Norepinephrine and Serotonin Can Modulate the Behavior of the Probiotic Enterococcus faecium NCIMB10415 towards the Host: Is a Putative Surface Sensor Involved?

The human gut microbiota has co-evolved with humans by exchanging bidirectional signals. This study aims at deepening the knowledge of this crucial relationship by analyzing phenotypic and interactive responses of the probiotic Enterococcus faecium NCIMB10415 (E. faecium SF68) to the top-down signals norepinephrine (NE) and serotonin (5HT), two neuroactive molecules abundant in the gut. We treated E. faecium NCIMB10415 with 100 µM NE and 50 µM 5HT and tested its ability to form static biofilm (Confocal Laser Scanning Microscopy), adhere to the Caco-2/TC7 monolayer, affect the epithelial barrier function (Transepithelial Electrical Resistance) and human dendritic cells (DC) maturation, differentiation, and cytokines production. Finally, we evaluated the presence of a putative hormone sensor through in silico (whole genome sequence and protein modelling) and in vitro (Micro-Scale Thermophoresis) analyses. The hormone treatments increase biofilm formation and adhesion on Caco-2/TC7, as well as the epithelial barrier function. No differences concerning DC differentiation and maturation between stimulated and control bacteria were detected, while an enhanced TNF-α production was observed in NE-treated bacteria. Investigations on the sensor support the hypothesis that a two-component system on the bacterial surface can sense 5HT and NE. Overall, the data demonstrate that E. faecium NCIMB10415 can sense both NE and 5HT and respond accordingly.

PHA Production from Cheese Whey and "Scotta": Comparison between a Consortium and a Pure Culture of Leuconostoc mesenteroides.

It is urgent to expand the market of biodegradable alternatives to oil-derived plastics owing to (i) increasingly limited oil availability/accessibility, and (ii) the dramatic impact of traditional plastics on aquatic life, the food chain, all Earth ecosystems, and ultimately, human health. Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that can be obtained through microbial fermentation of agro-industrial byproducts, e.g., milk and cheese whey. Here, the PHA-accumulating efficiency of a mixed microbial culture (MMC, derived from activated sludges) grown on dairy byproducts (cheese and scotta whey) was measured. Bioreactor tests featuring temperature and pH control showed that both scotta and pre-treated Toma cheese whey could be used for PHA production by MMC, although scotta cheese whey supported higher PHA yield and productivity. The advantages of open MMCs include their plasticity and versatility to fast changing conditions; furthermore, no growth-medium sterilization is needed prior to fermentation. However, the use of pure cultures of efficient PHA producers may support better metabolic performances. Therefore, PHA-producing strains were isolated from a MMC, leading to the satisfactory identification of two bacterial strains, Citrobacter freundii and Leuconostoc spp., whose ability to accumulate PHAs in synthetic media was confirmed. A more detailed investigation by mass spectrometry revealed that the strain was L. mesenteroides. Although the validation of L. mesenteroides potential to produce PHA through fermentation of agro-industrial byproducts requires further investigations, this is the first study reporting PHA production with the Leuconostoc genus.

Clostridium cellulovorans Proteomic Responses to Butanol Stress.

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties.

BACKGROUND: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. METHODS: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. RESULTS: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. CONCLUSIONS: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.

Environmental Conditions Affecting GABA Production in Lactococcus lactis NCDO 2118.

GABA (γ-aminobutyric acid) production has been widely described as an adaptive response to abiotic stress, allowing bacteria to survive in harsh environments. This work aimed to clarify and understand the relationship between GABA production and bacterial growth conditions, with particular reference to osmolarity. For this purpose, Lactococcus lactis NCDO 2118, a GABA-producing strain, was grown in glucose-supplemented chemically defined medium containing 34 mM L-glutamic acid, and different concentrations of salts (chloride, sulfate or phosphate ions) or polyols (sorbitol, glycerol). Unexpectedly, our data demonstrated that GABA production was not directly related to osmolarity. Chloride ions were the most significant factor influencing GABA yield in response to acidic stress while sulfate ions did not enhance GABA production. We demonstrated that the addition of chloride ions increased the glutamic acid decarboxylase (GAD) synthesis and the expression of the gadBC genes. Finally, under fed-batch conditions in a complex medium supplemented with 0.3 M NaCl and after a pH shift to 4.6, L. lactis NCDO 2118 was able to produce up to 413 mM GABA from 441 mM L-glutamic acid after only 56 h of culture, revealing the potential of L. lactis strains for intensive production of this bioactive molecule.

