Acquisition and Analysis of Bacteriophage-insensitive Mutants of <i>Streptococcus thermophilus</i> Strain Isolated in Japan

<p> Bacteriophage of lactic acid bacteria often causes a problem in the industrial manufacturing of fermented dairy products such as yogurt and cheese. Phage-induced bacterial cell lysis leads to fermentation failure. One of the standard approaches to control phage infections is to prepare several starter cultures with different phage resistance and rotate them regularly. Besides, generating bacteriophage insensitive mutants (BIM) has been shown to be an effective approach against phage infections. We have been isolating and collecting lactic acid bacteria strains from environmental samples in various parts of Japan. To apply these strains as starter cultures for new fermented dairy products, it is important to take measures of minimizing the risk of phage infection in advance. In this study, we tried to generate BIMs of Streptococcus thermophilus strain isolated from raw milk in Japan and investigated the mechanism of phage resistance acquisition. We obtained a total of 12 BIMs of S. thermophilus ME-842 by co-culturing with three phage strains. By the draft genome sequence of BIMs, we found that all BIMs acquired novel spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region. In addition, we found that BIMs were resistant not only to the phage strain co-cultured, but also to other phage strains that had the same sequence in their genomes as the newly acquired spacer in the CRISPR regions of BIMs. This study provides the basic information on generating and analyzing BIMs of S. thermophilus for reducing the risk of phage infection.</p>