MONITORING OF GENETICALLY-MODIFIED LACTOCOCCUS-LACTIS IN GNOTOBIOTIC AND CONVENTIONAL RATS BY USING ANTIBIOTIC-RESISTANCE MARKERS AND SPECIFIC PROBE OR PRIMER BASED METHODS

Germ-free as well as conventional rats were dosed by proteinase-defici ent Lactococcus lactis Bu-2-60 as a potential recipient strain to moni tor colonization and lateral gene transfer. Lactococcus lactis MG1820 (pLMP1), Lactococcus lactis MG1820 (pAM beta 1), or Lactococcus lactis MG1820 (pLMP1, pAM beta 1) were applied as potential donor strains in different experiments. Lactococcus lactis MG1820 (pLMP1) contained a genetically modified proteinase gene, whereas Lactococcus lactis MG182 0 (pAM beta 1) carried the highly conjugative broad host-range plasmid pAM beta 1. Lactococcus lactis MG1820 (pLMP1, pAM beta 1) was constru cted as a potential donor for both plasmids. Faecal and intestinal sam ples were analysed for the presence of the initially introduced strain s and for lateral gene transfer of the modified proteinase gene and/or the plasmid pAM beta 1 by applying conventional antibiotic resistance based screening techniques, in situ colony hybridization with specifi c probes, and diagnostic polymerase chain reaction. The germ-free rats were readily colonized by all administered strains, whereas the conve ntional rats were colonized only transiently. Lateral transfer could n ot be observed for the modified proteinase gene. However, the plasmid pAM beta 1 was transferred to the lactococcal recipient strain and in one case to a strain or close relative of Enterococcus faecalis of the indigenous intestinal flora of a conventional rat.