The Russian Doll Model: How Bacteria Shape Successful and Sustainable Inter-Kingdom Relationships.

Successful inter-kingdom relationships are based upon a dynamic balance between defense and cooperation. A certain degree of competition is necessary to guarantee life spread and development. On the other hand, cooperation is a powerful tool to ensure a long lasting adaptation to changing environmental conditions and to support evolution to a higher level of complexity. Bacteria can interact with their (true or potential) parasites (i.e., phages) and with their multicellular hosts. In these model interactions, bacteria learnt how to cope with their inner and outer host, transforming dangerous signals into opportunities and modulating responses in order to achieve an agreement that is beneficial for the overall participants, thus giving rise to a more complex "organism" or ecosystem. In this review, particular attention will be addressed to underline the minimal energy expenditure required for these successful interactions [e.g., moonlighting proteins, post-translational modifications (PTMs), and multitasking signals] and the systemic vision of these processes and ways of life in which the system proves to be more than the sum of the single components. Using an inside-out perspective, I will examine the possibility of multilevel interactions, in which viruses help bacteria to cope with the animal host and bacteria support the human immune system to counteract viral infection in a circular vision. In this sophisticated network, bacteria represent the precious link that insures system stability with relative low energy expenditure.

Clostridium cellulovorans metabolism of cellulose as studied by comparative proteomic approach.

Clostridium cellulovorans is among the most promising candidates for consolidated bioprocessing (CBP) of cellulosic biomass to liquid biofuels (ethanol, butanol). C. cellulovorans metabolizes all the main plant polysaccharides and mainly produces butyrate. Since most butyrate and butanol biosynthetic reactions from acetyl-CoA are common, introduction of single heterologous alcohol/aldehyde dehydrogenase can divert the branching-point intermediate (butyryl-CoA) towards butanol production in this strain. However, engineering C. cellulovorans metabolic pathways towards industrial utilization requires better understanding of its metabolism. The present study aimed at improving comprehension of cellulose metabolism in C. cellulovorans by comparing growth kinetics, substrate consumption/product accumulation and whole-cell soluble proteome (data available via ProteomeXchange, identifier PXD015487) with those of the same strain grown on a soluble carbohydrate, glucose, as the main carbon source. Growth substrate-dependent modulations of the central metabolism were detected, including regulation of several glycolytic enzymes, fermentation pathways (e.g. hydrogenase, pyruvate formate lyase, phosphate transacetylase) and nitrogen assimilation (e.g. glutamate dehydrogenase). Overexpression of hydrogenase and increased ethanol production by glucose-grown bacteria suggest a more reduced redox state. Higher energy expenditure seems to occur in cellulose-grown C. cellulovorans (likely related to overexpression and secretion of (hemi-)cellulases), which induces up-regulation of ATP synthetic pathways, e.g. acetate production and ATP synthase. SIGNIFICANCE: C. cellulovorans can metabolize all the main plant polysaccharides (cellulose, hemicelluloses and pectins) and, unlike other well established cellulolytic microorganisms, can produce butyrate. C. cellulovorans is therefore among the most attractive candidates for direct fermentation of lignocellulose to high-value chemicals and, especially, n-butanol, i.e. one of the most promising liquid biofuels for the future. Recent studies aimed at engineering n-butanol production in C. cellulovorans represent milestones towards production of biofuels through one-step fermentation of lignocellulose but also indicated that more detailed understanding of the C. cellulovorans central carbon metabolism is essential to refine metabolic engineering strategies towards improved n-butanol production in this strain. The present study helped identifying key genes associated with specific catabolic reactions and indicated modulations of central carbon metabolism (including redox and energy balance) associated with cellulose consumption. This information will be useful to determine key enzymes and possible metabolic bottlenecks to be addressed towards improved metabolic engineering of this strain.

Antimicrobial Potential of Food Lactic Acid Bacteria: Bioactive Peptide Decrypting from Caseins and Bacteriocin Production.

