Green One-Step Synthesis of Silver Nanoparticles Obtained from Schinus areira Leaf Extract: Characterization and Antibacterial Mechanism Analysis.

The increased emergence of antibiotic-resistant bacteria is a serious health problem worldwide. In this sense, silver nanoparticles (AgNPs) have received increasing attention for their antimicrobial activity. In this context, the goal of this study was to produce AgNPs by a green synthesis protocol using an aqueous leaf extract of Schinus areira as biocomposite to later characterize their antimicrobial action. The nanomaterials obtained were characterized by UV‒vis spectroscopy, DLS, TEM, and Raman, confirming the presence of quasi-spherical AgNPs with a negative surface charge and diameter around 11 nm. Afterward, the minimum inhibitory and bactericidal concentration of the AgNPs against Staphylococcus aureus and Escherichia coli were obtained, showing high antibacterial activity. In both of the examined bacteria, the AgNPs were able to raise intracellular ROS levels. In E. coli, the AgNPs can harm the bacterial membrane as well. Overall, it can be concluded that it was possible to obtain AgNPs with colloidal stability and antibacterial activity against Gram-positive and Gram-negative bacteria. Our findings point to at least two separate mechanisms that can cause cell death, one of which involves bacterial membrane damage and the other of which involves intracellular ROS induction.

Antimicrobial properties of the essential oil of Schinus areira (Aguaribay) against planktonic cells and biofilms of S. aureus.

The essential oil (EO) of Schinus areira L. (Anacardiaceae) leaves has shown antibacterial activity against Staphylococcus aureus. In this study, we aimed to unravel the mechanisms of its antibacterial action by using bacterial cells and model membranes. First, the integrity of the S. aureus membrane was evaluated by fluorescence microscopy. It was observed that there was an increase in the permeability of cells that was dependent on the EO concentration as well as the incubation time. For a deep comprension of the action of the EO on the lipids, its effect on the membrane fluidity was evaluated on DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine): DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-1'-rac-glycerol) (5:1) liposomes by dynamic light scattering and by using Laurdan doped liposomes. The results indicate that EO produces changes in lipid membrane packing, increasing the fluidity, reducing the cooperative cohesive interaction between phospholipids and increasing access of water or the insertion of some components of the EO to the interior of the membrane. In addition, the potential effect of EO on intracellular targets, such as the increase of cytosolic reactive oxygen species (ROS) and DNA damage, were analyzed. The EO was capable of increasing the production of ROS as well as inducing a partial DNA degradation. Finally, the effect of EO on S. aureus biofilm was tested. These assays showed that EO was able to inhibit the biofilm formation, and also eradicate preformed biofilms. The results show, that the EO seems to have several bacterial targets involved in its antibacterial activity, from the bacterial membrane to DNA. Furthermore, the antibacterial action affects not only planktonic cells but also biofilms; reinforcing the potential application of this EO.

Insecticidal activity of the essential oil of Schinus areira against Rhipibruchus picturatus (F.) (Coleoptera: Bruchinae), and its inhibitory effects on acetylcholinesterase.

During the storage of Prosopis alba pods, substantial quantitative and qualitative losses were observed. One of the main factors is the seed beetle Rhipibruchus picturatus. A key strategy to develop new pest control management is the use of essential oils (EOs) due they are efficient, less toxic, and less persistent in the environment compared to synthetic pesticides. In this context, seeds and leaves of Schinus areira L. (Anacardiaceae) EOs and Citrus spp. EO were studied in the present work. In the leaves of S. areira EO, 1-epi-cadinol, sesquiterpenoid alcohol, was the major compound. On the other hand, the main compounds of the EO extracted from S. areira seeds are the monoterpenes sabinene, and α-pinene. Finally, in the Citrus EO, limonene is the principal component. The three EOs obtained exhibited insecticidal activity against R. picturatus, being the first report of the use of EOs against this insect pest. The best insecticidal results were obtained with the leaves of S. areira EO. Moreover, this EO inhibits the acetylcholinesterase enzyme in vitro assays. Molecular docking studies on acetylcholinesterase (AChE) suggest that the main components of the leaves of S, areira EOs, bind to the active site of the enzyme, in good agreement with in vitro competitive inhibition against AChE observed for this EO. The data obtained demonstrate the potential use of Schinus areira EOs in the development of new storage pest control strategies.

