A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa

Abstract

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

Keywords: aminoglycosides; antibiotics; iron; membranes; reactive oxygen species; redox stress; silver.

Fig 1

AGXX394 is more efficient in killing P. aeruginosa than silverdene and silver nitrate. Overnight PA14 cultures were diluted ~25-fold into MOPSg media (OD₆₀₀ = 0.1) and treated for 3 h with 25 µg/mL AgNO₃ (black square), silverdene (gray square), and AGXX394 (red square), respectively. Colony survival was evaluated every hour by serially diluting samples and plating them onto Luria-Bertani broth (LB agar. Percentage of survival was calculated relative to the untreated control (n = 4, ±SD).

Fig 2

AGXX exponentially increases the activity of aminoglycosides against P. aeruginosa. In our time-killing assay, overnight PA14 cultures were diluted ~25-fold (OD₆₀₀ = 0.1) into MHB and exposed to 75 µg/mL AGXX720C (black bars), a sublethal concentration of the indicated antibiotic (gray bar), or the combined treatment of both (red bars) for 3 h. Samples were taken every 60 min, serial diluted, plated on LB agar, and incubated for 20 h for CFU counts. Percentage of survival was calculated relative to the untreated control for: (A) 35 ng/mL ciprofloxacin and 100 ng/mL nalidixic acid, respectively; (B) 78 µg/mL carbenicillin and 0.156 µg/mL imipenem, respectively; (C) 125 µg/mL trimethoprim; (D) 1.5 µg/mL polymyxin B; and (E) 0.4 µg/mL gentamicin, 2.0 µg/ml amikacin, 1.0 µg/mL tobramycin, and 3 µg/mL streptomycin, respectively. All experiments were performed in at least three biological replicates and error bars represent mean (±SD. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test, calculated relative to cultures treated with antibiotics alone).

Fig 3

AGXX increases the sensitivity of P. aeruginosa strain PA14 to kanamycin. Overnight PA14 cultures were diluted ~25-fold into Mueller-Hinton media (OD₆₀₀ = 0.1) and either left untreated or exposed to 75 µg/mL AGXX720C, 50 µg/mL kanamycin, or the combination thereof for 3 h. Samples were taken every 60 min, serial diluted, and plated on LB agar for CFU counts (n = 4, ±S.D).

Fig 4

The combination of sublethal concentrations of AGXX and gentamicin increases ROS levels and causes DNA damage and protein aggregation. Mid-log PA14 cells were treated with sublethal concentrations of Gm (0.25 µg/mL), AGXX720C (50 µg/mL), the combination thereof, or left untreated. (A) Intracellular ROS levels were quantified by H₂DCFDA fluorescence. 50 mM thiourea was used as a ROS quencher (n = 3, ±SD). (B) Samples were serially diluted in PBS after 60 min of incubation, spot-titered onto LB agar and incubated for 20 h. One representative of three independent experiments with similar outcomes. (C and D) Cells were strained with (C) 10 µM PO1 and (D) 10 µM HPF for 60 min and fluorescence was measured via flow cytometry (n = 3, ±SD). (E) The induction of sulA (white bar) and ibpA (black bar) transcript levels was determined by qRT-PCR. Gene expression was normalized to the housekeeping gene rrsD and calculated as fold changes based on expression levels in the untreated control (n = 3, ±SD; one-way ANOVA, Dunnett’s posttest; ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Fig 5

Anaerobic growth and antioxidant systems provide protection against ROS-mediated damage caused by a combinational treatment of AGXX and aminoglycosides. (A) PA14 were diluted approximately 1,000-fold (OD₆₀₀ = 0.002) in MHB supplemented with 1% KNO₃ and grown under aerobic (black bars) and anaerobic (gray bars) conditions, respectively. At OD₆₀₀ ~0.2, cultures were treated with either 0.5 µg/mL or 1.0 µg/mL Gm alone or in combination with 75 µg/mL AGXX720C for 4 h. Cells were subsequently plated onto LB agar for 20 h at 37°C to enumerate surviving colonies (n = 3, ±SD). (B) Overnight cultures of PA14 wild-type and mutant strains with MrT7 transposon insertions in oxyR, katA, katB, and dps were diluted into MOPSg to an OD₆₀₀ = 0.01 and grown under aerobic conditions until OD₆₀₀ = 0.1. Cultures were either left untreated (gray bars) or treated with a combination of 0.25 µg/mL tobramycin and 25 µg/mL AGXX720C (white bars). Bacterial survival was quantified after 2 h by serially diluting cells in PBS and plating on LB agar for 20 h at 37°C (n = 3, ±SD; Student t-test, *P < 0.05).

