The enterococci have traditionally been used as
indicators of faecal contamination
because they are common inhabitants of the human and
animal intestinal tract. In
addition, some strains are well documented as opportunistic
pathogens, and have
been implicated in endocarditis, infant diarrhoea and
other conditions. However,
other strains are widespread in foods, particularly in
milk and dairy products, where
they are considered desirable microflora. In fact, through
their proteolytic and
lipolytic abilities, they play an important role in cheese
ripening, contributing to the
development of the organoleptic properties characteristic
of certain cheeses (Villani et al. 1993).Production of antimicrobial substances is one of the
mechanisms by which
microorganisms can exert a probiotic effect in a host.
In this context, a significant
number of bacteriocin-producing enterococci of dairy
origin have been isolated in
recent years (Giraffa, 1995). Production of these
antimicrobial peptides or proteins
is a common phenotype among lactic acid bacteria, and
this is the application of
enterococcal bacteriocins of special interest in dairy
systems. Firstly, they show
activity against a broad spectrum of spoilage and
pathogenic organisms of concern
in dairy industries, such as Listeria monocytogenes.
Secondly, they are inactivated by
human gastric enzymes but not by some enzymes that, like
rennet, are frequently
used in dairy plants. Finally, their marked heat stability
enables them to be used in
a wide variety of dairy products (Giraffa, 1995).The genes that encode the biosynthesis of some enterocins
or enterococcins, such
as enterocin AS-48 (Martínez-Bueno et al. 1994),
have been sequenced, allowing their
rapid detection by molecular biology techniques such as
the polymerase chain
reaction (PCR) (Joosten et al. 1997). Enterococcal
bacteriocins that have been
genetically characterized have been shown to be
plasmid-encoded (Clewell, 1993).In this paper, we report a simple method for the isolation
of plasmid DNA from
dairy enterococci, using a combination of lysozyme and
glass beads (Frère, 1994;
Reinkemeier et al. 1996) to achieve cell lysis.
Plasmid DNA was used in dot-blot and
Southern hybridization analyses to identify enterocin
AS-48-encoding dairy
enterococci by using a specific PCR-generated probe. In
addition, a more rapid
detection method based on colony hybridization was also developed.