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In a previous study, the authors isolated lactic acid bacteria from breast milk of healthy mothers. Since some of the identified isolates belonged to the species Enterococcus faecium, the objective of this work was to evaluate their safety. The enterococcal strains were screened by polymerase chain reaction (PCR) and Southern hybridization for the presence of virulence determinants. The potential of the strains to acquire plasmids by conjugation was investigated by screening for genes involved in conjugation processes. Parallel, phenotypic assays were performed. Presence of genes conferring resistance to vancomycin was assessed by PCR. PCR amplifications and Southern hybridizations revealed that all the strains were clear of the majority of potential virulence determinants. None of the strains showed gelatinase activity, hemolysin production, or aggregation phenotype, and none carried the vanA or vanB genes. These findings suggest that milk of healthy mothers may be a source of avirulent E faecium isolates to the newborns.

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Food-grade heterologous production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains, previously selected because of their technological properties for cheese making, was achieved. Plasmid pGA1, which contains the complete pediocin operon under the control of the strong P32 promoter and is devoid of any antibiotic marker, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). Transformation of L. lactis ESI 515 with pGA1 did not affect its ability to produce nisin. The antimicrobial activity of the pediocin-producing transformants on the survival of Listeria innocua SA1 during cheese ripening was also investigated. Cheeses were manufactured from milk inoculated with 1% of the lactic culture and with or without approximately 4 log CFU/ml of the Listeria strain. L. lactis ESI 153, L. lactis ESI 515, and their transformants (L. lactis GA1 and GA2, respectively) were used as starter cultures. At the end of the ripening period, counts of L. innocua in cheeses made with the bacteriocin-producing lactococcal strains were below 50 CFU/g in the L. lactis GA1 cheeses and below 25 CFU/g in the L. lactis GA2 ones, compared with 3.7 million CFU/g for the controls without nisin or pediocin production.

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A novel method for genetic labelling of specific lactic acid bacteria strains was developed. The approach implied the transformation of the hosts with a plasmid containing a heterologous DNA fragment. The sequence of a DNA fragment that has been used to label a variety of genetically modified (GM) soya was used to design a forward primer and three reverse primers yielding PCR products recognisable by their sizes. Stability of the recombinant plasmid in the transformed strains was studied by PCR, and the results varied significantly depending on the strain. To test the usefulness of the DNA label to study in vivo properties of probiotic bacteria, such as viability after transit through the digestive tract, mice were orally inoculated with a genetically-labelled Enterococcus faecium strain. Later, their faeces were aseptically collected and the genetically-labelled strain was detected among the colonies that grew on MRS agar.

In this work, heterologous production of pediocin PA-1 in Lactococcus lactis ESI 153 and ESI 515 (Nis + ), two strains selected because of their technological properties for cheesemaking, was achieved after transformation with plasmids pMC117, pRK119 and pCNC1, which contain the complete pediocin operon under the control of the strong P32 promoter. The pediocin production of the L. lactis ESI 153 derivatives containing pRK119 or pCNC1 was higher (approximately 165%) than that achieved by the natural pediocin PA-1 producer Pediococcus acidilactici 347. In the case of the L. lactis ESI 515 derivatives, those containing pRK119 or pCNC1 showed a pediocin production level similar (95-100%) to that of P. acidilactici 347.