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.

Influence of Catecholamines (Epinephrine/Norepinephrine) on Biofilm Formation and Adhesion in Pathogenic and Probiotic Strains of Enterococcus faecalis

“…Four E. faecalis strains were used in this study, two pathogenic strains: MMH594 ( Huycke et al, 1991 ) and V583 ( Sahm et al, 1989 ), isolated from blood, and two probiotic strains: OB15 isolated from Rigouta, a Tunisian traditional fermented dairy product ( Baccouri et al, 2019 ) and Symbioflor 1 DSM 16431 isolated from a stool specimen of an healthy human adult (Symbiopharm, Herborn, Germany). Bacteria were routinely grown at 37°C under static conditions in brain heart infusion (BHI).…”

“…In this context, the aim of our work was to evaluate the effect of the catecholamine stress hormones, epinephrine (Epi), and norepinephrine (NE) on some Enterococcus s trains. Four E. faecalis strains were included in this study: E. faecalis MMH594 and E. faecalis V583, pathogenic strains of clinical origin, E. faecalis Symbioflor 1 clone DSM 16431, a pharmaceutical probiotic, and E. faecalis OB15, a probiotic strain previously isolated from Tunisian rigouta ( Baccouri et al, 2019 ). Epi was found to modulate the formation of biofilm (biovolume and thickness) in E. faecalis , whether pathogens or probiotics.…”

Draft Genome Sequence of Enterococcus faecalis Strain OB15, a Probiotic Strain Recently Isolated from Tunisian Rigouta Cheese

“…Recently, we isolated E. faecalis OB15 from rigouta, a popular traditional Tunisian fermented cheese that is often prepared by boiling acidified cheese whey from cow’s milk ( 7 ). Physiological and genomic analyses demonstrated that E. faecalis OB15 has met all of the principle requirements for and has properties of an efficient probiotic and could be a reliable candidate for future use in the food or feed industry ( 7 ).…”

“…Several strains are known to be opportunistic pathogens and are recognized among the major etiological agents of nosocomial and other human infections (endocarditis and urinary tract infections), due to their ability to acquire resistance to a wide range of antibiotics and the presence of virulence determinants (2, 3). In contrast, other strains have potential benefits for human health and are currently used as starter cultures in manufacturing cheese (4, 5), as bacteriocin (enterocins) producers in food preservation, and as potential probiotics in the food industry (1, 6).Recently, we isolated E. faecalis OB15 from rigouta, a popular traditional Tunisian fermented cheese that is often prepared by boiling acidified cheese whey from cow's milk (7). Physiological and genomic analyses demonstrated that E. faecalis OB15 has met all of the principle requirements for and has properties of an efficient probiotic and could be a reliable candidate for future use in the food or feed industry (7).Genomic DNA of E. faecalis OB15 was extracted from an overnight culture in MRS broth at 37°C, under static conditions, with the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, France), following the manufacturer's recommendations.…”

Safety and Microbiological Quality

“…In addition, they produce bile salt hydrolases and are resistant to low pH, and are therefore able to stay alive until they reach the intestinal tract. However, these two Gram-positive lactic acid bacteria have been implicated in infectious diseases and a careful examination of antibiotic susceptibility, virulence, and absence of hemolysis and cytotoxicity is required before their use as starters and possibly probiotics [8][9][10].…”