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

QD synthesis and characterization

CdTe-2.4 QDs were synthesized and filtered for experiments, as described in the study by Courtney et al. (16). Size distribution analysis of the used particles was obtained from transmission electron micrographs (fig. S1) using ImageJ (d = 2.8 ± 0.3 nm). Images were acquired using a Phillips CM 100 transmission electron microscope at an acceleration of 80 kV. Samples were prepared using Cu-Formvar grids, which were made hydrophilic using a glow-discharge plasma treatment.

Culture conditions

For nonclinical isolate bacterial growth, liquid LB broth (2% LB; L3022, Sigma-Aldrich) or solid LB (2% LB and 1.5% agar; 214010) was used for all experiments. For clinical isolate growth liquid, cation-adjusted Mueller Hinton broth (CAMHB) (212322, Becton, Dickinson and Company) or solid CAMHB (1.5% agar) was used. Dulbecco modified Eagle’s medium (Sigma-Aldrich) supplemented with fetal bovine serum (10%), l-glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM), and Hepes (10 mM) was used for HeLa infection assays and maintenance. All cells were incubated at 37°C during growth. HeLa infection studies were carried out with 5% CO₂, and liquid bacterial cultures were grown with shaking at 225 rpm. Worms were grown on solid nematode growth medium (NGM) [2.3% N1000 powder (United States Biological), 25 mM phosphate buffer, 1 mM CaCl₂, and 1 mM MgSO₄] for propagation and during infection. Worms were grown in liquid S medium {1 liter of S Basal [0.585% NaCl, 0.1% K₂HPO₄, 0.6% KH₂PO₄, and cholesterol (5 mg/liter)], 10 ml of 1 M potassium citrate, 10 ml of trace metals solution, 3 ml of 1 M CaCl₂, and 3 ml of 1 M MgSO₄} during treatment.

Bacterial strains

Clinical strains were obtained from N. Madinger at the University of Colorado Anschutz Medical Campus and were stored in 10% glycerol at –80°C for long-term storage. Biological replicates were started in liquid medium from individual, single colonies off of solid plates and grown for 16 hours before starting the experiments. OD was measured with a Tecan GENios at 562 nm with a bandwidth of 35 nm. All bacterial strains used are in tables S1 and S3.

Cloning of sodB overexpression plasmid

The SodB gene, including the RBS, was cloned from the E. coli MG1655 genome and inserted to the Bam HI and Mlu I sites of the pZE21MCS plasmid (EXPRESSYS) for the expression in E. coli by the P_LtetO-1 promoter. The control plasmid was the pZE21MCS plasmid in E. coli MG1655 without insert. Plasmids were cloned into chemically competent E. coli MG1655 and maintained with kanamycin (25 μg/ml).

GIC₅₀ measurement

Overnight cultures of clinical isolates were diluted to a 0.5 McFarland standard in the media with respective test concentrations of antibiotic. Cultures were grown for 24 hours in 384-well microplates. After 24 hours of growth, Resazurin sodium salt (Sigma-Aldrich) solution was added, and the reaction was monitored to measure fluorescence every 5 min for 4 hours at 37°C with shaking at 225 rpm using 485/610-nm filters. The slope of Resazurin fluorescence was used as a quantitative measure of cell metabolism. The GIC₅₀ was determined as the lowest concentration of antibiotic that caused a 50% or greater reduction in slope compared to the same biological replicate in no treatment. GIC₅₀’s were compared to sensitive/resistant breakpoints from 2016/2017 CLSI guidelines or literature (34, 35) shown in table S4.

Combinatorial experiments

Five antibiotic concentrations were chosen for each strain so that the levels tested would be below the GIC₅₀, near the CLSI or defined breakpoint, and near the GIC₅₀. Concentrations tested for each strain can be seen in table S2, with breakpoints shown in table S4. Concentrations of CdTe-2.4 were held constant for all strains at 12.5, 25, and 50 nM. Using these metrics, three biological replicates were tested from each strain with 15 combination test conditions as well as monotherapy controls and a no-treatment condition. Clinical strains were diluted 1:100 from the overnight cultures, into the test condition. Optical density was measured every 30 min for the first 3 hours and every hour subsequently until 8 hours. The optical density at 8 hours normalized to no treatment was used in the Bliss Independence model (27) to determine the combinatorial effect. We used optical density at 8 hours instead of growth rate because of the two-phase growth many of our conditions demonstrated, which yielded multiple growth rates. The optical density at 8 hours was normalized to account for the difference in starting cell viability and biological replicate colony variation.

