Research ArticleANTIBIOTICS

Potentiating antibiotics in drug-resistant clinical isolates via stimuli-activated superoxide generation

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Science Advances  04 Oct 2017:
Vol. 3, no. 10, e1701776
DOI: 10.1126/sciadv.1701776
  • Fig. 1 Light-activated QDs engineered to produce superoxide in MDR isolates.

    (A) MDR clinical isolates for this study demonstrated high resistance to different classes of antibiotics. Sensitive (blue line)/resistant (red line) breakpoint minimum inhibitory concentrations; filled diamonds are biological replicates, and the open diamond represents replicate average. (B) Schematic showing MDR bacteria inhibited with previously ineffective concentrations of antibiotics, with superoxide-producing CdTe-2.4 potentiation of antibiotic activity. (C) Superoxide or hydroxyl adducts identified and measured by EPR in (C) and (D) (left). Confirmation of superoxide production from CdTe-2.4 by signal quenching upon the addition of SOD (middle). Hydroxyl signal is observed upon the addition of iron as Fenton chemistry occurs in solution. Production of superoxide by CdTe-2.4 and dismutation to hydroxyl as a function of time (right). (D) Concentration dependence of ROS production from CdTe-2.4. Dark CdTe-2.4 spectra are subtracted for (C) and (D). (E and F) Evidence of superoxide production by CdTe-2.4 in vitro. Overexpression (left) or deletion (right) of sodB in E. coli reduced or increased the inhibitory effect of CdTe-2.4, respectively, compared to control or wild-type (WT) strain, shown as normalized optical density at 24 hours (E). Corresponding growth curves are shown in (F). no trt, no treatment. (G) Micrographs of MDR bacterial clinical isolates with 100 nM CdTe-2.4 in light or dark and treated with DCFH-DA demonstrating light-activated ROS intracellularly. *P < 0.05.

  • Fig. 2 QDs potentiate bactericidal and bacteriostatic antibiotic activity and lower antibiotic GIC50 values.

    (A) Growth curves of strains under respective treatments demonstrating increased growth inhibition upon combination of antibiotic and CdTe-2.4. (B) Evaluation of CdTe-2.4 and antibiotic synergistic interaction using the Bliss Independence model. S > 0 (red scale) indicates a synergistic interaction, where S >> 0 is the higher deviation from no interaction between treatments. S < 0 indicates antagonistic interaction (gray scale). CLI, clindamycin; CIP, ciprofloxacin; CHL, chloramphenicol; STR, streptomycin. (C) Histogram of S values for all combinations of antibiotic and CdTe-2.4 across all clinical isolates tried in this investigation (n = 271; left). The S value distribution average is significantly higher than 0 (P < 0.05; right-tailed t test). Demonstration of increased potentiation of antibiotic activity with increasing CdTe-2.4 concentration (right). At constant antibiotic concentration, the addition of greater CdTe-2.4 increases the S value, indicating a more synergistic relationship. S values shown in (B) and (C) are the average of three biological replicates. (D) GIC50 of respective antibiotic with the addition of CdTe-2.4 at various concentrations. The addition of CdTe-2.4 potentiates the activity of antibiotics to allow for successful inhibition of 50% or greater at or below senstive (blue line)/resistant (red line) breakpoint values. The effect is seen as a sharp decrease in GIC50 corresponding with the increased addition of CdTe-2.4.

  • Fig. 3 Increased inhibition of bacteria in infection models with the addition of stimuli-activated superoxide.

    (A) Micrographs of uninfected and Salmonella Typhimurium–infected HeLa cells [composite images, red, is MitoTracker (mitochondrial voltage indicator); blue, DAPI for nuclei; green, GFP-expressing SL1344 Salmonella Typhimurium]. (B) Effect of monotherapies on Salmonella Typhimurium load (CFU per milliliter) (top axis, CdTe-2.4; bottom axis, ciprofloxacin) in Salmonella Typhimurium–infected HeLa cells. (C) Addition of CdTe-2.4 to ciprofloxacin treatment significantly reduces intracellular Salmonella Typhimurium (CFU per milliliter) compared to antibiotic alone. (D) Reduction in Salmonella Typhimurium CFU in HeLa cells as a function of adding CdTe-2.4 in the presence of constant ciprofloxacin concentration. (E) Effect of monotherapies on MRSA load (CFU per milliliter) (top axis, CdTe-2.4; bottom axis, chloramphenicol) in MRSA-infected HeLa cells. (F) Addition of CdTe-2.4 to chloramphenicol treatment significantly reduces MRSA compared to antibiotic alone. (G) Heat map with log-scale coloring showing the effect of CdTe-2.4 and chloramphenicol combination on MRSA CFU per milliliter in HeLa infection. For (B) to (G), CFU per milliliter data shown are the average of three biological replicates and are represented and analyzed as normalized to no treatment (fig. S16). (H) SYTOX orange viability stain used to determine live and dead C. elegans. (I) Survival of C. elegans infected with Salmonella Enteritidis with mono- and combinatorial therapy. The percent survival of C. elegans with combination therapy is higher than that with monotherapy and no treatment. n = 2 biological replicates composed of >28 nematodes per condition per biological replicate. *P < 0.05 compared to no treatment, antibiotic, and CdTe-2.4 only.

  • Fig. 4 Modeling skin depth for effective QD antibiotic potentiation.