Review of antiviral peptides for use against zoonotic and selected non-zoonotic viruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

25-Hydroxycholesterol Effect on Membrane Structure and Mechanical Properties.

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.

Zeta potential beyond materials science: Applications to bacterial systems and to the development of novel antimicrobials.

This review summarizes the theory of zeta potential (ZP) and the most relevant data about how it has been used for studying bacteria. We have especially focused on the discovery and characterization of novel antimicrobial compounds. The ZP technique may be considered an indirect tool to estimate the surface potential of bacteria, a physical characteristic that is key to maintaining optimal cell function. For this reason, targeting the bacterial surface is of paramount interest in the development of new antimicrobials. Surface-acting agents have been found to display a remarkable bactericidal effect and have simultaneously revealed a low tendency to trigger resistance. Changes in the bacterial surface as a result of various processes can also be followed by ZP measurements. However, due to the complexity of the bacterial surface, some considerations regarding the assessment of ZP must first be taken into account. Evidence on the application of ZP measurements to the characterization of bacteria and biofilm formation is presented next. We finally discuss the feasibility of using the ZP technique to assess antimicrobial-induced changes in the bacterial surface. Among these changes are those related to the interaction of the agent with different components of the cell envelope, membrane permeabilization, and loss of viability.

Antibacterial activity of prenylated benzopyrans from Peperomia obtusifolia (Piperaceae).

Peperomia obtusifolia is a herbaceous perennial plant native to the Americas reported as a traditional medicine to treat snake bites and as a skin cleanser. The bioassay-guided fractionation of crude extracts from aerial parts of P. obtusifolia against a panel of clinically important fungi and bacteria, showed that hexane and dichloromethane extracts demonstrated selective bacterial inhibition, allowing the isolation of the known compounds peperobtusin A (1), and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3"-methyl-2"-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2H-1-benzopyran-6-carboxylic acid (2) from dichloromethane extract. Compound 2 was active against Gram-positive bacteria including community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates and an Enterococcus faecalis vancomycin-resistant strain, with minimal inhibitory concentration (MIC) values of 4 µg/mL (10.8 µM) and 8 µg/mL (21.6 µM) respectively. The interaction of compound 2 with the bacterial membrane was demonstrated by means of Zeta potential experiments on S. aureus, then confirming the membrane damage by fluorescent microscopy experiments.

Chemical composition of Schinus areira essential oil and antimicrobial action against Staphylococcus aureus.

The antimicrobial activities of plant extracts have formed the basis of many alternative medicines. In this context, the genus Schinus L. (Anacardiaceae), exhibits many traditional uses in medicine. However, a few studies on the antimicrobial properties of Schinus areira essential oils were conducted. The essential oil from S. areira leaves from Santiago del Estero was obtained by hydrodistillation and twenty-eight compounds were identified using CG-MS-EI spectrometry. The sesquiterpenoid alcohol 1-epi-cadinol was the major compound, followed by δ-cadinene, alloaromadendrene, ß-pinene, ß-caryophyllene, and γ-cadinene. The essential oil obtained exhibited antimicrobial activity against Staphylococcus aureus, showing a bacteriostatic activity at 64 µg/mL and bacteriolytic activity at 256 µg/mL; in contrast, non antibacterial effect was observed in Escherichia coli in the assayed conditions. The antibacterial activity was accompanied by significant changes in Zeta potential on the S. aureus surface. The data obtained suggest that the essential oil of S.areira leaves presents potential use in pharmaceutical industries.

Unravelling the mechanism of action of "de novo" designed peptide P1 with model membranes and gram-positive and gram-negative bacteria.

In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the "de novo" designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium. All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.

Interactions of "de novo" designed peptides with bacterial membranes: Implications in the antimicrobial activity.

Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level.

