Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain.

Enterococcus faecalis UGRA10, a new AS-48-producer strain, has been isolated from a Spanish sheep's cheese. The inhibitory substance produced by E. faecalis UGRA10 was purified and characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirming its identity with AS-48 enterocin (7.150 Da). Subsequent genetic analysis showed the existence of the as-48 gene cluster on a plasmid of approximately 70-kb. The UGRA10 strain was examined for safety properties such as enterococci virulence genes, biogenic amine production, and antibiotic resistance. As for most E. faecalis strains, PCR amplification revealed the existence of gene encoding for GelE, Asa1, Esp, EfaA, and Ace antigens and for tyrosine decarboxylase. This strain was sensitive to most of the antibiotics tested, being resistant only to aminoglycosides, lincosamide, and pristinamicins. In addition, UGRA10 developed an ability to form biofilms and to adhere to Caco 2 and HeLa 229 cells. More interestingly, this strain shows a high ability to interfere with the adhesion of Listeria monocytogenes to Caco 2 cells. Altogether, the results suggest that this broad-spectrum bacteriocin-producing strain has biotechnological potential to be developed as a protective agent in food preservation and as a probiotic.

Safety and potential risks of enterococci isolated from traditional fermented capers.

A collection of 17 enterococci isolates obtained from fermentations of capers (the fruits of Capparis sp.) were investigated for incidence of known virulence determinants, antibiotic resistance and production of biogenic amines. Molecular identification revealed the presence of Enterococcus faecium (nine isolates), Enterococcus faecalis (4), E. avium (3) and Enterococcus casseliflavus/flavescens (1). Alpha-haemolytic activity was detected in two E. avium and one E. faecalis isolates, and beta-haemolytic activity was detected in E. casseliflavus/flavescens. The haemolytic component cylB was detected by PCR amplification in three non-haemolytic isolates and in E. casseliflavus/flavescens. The collagen adhesin ace gene and the endocarditis associated antigen gene efaA(fm) were detected in two isolates each. Genes encoding sex pheromone precursors (cpd, cob, ccf) were detected in E. faecalis and E. casseliflavus/flavescens. Other presumed virulence genes (agg, gelE, cylM, cylA and efaA(fs)) were not detected. All isolates were resistant to rifampicin, erythromycin and ciprofloxacin, and some were also resistant to quinupristin/dalfopristin, tetracycline, levofloxacin, gentamicin and streptomycin. Vancomycin resistance was not detected. Tyrosine decarboxylation was detected in all E. faecium isolates. Given the high resistance of enterococci to environmental conditions, and their implication in opportunistic infections, the incidence of potential virulent enterococci in foods (especially those of a higher risk-like home-made foods) should be carefully studied.

Characterization of functional, safety, and gut survival related characteristics of Lactobacillus strains isolated from farmhouse goat's milk cheeses.

A set of 80 Lactobacillus strains (36 Lactobacillus plantarum and 44 Lactobacillus paracasei) isolated from Spanish farmhouse cheeses have been studied as to their functional and safety properties and their survival under gut-related conditions. None of these 80 Lactobacillus strains were able to hydrolyse starch. A high percentage of L. plantarum and L. paracasei strains were, however, capable of hydrolysing casein (86.1% and 68.2% respectively). For the other characteristics investigated, L. plantarum strains generally had more positive responses than L. paracasei. The latter strains tested negative for most of these characteristics, with the exception of stachyose hydrolysis, which was positive in six strains of L. paracasei. A high percentage (91.7%) of L. plantarum produced haemo-dependent catalase. Phytase was present in 10 L. plantarum and in 2 L. paracasei. Most L. plantarum (83.3%) but no L. paracasei hydrolysed bile salts. All strains were completely resistant to a challenge of pH3, but many showed a loss of viability after a subsequent exposure to 0.3% oxgall; in fact, only one L. paracasei strain and 33 L. plantarum strains (91.67%) were tolerant to both stresses. L. plantarum Mb25 and L. plantarum Mb26 were the most adherent to Caco-2 cells (adherence percentages of 36 and 7% respectively). These two strains were also the most adherent to HeLa 229 cells, with 19.3 and 16.0% adhesion respectively. The Mb26 strain inhibited the adhesion of Listeria monocytogenes to Caco-2 cells when added simultaneously to Listeria and also when added 1h before the pathogen (21.0% and 51.6% adhesion inhibition, respectively). Production of H2O2 was detected in 38.9% of L. plantarum strains and in 9.1% of L. paracasei. Twelve L. plantarum and eight L. paracasei strains produced bacteriocin-like inhibitors. PCR amplifications of several plantaricin genes suggest that all the bacteriocinogenic strains may produce plantaricin E/F and some may also manufacture the plantaricin J/K. The nine L. plantarum strains assayed for antibiotic resistance were resistant to ciprofloxacin (MIC>2 µg/ml), vancomycin (MIC>16 µg/ml), and teicoplanin (MIC>16 µg/ml). Moreover, some strains showed intermediate resistance to penicillin, tetracycline, rifampicin, and levofloxacin. We conclude that farmhouse cheeses are good sources of biotechnologically relevant lactobacilli and that the L. plantarum species shows better biotechnological properties than L. paracasei. This can be deduced from the finding of a high percentage of strains of L. plantarum that exhibit remarkable functional and inhibitory properties and high abilities to survive in gut-related conditions, which can be further developed for biotechnological applications.