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Detection of enterocin AS-48-producing dairy enterococci by dot-blot and colony hybridization

  • EVA RODRÍGUEZ (a1), MARIA I. MARTÍNEZ (a2), MARGARITA MEDINA (a1), PABLO E. HERNÁNDEZ (a2) and JUAN M. RODRÍGUEZ (a2)...

Abstract

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.

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Detection of enterocin AS-48-producing dairy enterococci by dot-blot and colony hybridization

  • EVA RODRÍGUEZ (a1), MARIA I. MARTÍNEZ (a2), MARGARITA MEDINA (a1), PABLO E. HERNÁNDEZ (a2) and JUAN M. RODRÍGUEZ (a2)...

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