Fungal Mycobiome of Mature Strawberry Fruits (Fragaria x ananassa Variety 'Monterey') Suggests a Potential Market Site Contamination with Harmful Yeasts.

An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to investigate the diversity of fungi associated with mature strawberries collected from a volcanic orchard and open-air market stands. Based on the Kruskal-Wallis test, no statistically significant differences were observed in both non-phylogenetic and phylogenetic alpha diversity indices. According to beta diversity analyses, significant differences in fungal communities were found between groups (orchard vs. market). Taxonomic assignment of amplicon sequence variables (ASVs) revealed 7 phyla and 31 classes. The prevalent fungal phyla were Basidiomycota (29.59-84.58%), Ascomycota (15.33-70.40%), and Fungi-phy-Insertae-sedis (0.45-2.89%). The most predominant classes among the groups were Saccharomycetes in the market group, and Microbotryomycetes and Tremellomycetes in the orchard group. Based on the analysis of microbiome composition (ANCOM), we found that the most differentially fungal genera were Hanseniaspora, Kurtzmaniella, and Phyllozyma. Endophytic yeasts Curvibasidium cygneicollum were prevalent in both groups, while Candida railenensis was detected in fruits originating only from the market. In addition, Rhodotorula graminis (relative abundance varying from 1.7% to 21.18%) and Papiliotrema flavescens (relative abundance varying from 1.58% to 16.55%) were detected in all samples regardless of origin, while Debaryomyces prosopidis was detected in samples from the market only, their relative abundance varying with the sample (from 0.80% to 19.23%). Their role in fruit quality and safety has not been yet documented. Moreover, several clinically related yeasts, such as Meyerozyma guilliermondii and Candida parapsilosis, were detected in samples only from the market. Understanding the variety and makeup of the mycobiome in ripe fruits during the transition from the orchard to the market is crucial for fruit safety after harvest.

Preventing microbe colonization on avocado (Persea nubigena var. guatemalensis) through metabiotic treatment, a promising postharvest safety improvement.

Introduction: Lactic acid bacteria (LAB) produce various metabolites (i.e. metabiotics) with inhibitory capacity towards harmful foodborne pathogens. Methods: This study aimed to design several antimicrobial formulations based on metabiotics obtained from different native LAB species (Lactobacillus pentosus UTNGt5, Lactococcus lactis UTNGt28, and Weissella cibaria UTNGt21O) and to detect the possible mode of action towards two multidrug resistant Staphylococcus spp. strains isolated from avocado (Persea nubigena var. guatemalensis) fruits. Additionally, the formulation with the highest inhibitory activity was tested ex vitro on avocados at the immature (firm) ripeness stage to evaluate their effect on microorganisms' growth and fruit quality attributes post-harvest. Results and discussion: Out of the top five formulations showing the highest bactericidal effect in vitro at their minimum inhibitory concentration (1 x MIC) on both Staphylococcus spp. targets one candidate annotated P11 (consisting of UTNGt21O and UTNGt28; 1:3, v/v) was selected. Co-cultivation of Staphylococcus strains with P11 formulation results in cell viability reduction by 98%, by impairing the integrity of the cell membrane inducing cytoplasm molecule content leakage, protein profile changes, and finally bacterial death. Even though the total coliforms, Staphylococcus spp., Enterobacter spp., molds, and yeasts counts were not fully eliminated by day 13 of storage, a statistically significant reduction (p < 0.05) in viable cell counts were observed by day 8 upon the P11 treatment compared with non-treated control (C) and treated with a commercial disinfectant (T1) samples, suggesting that P11 formulation inhibited microbial colonization during storage. Likewise, no visible dark spots were observed on the mesocarp (pulp) upon the treatment with P11, whereas T1 and C fruits showed greater dark spots on the pulp as indicative of damage. The quality attributes, such as pH, total soluble solids, total titratable acidity, antioxidant capacity, and total polyphenol content, were not affected by the treatment. Principal Component Analysis (PCA) conducted on these five variables showed a clear separation of samples according to the maturity stage regardless of the treatment. Conclusion: These results suggest that the active metabolites from LAB strains might create a barrier between the exocarp and mesocarp, inhibiting the microorganisms colonization, reducing fruit damage, and lengthening the fruit quality and safety after harvest.

Bacterial community changes in strawberry fruits (Fragaria × ananassa variety "Monterey") from farm field to retail market stands, an indicator of postharvest contamination.

