Antibiotic resistance has been increasingly described among bacterial species including oral microorganisms. Despite the magnitude of this problem there is still a critical gap in the knowledge base that centers on the frequency and mechanism of spread of resistance genes in microbial biofilms. Resistance genes are often carried on mobile genetic elements capable of interspecies transmission. Conjugation, i.e. the transfer of mobile elements by direct cell-cell contact is assumed to be the most effective mechanism of genetic transfer. Our long-term goal is to monitor the potential, the extent, and the dynamics of conjugative gene transfer in oral biofilms in situ. Current methods applied to the detection of gene transfer are either limited to the culturable portion of the biofilm and/or require the use of a combination of genetic and molecular techniques. These procedures are tedious and of limited use when assessing conjugative gene transfer in dental biofilms. The objective of this application is to design a simple broad-host-range genetic tool based on a DNA inversion system that will allow for the non-intrusive visual tracking of mobile genetic elements in multispecies biofilms. This tool will be applicable to the in situ study of conjugal gene transfer between Gram-negative and Gram-positive bacteria. The objective of this application will be accomplished by pursuing the following two aims: i) The construction of the genetic tool and its insertion into broad-hostrange mobile DNA elements, and ii) the application of this tool to monitor conjugative transfer in an oral biofilm model. The availability of this new genetic tool will facilitate future assessment on the true level of conjugative transfer in situ and its associated increase in antibiotic resistance. Such information could potentially aid in developing strategies that target conjugation to minimize the spread and break the resistance cycle. ? ? ?
