Isolation and species delineation of genus Bifidobacterium using PCR-RFLP of partial hsp60 gene fragment

In the present study, RFLP fingerprinting of PCR amplified partial hsp60 gene fragment was applied to characterize Bifidobacterium species isolated from different sources of animal and human origin. A total of 93 isolates of genus Bifidobacterium were confirmed based on various biochemical and molecular approaches targeting transaldolase and genus specific primers. Twelve standard Bifidobacterium strains were also included in this study and RFLP pattern based validation was inferred with three different restriction enzymes (HaeIII, Hin1l and MaeII). Cluster analysis of PCR-RFLP patterns of HaeIII restriction analysis grouped all the isolates into 9 clusters. Each cluster represented a single species as compared to standard strains except one for which no placement was obtained; further characterized to be B. ruminantium. Hin1l and MaeII were not able to provide a clear species differentiation and rather grouped different species into one cluster as compared to HaeIII, while none of the patterns able to distinguish in between B. longum subsp. longum and B. longum subsp. infantis. This study indicates that PCR-RFLP of hsp60 gene fragment with HaeIII is more discriminatory and hence a suitable tool for differentiation and identification of Bifidobacterium species upto species level.