Development of a Lactobacillus specific T-RFLP method to determine lactobacilli diversity in complex samples

Terminal restriction fragment length polymorphism (T-RFLP) analysis has been widely used for studying microbial communities. However, most T-RFLP assays use 16S rDNA as the target and are unable to accurately characterize a microbial subpopulation. In this study, we developed a novel T-RFLP protocol based on Lactobacillus hsp60 to rapidly characterize and compare lactobacilli composition. The theoretical terminal restriction fragment (TRF) profiles were calculated from 769 Lactobacillus hsp60 sequences from online databases. In silico digestion with restriction endonucleases AluI and TacI on hsp60 amplicons generated 83 distinct TRF patterns, of which, 70 were species specific. To validate the assay, five previously sequenced lactobacilli were cultured independently, mixed at known concentrations and subjected to analysis by T-RFLP. All five strains generated the predicted TRFs and a qualitative consistent relationship was revealed. We performed the T-RFLP protocol on fecal samples from mice fed 6 different diets (n = 4). Principal component analysis and agglomerative hierarchical clustering revealed that the lactobacilli community was strongly connected to dietary supplementation. Our study demonstrates the potential for using Lactobacillus specific T-RFLP to characterize lactobacilli communities in complex samples.