Untargeted Metabolomics for Unraveling the Metabolic Changes in Planktonic and Sessile Cells of Salmonella Enteritidis ATCC 13076 after Treatment with Lippia origanoides Essential Oil

Abstract

Nontyphoidal Salmonella species are one of the main bacterial causes of foodborne diseases, causing a public health problem. In addition, the ability to form biofilms, multiresistance to traditional drugs, and the absence of effective therapies against these microorganisms are some of the principal reasons for the increase in bacterial diseases. In this study, the anti-biofilm activity of twenty essential oils (EOs) on Salmonella enterica serovar Enteritidis ATCC 13076 was evaluated, as well as the metabolic changes caused by Lippia origanoides thymol chemotype EO (LOT-II) on planktonic and sessile cells. The anti-biofilm effect was evaluated by the crystal violet staining method, and cell viability was evaluated through the XTT method. The effect of EOs was observed by scanning electron microscopy (SEM) analysis. Untargeted metabolomics analyses were conducted to determine the effect of LOT-II EO on the cellular metabolome. LOT-II EO inhibited S. Enteritidis biofilm formation by more than 60%, without decreasing metabolic activity. Metabolic profile analysis identified changes in the modulation of metabolites in planktonic and sessile cells after LOT-II EO treatment. These changes showed alterations in different metabolic pathways, mainly in central carbon metabolism and nucleotide and amino acid metabolism. Finally, the possible mechanism of action of L. origanoides EO is proposed based on a metabolomics approach. Further studies are required to advance at the molecular level on the cellular targets affected by EOs, which are promising natural products for developing new therapeutic agents against Salmonella sp. strains.

Keywords: Salmonella; antimicrobial agent; biofilm; essential oil; metabolomics; microbial resistance; planktonic cells.

Figure 1

Effect of cell viability of sessile cells of S. Enteritidis when treated with different concentrations of essential oils. Data are expressed as mean ± SD (n = 3). (* p < 0.01, ** p < 0.001, *** p < 0.0001).

Figure 2

Micrographs of biofilm of S. Enteritidis on ground glass coupons obtained by scanning electron microscopy (SEM). (a,b) Non-treated biofilm of S. Enteritidis (control). (c,d) Biofilm of S. Enteritidis treated with subinhibitory concentrations of LOT-II EO. Arrows highlight the areas of the extracellular matrix.

Figure 3

Release of intracellular material from S. Enteritidis treated with different EO concentrations. (a) Protein release concentration expressed in µg/mL. (b) Nucleic acid release measured at 260 nm. Data are expressed as the mean ± SD (n = 3). (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Scoring plots representing PCA and PLS-DA results based on LC/MS data on planktonic cells of S. Enteritidis with and without treatment with LOT-II EO. (a,b) PCA with data obtained from negative and positive ion acquisition modes, respectively. (c,d) PLS-DA with data obtained from negative and positive ion acquisition modes, respectively. Control (red color) and treated cells (blue color).

Figure 5

Heatmap plot of the most representative metabolites found to be affected on bacterial cells after treatment with LOT-II EO on (a) planktonic and (b) sessile cells. Row: metabolites; columns: samples.

Figure 6

Possible metabolic pathways altered in planktonic cells of S. Enteritidis after LOT-II EO treatment. Metabolites colored blue and red indicate up- and downmodulated, respectively, in treated cell samples.

Figure 7

Scoring plots representing PCA and PLS-DA results based on LC/MS data of sessile cells of S. Enteritidis cells with and without treatment with LOT-II EO. (a,b) PCA with data obtained from negative and positive ion acquisition modes, respectively. (c,d) PLS-DA with data obtained by negative and positive ion acquisition modes, respectively. Control (red color) and treated cells (blue color).

Figure 8

Possible metabolic pathways altered in sessile cells of S. Enteritidis after LOT-II EO treatment. Metabolites colored blue and red indicate up- and downmodulated metabolites, respectively, on treated samples.