Background: Strawberry (Fragaria × ananassa) fruits are vulnerable to bacterial contamination; some species are pathogenic and can affect human health. Comprehending the bacterial composition and diversity at different ripe stages is a key determinant of the fruit health, productivity, and quality. Methodology: An amplicon metagenomic approach on the 16S rRNA region was used to identify the bacterial diversity in exocarp of fruits collected from a farm field at two ripe stages: breaking (white, phase two) and ripe (red, phase four) and purchased from different retail market stands at ripe (red, phase four, ready-to-eat) stage. Besides, the fruit quality was assessed. Results: Strawberries carries a high microorganisms diversity, with Pseudomonaceae, Yearsiniaceae, and Hafniaceae being the most abundant families across the samples. Among the groups, Pseudomonaceae and Clostridiaceae were the most abundant families at breaking (phase two) and ripe (phase four), whereas Yearsiniaceae, Hafniaceae, Aeromonadaceae, and Streptococcaceae were the most abundant families in the market group. Although samples from group four-field and market were at the same ripe stage, the bacterial species composition was divergent. Serratia spp. were prevalent (above 60%) in samples collected from the market group, and Pseudomonas (above 70%) species were mostly found in the samples collected from the field settings regardless of the phase. Besides, Escherichia coli and Salmonella enterica were detected in the ready-to-eat samples from both the field and the market, while Enterococcus gallinarum was detected in the samples that originated from the market. Interestingly, Shewanella putrefaciens and Shewanella profunda, two human opportunistic pathogens, were detected in the fruits from the market only. According to alpha and beta diversity analyses, strawberry fruits displayed significant differences (P < 0.05) in bacterial communities within the ripe group, with the samples from the market showing the most bacterial diversity. Although we do not directly correlate the quality attributes with bacterial diversity, the results indicated a clear separation between groups according with their ripe stage and origin. Conclusion: This study provides a comprehensive framework of the bacterial diversity throughout the transition from unripe to ripe strawberries which may aid in the development of preventative measures to manage the postharvest contamination.

Microbiota and its antibiotic resistance profile in avocado Guatemalan fruits (Persea nubigena var. guatemalensis) sold at retail markets of Ibarra city, northern Ecuador.

Introduction: Avocados are typically sold in unsanitary conditions at the retail markets in Ecuador, which can raise the risk of microbial contamination. These microorganisms could exhibit multi-antibiotic resistance (MAR), being a serious threat concern to human health. In this study, we aimed to evaluate the microbiota and its antibiotic resistance profile in avocado Guatemalan fruits (Persea nubigena var. guatemalensis), at ripe stage: immature, firm light green (ready to eat in 4 days), peel (AFPE) and pulp (AFPU), and mature intense green (ready to eat) peel (AMPE) and pulp (AMPU), to gain baseline information on the prevalence of MAR bacteria. Methods: Culture-independent (16S rRNA metagenomics) and culture-dependent approach (to detect specific indicator microorganisms) were used. Moreover, antibiotic susceptibility of selected target indicator bacteria was assessed providing information about the antibiotic resistance (AR) among the groups. Results: Based on 16S rRNA gene metagenomic analysis, over 99.78% of reads were classified as bacteria in all samples. Shannon diversity index varies from 1.22 to 2.22, with the highest bacterial population assigned to AFPE samples (1327 species). The highest microbial counts of indicator Staphylococcus spp. (STAPHY), Enterobacter spp. (ENT), and Listeria spp. (LIST), were detected in AMPE samples. Thirty percent of the selected STAPHYs, and 20.91% of Enterobacter (ENT) clones were resistant to various classes of antibiotics. The MAR index varies between 0.25 to 0.88 and was clone-, and fruit ripe stage-dependent. Conclusions: The results indicated that ready to eat avocados contained detectable levels of MAR bacteria, including methicillin resistant (MR)-STAPHY, which may act as a potential vector for the spread of antibiotic resistance. To achieve the increase of the production and marketing of Fuerte cultivar in Ecuador, it is vitally important to consider valuable strategies to protect the fruits at the early ripe stage in future. Thus, it is crucial to set up efficient control measures and develop coordinated strategies to guarantee the microbiological quality of the food.

Inhibitory-like Substances Produced by Yeasts Isolated from Andean Blueberries: Prospective Food Antimicrobials.

