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Winemaking is a complex process involving two successive fermentations: alcoholic fermentation, by yeasts, and malolactic fermentation (MLF), by lactic acid bacteria (LAB). During MLF, LAB can contribute positively to wine flavor through decarboxylation of malic acid with acidity reduction and other numerous enzymatic reactions. However, some microorganisms can have a negative impact on the quality of the wine through processes such as biogenic amine production. For these reasons, monitoring the bacterial community profiles during MLF can predict and control the quality of the final product. In addition, the selection of LAB from a wine-producing area is necessary for the formulation of native malolactic starter cultures well adapted to local winemaking practices and able to enhance the regional wine typicality. In this sense, molecular biology techniques are fundamental tools to decipher the native microbiome involved in MLF and to select bacterial strains with potential to function as starter cultures, given their enological and technological characteristics. In this context, this work reviews the different molecular tools (both culture-dependent and -independent) that can be applied to the study of MLF, either in bacterial isolates or in the microbial community of wine, and of its dynamics during the process.

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Argentina is the fifth world-wide wine producer, with an area of emerging importance in the Southwest of Buenos Aires Province, where climatic conditions are rather challenging. We studied the variations in soil and wine bacterial diversity through three consecutive vintages, and how climatic conditions affected said diversity. During the years of our study there were two harsh climatic events, a prolonged drought that extended over two vegetative periods, and an unseasonable spring frost in 2017. We found that the bacterial diversity reacted to these climatic events, given that there was a shift in the taxa exclusive to soil and wine, and shared by both, through time. Our results show a core of microorganisms in soil as well as in wine, belonging to different phyla that are conserved across the vintage years. A trend to an enrichment in Actinobacteria was detected in soil samples, whereas a high relative abundance of the Acetobacteraceae family and a scarcity of Lactic Acid Bacteria (LAB) were detected in the wine samples. We believe our results contribute to a better understanding of the impact of climatic conditions on the soil and wine microbiota, and can provide vintners with valuable knowledge for improving their wine production.

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The aim of this work was to obtain freeze-dried biomass of the native Patagonian Lactiplantibacillus plantarum strain UNQLp 11 from a whey permeate (WP)-based medium and compare it with the growth in commercial MRS broth medium. Survival and activity of the freeze-dried Lb. plantarum strain were investigated after inoculation in wine as a starter culture for malolactic fermentation (MLF). The effect of storage and rehydration condition of the dried bacteria and the nutrient supplementation of wine were also studied. The freeze-dried cultures from WP and those grown in MRS showed similar survival results. Rehydration in MRS broth for 24 h and the addition of a rehydration medium to wine as nutrient supplementation improved the survival under wine harsh conditions and guaranteed the success of MLF. Storage at 4 °C under vacuum was the best option, maintaining high cell viability for at least 56 days, with malic acid consumption higher than 90% after 7 days of inoculation in a wine-like medium. These results represent a significant advance for sustainable production of dried malolactic starter cultures in an environmentally friendly process, which is low cost and easy to apply in winemaking under harsh physicochemical conditions.

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Soil microbiomes, as a primary reservoir for plant colonizing fungi and bacteria, play a major role in determining plant productivity and preventing invasion by pathogenic microorganisms. The use of 16S rRNA and ITS high-throughput amplicon sequencing for analysis of complex microbial communities have increased dramatically in recent years, establishing links between wine specificity and, environmental and viticultural factors, which are framed into the elusive terroir concept. Given the diverse and complex role these factors play on microbial soil structuring of agricultural crops, the main aim of this study is to evaluate how external factors, such as vintage, vineyard location, cultivar and soil characteristics, may affect the diversity of the microbial communities present. Additionally, we aim to compare the influence these factors have on the structuring of bacterial and fungal populations associated with Malbec grapevine rhizosphere with that of the more widespread Cabernet Sauvignon grapevine cultivar. Samples were taken from Malbec and Cabernet Sauvignon cultivars from two different vineyards in the San Juan Province of Argentina. Total DNA extracts from the rhizosphere soil samples were sequenced using Illumina's Miseq technology, targeting the V3-V4 hypervariable 16S rRNA region in prokaryotes and the ITS1 region in yeasts. The major bacterial taxa identified were Proteobacteria, Bacteroidetes and Firmicutes, while the major fungal taxa were Ascomycetes, Basidiomycetes, Mortierellomycetes and a low percentage of Glomeromycetes. Significant differences in microbial community composition were found between vintages and vineyard locations, whose soils showed variances in pH, organic matter, and content of carbon, nitrogen, and absorbable phosphorus.

