Conventional and molecular methods for understanding probiotic bacteria functionality in gastrointestinal tracts

The recent successes of probiotic application to limit colonization of foodborne pathogens in the gastrointestinal tracts of food animals ensures continued commercialization and widespead use of such cultures. Given that the the fermentation response and ecological balance of the probiotic consortium appears to be essential for the effectiveness of the cultures, it is essential to develop a methodology to accurately identify and quantitate these organisms during commercial production as well as successful in vivo colonization after administration. However, if further optimization of the effectiveness of defined cultures is to be achieved, methods to assess expression of key metabolic processes occurring during establishment of the probiotic culture as well as its subsequent ability to limit foodborne pathogen colonization are needed. Conventional methods to study individual probiotic gastrointestinal organisms include selective plating to identify specific nutritional groups, but the requirement of strict anaerobiosis for the obligate anaerobic members of these cultures can confound sample handling and preparation. Immunological methods can circumvent some of these problems but are somewhat limited for assessing functionality. The main advantage of using molecular tools is that the genetic diversity of the microflora, as well as their gene activity data are obtainable, both at the community level and at the single species level. Methods are currently available that permit studying individual members of microbial consortia, fluxes in community diversity, spatial distribution of consortia members, and the expression of specific microbial genes within communities. These methods involve the utilization of both DNA- and RNA-targeted probes, gene amplification protocols, and mRNA analysis. The study of mechanisms and functionality can only enhance the potential of probiotic cultures for limiting foodborne pathogen colonization.