Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. Genotyping is a molecular tool widely used to study microbial diversity because it is able to fingerprinting specific DNA patterns that are characteristic for a single strain. In the present study we describe the optimization of an AFLP (Amplified Fragment Length Polymorphism) protocol in terms of primer combinations and experimental conditions to differentiate S. thermophilus strains at intraspecific level. Moreover the fingerprinting resolution of RAPD (Random Amplified Polymorphic DNA) and AFLP was evaluated and compared.
Seventy-four S. thermophilus from different collections and isolation sources, 2 Lactococcus garvieae, 1 Enterococcus faecalis, 1 Lactococcu lactis subsp. cremoris and LMG 6896 as reference strain were used in this study. Primer OPI-2MOD RAPD was choosen for RAPD characterization. The AFLP procedure was performed by means of the AFLP Microbial fingerprinting Kit (Applied Biosystem-Pe Corporation, California U.S.A.) with some modifications.
The RAPD analysis of the 78 strains studied showed 15 different clusters, for the majority constituted by 5-6 biotypes at most. The AFLP technique was optimized by using 200ng of template DNA and the preselective and selective primers combination EcoRIO/MseIO-EcoRIA/MseIA. The AFLP dendrogram was characterized by the presence of 46 clusters with 30 clusters formed by single strains that showed a unique pattern. The 4 strains belonging to other species were genetically differentiated from S. thermophilus strains only thanks to AFLP comparison.
The overall data suggest that genotypic characterization performed by AFLP, provide a better view of microbial diversity of S. thermophilus indicating that RAPD is less discriminating than AFLP. In this context the optimization of an AFLP protocol to differentiate S. thermophilus at intraspecific level could be considered a novel application and should facilitate further use of this technique to study food bacteria with technological purpose.