FIG. 4.

Inhibition of Stx2 production by factors secreted by B. thetaiotaomicron. (A) Stx2 synthesis in media conditioned with the main species of human microbiota. Stx2 concentrations were measured after 24 h of growth in conditioned media and given as a percentage of Stx2 production in the nonconditioned medium (NC). The strains used to prepare the conditioned media are as indicated: R.f, Roseburia faecis CC111; R.c, Ruminococcus callidus ATCC 27760; L.r, Lactobacillus reuteri DSM 20016; C.l, Clostridium leptum DSM 753; C.c, Clostridium coccoides DSM 935; B.sp, Bacteroides sp. strain CC134; B.b, Bifidobacterium breve IPC 6469T; and B.t, Bacteroides thetaiotaomicron VIP 5482. (B) Stx2 synthesis in B. thetaiotaomicron-conditioned medium as a percentage of Stx2 synthesis in the nonconditioned medium at 6 h and 24 h (n = 4). *, a P value of <0.05 for the unconditioned versus the conditioned medium. (C) 16S rRNA genes specific to B. thetaiotaomicron is detected in the four microbiota. Total DNA was extracted from each microbiota and from pure cultures of Bacteroides species as controls, and then 10 ng was submitted to PCR using primers specific to B. thetaiotaomicron. Lanes 1 and 12, molecular mass ladder; lanes 2 to 5, PCR from human microbiota (lane 5, microbiota from the child); lane 6, PCR from pure culture of Bacteroides eggerthii; lane 7, from pure culture of Bacteroides fragilis; lane 8, from pure culture of Bacteroides ovatus; lane 9, from pure culture of Bacteroides vulgatus; lane 10, negative control (no DNA); lane 11, from pure culture of B. thetaiotaomicron. (D) Stx2 synthesis in fractionated nonconditioned and B. thetaiotaomicron-conditioned medium at 24 h. *, a P value of <0.05 for the unconditioned versus the conditioned medium. Data represent the mean ± SEM of at least three independent experiments performed in duplicate.