Natural agents from microorganisms have emerged as suitable options to replace chemical preservatives in foods. In this study, the antibacterial activity of cell-free supernatant (CFS) from five native yeasts (Saccharomyces cerevisiae Lev6 and Lev30, C. pseudointermedia Lev8, Candida intermedia Lev9, C. parapsilosis Lev15) and the reference S. boulardi SSB, was evaluated against some indicator food pathogens. The generation of antimicrobials was reliant on strain-, and sugar-supplemented media, which supported yeast growth established at 30 °C and 200 rpm for 48 h. Treatment with proteinase K and catalase was unable to completely abolish the inhibitory effect, indicating that the active components are likely complex combinations of acids, proteins, hydrogen peroxide, and other metabolites. Although there was no impact on Listeria monocytogenes, exposure to CFS and extracellular fractions obtained through precipitation with methanol (PPm) at 120 °C for 60 min significantly (p < 0.05) increased the inhibitory activity against Escherichia coli, Salmonella enterica, Kosakonia cowanii, and Staphylococcus aureus, indicating that the inhibitory activity was stimulated by heat. Likewise, a synergistic inhibitory action against Listeria was obtained following the pretreatment of PPm with EDTA (ethylenediaminetetraacetic acid). These activities were yeast strain-dependent, with Lev6, Lev8, and Lev30 showing the highest activity. In addition, a heat-stable low-molecular-mass molecule under 5 kDa was detected in Lev30. Further research is required to evaluate the mode of action and characterize the composition of the released molecules in the CFS in order to develop a novel biocontrol agent based on yeasts.

Metabiotics Signature through Genome Sequencing and In Vitro Inhibitory Assessment of a Novel Lactococcus lactis Strain UTNCys6-1 Isolated from Amazonian Camu-Camu Fruits.

Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.

Beta-Lactam Antibiotic Resistance Genes in the Microbiome of the Public Transport System of Quito, Ecuador.

Multidrug-resistant bacteria present resistance mechanisms against ß-lactam antibiotics, such as Extended-Spectrum Beta-lactamases (ESBL) and Metallo-ß-lactamases enzymes (MBLs) which are operon encoded in Gram-negative species. Likewise, Gram-positive bacteria have evolved other mechanisms through mec genes, which encode modified penicillin-binding proteins (PBP2). This study aimed to determine the presence and spread of ß-lactam antibiotic resistance genes and the microbiome circulating in Quito's Public Transport (QTP). A total of 29 station turnstiles were swabbed to extract the surface environmental DNA. PCRs were performed to detect the presence of 13 antibiotic resistance genes and to identify and to amplify 16S rDNA for barcoding, followed by clone analysis, Sanger sequencing, and BLAST search. ESBL genes blaTEM-1 and blaCTX-M-1 and MBL genes blaOXA-181 and mecA were detected along QPT stations, blaTEM being the most widely spread. Two subvariants were found for blaTEM-1, blaCTX-M-1, and blaOXA-181. Almost half of the circulating bacteria found at QPT stations were common human microbiota species, including those classified by the WHO as pathogens of critical and high-priority surveillance. ß-lactam antibiotic resistance genes are prevalent throughout QPT. This is the first report of blaOXA-181 in environmental samples in Ecuador. Moreover, we detected a new putative variant of this gene. Some commensal coagulase-negative bacteria may have a role as mecA resistance reservoirs.

Pathogenic Microorganisms Linked to Fresh Fruits and Juices Purchased at Low-Cost Markets in Ecuador, Potential Carriers of Antibiotic Resistance.

The pathogenic microorganisms linked to fresh fruits and juices sold out in retail low-cost markets raise safety concerns as they may carry multidrug-resistant (MDR) genes. To evaluate the microbiological quality and safety of highly consumed fruits and derivatives in Imbabura Province, Ecuador, ready-to-eat strawberries (5 independent batches; n = 300 samples), and gooseberries (5 separate batches; n = 500 samples), purchased from a local fruit farm grower and low-cost retail market, along with 20 different natural fruit- and vegetables-based juices (3 independent batches; n = 60 samples) purchased from food courts located within the low-cost markets were analyzed. Bacteriological analysis showed that the microbial quality was lower as several indicators (n = 984) consisting of total coliforms (TCOL), total aerobes (AEROB), Enterobacter spp. (ENT), Shigella spp., (SHIGA), yeasts (YE), and molds (M) were detected. Staphylococcus spp. (STAPHY) was found in both fruits regardless of origin, while Escherichia coli (EC) isolates were found in strawberries but not gooseberries. Salmonella spp. (SALM) were detected in juices only. Antibiotic susceptibility testing showed multidrug resistance of several isolates. The hemolytic pattern revealed that 88.89% of EC and 61.11% of ENT isolates were beta-hemolytic. All STAPHY isolates were beta-hemolytic while SALM and SHIGA were alpha-hemolytic. Plasmid curing assay of MDR isolates (ENT, EC, SALM, and STAPHY) showed that the antibiotic resistance (AR) was highly indicative of being plasmid-borne. These results raise concerns about the consumption of MDR bacteria. However, good agricultural and industrial practices, behavioral change communication, and awareness-raising programs are necessary for all stakeholders along the food production and consumption supply chain.