C. elegans infection experiments

C. elegans CF512 [(fer-15(b26); fem-1(hc17)] were used for infection experiments. CF512 do not replicate when grown at 25°C to allow for control of the worm count and determination of live and dead populations from starting t = 0. E. coli op50 was used both as the food source before infection and as the control strain during infection experiments. During the treatment phase of infection experiments, worms were suspended in standard liquid S medium, and all wash steps were carried out with standard M9 buffer (36). NGM plates were seeded with E. coli op50 or MDR strains by plating 100 μl of overnight culture onto NGM and incubating at 37°C for 8 hours. Worms were chunked onto NGM plates seeded with E. coli op50 and allowed to grow for 10 to 14 days at 15°C to allow for egg production. The eggs were then collected using NaOH and bleach and transferred to NGM and incubated at 25°C. After 24 hours, hatched worms were transferred to NGM seeded with op50 and grown at 25°C to allow for adult worms to grow without reproduction for 48 hours. Adult worms were collected and washed twice before being transferred to NGM seeded with infection strains. Infection on solid plates was carried out for 3 days. After infection, worms were collected and washed three times (30 s at 600g) before being transferred to S medium, with respective treatment conditions in 100 μl of cultures in 96-well plates at 25°C to limit reproduction. Every 24 hours, worms were monitored for morphology, and the medium was changed to refresh CdTe and antibiotic. At the end of the treatment period, worms were stained with 0.5 μM SYTOX orange (S11368, Thermo Fisher Scientific) for 30 min before imaging and counting. Images were acquired, and counting was performed using an EVOS FL microscope and analyzed using ImageJ. Forty-six clinical isolates were screened to choose the S. enterica serovar Enteritidis strain used in experiments (S48; fig. S17).

EPR spectroscopy

For all EPR measurements, we used continuous-wave X-band EPR spectroscopy. QD samples were prepared for EPR measurements by filtering, as described above, and resuspended in water (pH 11). One hundred microliters of this solution was then mixed with 1 μl of DMPO (Dojindo) and sequestered from light exposure. Quartz capillaries were filled with the CdTe-DMPO mixture and measured in a Bruker Elexsys E 500 spectrometer equipped with an SHQE resonator. Exposure to ambient light was greatly minimized by preparing and recording every measurement in a dark room. A dark background was measured for each sample, which consisted of the average of 10 scans using a 200-G scan range (0.05-G resolution) centered on 3515 G with a microwave attenuation of 16 dB and power of 5 W and was subtracted from the light signal as negligible (fig. S1). Time-dependent scans were conducted over the same 200-G scan range (20.48 s) with a delay of 100 ms between scans. The SiO₂ E′ defect was present in all measurements but was subtracted during the analysis as part of the dark signal. The sample was then exposed to 9-mW/cm² white light and immediately remeasured to obtain the photoactivated spectrum. For confirmation of superoxide production, conditions were as follows: 10% (v/v) dimethyl sulfoxide in double distilled water (DDW), 1 to 2 mg of the SOD enzyme (Sigma-Aldrich) in 100 μl of DDW, or the degassed water was prepared by bubbling nitrogen through DDW for 1 hour. Each spectrum containing multiple species was simulated using the SpinFit module of the Bruker Xepr software (version 2.6b 149) to identify the radical adducts. The initial fit parameters were as follows: DMPO-OH, a_N = 14.90 G and a_H^β = 14.93 G; DMPO-OOH, a_N = 14.2 G, a_H^β = 11.4 G, and a_H^γ1 =1.2 G. By providing a fixed and known active sample volume in each capillary, the concentration of DMPO adducts was calculated from the total number of spins detected using the Bruker SpinFit software. This software-reported concentration corresponded to the ROS generated multiplied by the number of capillaries in the cavity. Concentrations of ROS species are reported above, in the main text, for one capillary. Data in Fig. 1B (middle) are time-dependent EPR scan; successive scans were each saved as slices, and we averaged the results of the SpinFit from three replicate experiments. Data in Fig. 1B (right) show 10 successive scans that were averaged to minimize error.

EPR was used to evaluate the effects of SOD and consequent hydroxyl radical formation through Fenton chemistry. CdTe-2.4 was filtered and washed as detailed above and redispersed in M9 medium to eliminate pH effects when combined with SOD. The stock of SOD contained 1 mg/ml of the enzyme and 0.1 M potassium phosphate. For the lysozyme control in fig. S1, 1 mg/ml of lysozyme was used. A 1 M solution of ferrous iron sulfate (FeSO₄) in M9 was used as a source of iron. Spectra were first measured in light and dark, as described above, for CdTe-2.4 alone. The resulting spectra were smoothed using a five-point moving average and are shown in Fig. 1C (middle).

