properties of commercial orange (Citrus sinensis (L.) Osbeck) essential oils obtained using the cold-press method (EOP) and the cold-press method followed by steam distillation (EOPD). The chemical compositions of the volatilizable fractions, determined by gas chromatography-mass spectrometry, were similar in both samples. A relatively large amount of γ-terpinene was found in the EOPD (1.75%) as compared to the EOP (0.84%). Monoterpene hydrocarbons with limonene (90.4-89.8%) followed by myrcene (3.2-3.1%) as the main compounds comprised the principal phytochemical group. The non-volatile phenolics were eight times higher in the EOP than in the EOPD. Several assays with different specificity levels were used to study the antioxidant activity. Although both essential oils presented similar reducing capacities, the radical elimination ability was higher for the EOP. Regarding the antipathogenic properties, the EOs inhibited the biomass and cell viability of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. Furthermore, both EOs similarly attenuated the production of elastase, pyocyanin, and quorum-sensing autoinducers as assessed using Gram-negative bacteria. The EOP and EOPD showed important antioxidant and antipathogenic properties, so they could represent natural alternatives to extend the shelf life of food products by preventing oxidation and contamination caused by microbial spoilage.

Influence of phenolic compounds on the growth and arginine deiminase system in a wine lactic acid bacterium.

The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.

Bioactive sesqui- and diterpenoids from the Argentine liverwort Porella chilensis.

Four fusicoccane-type diterpenoids (1-4), including the new 1 and 2; four pinguisane-type sesquiterpenoids (5-8); and two aromadendrane-type sesquiterpenoids (9 and 10) were isolated from an Argentine collection of the endemic liverwort Porella chilensis. The biofilm formation of the human pathogen Pseudomonas aeruginosa was inhibited by compounds 3 (53 and 47%), 9 (45 and 41%), and 10 (48 and 37%) at 50 and 5 µg/mL, respectively. Compounds 3, 9, and 10 also produced a slight decrease in bacterial growth and interfere with the process of quorum sensing at the same doses.

In vivo and in vitro antibacterial activity of acanthospermal B, a sesquiterpene lactone isolated from Acanthospermum hispidum.

Acanthospermal B (AcB), the major sesquiterpene lactone (SL) of Acanthospermum hispidum, an herb widely spread in Argentina, is a selective antibacterial agent against Enterococcus faecalis and Staphylococcus aureus, but inactive on Gram-negative and Lactobacillus. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main microorganisms involved in human chronic infection. A balb\c mouse skin infection model was developed to reproduce the lesions caused by acute and chronic infections produced by MRSA. After determination of the maximum concentration of AcB unable to produce tissular injury after intradermal injection, the anti-MRSA effect of AcB was evaluated on skin, liver and spleen tissues of infected mice. AcB, at doses of 2.5 mg/kg, produced a ten times decrease of MRSA growth in skin infection. In addition, the same dose prevented the dissemination to liver and/or spleen. AcB also displayed a bacteriostatic effect, in vitro, on MRSA cultures at 50 µg/mL that seems to be caused by partial denaturation of total bacterial DNA and/or inhibition of the PCR reaction in not denaturized DNA. Finally, total MRSA cell wall lysis occurred at a concentration of 100 µg/mL of AcB after 2 h of exposure.

The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

Antibacterial prenylated acylphloroglucinols from the fern Elaphoglossum yungense.

Seven new prenylated acylphloroglucinols, yungensins A-G, were isolated from the diethyl ether extract of the scaly rhizomes and roots of an Argentine collection of the fern Elaphoglossum yungense. All the compounds contained a geranyl group attached to the filicinic acid-type residue. The diethyl ether extract and yungensins A-F displayed antibacterial effects against Staphylococcus aureus and Pseudomonas aeruginosa with MICs ranging from 10 to 200 µg/mL. All tested acylphloroglucinols, except for yungensin D, caused bacteriolysis of S. aureus at MBC or higher doses (50-200 µg/mL). At 10 µg/mL, the ether extract as well as yungensins A, B, and D-F altered the biofilm production in both microoganisms.

Grapefruit essential oils inhibit quorum sensing of Pseudomonas aeruginosa.

Citrus essential oils are used in food to confer flavor and aromas. The citrus essential oils have been granted as GRAS and could be used as antimicrobial additives to control bacterial quorum sensing from potential food bacterial pathogens. The chemical composition and inhibitory activity of Citrus paradisi (grapefruit) essential oils obtained by cold-pressed method (EOP) and cold-pressed method followed by steam distillation, against Pseudomonas aeruginosa were determined. The GC-MS analyses of the oil indicated the amount of the essential oil components was highest with D-limonene in both cases. However, the extraction method modified the chemical composition. EOP had higher amount of coumarins and flavonoid as well as less oxygenated terpenoids. At 0.1 mg/mL essential oils were not able to modify the bacterial development but inhibited the P. aeruginosa biofilm production between 52% and 55%, sessile viability between 45% and 48%, autoinducer production and elastase activity between 30% and 56%. Limonene was less effective at inhibiting P. aeruginosa than the essential oils, suggesting a synergistic effect of the minor components. According to our results, grapefruit essential oils could be used as a food preservative to control P. aeruginosa virulence.

Volatiles from Subtropical Convolvulaceae That Interfere with Bacterial Cell-to-Cell Communication as Potential Antipathogenic Drugs.

