Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

Abstract

The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ⌬fslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ⌬feoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 M gallium and 10 g/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo.F rancisella tularensis is the etiological agent of the zoonotic disease tularemia. The bacterium has the capability to infect via many different routes, and the most common clinical manifestation is through vector-borne transmission, which leads to ulceroglandular tularemia (1). Another common route is through inhalation, which leads to respiratory tularemia, the most serious form of the disease (1). There are several subspecies of F. tularensis, and of these, the two clinically important forms are F. tularensis subsp. holarctica and F. tularensis subsp. tularensis (2). The latter is the more virulent form, and in the preantibiotic era, the respiratory form had a case-fatality rate of more than 50%. F. tularensis is a facultative intracellular bacterium capable of infecting many forms of cells; however, by far the most studied interaction is between F. tularensis and monocytic cells (3). As for other intracellular bacteria, the uptake of iron is critical for the successful replication, but in this regard F. tularensis is unusual since previous studies have identified only two iron uptake systems; the ferrous iron (feo) and the ferric siderophore (fsl) systems (4). The exact roles and contributions of these systems for efficient intramacrophage replication of the highly virulent...

Development of Gallium(III) as an Antimicrobial Drug Targeting Pathophysiologic Iron Metabolism of Human Pathogens

“…They also found that gallium enhanced the effectiveness of gentamicin. 112 Boza et al examined the activity of a series of metalloporphyrins, including GaPP and several other gallium porphyrins, such as GaMP, and gallium hematoporphyrin (GaHPIX), against the sexually transmitted bacteria Neisseria gonorrhoeae and Haemophilus ducreyi. GaPP had an average minimal growth inhibitory concentration (MGIC) of 8 μg/mL while GaMP and GaHPIX had MGICs of 32 and 16 μg/mL, respectively.…”

“…129 Lindgren and Sjostedt used a combination of gentamicin and gallium citrate to treat bone marrow-derived macrophages infected with the SCHU 4 strain of F. tularensis. 112 By using them together, the compounds exerted a much greater bactericidal effect than either compound individually, seeing a 98% inhibition of growth compared to that of the control and displaying a high degree of synergism (FICI < 0.05). When mice infected with the LVS strain of F. tularensis were treated with gentamicin (0.2 mg/mouse) and/or gallium (0.6 mg/ mouse) or a PBS control, the combination-treated mice saw a drastic reduction in the number of bacteria observed in the liver compared to both the mock and singly treated mice.…”

Genetic Determinants of Antibiotic Resistance in Francisella

“…After a time for incubation (18-24 h), bacterial cells are quantified either via eukaryotic cell lysis and cell plating (if possible) or a variety of other quantification methods based upon the bacterium in question. Such an assay was performed to demonstrate the synergistic activity of gallium to gentamicin against intracellular F. tularensis SchuS4 (Lindgren and Sjostedt, 2016).This method has been performed with Francisella and shows reasonable predictive power of patient outcome when compared to clinical data (Maurin et al, 2000). A more recent method developed by Sutera et al involves a dye uptake assay to quickly and quantitatively measure the susceptibility of intracellular Francisella to extracellular antibiotics.…”

Iron and Virulence in Francisella tularensis

“…As might be predicted, growth of F. tularensis is inhibited by gallium, which competes with ferric iron for uptake and also interferes with iron-dependent biological processes (Olakanmi et al, 2010; Lindgren and Sjöstedt, 2016). Inhibition of the iron-associated enzymes catalase and superoxide dismutase leads to increased susceptibility to oxidative stress (Bakshi et al, 2006; Lindgren et al, 2007; Olakanmi et al, 2010; Binesse et al, 2015).…”