Fortification of cocoa semi-skimmed milk formulations with native lactic acid bacteria: Cell viability, physicochemical and functional properties for developing novel foods.

This study aimed to evaluate several cocoa semi-skimmed milk formulations (CSMFs) as potential carriers of native lactic acid bacteria (LAB) strains to obtain novel probiotic beverages (PBs) with improved technological and functional characteristics, and satisfactorily organoleptic acceptance. The viability of two native LAB (Lactiplantibacillus plantarum UTNGt2 and Lactiplantibacillus pentosus UTNGt5) was assessed in comparison with two references (Lactococcus lactis subsp. lactis ATCC11474 and Limosilactobacillus reuteri DSM17938) strains in supplemented CSMFs throughout storage with refrigeration. The optimum conditions to produce novel beverages supplemented with native LAB were pH 6.6, 42°C, and 1 h of fermentation. Moreover, the effect of LAB strains fortification on pH, titratable acidity, total solids (°Brix), total polyphenolic compounds (TPC), antioxidant capacity (AOX), and ascorbic acid content (AAC), total proteins and fat, at initial and final storage was evaluated. The addition of two native LAB strains did alter the physicochemical quality of CSMFs to a lesser extent, where the bioactive molecules improved significantly (p < 0.05) with the increase of cocoa concentration and depending on the supplied strain. Although a statistically significant (p < 0.05) decrease in cell counts was recorded during storage, the LAB cells were found to be viable up to 21 days of storage at 4°C (>6 logCFU/ml), which is sufficient in number to prove their stability in vitro. Overall organoleptic results suggested that LAB supplementation had a significant impact on sensory attributes with satisfactory acceptability (>78%) of PBs containing the native strains and 1-2% cocoa, while CSMFs counterparts were less appreciated (40%) as perceived off-flavor. It appears that supplying bacteria to CSMF preserves flavor in the final product. Furthermore, the final beverages were free of harmful bacteria; thus, they comply with consumer safety regulations. This study concludes that CSMF can be used as a carrier of native LAB strains, maintaining cell viability, unaltered physicochemical properties, and improved functional and sensory characteristics, for which final beverages can be regarded as functional food. From the application standpoint, these formulations are an alternative to delivering native LAB strains and could help the cocoa and dairy industry to develop more attractive products for the growing regional market.

Probiotic Characteristics and Antimicrobial Potential of a Native Bacillus subtilis Strain Fa17.2 Rescued from Wild Bromelia sp. Flowers.

In the present study, we identified the Bacillus subtilis strain annotated Fa17.2 isolated from Bromelia flower inflorescences collected from the subtropical humid mesothermal region, Santo Domingo de Los Tsachilas Province, Ecuador. The probiotic capacity and antimicrobial potential against four foodborne pathogens were assessed. The cell culture of Fa17.2 is highly resistant to synthetic gastric acid (pH 2.5, 3.0, and 3.5), bile salts (0.3%), tolerating different sodium chloride concentrations (1, 3, and 5%), and growth conditions (15 °C and 45 °C), suggesting its potential probiotic features. The isolate showed no antibiotic resistance and was considered safe as no hemolysis was detected on sheep blood agar. The optimum medium for bacterial growth and the release of antimicrobial compounds was MRS with 10% glucose. The active components released in the neutralized crude extract (NCE) were insensitive to organic solvents, surfactants, and nonproteolytic enzymes and sensitive to proteolytic enzymes suggesting their proteinaceous nature. The antimicrobial activity was enhanced by heat and maintained active over a wide range of pH (2.0-8.0). Moreover, the crude extract (CE) showed inhibitory activity against several Gram-negative and Gram-positive bacteria. The molecular weight of partially purified precipitated bacteriocin-like substances (BLISs) was about 14 kDa in 20% Tricine-SDS-PAGE. The CE obtained from Fa17.2 inhibits the growth of four foodborne pathogens, Staphylococcus aureus, Escherichia coli, Kosaconia cowanii, and Shigella dysenteriae, which implies its potential as an antimicrobial producer strain.

Bioprospecting of Ribosomally Synthesized and Post-translationally Modified Peptides Through Genome Characterization of a Novel Probiotic Lactiplantibacillus plantarum UTNGt21A Strain: A Promising Natural Antimicrobials Factory.

