Promises and failures of gallium as an antibacterial agent

Abstract

The inexorable rise in the incidence of antibiotic resistance in bacterial pathogens, coupled with the disappointingly low discovery rate of new and clinically useful of antibiotics, has focused the attention on search for alternatives to conventional antibiotics. Given the crucial role of iron in bacterial physiology and pathogenicity, iron metabolism has been evaluated as a possible drug target. Due to the chemical similarity between gallium and iron, gallium acts as an iron-mimetic which substitutes for iron in several enzymes, thereby perturbing bacterial metabolism. In the last years, the repurposing of existing (FDA-approved) drugs for new clinical applications has become a major research area in drug discovery. In this view, the transition metal gallium [Ga(III)], that has a long history as a diagnostic and chemotherapeutic agent, was recently repurposed as an antibacterial agent. Gallium has no proven function in biological systems, but acts as an iron-mimetic by replacing iron in redox enzymes, impairing their function and ultimately hampering cell growth. To investigate the effect of gallium on planktonic and biofilm cells, the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii were used as model organisms. Both species represent one of the most frequent cause of hospital-acquired infection (i.e., pneumonia, surgical site infection, urinary tract infection, and bloodstream infection) and are intrinsically resistance to many classes of antibiotics. Consequently, the treatment of patients infected with these pathogens has become very problematic. In the first part of the thesis, the possibility of improving the anti-P. aeruginosa potency of gallium by complex formation with a variety suitable carriers that are actively taken up by bacterium, was investigated. It was observed that Ga(III), complexed with the pyochelin siderophore, was more efficient than Ga(NO3)3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga(III) entrance into the cell through the pyochelin translocon. Since bacteremia represents a frequent complication of P. aeruginosa infections, it was also investigated whether Ga(III) would also exert its antibacterial activity in human serum. It was observed that the growth of P. aeruginosa in complement-free human serum depended on the ability of individual strains to produce proteases that cleave serum proteins, including transferrin, ultimately causing iron release and stimulation of bacterial growth. In fact, strains displaying high proteolytic activity were less susceptible than low proteolytic strains to Ga(NO3)3 in human serum. This phenomenon should be taken into account when assessing the antibacterial efficacy of Ga(NO3)3, since the ability of bacteria to retrieve iron from the host would counteract growth inhibition based on iron mimetism. The high Ga(NO3)3 concentration required to inhibit P. aeruginosa growth in serum poses a limitation to the potential of Ga(NO3)3 in the treatment of P. aeruginosa bloodstream infections, and highlights the need for appropriate in vitro condition to assess growth inhibition by an iron mimetic. The ability of A. baumannii to adhere and form biofilm on on both biotic and abiotic surface has been demonstrated and it allows to A. baumannii to survive in the hospital environment for long time. Biofilm-associated infection are very difficult to treat, because the biofilm mode of growth reduces antibiotic susceptibility as result of reduced bacterial metabolism. Taken together, these observations prompted us to investigated the anti-biofilm properties of gallium (provided as dicitrate salt, Ga-CIT2) against different A. baumannii strains. It was observed that biofilm formation was moderately inhibited by Ga-CIT2 while pre-formed biofilms were disrupted by ca. 50%. Qualitative experiments have shown that Ga-CIT2 was ineffective in disintegrating the biofilm matrix, which is mainly constituted by exopolysaccharides. Interestingly, the Ga-CIT2 concentrations required to inhibit and disrupt A. baumannii biofilm were much lower in human serum than in iron-poor laboratory medium. Although data obtained in human serum raise new hope for gallium treatment of systemic A. baumannii infections, the potential use gallium in coating of implants and medical devices should also be considered, given the propensity of A. baumannii to persist on biotic and abiotic surfaces. Physiochemical modification of the biomaterial surface to create anti-adhesive surfaces and incorporation of antimicrobial agents into medical device polymers are regarded as the last resort strategy to preserve medical device or surface contaminations that are considered hallmarks of many types of nosocomial infections In the worrisome scenario of expanding drug resistance among Gram-negative bacterial pathogens, particularly A. baumannii and P. aeruginosa, our findings on the antibacterial and antibiofilm activity of gallium could open the way to valuable therapeutic options to cure or prevent otherwise untreatable bacterial infections.