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

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Terminal restriction fragment length polymorphism (T-RFLP) is a common method for microbial community analysis.
• Existing T-RFLP assays targeting 16S rDNA often lack the precision to fully characterize specific microbial subpopulations.
• Accurate characterization of specific bacterial groups, like Lactobacillus, is crucial for understanding their roles in various environments.

Purpose of the Study:

• To develop a novel T-RFLP protocol utilizing the Lactobacillus hsp60 gene for enhanced characterization of lactobacilli communities.
• To establish a species-specific T-RFLP assay for rapid and accurate identification and comparison of Lactobacillus species.
• To investigate the influence of dietary interventions on the composition of lactobacilli communities in a murine model.

Main Methods:

• In silico analysis of 769 Lactobacillus hsp60 sequences to predict theoretical terminal restriction fragment (TRF) profiles.
• In silico digestion using restriction endonucleases AluI and TacI to identify distinct TRF patterns.
• Experimental validation using cultured Lactobacillus strains at known concentrations.
• Application of the developed T-RFLP protocol to fecal samples from mice under different dietary conditions.

Main Results:

• The in silico analysis predicted 83 distinct TRF patterns from Lactobacillus hsp60 sequences, with 70 being species-specific.
• Experimental validation confirmed the accuracy of the T-RFLP assay, with all tested strains producing predicted TRFs.
• Analysis of mouse fecal samples revealed significant correlations between dietary supplementation and the composition of the lactobacilli community.
• Principal component analysis and hierarchical clustering demonstrated diet-driven shifts in lactobacilli populations.

Conclusions:

• The developed Lactobacillus hsp60-based T-RFLP protocol offers a rapid and accurate method for characterizing lactobacilli communities.
• This assay provides a valuable tool for studying the dynamics of Lactobacillus populations in complex biological samples.
• Dietary interventions can significantly modulate the composition of the lactobacilli gut microbiota, highlighting the interplay between diet and microbial ecology.