The present work describes the genome sequencing and characterization of a novel Lactiplantibacillus plantarum strain assigned UTNGt21A isolated from wild Solanum quitoense (L.) fruits. In silico analysis has led to identifying a wide range of biosynthetic gene clusters (BGCs) and metabolic compounds. The genome had a total of 3,558,611 bp with GC of 43.96%, harboring 3,449 protein-coding genes, among which 3,209 were assigned by the EggNOG database, and 240 hypothetical proteins have no match in the BLASTN database. It also contains 68 tRNAs, 1 23S rRNA, 1 16S rRNA, 6 5S rRNA, and 1 tmRNA. In addition, no acquired resistance genes nor virulence and pathogenic factors were predicted, indicating that UTNGt21A is a safe strain. Three areas of interest (AOI) consisting of multiple genes encoding for bacteriocins and ABC transporters were predicted with BAGEL4, while eight secondary metabolite regions were predicted with the antiSMASH web tool. GutSMASH analysis predicted one metabolic gene cluster (MGC) type pyruvate to acetate-formate, a primary metabolite region essential for anaerobe growth. Several lanthipeptides and non-ribosomal peptide synthetase (NRPS) clusters were detected in the UTNGt21A but not the reference genomes, suggesting that their genome diversity might be linked to its niche-specific lineage and adaptation to a specific environment. Moreover, the application of a targeted genome mining tool (RiPPMiner) uncovered a diverse arsenal of important antimicrobial molecules such as lanthipeptides. Furthermore, in vitro analysis indicated that the crude extract (CE) of UTNGt21A exerted a wide spectrum of inhibition against several pathogens. The results indicated that the possible peptide-protein extract (PC) from UTNGt21A induces morphological and ultrastructural changes of Salmonella enterica subsp. enterica ATCC51741, compatible with its inhibitory potential. Genome characterization is the basis for further in vitro and in vivo studies to explore their use as antimicrobial producers or probiotic strains.

Decoding the Gene Variants of Two Native Probiotic Lactiplantibacillus plantarum Strains through Whole-Genome Resequencing: Insights into Bacterial Adaptability to Stressors and Antimicrobial Strength.

In this study, whole-genome resequencing of two native probiotic Lactiplantibacillus plantarum strains-UTNGt21A and UTNGt2-was assessed in order to identify variants and perform annotation of genes involved in bacterial adaptability to different stressors, as well as their antimicrobial strength. A total of 21,906 single-nucleotide polymorphisms (SNPs) were detected in UTNGt21A, while 17,610 were disclosed in the UTNGt2 genome. The comparative genomic analysis revealed a greater number of deletions, transversions, and transitions within the UTNGt21A genome, while a small difference in the number of insertions was detected between the strains. A divergent number of types of variant annotations were detected in both strains, and categorized in terms of low, moderate, and high modifier impact on the protein effectiveness. Although both native strains shared common specific genes involved in the stress response to the gastrointestinal environment, which may qualify as a putative probiotic (bile salt, acid, temperature, osmotic stress), they were different in their antimicrobial gene cluster organization, with UTNGt21A displaying a complex bacteriocin gene arrangement and dissimilar gene variants that might alter their defense mechanisms and overall inhibitory capacity. The genome comparison revealed 34 and 9 genomic islands (GIs) in the UTNGt21A and UTNGt2 genomes, respectively, with the overrepresentation of genes involved in defense mechanisms and carbohydrate utilization. In addition, pan-genome analysis disclosed the presence of various strain-specific genes (shell genes), suggesting a high genome variation between strains. This genome analysis illustrates that the bacteriocin signature and gene variants reflect a niche-inherent pattern. These extensive genomic datasets will guide us to understand the potential benefits of the native strains and their utility in the food or pharmaceutical sectors.

Combinations of Peptide-Protein Extracts from Native Probiotics Suppress the Growth of Multidrug-Resistant Staphylococcus aureus and Citrobacter freundii via Membrane Perturbation and Ultrastructural Changes.