New insights into novel Escherichia coli colistin-resistant strains isolated from Argentina.

Colistin is a polymyxin antibiotic (polymyxin E) that has in recent years re-emerged as an option for treatment of multidrug-resistant bacteria. Recently, the re-introduction of colistin resulted in the appearance of colistin-resistant bacteria, which is usually caused by LPS modifications. The fact that this modification is mediated by a plasmid carrying the mcr-1 gene, implies a horizontal transfer of colistin resistance. In Argentina, the National Reference Laboratory in Antimicrobial Resistance (NRLAR), has recently screened several bacteria for the MCR-1 plasmid, detecting nine Escherichia coli isolates carrying the plasmid with the mcr-1 gene, among others. In this context, we proposed to assess the effect of surface charge modifications induced by the plasmid MCR-1 and its impact on the resulting colistin resistance in two clinical isolates of colistin-resistant E. coli. Using zeta potential assays, we confirmed the reduction of negative charge exposure on clinical isolates compared to the reference strain of E. coli. In addition, through permeabilization assays, we were able to correlate this reduction in charge exposure with the extent of damage to the bacterial membrane. The fact that this surface charge modification through substitution of lipid A is plasmid encoded, represents an important concern for future antimicrobial peptide drug development.

Synergistic and antibiofilm activity of the antimicrobial peptide P5 against carbapenem-resistant Pseudomonas aeruginosa.

In the search for new antimicrobial molecules, antimicrobial peptides (AMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane disruption and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing research and clinical interest. The AMP P5 has been previously designed as a cationic linear α-helical sequence, being its antimicrobial and hemolytic properties also evaluated. In this work, we analyzed the feasibility of using P5 against a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa, one of the most common and risky pathogens in clinical practice. After antimicrobial activity confirmation in in vitro studies, synergistic activity of P5 with meropenem was evaluated, showing that P5 displayed significant synergistic activity in a time kill curve assay. The ability of P5 to permeabilize the outer membrane of P. aeruginosa can explain the obtained results. Finally, the antibiofilm activity was investigated by viability analysis (MTT assay), crystal violet and confocal imaging, with P5 displaying mild biofilm inhibition in the range of concentrations tested. Regarding biofilm disruption activity, P5 showed a higher efficacy, interfering with biofilm structure and promoting bacterial cell death. Atomic force microscope images further demonstrated the peptide potential in P. aeruginosa biofilm eradication, confirming the promising application of P5 in multi-resistant infections therapeutics.

Combining 25-Hydroxycholesterol with an HIV Fusion Inhibitor Peptide: Interaction with Biomembrane Model Systems and Human Blood Cells.

The fusion between the viral and the target cell membrane is a crucial step in the life cycle of enveloped viruses. The blocking of this process is a well-known therapeutic approach that led to the development of the fusion inhibitor peptide enfuvirtide, clinically used against human immunodeficiency virus (HIV) type 1. Despite this significant advance on viral treatment, the appearance of resistance has limited its clinical use. Such a limitation has led to the development of other fusion inhibitor peptides, such as C34, that present the same structural domain as enfuvirtide (heptad repeat sequence) but have different functional domains (pocket-binding domain in the case of C34 and lipid-binding domain in the case of enfuvirtide). Recently, the antiviral properties of 25-hydroxycholesterol were demonstrated, which boosted the interest in this oxysterol. The combination of two distinct antiviral molecules, C34 and 25-hydroxycholesterol, may help to suppress the emergence of resistant viruses. In this work, we characterized the interaction of the C34-25-hydroxycholesterol conjugate with biomembrane model systems and human blood cells. Lipid vesicles and monolayers with defined lipid compositions were used as biomembrane model systems. The conjugate interacts preferentially with membranes rich in sphingomyelin (a lipid enriched in lipid rafts) and presents a poor partition to membranes composed solely of phosphatidylcholine and cholesterol. We hypothesize that cholesterol causes a repulsive effect that is overcome in the presence of sphingomyelin. Importantly, the peptide shows a preference for human peripheral blood mononuclear cells relative to erythrocytes, which shows its potential to target CD4+ cells. Antiviral activity results against different wild-type and drug-resistant HIV strains further demonstrated the potential of C34-HC as a good candidate for future studies.

