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

The recent successes of probiotic application to limit colonization of food borne pathogens in the gastrointestinal tracts of food animals ensures cont inued commercialization and widespead use of such cultures. Given that the the fermentation response and ecological balance of the probiotic consortiu m appears to be essential for the effectiveness of the cultures, it is esse ntial to develop a methodology to accurately identify and quantitate these organisms during commercial production as well as successful in vivo coloni zation after administration. However, if further optimization of the effect iveness 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 colo nization are needed. Conventional methods to study individual probiotic gas trointestinal organisms include selective plating to identify specific nutr itional groups, but the requirement of strict anaerobiosis for the obligate anaerobic members of these cultures can confound sample handling and prepa ration. 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 a t the single species level. Methods are currently available that permit stu dying individual members of microbial consortia, fluxes in community divers ity, spatial distribution of consortia members, and the expression of speci fic microbial genes within communities. These methods involve the utilizati on of both DNA- and RNA-targeted probes, gene amplification protocols, and mRNA analysis. The study of mechanisms and functionality can only enhance t he potential of probiotic cultures for limiting foodborne pathogen coloniza tion.