Antibiotics Are Man's Greatest Invention

Reactive oxygen species induce antibiotic tolerance during systemic Staphylococcus aureus infection

Macrophage growth and infection

J774A.1 murine macrophage-like cells (ATCC, TIB-67) were cultured in high-glucose Dulbecco’s modified essential media (Gibco) supplemented with 10% fetal bovine serum (FBS; Milipore), non-essential amino acids (Gibco), sodium pyruvate (Gibco) and l-glutamine (Gibco) in a humidified incubator at 37 °C and 5% CO2. For infection, the macrophages were seeded in 24-well plates at a density of 2 × 105 cells per well in minimum essential medium (MEM; Gibco) supplemented with 10% FBS and l-glutamine. We found respiratory burst to be sensitive to the media the cells were cultured in as well as the confluency history of the cells. Cells that were more than 90% confluent no longer reliably generated reactive species in response to S. aureus. To stimulate the J774A.1 cells, 500 ng ml−1 LPS from Escherichia coli O55:B5 (Sigma) and 20 ng ml−1 recombinant murine IFNγ (Peprotech; LPS + IFNγ) were added to supplemented MEM for 20 h31. THP-1 human monocytes were cultured in RPMI (Gibco) supplemented with 10% FBS, l-glutamine and 0.05 mM 2-mercaptoethanol. To differentiate monocytes to macrophage-like cells, THP-1 cells were cultured in 24-well plates at a density of 2 × 105 cells per well in supplemented RPMI with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 3 d. For infection, the medium containing PMA was removed and the cells were washed with PBS. Supplemented RPMI (without PMA) was added to cells 16 h before infection.

Wild-type and mutant S. aureus HG003 strains were used to infect macrophages at a multiplicity of infection of 25 (approximately 5 × 106 c.f.u. per well). Where indicated, the cells were pretreated with 20 µM BHA (Sigma) or 10 µM VAS2870 (Cayman Chemicals) for 1 h before infection. The 24-well plates were centrifuged at 1,200g for 2 min to bring the bacteria into contact with the macrophages. The cells were washed once with PBS at 30 min post infection. Fresh medium containing 30 µg ml−1 gentamicin (Fisher) was added to kill the extracellular bacteria32, and 10 µg ml−1 rifampicin (Fisher) or 40 µg ml−1 ciprofloxacin (Acros Organics) were added to the appropriate wells. At the indicated times, 200 µl Triton X-100 (0.1%) was added to each well for 5 min at 37 °C to selectively lyse the macrophages and release the bacteria. PBS (800 µl) was then added to each well, and the lysates were suspended by pipetting, serially diluted in 1% NaCl and plated to enumerate the surviving bacteria. The survival percentage after rifampicin treatment was determined by comparing the number of surviving bacteria after 4 h (J774) or 6 h (THP-1) of rifampicin treatment to the survivors of the untreated group at the corresponding time point (that is, 4 or 6 h). The averages and s.d. of three biological replicates are shown (n = 3). The cell lines were obtained from the Tissue Culture Facility at the UNC Lineberger Comprehensive Cancer Center. We did not authenticate or test the cells for mycoplasma contamination. Statistical significance was calculated using an unpaired two-tailed Student’s t-test or a one- or two-way ANOVA with Sidak’s multiple comparison test, as described in the figure legends.