Lactic acid bacteria (LAB) potential in the food industry and in the biotechnological sector is a well-established interest. LAB potential in counteracting especially food-borne infections has received growing attention, but despite being a road full of promises is yet poorly explored. Furthermore, the ability of LAB to produce antimicrobial compounds, both by ribosomal synthesis and by decrypting them from proteins, is of high value when considering the growing impact of multidrug resistant strains. The antimicrobial potential of 14 food-derived lactic acid bacteria strains has been investigated in this study. Among them, four strains were able to counteract Listeria monocytogenes growth: Lactococcus lactis SN12 and L. lactis SN17 by high lactic acid production, whereas L. lactis 41FLL3 and Lactobacillus sakei I151 by Nisin Z and Sakacin P production, respectively. Strains Lactococcus lactis MG1363, Lactobacillus rhamnosus 17D10 and Lactobacillus helveticus 4D5 were tested and selected for their potential attitude to hydrolyze caseins. All the strains were able to release bioactive peptides with already known antimicrobial, antihypertensive and opioid activities. These features render these strains or their bioactive molecules suitable for use in food as biocontrol agents, or as nutraceutical supplements to treat mild disorders such as moderate hypertension and children insomnia. These results highlight once again that LAB potential in ensuring food safety, food nutraceutical value and ultimately in favoring human health is still underexplored and underexploited.

The Less Expensive Choice: Bacterial Strategies to Achieve Successful and Sustainable Reciprocal Interactions.

Bacteria, the first organisms that appeared on Earth, continue to play a central role in ensuring life on the planet, both as biogeochemical agents and as higher organisms' symbionts. In the last decades, they have been employed both as bioremediation agents for cleaning polluted sites and as bioconversion effectors for obtaining a variety of products from wastes (including eco-friendly plastics and green energies). However, some recent reports suggest that bacterial biodiversity can be negatively affected by the present environmental crisis (global warming, soil desertification, and ocean acidification). This review analyzes the behaviors positively selected by evolution that render bacteria good models of sustainable practices (urgent in these times of climate change and scarcity of resources). Actually, bacteria display a tendency to optimize rather than maximize, to economize energy and building blocks (by using the same molecule for performing multiple functions), and to recycle and share metabolites, and these are winning strategies when dealing with sustainability. Furthermore, their ability to establish successful reciprocal relationships by means of anticipation, collective actions, and cooperation can also constitute an example highlighting how evolutionary selection favors behaviors that can be strategic to contain the present environmental crisis.

Detailed Soluble Proteome Analyses of a Dairy-Isolated Enterococcus faecalis: A Possible Approach to Assess Food Safety and Potential Probiotic Value.

Enterococci are common inhabitants of the gastrointestinal tracts of humans and animals and thanks to their capability to tolerate different environmental conditions and their high rates of gene transfer, they are able to colonize various ecological niches, as food matrices. Enterococcus faecalis bacteria are defined as controversial microorganisms. From one side they are used as food starters, bio-control agents and probiotics to improve human or animal health. From the other side, in the last two decades enterococci have emerged as important nosocomial pathogens, because bearing high-level of resistance to antibiotics and several putative virulence factors. In this study, the soluble proteome quantitation data (LC-MS/MS) of the food-isolated strain E. faecalis D27 (dairy-isolate) was compared with the soluble proteome quantitation data of the pathogenic E. faecalis UW3114 (urinary tract infection isolate) and with the one of the health promoting strain E. faecalis Symbioflor1, respectively. The comparison of cytosolic protein expression profiles highlighted statistically significant changes in the abundance of proteins mainly involved in specific metabolic pathways, nutrient transport, stress response, and cell wall modulation. Moreover, especially in the dairy isolate and the clinical isolate, several proteins with potential pathogenic implications were found, such as serine proteases, von Willebrand factor, serine hydrolase with beta lactamase activity, efflux transporter, and proteins involved in horizontal gene transfer. The analysis of the extracellular proteome provided interesting results concerning proteins involved in bacterial communication, such as pheromones and conjugative elements and also proteins able to interact with human components. The phenotypic characterization evaluating (i) biofilm formation (ii) hemolytic activity on blood agar plates (iii) protease activity (iv) gelatinase (v) antibiotic resistance pattern, enabled us to elucidate the risks associated with the poor characterized foodborne E. faecalis D27.

Treatment with selenium-enriched Saccharomyces cerevisiae UFMG A-905 partially ameliorates mucositis induced by 5-fluorouracil in mice.

