Carbonyl Cyanide 3-Chloro Phenyl Hydrazone (CCCP) Restores the Colistin Sensitivity in Brucella intermedia

Brucella intermedia (formerly Ochrobactrum intermedium), a non-fermentative bacterium, has been isolated from animals and human clinical specimens. It is naturally resistant to polymyxins, including colistin (CO), and may cause opportunistic infections in humans. We isolated six Brucella intermedia strains from Senegalese monkey stool. In order to determine whether an efflux pump mechanism was involved in CO resistance in B. intermedia, we evaluated the effects of verapamil (VRP), reserpine (RSP), phe-arg beta-naphthylamide dihydrochloride (PA beta N) and carbonyl cyanide 3-chloro phenyl hydrazone (CCCP), four efflux pump inhibitors, on these colistin-resistant strains. Using the broth microdilution method, a CO and CCCP combination of 2 mu g/mL and 10 mu g/mL, respectively, significantly reduced the CO minimal inhibitory concentration (MIC) of B. intermedia, supporting an efflux pump mechanism. In contrast, VRP, PA beta N and RSP did not restore CO susceptibility. A time kill assay showed a bactericidal effect of the CO-CCCP combination. Genomic analysis revealed a potential implication in the CO resistance mechanism of some conserved efflux pumps, such as YejABEF, NorM and EmrAB, as previously reported in other bacteria. An inhibitory effect of the CO-CCCP combination was observed on biofilm formation using the crystal violet method. These results suggest that the intrinsic CO resistance in Brucella intermedia is linked to an efflux pump mechanism and that the synergistic effect of CO-CCCP may open a new field to identify new treatments to restore antibiotic efficacy in humans.

Automated summary

Brucella intermedia, a non-fermentative bacterium, has intrinsic resistance to polymyxins, including colistin. Efflux pump inhibitors such as verapamil, reserpine, phe-arg beta-naphthylamide dihydrochloride, and carbonyl cyanide 3-chloro phenyl hydrazone were evaluated on colistin-resistant strains of B. intermedia, and it was found that the combination of CO and CCCP significantly reduced the colistin minimal inhibitory concentration, indicating the involvement of an efflux pump mechanism. Inhibitory effects on biofilm formation and a bactericidal effect were also observed.