A fast PCR-based method for the characterization of prophage profiles in strains of the Lactobacillus casei group

Lysogeny is widespread among Lactobacillus strains of the casei group (L. casei, L. paracasei and L. rhamnosus), and prophages account for most strain-specific DNA. Numerous PCR based methods have been developed to detect free phages of lactic acid bacteria, but they do not take in consideration prophages. In this study, a new PCR method for the detection of lysogeny was developed using genome sequences of L. casei group strains (including BL23) and bacteriophages. Nine pairs of primers were designed to selectively amplify the highly conserved prophage iA2 (pairs #1-#3) and fragments of two groups phages of temperate origin: C_L1/C_L2/iLp1308/iLp84 (pairs #4 and #5) and Lrm1/J-1/PL-1/A2/AT3/Lc-Nu (pairs #6 to #9). Forty-nine strains of the casei group were subjected to PCR. Strains containing remnants of lytic phages outnumbered those containing iA2-related prophages. The combination of pair #2, annealing on the terminase large subunit (TLS), and pair #3, annealing on the helicase (forward) and a non-coding region (reverse), showed the best diagnostic performance for iA2-like prophages. For the assessment of remnants of phages C_L1/C_L2/iLp1308/iLp84, pair #4 (annealing on the TLS) was preferred over pair #5 (portal protein). Detection of phages Lrm1/J-1/PL-1/A2/AT3/Lc-Nu was optimal with primers of pair #6, designed on non-coding regions of phage genomes; pair #6 also evidenced a high conservation of certain prophage remnants. Overall, our PCR-based method successfully detected and discriminated groups of prophages or remnants in L. casei group strains.