ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
