Immunomagnetic-bead enriched culturomics (IMBEC) for isolating pathobionts from feces of colorectal cancer patients

Culturomics employs various cultivating conditions to obtain different types of bacteria and new species. However, current culturomics lacks a highly efficient method for isolating specific pathobionts. Immunomagnetic bead technology, which uses magnetic beads conjugated with antibodies for capturing the antigen to realize enrichment of the targets, has been employed as an alternative method. In this study, we developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), in which magnetic bead-conjugated antibodies purified from the fecal samples of patients with colorectal cancer (CRC) were used to enrich and isolate potential pathobionts. A protocol for enriching potential pathobionts via immunomagnetic capture was developed by optimizing the concentrations of coupling reagents, NaCl, and detergent. The efficacy of pathobiont enrichment was compared between antibody-coated magnetic beads (antibody group) and nonconjugated blank magnetic beads (blank group). To determine the proinflammatory potential of isolates from both groups, we investigated their ability to induce cytokine production in THP-1 macrophages. This protocol was employed for isolating bacteria from 10 fecal samples of patients with CRC, which were simultaneously compared with those isolated from the blank group. A total of 209 bacterial species were isolated from both groups, including 173 from the antibody group, 160 from the blank group, and 124 from both groups. Bacteria isolated from the antibody group produced more proinflammatory cytokines than those isolated from the blank group. IMBEC is a promising method for relatively specific isolation of potential pathobionts for a particular disease of interest. We developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), which can obtain potential pathobionts from the precultivated colorectal cancer (CRC) fecal samples. We optimized the IMBEC process from the perspective of coupling reagents and magnetic beads washing lotion. Not only in CRC but it has also been reported that antibodies produced by pathobiont strains exist in the blood of patients with other diseases, so the method of precise isolation of disease-related strains by IMBEC is expected to be a powerful method to accurately isolate pathobiont strains.Highlightsimage We have developed a novel method called immunomagnetic bead-enriched culturomics (IMBEC) to enrich and isolate colorectal cancer-related pathobionts. This method combines immunomagnetic beads and culturomics technology.IMBEC can be targeted to isolate more bacterial species associated with cancer or inflammation, and can be accurate to the strain level.Bacterial DNA is not only detected in the blood of colorectal cancer patients but also in the serum of patients with other diseases, so IMBEC can also be employed in other diseases to isolate pathobionts.