Studies on interaction of green silver nanoparticles with whole bacteria by surface characterization techniques.

The use of silver nanoparticles (AgNPs) with their novel and distinct physical, chemical, and biological properties, has proven to be an alternative for the development of new antibacterial agents. In particular, the possibility to generate AgNPs coated with novel capping agents, such as phytomolecules obtained via a green synthesis (G-AgNPs), is attracting great attention in scientific research. Recently, we showed that membrane interactions seem to be involved in the antibacterial activity of AgNPs obtained via a green chemical synthesis using the aqueous leaf extract of chicory (Cichorium intybus L.). Furthermore, we observed that these G-AgNPs exhibited higher antibacterial activity than those obtained by chemical synthesis. In order to achieve the green AgNPs mode of action as well as their cellular target, we aimed to study the antibacterial activity of this novel green AgNPs against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The effect of the G-AgNPs on the bacterial surface was first evaluated by zeta potential measurements and correlated with direct plate count agar method. Afterwards, atomic force microscopy was applied to directly unravel the effects of these G-AgNPs on bacterial envelopes. Overall, the data obtained in this study seems correlate with a multi-step mechanism by which G-AgNPs-lipid membrane interactions is the first step prior to membrane disruption, resulting in antibacterial activity.

Lactobacillus plantarum as a malolactic starter culture in winemaking: a new (old) player?

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO2, which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking.

Zeta potential changes of Saccharomyces cerevisiae during fermentative and respiratory cycles.

Saccharomyces cerevisiae is a type of yeast, widely used in diverse biotechnological food-beverage processes. Although the performance of an industrial fermentation process depends largely on the number of cells, it is necessary to consider the physiological state of the cultures. In this context, the aim of this study was to determine in a yeast culture how factors such as growth conditions affect surface properties at the different growth stages. Our results show that, S. cerevisiae spp. exhibits different zeta potential mean values along the exponential, post-diauxic and stationary growth phases. In addition, there were differences depending on whether they are in aerobic or anaerobic conditions. When the effect of pH on the media was studied, a different dependence of zeta potential at each stage reveals that in the living cells the surface potential depends on the interaction between secreted acids and the constituents of the surfaces, according to the growth conditions. In order to have a view at the cellular level, the zeta potential on individual cells by optical microscopy has been determined at different stages of culture in aerobic and anaerobic conditions. This single-cell method allows for the identification and following of the development of different cell subpopulations during each growth stage. Furthermore, the behavior of the dead cells provided evidence to relate the large negatively charged population with cell wall damage. Overall, the results obtained in the present work represent an important milestone for a novel application of zeta potential technique on yeast.

Study of the interactions of bovine serum albumin with a molybdenum(II) carbonyl complex by spectroscopic and molecular simulation methods.

Therapy with inhaled carbon monoxide (CO) is being tested in human clinical trials, yet the alternative use of prodrugs, CO-Releasing Molecules (CORMs), is conceptually advantageous. These molecules are designed to release carbon monoxide in specific tissues, in response to some locally expressed stimulus, where CO can trigger a cytoprotective response. The design of such prodrugs, mostly metal carbonyl complexes, must consider their ADMET profiles, including their interaction with transport plasma proteins. However, the molecular details of this interaction remain elusive. To shed light into this matter, we focused on the CORM prototype [Mo(η5-Cp)(CH2COOH)(CO)3] (ALF414) and performed a detailed molecular characterization of its interaction with bovine serum albumin (BSA), using spectroscopic and computational methods. The experimental results show that ALF414 partially quenches the intrinsic fluorescence of BSA without changing its secondary structure. The interaction between BSA and ALF414 follows a dynamic quenching mechanism, indicating that no stable complex is formed between the protein Trp residues and ALF414. The molecular dynamics simulations are in good agreement with the experimental results and confirm the dynamic and unspecific character of the interaction between ALF414 and BSA. The simulations also provide important insights into the nature of the interactions of this CORM prototype with BSA, which are dominated by hydrophobic contacts, with a contribution from hydrogen bonding. This kind of information is useful for future CORM design.