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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.

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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.

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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.

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Cationic antimicrobial peptides (AMPs) are short linear amino acid sequences, which display antimicrobial activity against a wide range of bacterial species. They are promising novel antimicrobials since they have shown bactericidal effects against multiresistant bacteria. Their amphiphilic structure with hydrophobic and cationic regions drives their interaction with anionic bacterial cytoplasmic membranes, which leads to their disruption. In this work two synthetic designed AMPs, P5 and P6.2, which have been previously analyzed in their ability to interact with bacterial or eukaryotic membranes, were evaluated in their anti-biofilm and in vivo antibacterial activity. In a first step, a time-kill kinetic assay against P. aeruginosa and S. aureus and a curve for hemolytic activity were performed in order to determine the killing rate and the possible undesirable toxic effect, respectively, for both peptides. The biofilm inhibitory activity was quantified at sub MIC concentrations of the peptides and the results showed that P5 displayed antibiofilm activity on both strains while P6.2 only on S. aureus. Scanning electron microscopy (SEM) of bacteria treated with peptides at their MIC revealed protruding blisters on Gam-negative P. aeruginosa strain, but almost no visible surface alteration on Gram-positive S. aureus. These micrographs highlighted different manifestations of the membrane-disrupting activity that these kinds of peptides possess. Finally, both peptides were analyzed in vivo, in the lungs of neutropenic mice previously instilled with P. aeruginosa. Mice lungs were surgically extracted and bacteria and pro-inflammatory cytokines (IL-ß, IL-6 and TNF-α) were quantified by colony forming units and ELISA, respectively. Results showed that instillation of the peptides produced a significant decrease in the number of living bacteria in the lungs, concomitant with a decrease in pro-inflammatory cytokines. Overall, the results presented here suggest that these two new peptides could be good candidates for future drug development for anti-biofilm and anti-infective therapy.

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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.

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The production of malolactic starter cultures requires the obtention of suitably large biomass at low-cost. In this work it was possible to obtain a good amount of biomass, at laboratory scale, of two enological strains of Lb. plantarum, by formulating a culture medium based on whey permeate (WP), a by-product of the cheese industry usually disposed as waste, when this was supplemented with yeast extract (Y), salts (S) and Tween 80 (T) (WPYST). Bacteria grown in WPYST medium exhibited good tolerance to stress conditions of synthetic wine (pH 3.5, ethanol 13% vol/vol). However, when WPYST was added with 8% vol/vol ethanol, cultures inoculated in synthetic wine, showed a lower viability and capacity to consume L-malic acid than when they were cultured in WPYST without ethanol. Subsequently, strains grown in WPYST were inoculated in sterile wine samples (final stage of alcoholic fermentation) of the red varietals Merlot and Pinot noir, and incubated at laboratory scale. Cultures from WPYST, inoculated in Pinot noir wine, showed a better performance than bacteria grown in MRS broth, and exhibited a consumption of L-malic acid higher than 90%. However, cultures from WPYST or from MRS broth, inoculated in sterile Merlot wine, showed a lower survival. This study allowed the formulation of a low-cost culture medium, based on a by-product of the food industry, which showed to be adequate for the growth of two enological strains of Lb. plantarum, suggesting their potentiality for application in the elaboration of malolactic starter cultures.

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