Fig 6

The synergy between AGXX and aminoglycosides on PA14 killing is in parts mediated by a disruption in iron homeostasis. Overnight PA14 cultures were diluted into MOPSg and incubated under aerobic conditions until exponential phase was reached. (A) Cells were left untreated or treated with 100 µg/mL AGXX720C, 0.6 µg/mL Gm, or the combination thereof for 1 h. Aconitase activities were determined in crude extracts (n = 4, ±SD). One-way ANOVA, Dunnett’s posttest; ns = P > 0.05, ** P < 0.01. (B) Cultures were either left untreated (black square) or treated with 0.25 µg/mL Gm (grey diamond), 50 µg/mL AGXX720C (blue circle), or the combination thereof (red triangle) for 3 h. Survival was determined each hour by serially diluting samples in PBS and plating onto LB agar for overnight growth. The impact of free iron on the increased killing by AGXX/Gm cotreatments was tested by the absence (red triangle) and presence (purple diamonds) of 125 µM 2′,2′ bipyridyl (n = 3, ±SD). Student’s t-test, *P < 0.05.

Fig 7

Combined AGXX and aminoglycoside treatment induces significant membrane damage. PA14 cells grown to mid-log phase in MOPSg media were left untreated or treated with sublethal concentrations of Gm (0.25 µg/mL), AGXX720C (50 µg/mL), and the combination thereof. Cells were harvested after 1 h of treatment, washed in PBS, and stained with (A) 10 µM NPN dye and (B) 0.5 µM PI. Fluorescence intensities were determined at excitation/emission wavelengths of (A) 350/420 nm and (B) 535/617 nm, respectively (n = 4, ±SD). One-way ANOVA, Dunnett’s posttest; ns = P > 0.05, *P < 0.05, ****P < 0.0001. (C) Samples were washed in PBS, stained with PI/Syto9, incubated in the dark for 15 min at room temperature, mounted onto a glass slide with 1% agarose, and imaged at 63× magnification via inverted confocal microscopy. One representative of three independent experiments with similar outcomes.

Fig 8

AGXX increases aminoglycoside uptake and lethality through increased activity of the PMF. (A) Mid-log PA14 cells were treated with 1.0 µg/mL TR-Gm, 1.0 µg/mL TR-Gm + 50 µg/mL AGXX720C, and 1.0 µg/mL TR-Gm + 2.0 µg/mL polymyxin B (PMB) for 1 h, respectively. TR-Gm uptake was measured via flow cytometry. (B) Mid-log phase PA14 was left untreated (control) or exposed to 0.25 µg/mL Gm, 50 µg/mL AGXX720C, or 0.25 µg/mL Gm + 50 µg/mL AGXX720C for 3 h. Samples were serial diluted, plated on LB agar, and incubated for 20 h for CFU counts. To test the impact of the PMF on the killing of a combination of AGXX and aminoglycosides, PA14 were pretreated with or without 50 µM CCCP prior to AGXX/Gm exposure (n = 3, ±SD). Student’s t-test, ** P < 0.01.

Fig 9

Proposed model for the synergy between AGXX and aminoglycoside antibiotics. The antimicrobial action of AGXX is mediated by O₂ ⁻ and H₂O₂, which are generated in a redox cycle between Ag and Ru^x+1. The combination of AGXX and aminoglycosides concertedly increases endogenous ROS levels. As such anaerobic growth suppressed the synergistic interaction between the two antimicrobials at lower Gm concentrations. Potentially facilitated by a release of silver ions, the increased ROS level may disrupt iron-sulfur clusters in metabolic enzymes such as aconitase, resulting in the release of free iron, which ultimately triggers hydroxyl radical (•OH) formation in a Fenton reaction. Increasing ROS levels can inflict macromolecule damage such as DNA damage and protein aggregation, contributing to increased killing as observed for P. aeruginosa that were treated with a combination of AGXX and aminoglycosides. The synergistic effect between AGXX and aminoglycosides is also mediated by an increased uptake and the cellular accumulation of aminoglycosides, which can be attenuated by disrupting the bacterial membrane potential with ionophores such as CCCP.