DCFH-DA imaging

Respective strains were diluted 1:10 from overnight cultures into CAMHB and treated with respective concentrations of CdTe-2.4 for 1 hour in dark or light. Cells were pelleted and resuspended in phosphate-buffered saline (PBS) with DCFH-DA and incubated for 5 min before cells were imaged on glass slides with coverslips on an EVOS FL microscope. Images were processed identically for all conditions using ImageJ.

Gentamicin protection assays

HeLa cells were seeded at 1.0 × 10⁴ cells per well in 96-well tissue culture plates and grown for 24 hours. STm wild-type strain SL1344 with chromosomal rpsM::GFP (30) was grown overnight, diluted 1:10, and grown for 4 hours in LB before infections. Streptomycin antibiotic selection was used at 30 μg/ml. Salmonella Typhimurium in Gibco PBS was added at a multiplicity of infection of 30:1 (bacteria/HeLa) and centrifuged for 5 min at 500g. After 45 min, cells were incubated for a further 1.25 hours at 37°C in fresh medium supplemented with gentamicin (40 μg/ml) to kill extracellular bacteria. Medium was then exchanged for fresh medium with treatment conditions supplemented with gentamicin (40 μg/ml) to inhibit extracellular bacterial growth. At 18 hours after infection, the wells were washed three times with 300 μl of PBS, and cells were lysed with 30 μl of 0.1% Triton X-100 for 15 min at room temperature. After 15 min, 270 μl of PBS was added to each well (serving as 100-fold dilution), serially diluted, plated onto 2% LB, 1.5% agar, and streptomycin (40 μg/ml), and then incubated at 37°C for 16 hours to enumerate CFU per milliliter. For staining and imaging, wells were stained with 100 nM MitoTracker, followed by fixation with 16% paraformaldehyde for 15 min. Wells were then washed twice with PBS, stained with DAPI for 20 min, washed twice with PBS, and stored in 100 μl of 90% glycerol and 1× PBS before imaging and/or storage at 4°C in dark. Images were acquired using an EVOS FL microscope and analyzed using ImageJ.

S. aureus and HeLa coculture

HeLa cells were seeded as described above. MRSA was grown overnight to saturation and added to HeLa at a multiplicity of infection of 5:1 (bacteria/HeLa) in medium supplemented with respective treatment conditions. At 24 hours after infection, the wells were agitated using pipetting, and 10 μl of the medium was transferred to PBS (serving at 10-fold dilution), serially diluted, plated on to 2% LB and 1.5% agar, and incubated at 37°C for 16 hours to enumerate CFU per milliliter.

Lactate dehydrogenase cytotoxicity assay

HeLa cells were seeded as described above. Cells were treated with respective conditions and incubated for 18 hours. Fifty microliters of the supernatant was used to determine lactate dehydrogenase (LDH) release as a measure of cytotoxicity using the Pierce LDH cytotoxicity assay kit. Cells lysed with the supplied lysis buffer served as a positive control.

Skin depth modeling

To estimate the penetration of light, excitation of CdTe-2.4, and potentiation of antibiotics in skin, we used in vivo absorption coefficients of the human skin, separated into epidermis and dermis, from Lister et al. (31). The light source for calculations of skin depth was an LED sheet with emission at 520 nm with an intensity of 1.6 mW/cm². We assumed that 160 nM CdTe-2.4 was dispersed in the skin, which had low toxicity in our LDH assay of human epithelial cells (HeLa). The skin depths where CdTe-2.4 superoxide flux and potentiation would be equal to 12.5, 25, and 50 nM in broth, 1.47, 1.32, and 1.16 cm, respectively, were calculated and then used to graph GIC₅₀ and GIC₇₅ skin depth isosurfaces (Fig. 4 and fig. S18). We then used an exponential decay fit for the inhibition data at each antibiotic concentration to interpolate or extrapolate skin depths for GIC₅₀ and GIC_90. (Fig. 4 and fig. S19).

Error and significance analysis

Error is represented as SD between technical replicates or SEM for biological replicates. Significance is defined as P < 0.05 and denoted by an asterisk (*) unless otherwise stated. Significance was determined using Student’s t tests, with a 95% confidence interval, unless otherwise stated.