Conjugal transfer to L. mesenteroides M7-1 was only obtained with pRE25, albeit at very low frequency (Table 4). Gene transfer from RE25 to L. mesenteroides M7-1 has been observed before at low frequencies (Devirgiliis et al., 2009), and so the unsuccessful transfer of pRE25* from E. faecalis to L. mesenteroides is probably due to the naturally occurring low efficiency of gene transfer between these species No transconjugants were obtained with E. faecalis Lapatinib ic50 1528, Lactobacillus fermentum ROT1, and Staphylococcus aureus VG1 as recipients (Table 1), most probably due to plasmids incompatible to pRE25 present in those strains. The comparison of pRE25* with its parental plasmid pRE25 Selumetinib solubility dmso revealed

that the inserted 2.7-kb sequence did not affect the copy number of pRE25*, nor did it have a major impact on its conjugational potential. Furthermore, both pRE25* and the gfp marker were stable, showing that E. faecalis CG110/gfp/pRE25* is suitable as a marker tool to examine horizontal ABR gene transfer

in complex microbial communities using elevated experimental durations. After construction and characterization of E. faecalis CG110/gfp/pRE25*, the tool was tested in a complex microbial background for its functionality. Fresh overnight cultures of the donor strain E. faecalis CG110/gfp/pRE25*, the recipient strain L. monocytogenes 10403S, and the transconjugant L. monocytogenes 10403S/pRE25* (Table 1) were mixed at different transconjugants to donor ratios ranging from 0.2 : 1 to 2000 : 1 in complex microbiota background. The composition of this microbiota was determined by qPCR and consistent with the main groups usually encountered in infant feces (Laboratory of Food Biotechnology, ETH Zurich, unpublished data). Subsequently, donor and transconjugants were quantified by real-time PCR and plate counts. The

ratio of pRE25* to gfp quantified by real-time PCR was plotted against the ratio calculated from plate counts and showed linear correlation coefficient (R2 of >0.99) over a pRE25*/gfp ratio of more than three orders of magnitude (Fig. 2). Furthermore, differences as crotamiton low as 0.2 transconjugants per donor were detectable by qPCR, thereby elaborating the detection limit of the method. This demonstrates that the genetic markers of E. faecalis CG110/gfp/pRE25* can be quantified in complex backgrounds by qPCR and that E. faecalis CG110/gfp/pRE25* is indeed a suitable tool for quantification of HGT. Even though new technologies, for example metagenomic sequencing, yield a deep insight into the human microbiome (Qin et al., 2010), general links between DNA sequences and their transmission route within the microbiota cannot be established using such methods, making use of tagged strains and genes insurmountable for mechanistic studies. The novel strain E.