Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis

Abstract

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 μg/mL) and vancomycin (MIC = 2 μg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

Keywords: Enterococcus faecalis; Rigouta; Testouri cheese; antibiotic resistance; genomic analysis; probiotics; safety; virulence.

FIGURE 1

Dendrograms (representations of hierarchical cluster analysis) of the Tunisian dairy isolates Enterococcus faecalis OB14 and E. faecalis OB15,(A) compared to E. faecalis Symbioflor 1 DSM 16431 and (B) to the reference strains of the Biotyper library.

FIGURE 2

(A) Heat-map of Average Nucleotide Identity (ANI) between Enterococcus faecalis OB14, E. faecalis OB15 and E. faecalis Symbioflor 1 DSM 16431. ANI values (%) and coverage (between brackets) are also indicated. (B) Pairwise alignment between the reference strain Symbioflor 1 and the related strain OB15 using the MAUVE software. Colored blocks outline genome sequences that align to the other genome and are presumably homologous and internally free of genomic rearrangement (Locally Colinear Blocks or LCBs). White regions correspond to sequences that are not aligned and probably contain sequence elements specific to a genome. Blocks below the center line indicate regions that aligned in the reverse complement (inverse) orientation. The height of the profile within each LCB demonstrates the average degree of sequence conservation within an aligned region.

FIGURE 3

Hydrophobicity (A) and autoaggregation (B) of Enterococcus faecalis OB14, E. faecalis OB15 and E. faecalis Symbioflor 1 DSM 16431.

FIGURE 4

Quantification of IL-10 in the supernatants of Caco-2/TC7 cells after treatment with Enterococcus faecalis OB14, E. faecalis OB15 and E. faecalis Symbioflor 1 DSM 16431. ^∗P < 0.05 compared to Caco-2/TC7.

FIGURE 5

TER of Caco-2/TC7 cells exposed to Enterococcus faecalis OB14, E. faecalis OB15, and E. faecalis Symbioflor 1 DSM 16431. ^∗∗P < 0.01 compared to initial value.

FIGURE 6

Hemolysis assay (A) of Enterococcus faecalis OB14, E. faecalis OB15 and E. faecalis Symbioflor 1 DSM 16431 on Columbia agar containing 5% of sheep blood. Viability of Caco-2/TC7 cells (B) measured using the Neutral Red uptake assay and observed by microscopy after treatment with 10⁸ bacteria/mL of E. faecalis OB14, E. faecalis OB15 and E. faecalis Symbioflor 1 DSM 16431, compared to untreated Caco-2/TC7 cells.

FIGURE 7

Galleria mellonella survival following injection with Enterococcus faecalis OB14, E. faecalis OB15, E. faecalis Symbioflor 1 DSM 16431 or E. faecalis V19.