The occurrence of multidrug-resistant pathogens in the food chain causes health problems in humans, thus, research for novel antimicrobials to combat their growth is of interest. This study evaluates the antimicrobial potential of several combinations of peptide-protein extracts (PCs) consisting of peptide extracts from three native probiotic strains, Lactiplantibacillus plantarum UTNGt2, Lactococcus lactis UTNGt28, and L. plantarum UTNGt21A, alone or in combination with EDTA (ethylenediaminetetraacetic acid) against multidrug-resistant Staphylococcus aureus ATCC1026 and Citrobacter freundii UTNB3Sm1. Based on the antimicrobial assay, among the 19 tested PCs, two (PC11 and PC17) produced a greater zone of inhibition against both pathogens in vitro. Time-killing assays indicated the rapid death of S. aureus after exposure to PC11 and PC17, while C. freundii was rapidly inhibited by PC11 and PC1 (UTNGt2 only), suggesting that the inhibitory action is pathogen and dose-dependent of a particular molecule present in the extract. A marginal inhibitory effect was observed when the peptides were combined with EDTA. Transmission electron microscopy (TEM) revealed the structural membrane damage of both target strains upon interaction with individual peptide extracts. Different degrees of cell deformation, condensed cytoplasm, membrane blebbing, and ghost cell formation with visibly broken cell walls were observed in S. aureus. Likewise, the separation of the cytoplasmic membrane from the outer membrane, ghost cells, along with ovoid and deformed cells with undulated cell walls were observed for C. freundii. Furthermore, scanning electronic microscopy (SEM) analysis revealed different wrinkled and deformed cells covered by debris. A leakage of aromatic molecules was detected for both pathogens, indicating that PCs disrupted the cell wall integrity, inducing cell death. Given their inhibitory action and capacity to induce damage of the cytoplasmic membrane, the selected PCs may serve to slow bacterial growth in vitro; further research is required to prove their efficiency ex vitro to battle against food poisoning and subsequent human infection.

Next-Generation Sequencing for Whole-Genome Characterization of Weissella cibaria UTNGt21O Strain Originated From Wild Solanum quitoense Lam. Fruits: An Atlas of Metabolites With Biotechnological Significance.

The whole genome of Weissella cibaria strain UTNGt21O isolated from wild fruits of Solanum quitoense (naranjilla) shrub was sequenced and annotated. The similarity proportions based on the genus level, as a result of the best hits for the entire contig, were 54.84% with Weissella, 6.45% with Leuconostoc, 3.23% with Lactococcus, and 35.48% no match. The closest genome was W. cibaria SP7 (GCF_004521965.1) with 86.21% average nucleotide identity (ANI) and 3.2% alignment coverage. The genome contains 1,867 protein-coding genes, among which 1,620 were assigned with the EggNOG database. On the basis of the results, 438 proteins were classified with unknown function from which 247 new hypothetical proteins have no match in the nucleotide Basic Local Alignment Search Tool (BLASTN) database. It also contains 78 tRNAs, six copies of 5S rRNA, one copy of 16S rRNA, one copy of 23S rRNA, and one copy of tmRNA. The W. cibaria UTNGt21O strain harbors several genes responsible for carbohydrate metabolism, cellular process, general stress responses, cofactors, and vitamins, conferring probiotic features. A pangenome analysis indicated the presence of various strain-specific genes encoded for proteins responsible for the defense mechanisms as well as gene encoded for enzymes with biotechnological value, such as penicillin acylase and folates; thus, W. cibaria exhibited high genetic diversity. The genome characterization indicated the presence of a putative CRISPR-Cas array and five prophage regions and the absence of acquired antibiotic resistance genes, virulence, and pathogenic factors; thus, UTNGt21O might be considered a safe strain. Besides, the interaction between the peptide extracts from UTNGt21O and Staphylococcus aureus results in cell death caused by the target cell integrity loss and the release of aromatic molecules from the cytoplasm. The results indicated that W. cibaria UTNGt21O can be considered a beneficial strain to be further exploited for developing novel antimicrobials and probiotic products with improved technological characteristics.

Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications.

The genome characterization of the Lactiplantibacillus plantarum strain UTNGt2, isolated from wild copoazu or white cacao (Theobroma grandiflorum), is described. A total of 31 contigs is assembled with a total length of 3,264,448 bases, with all contigs matching the core genome of different groups in the database. The genome size is 3,540,752 bases with GC content of 44.53% and the genome repeat sequences constitute around 457,386 bases of the assembly. The UTNGt2 matches the Lactiplantibacillus plantarum genome with 99% identity. The genome contains 3115 genes, 3052 protein-coding genes, assigned with the EggNOG database. On the basis of the results, 745 proteins are classified with an unknown function, from which 128 proteins have no match in the BLASTN database. It also contains 57 tRNAs, 5 copies of 5S rRNA, and 1 copy of tmRNA. Based on gene prediction and annotation results, 9.4% of proteins are involved in carbohydrate transport and metabolism and 8.46% in transcription, 2.36% are responsible for defense mechanisms, 0.5% are responsible for the biosynthesis of secondary metabolites, transport, and catabolism, while 25.11% have an unknown function. The genome revealed the presence of genes involved in riboflavin and folate production, the presence of CRISPR/Cas genes, phage sequences, the absence of acquired antibiotics resistance genes, virulence, and pathogenic factors, suggesting that UTNGt2 is a safe strain. Its highly antimicrobial capacity is related to the presence of two bacteriocin clusters (class IIc) of the sactipeptide class (contig 4) and plantaricin E class (contig 22), as detected by the BAGEL 4 webserver. Several RiPP-like peptides (non-bactericidal ribosomally produced and post-translationally modified peptides), polyketides (PKs), and terpenes were predicted. Whole-genome sequencing analysis revealed that the UTNGt2 strain has diverse bacteriocins with a high inhibitory capacity, thus it is a bacteriocinogenic strain. Considering the safety profile, UTNGt2 is a nonpathogenic, nonvirulent strain with valuable biotechnological traits and can be further exploited for its probiotic and antimicrobial potential in the food industry or as a potential producer strain of antimicrobial peptides as an alternative to conventional antibiotics.

