, 1998). All E. coli strains were grown overnight in LB broth at 37 °C with aeration. Twenty microliters of cultures were mixed with or without 0.5% BE. The mixtures were then spread onto nematode growth media
agar plates (Hope et al., 1998). The plates were dried at 25 °C and immediately utilized for the assays. Twenty nematodes previously synchronized on the L4 stage were transferred to each plate and incubated at 25 °C. After every 24 h, live worms were scored. When the worms did not respond to being touched by a platinum wire pick, they were considered dead. Data are expressed as mean±SD. An unpaired Student’s learn more t-test was used to analyze the data. To compare differences among more than three groups, one way anova was used. A P-value of <0.05 was considered statistically significant. All the experiments were repeated for reproducibility. AI-2-mediated QS plays a major role in the virulence of E. coli O157:H7 (Sperandio et al., 2001; Sircili et al., 2004). To investigate the specific effect of the
BE on QS, we measured the level of AI-2 secreted by E. coli O157:H7 in response to the treatment with BE. When assayed using V. harveyi AI-2 reporter strain BB170, a decreasing level of AI-2 was detected in culture supernatants of E. coli O157:H7 Selumetinib cell line grown with increasing concentrations of BE. Figure 1a shows a dose-dependent decrease in AI-2 level upon treatment with BE. It is of note that AI-2 level was almost undetectable in the presence of 5% BE. AI-2 level at each treatment normalized to that obtained from growth with no BE (Fig. 1a). We then tested C. violaceum strain CV026, which produces violacein, a violet pigment, as a result of QS through its autoinducer N-hexanoyl homoserine lactone (McClean et al., 1997). Violacein production in the presence of BE was also gradually decreased in a dose-dependent manner (Fig. 1b), suggesting that BE is also capable of inhibiting QS of C. violaceum CV026. To rule out the possibility that reduced production of AI-2 is a consequence of decreased bacterial growth, we examined whether or not BE exhibited any adverse effects on bacterial growth. Figure 1c compares
the growth curves of E. coli O157:H7 during 8 h cultures in LB without or with 5% BE. In our experiments, stationary phase was achieved after ∼6 h of culture. Interleukin-2 receptor Growth of E. coli O157:H7 was elevated by the addition of BE (Fig. 1c). The bacterial culture reached OD600 nm of ∼5.0 after 6 h of growth in plain LB, whereas bacterial cell density reached OD600 nm of ∼5.7 in LB media amended with BE. Taken together, these results demonstrate that suppressed AI-2 production was not due to any secondary effects associated with retarded bacterial growth and occurred rather efficiently even at higher cell density. It has been reported that swarming motility is dependent on AI-2 signaling in E. coli O157:H7 (Sperandio et al., 2002). To test whether the reduced AI-2 synthesis by BE treatment is reflected in bacterial motility, a swarming motility assay was performed.