| Summary: | We demonstrate that <i>Blautia coccoides</i> JCM1395<sup>T</sup> has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, <i>Blautia coccoides</i> JCM1395<sup>T</sup> and <i>Bacteroides vulgatus</i> JCM5826<sup>T</sup> were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that <i>Blautia coccoides</i> JCM1395<sup>T</sup> efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to <i>Bifidobacterium</i> sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.
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