Effect of iron chelation on anti-pseudomonal activity of doxycycline.

BACKGROUND: Increasing resistance of microorganisms to antimicrobial agents is a growing concern and there is a lack of novel agents. This has stimulated the exploration of novel strategies for treatment of infection. OBJECTIVE: To investigate synergistic interactions between five tetracyclines and tobramycin with an iron chelator (CP762) against two reference strains and nine clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients. METHOD: Microdilution assays for minimal inhibitory concentration determination and checkerboard assays were used to assess synergy between antibiotics and CP762. Given the iron-binding capacity of tetracyclines, the binding of iron with doxycycline was investigated using Job's plot methodology. Synergy between the iron-bound form of doxycycline and CP762 was compared with that of unbound doxycycline and CP762. Enhancement of doxycycline anti-biofilm activity was also assessed. RESULTS: There was synergy between CP762 and all tetracyclines, except minocycline, against the reference strains but that against clinical isolates was variable. Synergy was not demonstrated for tobramycin against any of the strains tested. This led to the hypothesis that iron chelation preserves the binding of tetracyclines to the bacterial ribosome. Susceptibility to iron-bound doxycycline was decreased by two- to four-fold and synergistic interactions with the iron chelator were consistently more intense with iron-bound doxycycline than with doxycycline alone. The doxycycline-iron chelator combination also significantly reduced cell viability in established biofilms. CONCLUSION: The data in this study provide evidence that iron chelation enhances the anti-pseudomonal activity of tetracyclines, specifically doxycycline.

The small quinolone derived compound HT61 enhances the effect of tobramycin against Pseudomonas aeruginosa in vitro and in vivo.

HT61 is a small quinolone-derived compound previously demonstrated to exhibit bactericidal activity against gram-positive bacteria including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). When combined with the classical antibiotics and antiseptics neomycin, gentamicin, mupirocin and chlorhexidine, HT61 demonstrated synergistic bactericidal activity against both MSSA and MRSA infections in vitro. In this study, we investigated the individual antimicrobial activity of HT61 alongside its capability to potentiate the efficacy of tobramycin against both a tobramycin sensitive laboratory reference strain (PAO1) and tobramycin resistant clinical isolates (RP73, NN2) of the gram-negative bacteria Pseudomonas aeruginosa (P. aeruginosa). Using broth microdilution methods, the MICs of HT61 were assessed against all strains, as well as the effect of HT61 in combination with tobramycin using both the chequerboard method and bacterial time-kill assays. A murine model of pulmonary infection was also used to evaluate the combination therapy of tobramycin and HT61 in vivo. In these studies, we demonstrated significant synergism between HT61 and tobramycin against the tobramycin resistant P. aeruginosa strains RP73 and NN2, whilst an additive/intermediate effect was observed for P. aeruginosa strain PA01 which was further confirmed using bacterial time kill analysis. In addition, the enhancement of tobramycin by HT61 was also evident in in vitro assays of biofilm eradication. Finally, in vivo studies revealed analogous effects to those observed in vitro with HT61 significantly reducing bacterial load when administered in combination with tobramycin against each of the three P. aeruginosa strains at the highest tested dose (10 mg/kg).