Characterization of two safe Enterococcus strains producing enterocins isolated from Egyptian dairy products

Five bacterial cocci isolates were selected from a wide pool of 503 isolates collected from traditional Egyptian dairy products on the basis of their inhibitory activities against Lactobacillus brevis F145, Lactobacillus bulgaricus 340, Enterococcus faecium HKLHS, Listeria ivanovii ATCC, Listeria innocua CIP 80.11 and Listeria monocytogenes EGDe 107776. These 5 isolates were identified as E. faecium TX1330 and E. faecium E980 by 16S rDNA amplification and sequencing. The antibacterial activity of the two strains was not affected by treatment of the cell free culture supernatant with catalase but their activities disappeared completely when digested with protease K, a-chymotrypsin and trypsin. The antimicrobial substance was stable over a wide range of pH (2-10) and was active after heating at 100 C for 10 min. Bacteriocin yield in two strains reached a maximum (1,600 AU/ml) at the end of the exponential phase (6 h) and remained stable until the end of 24 h-incubation period when the medium reached pH 5.5. Maximal production of bacteriocin was obtained when growing the bacterial cells at temperatures ranging between 30 and 37 C. Bacteriocin production was unaffected when the bacterial cells grew under severe conditions of pH (9.6) and in high salt (6.5\% NaCl). Thanks to PCR gene amplification the bacteriocins produced by E. faecium TX1330 could be identified as enterocins A and B structural genes, while the bacteriocins produced by E. faecium E980 could be identified as enterocins P and L50A structural genes, which can be classified into two enterocin subclasses (IIa and IIc), respectively. PCR amplification demonstrated that the two studied strains did not contain virulence factors asal, cyl A and B, ace, efaAfs and espfm. These two strains were sensitive to most of the tested antibiotics but were resistant to tetracycline. E. faecium E980 was also resistant to chloramphenicol.