Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in Enterococcus italicus Strains of Dairy Origin Francesca Borgo ® Giovanni Ricci ® Karsten Arends ® Katarzyna Schiwon ® Elisabeth Grohmann ® Maria Grazia Fortina Received: 31 March 2009 / Accepted: 7 May 2009 / Published online: 30 May 2009 Ó Springer Science+Business Media, LLC 2009 Abstract Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 har- bouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal spe- cies was able to acquire antibiotic resistance by conjuga- tion, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid. Introduction Enterococci are one of the most controversial species of lactic acid bacteria (LAB) found in food products. They are naturally present in the gastrointestinal tract of mammals and therefore in soil and polluted waters [17, 22]. Because of their presence in these habitats, the enterococci enter the human food chain, where they can play different and in many cases controversial roles [9]. The main question related to the presence of enterococci in food is the high level of antibiotic resistance observed in these bacteria and their potential role as reservoirs and vehicles for the dis- semination of antibiotic resistance determinants via the food chain [14, 26]. In enterococci, resistance factors are often associated with mobile genetic elements, such as transposons or conjugative plasmids: Tn916, discovered in E. faecalis, Tn5233 in E. faecium, and conjugative plas- mids, such as pAMb1, pCF10, pIP501 and pRE25 [18, 24, 25]. These plasmids are also capable of mobilizing smaller plasmids and this fact may explain the dissemination of smaller plasmids carrying antibiotic resistance genes among Gram positive bacteria [12]. In previous studies, we isolated a new enterococcal spe- cies E. italicus from typical Italian cheeses [8] and investi- gated its antibiotic resistance and virulence profile [7]. We observed that E. italicus only possessed tetracycline resis- tance determinants, particularly the RP (ribosomal protec- tion proteins) genes tetS and tetM and the tetK gene encoding an EP (efflux pump protein). Interestingly, the strains tested did not contain the Tn916 conjugative transposon. Similar results have been reported for other strains of E. italicus [20]. In this study we investigated the potential of tetracycline resistance transfer of this new species, with the aim to know the real relationship between the presence of E. italicus in dairy products and food safety. F. Borgo Á G. Ricci Á M. G. Fortina (&) Department of Food Science and Microbiology, Industrial Microbiology Section, University of Milan, Via Celoria 2, 20133 Milan, Italy e-mail: grazia.fortina@unimi.it K. Arends Á K. Schiwon Á E. Grohmann Environmental Microbiology Genetics, University of Technology Berlin, Franklinstraße 29, 10587 Berlin, Germany 123 Curr Microbiol (2009) 59:261–266 DOI 10.1007/s00284-009-9428-5