Increasing chronic bacterial infections create an urgent need for new antimicrobial agents or strategies for their control. Targeting virulence is one of the alternative approaches to find new medicines to treat persistent infections due to bacteria with biofilm-phenotype which are more resistant to antibiotics than their planktonic counterparts having an extreme capacity for evading the host defences. A bioguided study of sixteen extracts from flowers and leaves of four subtropical Convolvulaceae species provided evidence of the occurrence of antipathogenic natural products active against Gram positive and negative bacteria. Particularly, volatile metabolites from Merremia dissecta creeper, a food and medicinal plant, were able to interfere with the Pseudomonas aeruginosa quorum sensing system by a strong decrease of N-acyl homoserine lactone (AHL) biosynthesis (63-75%), which attenuated the virulence factor expression like biofilm (55%) and elastase activity (up to 27%), key factors that enable the colonization and dissemination of the infection in the host. Control of the P. aeruginosa biofilm and the QS process by phytochemicals, such as (+) spathulenol, isolated from a bioactive extract of M. dissecta leaves would be a good strategy for the development of new and effective antipathogenic drugs.

Influence of ethanol and low pH on arginine and citrulline metabolism in lactic acid bacteria from wine.

The aim of this work was to study the effects of ethanol on cell growth and arginine and citrulline metabolism in two heterofermentative lactic acid bacteria from wine, and to determine their possible association with the formation of ethyl carbamate (EC), a carcinogenic compound. Lactobacillus hilgardii X1B is able to utilize arginine and citrulline, while Oenococcus oeni m can only use citrulline, a precursor of EC. Growth of both microorganisms was partially inhibited by 10 and 15% (v/v) ethanol. Specific arginine consumption by L. hilgardii increased when the pH value diminished from 6.5 to 3.8, but was not affected by an increasing ethanol concentration. However, the ethanol concentration affected the specific citrulline consumption of both microorganisms. Arginine metabolism by L. hilgardii X1B increased the amount of citrulline, thus allowing production of EC in the medium. Citrulline utilization by both microorganisms, at all pH values studied, indirectly inhibited the formation of EC; indeed, one of the precursors had practically disappeared after 48 h of incubation. Due to its ability to form precursors, L. hilgardii X1B has the potential to contribute to EC formation, whereas citrulline utilization by O. oeni m in the presence of ethanol may contribute to diminishing the formation of EC. Rapid degradation of citrulline in the presence of ethanol by O. oeni m is important from a toxicological point of view, because it is important to keep the EC levels as low as possible.

Differences between biogenic amine detection by HPLC methods using OPA and dansyl derivates.

Biogenic amines can be formed and degraded as a result of normal metabolic activity in animals, plants, and microorganisms and are usually produced by the decarboxylation of amino acids. Recent trends in food security are promoting an increasing search for trace compounds that can affect human health. Although they are present in fermented foods and beverages in low quantities, they exhibit interactions with normal human metabolism (e.g., having vasoactive or psychoactive properties) that justify the research on their presence in foods and the possible related toxicological effects that they may cause. Estimation of the biogenic amines histamine, tyramine, agmatine, putrescine, and cadaverine is important not only from the point of view of their toxicity, but also because they can be used as indicators of the degree of freshness or spoilage of food. Until recently, because of the difficulty in detecting and quantifying amines reliably we have had insufficient information about their occurrence in different types of foods and beverages. These problems are related to matrix interference (e.g., the presence of free amino acids) and the low levels at which the amines are found. Early techniques for the determination of biogenic amines in foods were based on thin-layer chromatography. More modern analytical techniques have since been developed that allow the acquisition of reliable quantitative data and better separation/resolution of various amines. The quantitative determination of biogenic amines is generally accomplished by overpressure-layer chromatography, high-performance liquid chromatography (HPLC), and gas chromatography. The use of reverse-phase column and precolumn derivatization was more efficient and faster than the conventional ion-exchange techniques. This study was conducted to evaluate two HPLC derivatization methods for quantitative determination of biogenic amines: the method described by Gonzales de Llano et al. for amino acid analysis and the method described by Eerola et al.

Comparative survey in Lactobacillus plantarum of the growth and metabolism of arginine and citrulline in different media.

Arginine deiminase activity increased in the presence of arginine in Lactobacillus plantarum strains N4 and N8 isolated from orange. The influence of citrulline and ornithine on arginine deiminase and ornithine transcarbamylase activities was strain-dependent. The growth and arginine and citrulline metabolism of L. plantarum were studied in the presence of tomato juice. Its addition enhances the growth in both strains. The specific amino acids utilization was inversely proportional to the initial glucose concentration. Arginine and citrulline addition to basal medium exerted a stimulatory effect on the growth of N4 strain, and this effect was observed only with citrulline in strain N8. The magnitude of this effect was lower in the presence of tomato juice.

Inhibition of quorum sensing in Pseudomonas aeruginosa by sesquiterpene lactones.

Six sesquiterpene lactones (SLs) of the goyazensolide and isogoyazensolide-type isolated from the Argentine herb Centratherum punctatum were evaluated on their ability to inhibit virulence factors of Pseudomonas aeruginosa ATCC 27853. Although compounds were not able to completely inhibit bacterial growth at 200µg/ml, the SLs do altered biofilm formation, elastase activity, and production of N-acyl-homoserinelactones (AHLs) which are known quorum sensing autoinducers at lower concentration. Compounds 2, 3, and 5 displayed significant inhibitory effects on P. aeruginosa biofilm formation at 0.5µg/ml being compound 3 (1.32µM) the most potent (42%). Compounds 2, 3, 4, 5 and 6, inhibited 39, 44, 42, 32 and 35% the production of AHLs at 100µg/ml and inhibited by more than 50% the elastase activity at 0.5µg/ml. Our results clearly indicated that sesquiterpene lactones are good candidates for the development of new antimicrobial agents acting not as bactericidal but as antipathogenic agents.

Influence of phenolic compounds on the growth and arginine deiminase system in a wine lactic acid bacterium

The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.