Interaction of green silver nanoparticles with model membranes: possible role in the antibacterial activity.

Silver nanoparticles (AgNPs) constitute a very promising approach for overcoming the emergence of antibiotic resistance bacteria. Although their mode of action could be related with membrane damage, the AgNPs-lipid membrane interaction is still unclear. In this sense, the present work investigated the interaction of model lipid membranes with AgNPs coated with different capping agents such as citrate (C-AgNPs) and phytomolecules (G-AgNPs) obtained via a green synthesis. The AgNPs-membrane interactions were evaluated studying i) the surface pressure changes on both zwitterionic (DMPC) and negatively charged (DMPC:DMPG) lipid monolayers, ii) the zeta potential and DLS of DMPC:DMPG liposomes and iii) Zeta potential on Escherichia coli membranes, incubated with this nanomaterials. The results showed that both negatively charged-AgNPs can interact with these lipid monolayers inducing an increase in the surface pressure but G-AgNPs presented a significantly higher affinity toward both monolayers in comparison with C-AgNPs. Zeta potential data confirmed again the interaction event showing that both DMPC:DMPG liposomes and E. coli bacteria became more negative with the addition of G-AgNPs. This increased net negative charge of the liposomes and E. coli allows to indicate an interfacial interaction where the green nanometal should keep adsorbed to the membrane via the insertion of aromatic/hydrophobic moieties of capping agents on the surface of AgNPs into the lipid bilayer. Summarizing, the AgNPs-membrane interaction should be an essential step in the antibacterial activity either because the membrane is the main target or by increasing the local concentration of silver from G-AgNPs accumulation which could cause the bactericidal effect.

Antimicrobial Peptides: Interaction With Model and Biological Membranes and Synergism With Chemical Antibiotics.

Antimicrobial peptides (AMPs) are promising novel antibiotics since they have shown antimicrobial activity against a wide range of bacterial species, including multiresistant bacteria; however, toxicity is the major barrier to convert antimicrobial peptides into active drugs. A profound and proper understanding of the complex interactions between these peptides and biological membranes using biophysical tools and model membranes seems to be a key factor in the race to develop a suitable antimicrobial peptide therapy for clinical use. In the search for such therapy, different combined approaches with conventional antibiotics have been evaluated in recent years and demonstrated to improve the therapeutic potential of AMPs. Some of these approaches have revealed promising additive or synergistic activity between AMPs and chemical antibiotics. This review will give an insight into the possibilities that physicochemical tools can give in the AMPs research and also address the state of the art on the current promising combined therapies between AMPs and conventional antibiotics, which appear to be a plausible future opportunity for AMPs treatment.

Cell surface damage and morphological changes in Oenococcus oeni after freeze-drying and incubation in synthetic wine.

The aim of the present study was to evaluate the effects of freeze-drying in the presence of trehalose as a cryoprotectant, followed by incubation in synthetic wine, on surface damage, viability and l-malic acid consumption of the oenological strain Oenococcus oeni UNQOe 73.2. After freeze-drying, no significant differences were observed in the number of viable cells (for both acclimated and non-acclimated cultures) respect to the fresh culture. In contrast, loss of viability was observed after wine incubation for 24 h, being acclimated freeze-dried cells the best conditions for this. After the preservation process, small changes in cell morphology were observed by Atomic Force Microscopy (AFM). The Zeta potential and AFM showed that 24 h of wine incubation was enough to induce several cell surface modifications. Plate count data allowed us to establish that surface damage is an important factor for loss of viability, regardless of the acclimation treatment. Although the number of surviving O. oeni cells decreased dramatically after incubation in synthetic wine for 15 days, the consumption of l-malic acid was higher than 70%, with freeze-dried cells showing a better performance than fresh cultures. These results demonstrate that O. oeni freeze-dried cultures could be applied to direct wine inoculation, to conduct malolactic fermentation, maintaining its technological properties and reducing the time and costs of the winemaking process.