a, Representative FISH staining of the ileum of mice from the indicated groups (n = 4 mice per group) to identify the bacterial 16S rRNA genes (red), co-stained with phalloidin (F-actin; green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Insets show higher magnification. Scale bars, 50 µm in the upper panels and 10 µm in the lower panels. b, Schematic drawing of the two-dimensional organoid microbiome interaction system used in this experiment. c,d, Representative scanning electron microscopy images (c) and the quantitative scoring of damaged intestinal epithelium with pores (>10 µm; epithelial pore) (d) of the intestinal epithelium after 8 h of culture of KP-P1 or KP-P5, enterohemorrhagic E. coli O157, a non-pore-forming K. pneumoniae strain JCM 1662T provided from RIKEN (KP-1662) and the PBS control. The middle and lower panels in c show each upper image at a higher magnification. Arrowheads indicate collapsed colonic monolayers. Microorganisms are indicated by the arrows. Scale bars, 20 µm in the top and middle panels and 2 µm in the lower panels. For c and d, n = 16 independent samples in the PBS group, n = 8 samples in the KP-P1, KP-P5 and KP-1662 groups and n = 24 samples in the O157 group. For d, data show the mean ± s.e.m. ****P < 0.0001 by ANOVA using Tukey’s multiple-comparison correction. e, Intestinal epithelial cells co-cultured with the indicated bacterial strain were stained with the anti-active caspase-3 antibody (green) and co-stained with phalloidin (F-actin; pink). Arrowheads represent epithelial pores, and the inset shows a higher magnification of an epithelial pore. Scale bars, 100 µm. Data are representative of four independent experiments. f, Comparative analysis of the whole genomes of the tested 13 K. pneumoniae strains revealed that 97 orthologous genes correlated with the pore-forming capacity, defined as present (orange) in more than 4 of 9 pore-forming K. pneumoniae strains (+) and defective (yellow) in all 4 non-pore-forming K. pneumoniae strains (−). Detailed information about the gene ID for each row is listed in Supplementary Table 4. ROS, reactive oxygen species; T6SS, type VI secretion system.