Peptide Extracts from Native Lactic Acid Bacteria Generate Ghost Cells and Spheroplasts upon Interaction with Salmonella enterica, as Promising Food Antimicrobials.

Protecting foods from contamination applying peptides produced by lactic acid bacteria is a promising strategy to increase the food quality and safety. Interacting with the pathogen membranes might produce visible changes in shape or cell wall damage. Previously, we showed that the peptides produced by Lactobacillus plantarum UTNGt2, Lactobacillus plantarum UTNCys5-4, and Lactococcus lactis subsp. lactis UTNGt28 exhibit a broad spectrum of antibacterial activity against several foodborne pathogens in vitro. In this study, their possible mode of action against the commensal microorganism Salmonella enterica subsp. enterica ATCC51741 was investigated. The target membrane permeability was determined by detection of beta-galactosidase release from ONPG (o-nitro-phenyl-L-D-galactoside) substrate and changes in the whole protein profile indicating that the peptide extracts destroy the membrane integrity and may induce breaks in membrane proteins to some extent. The release of aromatic molecules such as DNA/RNA was detected after the interaction of Salmonella with the peptide extract. Transmission electronic microscopy (TEM) micrographs depicted at least four simultaneous secondary events after the peptide extract treatment underlying their antimicrobial actions, including morphological alterations of the membrane. Spheroplast and filament formation, vacuolation, and DNA relaxation were identified as the principal events from the Gt2 and Cys5-4 peptide extracts, while Gt28 induced the formation of ghost cells by release of cytoplasmic content, filaments, and separation of cell envelope layers. Gel retarding assays indicate that the Gt2 and Gt28 peptide extracts are clearly binding the Salmonella DNA, while Cys5-4 partially interacted with Salmonella genomic DNA. These results increased our knowledge about the inhibitory mechanism employed by several peptide extracts from native lactic acid bacteria against Salmonella. Further, we shall develop peptide-based formulation and evaluate their biocontrol effect in the food chains.

A Novel Weissella cibaria Strain UTNGt21O Isolated from Wild Solanum quitoense Fruit: Genome Sequence and Characterization of a Peptide with Highly Inhibitory Potential toward Gram-Negative Bacteria.

A novel Weissella cibaria strain UTNGt21O from the fruit of the Solanum quitoense (naranjilla) shrub produces a peptide that inhibits the growth of both Salmonella enterica subsp. enterica ATCC51741 and Escherichia coli ATCC25922 at different stages. A total of 31 contigs were assembled, with a total length of 1,924,087 bases, 20 contig hits match the core genome of different groups within Weissella, while for 11 contigs no match was found in the database. The GT content was 39.53% and the genome repeats sequences constitute around 186,760 bases of the assembly. The UTNGt21O matches the W. cibaria genome with 83% identity and no gaps (0). The sequencing data were deposited in the NCBI Database (BioProject accessions: PRJNA639289). The antibacterial activity and interaction mechanism of the peptide UTNGt21O on target bacteria were investigated by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with different concentrations (1×, 1.5× and 2× MIC) of the peptide applied alone or in combination with chelating agent ethylenediaminetetraacetic acid (EDTA) at 20 mM. The results indicated a bacteriolytic effect at both early and late target growth at 3 h of incubation and total cell death at 6 h when EDTA was co-inoculated with the peptide. Based on BAGEL 4 (Bacteriocin Genome Mining Tool) a putative bacteriocin having 33.4% sequence similarity to enterolysin A was detected within the contig 12. The interaction between the peptide UTNGt21O and the target strains caused permeability in a dose-, time- response manner, with Salmonella (3200 AU/mL) more susceptible than E. coli (6400 AU/mL). The results indicated that UTNGt21O may damage the integrity of the cell target, leading to release of cytoplasmic components followed by cell death. Differences in membrane shape changes in target cells treated with different doses of peptide were observed by transmission electronic microscopy (TEM). Spheroplasts with spherical shapes were detected in Salmonella while larger shaped spheroplasts with thicker and deformed membranes along with filamentous cells were observed in E. coli upon the treatment with the UTNGt21O peptide. These results indicate the promising potential of the putative bacteriocin released by the novel W. cibaria strain UTNGt21O to be further tested as a new antimicrobial substance.