PURPOSE: Gastrointestinal mucositis is a major problem associated with cancer therapy. To minimize these deleterious effects, simultaneous administration of antioxidant components, such as selenium, can be considered. There is a growing interest in the use of yeasts because they are able to convert inorganic selenium into selenomethionine. In the present study, oral administration of Saccharomyces cerevisiae UFMG A-905 enriched with selenium was evaluated as an alternative in minimizing the side effects of 5FU-induced mucositis in mice. METHODS: Mice body weight, food consumption, faeces consistency and the presence of blood in faeces were assessed daily during experimental mucositis induced by 5-fluorouracil (5FU). Blood was used for intestinal permeability determination, and small intestine for oxidative stress, immunological and histopathological examination. RESULTS: The increased intestinal permeability observed with mucositis induction was partially reverted by S. cerevisiae and selenium-enriched yeast. Both treatments were able to reduce myeloperoxidase activity, but only selenium-enriched yeast reduced eosinophil peroxidase activity. CXCL1/KC levels, histopathological tissue damage and oxidative stress (lipid peroxidation and nitrite production) in the small intestine were reduced by both treatments; however, this reduction was always higher when treatment with selenium-enriched yeast was evaluated. CONCLUSIONS: Results of the present study showed that the oral administration of S. cerevisiae UFMG A-905 protected mice against mucositis induced by 5-FU, and that this effect was potentiated when the yeast was enriched with selenium.

Yeast distribution in Grignolino grapes growing in a new vineyard in Piedmont and the technological characterization of indigenous Saccharomyces spp. strains.

The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non-Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus, which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum, the yeast-like fungus Aerobasidium pullulans, Candida zemplinina, Pichia kluyveri, Candida californica, Curvibasidium cygneicollum, Meyerozima caribbica, Rhodotorula babjevae, Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus, potentially suitable as an ecotypical starter for territorial wines.

Cannabinoid Delivery Systems for Pain and Inflammation Treatment.

There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

'Ropy' phenotype, exopolysaccharides and metabolism: Study on food isolated potential probiotics LAB.

Lactic acid bacteria are fully recognized for their industrial applications among which the production and release of exopolysaccharides. In the present investigation, we screened fifteen Lactobacilli in order to find ropy strains, quantify exopolysaccharides and detect proteins specifically associated with the ropy-exopolysaccharide production. The highest ropy-exopolysaccharide producer (L. helveticus 6E8), was grown in stimulating and basal condition (10% and 2% lactose) and subjected to comparative proteomic analysis. The levels of 4 proteins were found significantly increased in the membrane fraction under stimulating conditions: a specific exopolysaccharide biosynthetic protein, a stress-induced protein, a protein involved in secretion and an ATP-synthase subunit. Conversely, several enzymes involved in anabolism and protein synthesis were decreased. These results suggest a general shift from growth to exopolysaccharide-mediated protection from the hyperosmotic environment. Due to the great interest in exopolysaccharides with novel features, the identification of these proteins could have implications for future improvements of industrial strains.

Back to the past-forever young: cutting-edge biochemical and microbiological tools for cultural heritage conservation.

Ancient documents and milestones of human history such as manuscripts and textiles are fragile and during aging undergo chemical, physical, and biological deterioration. Among the different causes of damage, also human intervention plays a role since some restoration strategies proved to be transient and/or they generated further damage. Outdoor monuments undergo deterioration since they are exposed to pollution, weathering, microbial attack (giving rise to undesired pigmentation, discoloration or true dissolution, corrosion, and overall decay), as well as man-made damage (i.e., graffiti). This review article reports the best-fitting strategies used to restore wall paintings, outdoor monuments, textiles, and paper documents to their ancient beauty by employing "soft" biobased approaches such as viable bacteria or suitable enzymes.

Back to the past: "find the guilty bug-microorganisms involved in the biodeterioration of archeological and historical artifacts".

Microbial deterioration accounts for a significant percentage of the degradation processes that occur on archeological/historical objects and artworks, and identifying the causative agents of such a phenomenon should therefore be a priority, in consideration of the need to conserve these important cultural heritage items. Diverse microbiological approaches, such as microscopic evaluations, cultural methods, metabolic- and DNA-based techniques, as well as a combination of the aforementioned methods, have been employed to characterize the bacterial, archaeal, and fungal communities that colonize art objects. The purpose of the present review article is to report the interactions occurring between the microorganisms and nutrients that are present in stones, bones, wood, paper, films, paintings, and modern art specimens (namely, collagen, cellulose, gelatin, albumin, lipids, and hydrocarbons). Some examples, which underline that a good knowledge of these interactions is essential to obtain an in depth understanding of the factors that favor colonization, are reported. These data can be exploited both to prevent damage and to obtain information on historical aspects that can be decrypted through the study of microbial population successions.