Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport

Elevated levels of mucins present in bronchiectatic airways predispose patients to bacterial infections and reduce the effectiveness of antibiotic therapies by directly inactivating antibiotics. Consequently, new antibiotics that are not inhibited by mucins are needed to treat chronic respiratory infections caused by Pseudomonas aeruginosa and Staphylococcus aureus. In these studies, we demonstrate that fosfomycin synergistically enhances the activity of tobramycin in the presence of mucin. The bactericidal killing of a novel 4:1 (wt/wt) combination of fosfomycin-tobramycin (FTI) is superior (>9 log 10 CFU/ml) relative to its individual components fosfomycin and tobramycin. Additionally, FTI has a mutation frequency resulting in an antibiotic resistance >3 log 10 lower than for fosfomycin and 4 log 10 lower than for tobramycin for P. aeruginosa. Mechanistic studies revealed that chemical adducts are not formed, suggesting that the beneficial effects of the combination are not due to molecular modification of the components. FTI displayed time-kill kinetics similar to tobramycin and killed in a concentration-dependent fashion. The bactericidal effect resulted from inhibition of protein biosynthesis rather than cell wall biosynthesis. Studies using radiolabeled antibiotics demonstrated that tobramycin uptake was energy dependent and that fosfomycin enhanced the uptake of tobramycin in P. aeruginosa in a dose-dependent manner. Lastly, mutants resistant to fosfomycin and tobramycin were auxotrophic for specific carbohydrates and amino acids, suggesting that the resistance arises from mutations in specific active transport mechanisms. Overall, these data demonstrate that fosfomycin enhances the uptake of tobramycin, resulting in increased inhibition of protein synthesis and ultimately bacterial killing.

Section: Discussionsupporting

confidence: 79%

Fosfomycin Enhances the Active Transport of Tobramycin in Pseudomonas aeruginosa

MacLeod

Velayudhan

Kenney

et al. 2012

“…This mutant appeared to be defective in streptomycin and gentamicin uptake because of reduced cytochrome oxidase activity. Two additional aminoglycoside-resistant mutants of P. aeruginosa have been described (4) in which decreased aerobic uptake of streptomycin and gentamicin were correlated with cytochrome d and nitrate reductase deficiencies and decreased terminal oxidase activity. Taber and Halfenger (18) isolated multiple aminoglycoside-resistant (mar) mutants of B. subtilis that showed cytochrome aa3 deficiencies and decreased kanamycin uptake.…”

Section: Resultsmentioning

confidence: 99%

Correlation between cytochrome aa3 concentrations and streptomycin accumulation in Bacillus subtilis

McEnroe¹,

Taber²

1984

Accumulation of aminoglycosides by Bacillus subtilis appears to require specific components of the electron transport chain. These components include cytochromes and the lipophilic quinone vitamin K2. The present study concerns the importance of cytochrome aa3, a terminal oxidase, in the uptake of streptomycin. Growth conditions have been established such that the concentration of cytochrome aa3 can be modified over a wide range; on defined minimal salts agar, the wild-type strain (RB1) and an strC mutant (RB95) synthesized cytochrome aa3 only when adequate amounts of Casamino Acids (Difco Laboratories, Detroit, Mich.) were present. A positive correlation between cytochrome aa3 levels and streptomycin accumulation was observed. The same correlation was seen when cytochrome aa3 was measured in relation to growth susceptibility. These correlations suggest that cytochrome aa3 Is necessary for accumulation of streptomycin by B. subtilis.

“…SCVs have a dysfunctional oxidative metabolism causing an alteration in the expression of virulence factors, a slow growth, and a loss of colony pigmentation (33). This dysfunctional oxidative metabolism was associated with a decreased susceptibility to aminoglycosides, because these antibiotics require the proton-motive force in order to penetrate the bacterium (8). This respiratory deficiency often is caused by mutations affecting the electron transport system, and several SCV isolates are auxotrophs for either hemin or menadione, which are needed to synthesize electron transport system components.…”

mentioning

confidence: 99%

Tomatidine Inhibits Replication of Staphylococcus aureus Small-Colony Variants in Cystic Fibrosis Airway Epithelial Cells

Mitchell

Gattuso

Grondin

et al. 2011

Small-colony variants (SCVs) often are associated with chronic Staphylococcus aureus infections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 g/ml), whereas the growth of normal S. aureus strains was not significantly altered by tomatidine (MIC, >16 g/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normal S. aureus strains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treated S. aureus at a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms of S. aureus.