Antagonistic activity of colombian yeasts against Salmonella Typhimurium and Escherichia coli O157

Description

The antimicrobial activity of 52 yeast isolates was evaluated against the pathogenic bacteria Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The hypothesis was that culture conditions of both the yeasts and the pathogenic bacteria will influence the antimicrobial activity of yeast. The yeasts were grown on modified yeast extract-malt agar (MYM) and modified yeast extract–peptone–glycerol agar (MYPG). While the enteropathogens were cultured on iron-deficient media (IDM) and Reasoner's 2A semisolid agar (R2A). At least three biological replicates consisting of 3 technical replicates were carried out for all treatments. Considering the results of the 31 yeast accessions displaying antimicrobial activity and their taxonomic identity, 8 isolates were selected for further experiments. The anti-biofilm activity was evaluated using polystyrene microplates. Cultures of enteropathogenic strains were prepared and mixed with lyophilized microbial extracts ((100 mg/mL) in the microplate wells. The stained biofilms were measured at 600 nm using a microtiter plate reader. Each treatment had eight replicates, and the experiment was repeated three times. To exhibit the presence of quorum quenching (QQ) compounds, Chromobacterium violaceum was used as biosensor. Violacein reduction was assessed by measuring optical density in liquid culture, following a protocol by Rajivgandhi et al. (2018). The supernatant was measured at 540 nm to compare violacein production with the control. Three biological replicates, each consisting of one technical replicate, were carried out. The growth of yeast under varying temperatures, pH levels, and osmotic shock was assessed using 96-well microplates. Yeast cell suspensions were prepared and added to Sabouraud dextrose broth (SDB). For temperature tests, the plates were incubated at 4, 25, 30, 35, and 40°C. For pH tests, SDB was adjusted to pH levels from 3 to 9. The osmotic shock was evaluated by supplementing SDB with glucose to create 50% and 60% solutions. Growth was measured at 405 nm after 48 hours of incubation, with experiments conducted in a completely randomized design with four replicates per treatment and repeated three times. To select the most competent yeasts for bioproduct development, data of all evaluated phenotypes were integrated into a composite index using a three-step process: (1) A univariate ANOVA was performed to evaluate yeast responses under specific conditions; (2) A factorial ANOVA was conducted to identify interactions and main effects of variables such as temperature, pH, and antimicrobial activity, followed by regression analysis to assess the impact of these conditions on yeast behavior; (3) An index was constructed using principal component analysis (PCA), selecting components that explained at least 70% of the total variance.

Steps to reproduce

Antimicrobial activity assays were performed using the spot technique as described by Acuña et al. (2017). The appearance of growth inhibition zones was considered a positive result for antimicrobial activity. Subsequently, images of the plates were captured using a Gel imaging system. Growth inhibitory zones were measured (cm2) using ImageJ v.1.52a software, and each plate's pixels/cm scale was adjusted. For taxonomic identification genomic DNA from yeast strains was extracted using a ZR Fungal/Bacterial DNA MiniPrep™ Kit, following the manufacturer’s instructions. The quality of the sequences was evaluated with the sangeranalyseR library on the R package, Subsequently, the consensus sequence for each yeast sample was generated with the same program (Chao et al., 2021). To identify the taxonomical classification of the yeasts, type material sequences were obtained from the NCBI database using BLAST (Johnson et al., 2008) and from the Mycobank database (Crous et al., 2004). Polystyrene microplates were used to evaluate the anti-biofilm activity following the methodology described by Amaya-Gómez et al. (2015). Crystal violet-stained biofilms were measured at 600 nm . One biological replicate included 8 wells per treatment. The experiment was repeated three times. Violacein reduction was measured by changes in the OD in liquid culture following the protocol described by Rajivgandhi et al. (2018). The supernatant was measured at 540 nm. The inhibition of violacein production was compared with the control treatment. Three biological replicates, each consisting of one technical replicate, were carried out. The ability of the yeast to grow under different temperatures and pH, and to withstand osmotic shock (water activity, Aw) was evaluated in 96-well microplates. Cell growth was measured at 405 nm using a Cytation3 microplate spectrophotometer. The yeast growth was determined by obtaining the average difference between the absorbance minus the blank (uninoculated medium) after 48 hours of incubation and the absorbance minus the blank at the initial time (time 0). A composite index was developed to identify the most competitive yeast isolates through a systematic process involving three main steps: 1. Univariate Analysis of Variance (ANOVA). 2) Factorial ANOVA and a regression analysis. 3) A principal component analysis was used to distill the data into a set of components that account for at least 70% of the total variance. The contribution of each variable to these key components was used as a weighting factor in a composite index. This index was then used to identify the yeast strains exhibiting the strongest biological activity and growth across the conditions tested.

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