Evaluation of marine bacteriocinogenic enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria.

Use of lactic acid bacteria (LAB) as probiotics may provide an alternative to the use of antibiotics in aquaculture. LAB strains isolated from wild fish viscera and skin were evaluated for bacteriocin production and safety aspects (lack of antibiotic resistance, production of virulence factors). 16S rRNA gene sequences revealed the presence of Enterococcus faecium (13 isolates) and Lactococcus lactis (3 isolates) from fish samples. Pulsed-field gel electrophoresis analyses of the 13 enterococci isolates showed that they were all clustered, with greater than 95% similarity. However, RAPD analysis revealed significant molecular diversity between enterococci strains. Six enterococci strains were chosen and evaluated for their antibacterial activities. These strains produced a bacteriocin-like substance and exhibited a broad spectrum of inhibition against pathogenic bacteria isolated from diseased fish, including Streptococcus parauberis, Vagococcus spp., and Carnobacterium maltaromaticum, and in particular against the Gram-negative bacteria Flavobacterium frigidarium, Vibrio pectenicida, V. penaeicida, and Photobacterium damselae. The inhibition activity towards bacterial indicator strains was at a maximum when bacteria were grown at 37°C. However, bacteriocin production was observed at 15°C after 12 h of incubation. Only structural genes of enterocins A and B were detected by PCR in the 6 enterococci strains, suggesting the production of these enterocins. In addition, these strains did not harbor any virulence factors or any significant antibiotic resistance, and they tolerated bile. Our results suggest that enterococci are an important part of the bacterial flora of fish and that some strains have the potential to be used as probiotics.