    (A) In vivo absorption coefficients for human epidermis and dermis (left) (31) and schematic of the external light illumination penetrating the two layers of skin (right). Our model assumes an epidermis depth of 0.007 cm, a 520-nm LED light for skin illumination, a concentration of 160 nM CdTe-2.4, and well-dispersed CdTe-2.4 and antibiotic. (B) Isosurfaces of GIC50 (red) and GIC75 (blue) for respective antibiotics and clinical isolates with predicted skin depths demonstrating bacterial growth inhibition as a function of skin depth. (C) Calculated or extrapolated skin depth (centimeters), using an exponential fit, where GIC50 or GIC90 inhibition occurs for respective clinical isolates and respective antibiotics at CLSI or lower levels (CRE E. coli/chloramphenicol at CLSI/8, ESBL KPN/streptomycin at CLSI/4, ESBL KPN/chloramphenicol at CLSI/8, MDR STm/chloramphenicol at CLSI/64, MDR STm/ciprofloxacin at CLSI/8, and MDR E. coli/chloramphenicol at CLSI/64).

  • Table 1 Antibiotic MIC and GIC50 (micrograms per milliliter) for respective strains and antibiotics.
    Clinical strainCeftriaxoneChloramphenicolClindamycinStreptomycinCiprofloxacin
    MICGIC50MICGIC50MICGIC50MICGIC50MICGIC50
    CRE E. coli>512512–1024>25632–64>648–64>256256–512>3216–32
    ESBL KPN>16,3842048–4096>2568–16>1616–64>6432–64>6432–64
    MDR STm>16,3841–4>82>264–128>832–641<0.125
    MDR E. coli>16,3848192–16,38484–8>25632–128>64128–512>3232

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/10/e1701776/DC1

    Supplementary Discussion

    fig. S1. QD characterization and EPR analysis.

    fig. S2. Growth curves for SodA and SodC deletion and SodA overexpression constructs and DCFH-DA controls.

    fig. S3. Chloramphenicol GIC50.

    fig. S4. Streptomycin GIC50.

    fig. S5. Ciprofloxacin GIC50.

    fig. S6. Clindamycin GIC50.

    fig. S7. Ceftriaxone GIC50.

    fig. S8. Growth curve of clinical strains subjected to treatment with different concentrations of streptomycin and CdTe-2.4.

    fig. S9. Growth curve of clinical strains subjected to treatment with different concentrations of ciprofloxacin and CdTe-2.4.

    fig. S10. Growth curve of clinical strains subjected to treatment with different concentrations of clindamycin and CdTe-2.4.

    fig. S11. Growth curve of clinical strains subjected to treatment with different concentrations of chloramphenicol and CdTe-2.4.

    fig. S12. Growth curve of clinical strains subjected to treatment with different concentrations of ceftriaxone and CdTe-2.4.

    fig. S13. Effect of antibiotics in combination with CdTe-2.4.

    fig. S14. S parameter heat maps for combinations of CdTe-2.4 and antibiotics.

    fig. S15. LDH assay results for HeLa cells under CdTe-2.4 treatment and MitoTracker staining.

    fig. S16. CFU per milliliter data for HeLa infection assay.

    fig. S17. Clinical strain screen for pathogen of C. elegans.

    fig. S18. Isosurfaces for respective antibiotic and clinical strain based on optical density at 8 hours normalized to no treatment.

    fig. S19. Inhibition of clinical isolates with CdTe-2.4 with varied light intensity.

    fig. S20. CdTe-2.4 superoxide production.

    table S1. Details for clinical isolates used in the study.

    table S2. Concentrations of antibiotics tested (micrograms per milliliter) for each clinical isolate bacterial strain in combination therapy.

    table S3. Nonclinically isolated E. coli strains used in studies.

    table S4. Sensitive/resistant breakpoints used for determining resistance of clinical strains.

    References (3741)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Discussion
    • fig. S1. QD characterization and EPR analysis.
    • fig. S2. Growth curves for SodA and SodC deletion and SodA overexpression constructs and DCFH-DA controls.
    • fig. S3. Chloramphenicol GIC50.
    • fig. S4. Streptomycin GIC50.
    • fig. S5. Ciprofloxacin GIC50.
    • fig. S6. Clindamycin GIC50.
    • fig. S7. Ceftriaxone GIC50.
    • fig. S8. Growth curve of clinical strains subjected to treatment with different concentrations of streptomycin and CdTe-2.4.
    • fig. S9. Growth curve of clinical strains subjected to treatment with different concentrations of ciprofloxacin and CdTe-2.4.
    • fig. S10. Growth curve of clinical strains subjected to treatment with different concentrations of clindamycin and CdTe-2.4.
    • fig. S11. Growth curve of clinical strains subjected to treatment with different concentrations of chloramphenicol and CdTe-2.4.
    • fig. S12. Growth curve of clinical strains subjected to treatment with different concentrations of ceftriaxone and CdTe-2.4.
    • fig. S13. Effect of antibiotics in combination with CdTe-2.4.
    • fig. S14. S parameter heat maps for combinations of CdTe-2.4 and antibiotics.
    • fig. S15. LDH assay results for HeLa cells under CdTe-2.4 treatment and MitoTracker staining.
    • fig. S16. CFU per milliliter data for HeLa infection assay.
    • fig. S17. Clinical strain screen for pathogen of C. elegans.
    • fig. S18. Isosurfaces for respective antibiotic and clinical strain based on optical density at 8 hours normalized to no treatment.
    • fig. S19. Inhibition of clinical isolates with CdTe-2.4 with varied light intensity.
    • fig. S20. CdTe-2.4 superoxide production.
    • table S1. Details for clinical isolates used in the study.
    • table S2. Concentrations of antibiotics tested (micrograms per milliliter) for each clinical isolate bacterial strain in combination therapy.
    • table S3. Nonclinically isolated E. coli strains used in studies.
    • table S4. Sensitive/resistant breakpoints used for determining resistance of clinical strains.
    • References (37–41)

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