cenocepacia K56-2 after 24 h of exposure. As shown in Fig. 2a, DHA exhibits a concentration-dependent bacteriostatic activity. Upon exposure to DHA, B. cenocepacia K56-2

cells aggregated and formed clusters (Fig. 2b). Moreover, the highest concentrations of DHA screened (50 and 100 mM) caused not only a significant growth inhibition (80–90%) but also death of B. cenocepacia K56-2 cells (8 log10-unit reduction of viable B. cenocepacia cells) (Fig. 2c). Therefore, these results indicate that DHA has a bacteriostatic/killing activity against B. cenocepacia K56-2. To further confirm the in vitro antibacterial effect of DHA (50 mM), we extended our analysis to one representative strain of each of the 17 Bcc species. In addition, see more we also

included two additional clinical isolates (J2315, AU1054) belonging to the B. cenocepacia species. Figure 3 demonstrates that although there is variation in the extent of the antibiotic effect observed, DHA significantly reduces the growth of all Bcc strains studied (40–100% inhibition). Burkholderia cenocepacia J2315, Burkholderia stabilis LMG14294 and Burkholderia anthinia AU1293 were particularly susceptible to DHA, while Burkholderia vietnamiensis PC259, Burkholderia pyrrocinia BC011 and Burkholderia lata 383 possessed the highest levels of resistance (Fig. 3). To determine whether RG7422 order the observed sensitivity/tolerance of the Bcc isolates to DHA was because of hydrophobic interactions with the bacterial cell membrane, the BATH assay was used (Rosenberg et al., 1980). As shown in Fig. 3, a direct relationship was not observed between the degree of cell surface hydrophobicity and DHA sensitivity/tolerance. The in vivo antimicrobial efficacy of DHA against B. cenocepacia was examined in a G. mellonella caterpillar model system.

To mimic a therapy with DHA, larvae were inoculated with a lethal dose of B. cenocepacia K56-2 followed by the administration of a single dose of DHA (50 mM: 190 mg kg−1), given 6 h after infection. The dose of DHA used was within the limits of dosage used in animal studies (Willumsen et al., 1993; Mizota et al., 2001). As shown in Fig. 4a, over a period of 5 days, the treatment with DHA, compared with an infected Clomifene control group, prolonged the survival of G. mellonella caterpillars (P < 0.01). Uninfected larvae were also inoculated with 50 mM of DHA, and 100% survival was observed after 5 days (Fig. 4a). We also monitored the growth of B. cenocepacia K56-2 in the hemolymph of infected larvae over a period of 24 h postinfection. We observed a reduced bacterial load (2 log10-unit reduction; P < 0.01) in treated group (administration of DHA) compared with control group (Fig. 4b). Finally, by using quantitative real-time RT–PCR, we determined the expression patterns of four immune-related G. mellonella genes encoding antimicrobial peptides at 10 and 21 h postinfection.