Probiotic Potential and Technological Properties of Bacteriocinogenic Lactococcus lactis Subsp. Lactis UTNGt28 from a Native Amazonian Fruit as a Yogurt Starter Culture.

A native Lactococcus lactis subsp. lactis UTNGt28 (GenBank accession no: MG675576.1) isolated from Amazonian fruit of the tropical Caimitillo (Chrysophyllum oliviforme) tree and the commercial strain Lactococcus lactis subsp lactis ATCC11454 (LacAT) were targeted ex vitro in whole milk in combination with Streptococcus thermophilus ATCC19258 to obtain a fermented probiotic beverage. Concomitant with cell viability determination during storage (28 days), the pH, titratable acidity, syneresis, protein and fat were evaluated. The results indicated that neither UTNGt28 nor LacAT displayed a high capacity to ferment whole milk and survive during storage; a statistically significant difference (p < 0.05) in cell viability was registered for UTNGt28 compared with LacAT when inoculated alone or in combination with S. thermophilus. A principal component analysis showed a clear difference between the yogurt formulations at day 1 and 28 of storage. The PC 1 explained 46.8% of the total variance (day 28), was loaded in the negative (-) direction with titratable acidity (% lactic acid), while the PC 2 explained 22.5% (day 1) with pH. PC 1 was loaded in the positive (+) direction with pH, cell viability, syneresis, fat and protein. Overall results indicated that UTNGt28 has the technological properties for further development of a new probiotic product.

Antimicrobial Peptides from Lactobacillus plantarum UTNGt2 Prevent Harmful Bacteria Growth on Fresh Tomatoes.

In a previous study, the antimicrobial peptides extracted from Lactobacillus plantarum UTNGt2 of wild-type fruits of Theobroma grandiflorum (Amazon) were characterized. This study aimed to investigate the antimicrobial mechanisms of peptides in vitro and its protective effect on fresh tomatoes. The addition of partially purified Gt2 peptides to the E. coli suspension cells at the exponential (OD605 = 0.7) growth phase resulted in a decrease with 1.67 (log10) order of magnitude compared to the control without peptide. A marginal event (< 1 log10 difference) was recorded against Salmonella, while no effect was observed when combined with EDTA, suggesting that the presence of a chelating agent interfered with the antimicrobial activity. The Gt2 peptides disrupted the membrane of E. coli, causing the release of ß-galactosidase and leakage of DNA/RNA molecules followed by cell death, revealing a bacteriolytic mode of action. The tomatoes fruits coated with Gt2 peptides showed growth inhibition of the artificially inoculated Salmonella cocktail, demonstrating their preservative potential.

Characterization of Bacteriocin-Producing Lactic Acid Bacteria Isolated from Native Fruits of Ecuadorian Amazon.

Tropical, wild-type fruits are considered biodiverse "hotspots" of microorganisms with possible functional characteristics to be investigated. In this study, several native lactic acid bacteria (LAB) of Ecuadorian Amazon showing highly inhibitory potential were identified and characterized. Based on carbohydrate fermentation profile and 16S rRNA gene sequencing, seven strains were assigned as Lactobacillus plantarum and one strain as Weissella confusa. Using agar-well diffusion method the active synthetized components released in the neutralized and hydroxide peroxide eliminated cell-free supernatant were inhibited by proteolytic enzymes, while the activity was maintained stable after the treatment with catalase, lysozyme, α-amylase and lipase suggesting their proteinaceous nature. The inhibitory activity was stimulated by acidic conditions, upon exposure to high heat and maintained stable at different ranges of sodium chloride (4-10%). The DNA sequencing analysis confirmed the presence of plw structural gene encoding for plantacirin W in the selected L. plantarum strains. Moreover, we showed that the active peptides of Cys5-4 strains contrast effectively, in a bactericidal manner, the growth of food borne E. coli UTNEc1 and Salmonella UTNSm2, with about tree fold reduction of viable counts at the early stage of the target cell growth. The results indicated that the bacteriocin produced by selected native lactic acid bacteria strains has elevated capacity to suppress several pathogenic microorganisms implying their potential as antimicrobial agents or food preservatives.