Antibiotic-survival assays

The S. aureus strains HG003 or COL were cultured aerobically in Mueller–Hinton broth (MHB; Oxoid) at 37 °C for approximately 16 h with shaking (225 r.p.m.). For the cell-wall-acting antibiotics, stationary cultures were diluted 1:1,000 in MHB and cultured to about 5 × 107 c.f.u. ml−1. For all other antibiotics, stationary cultures were diluted 1:100 in MHB and cultured to approximately 2 × 108 (HG003) or 5 × 108 c.f.u. ml−1 (COL). An aliquot was plated to enumerate the c.f.u. (Time 0) before the addition of antibiotics. Where indicated, the culture was incubated with 80 µM menadione (MP Biomedicals); 0.25 µg ml−1 mupirocin, acid; 4 mM N-acetyl cysteine; 150 mM thiourea; 5 mM paraquat; 120 mM hydrogen peroxide or 30 µg ml−1 chloramphenicol for 20 min before antibiotic challenge. In the case of acid stress, the medium was acidified to pH 4.5 with HCl for 20 min and then neutralized to pH 7 with NaOH before the addition of antibiotics. The following antibiotics were added at concentrations similar to the Cmax in humans at recommended dosing: 10 µg ml−1 rifampicin33, 2.34 µg ml−1 ciprofloxacin (Acros Organics)34, 50 µg ml−1 oxacillin (Acros Organics)35 and 50 µg ml−1 vancomycin (MP Biomedicals)36. An aliquot was removed at the indicated time points and washed with 1% NaCl. The cells were serially diluted and plated on tryptic soy agar to enumerate the survivors or tryptic soy agar containing 10 µg ml−1 rifampicin to enumerate the rifampicin-resistant mutants. The rifampicin-resistant mutants were detected at lower levels than the antibiotic-tolerant population (Extended Data Fig. 7), indicating that resistance did not contribute to the tolerance phenotype. We routinely used two time points to enumerate survivors (19 and 24 h after antibiotic challenge), as we previously found that susceptible S. aureus cells are killed and a stable sub-population of survivors emerges between 16 and 24 h of exposure to antibiotics12,37. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test or a one-way ANOVA with either Sidak’s or Dunnett’s multiple comparison test, as described in the figure legends.

ROS measurements

The luminescent probe L-012 (Wako Chemical Corporation) was used to measure ROS. For the in vitro ROS measurements, HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1. Where indicated, the cells were incubated with 80 µM menadione, 4 mM N-acetyl cysteine or 5.5 mM glucose for 20 min. Rifampicin (10 µg ml−1) was then added, a 100-µl aliquot was immediately removed and added to the wells of a white 96-well plate containing L-012 at a final concentration of 1 mM. Luminescence was recorded after 5 min using a Biotek Synergy H1 microplate reader. Aliquots were removed at additional time points (1, 2, 3 and 24 h) and luminescence was recorded as described above. The relative-luminescence-unit values of the samples treated with menadione (or menadione + NAC/glucose) were either provided as raw numbers or divided by those of the untreated (control) samples at each time point and expressed as the fold increase in ROS relative to the control. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test at each time point or a two-way ANOVA with Sidak’s multiple comparison test, as described in the figure legends. For the tissue culture cell assays, macrophages were seeded at 2 × 104 cells per well in white tissue-culture-treated 96-well plates. J774 cells were treated with LPS + IFNγ as described above. Where indicated, 20 µM BHA was added for 1 h before the addition of L-012. After incubation with BHA, the cells were washed three times with PBS. L-012 was diluted to 150 µM in Hanks’ balanced salt solution (Gibco). Luminescence was read immediately and recorded every 2 min for 1.5 h using a Biotek Synergy H1 microplate reader. Data shown are from the time point of peak luminescence of the control (untreated THP-1 or J774 cells stimulated with LPS + IFNγ). The averages and s.d. of at least three replicates are shown (J774, n = 4; THP-1, n = 3). Statistical significance was calculated using a one-way ANOVA with Sidak’s multiple comparison test or an unpaired two-tailed Student’s t-test, as described in the figure legends.

Growth curves

The S. aureus strain HG003 was cultured aerobically in MHB broth (Oxoid) for approximately 16 h at 37 °C with shaking (225 r.p.m.). Stationary cultures were diluted 1:100 in MHB and cultured to approximately 2 × 108 c.f.u. ml−1. Growth curves were established by c.f.u. or OD600 determination at the indicated times after the addition of stresses (80 µM menadione; 0.25 µg ml−1 mupirocin, acid; 4 mM N-acetyl cysteine; 5 mM paraquat; 120 mM hydrogen peroxide or 30 µg ml−1 chloramphenicol). In the case of acid stress, the media were acidified to pH 4.5 using HCl for 20 min and then neutralized to pH 7 with NaOH.

Oxygen consumption

HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1 and incubated with or without 80 µM menadione. After 2 h, the cells were pelleted and concentrated 3× in MHB. The samples were vigorously vortexed and immediately added to the borosilicate glass sample vessel of an Oxygraph Plus (Hansatech Instruments). The oxygen levels were allowed to equilibrate for 30 s at a stir speed of 5. Readings were recorded for the subsequent 300 s. The linear part of the graph (the first 100 s) was used to calculate the rate of oxygen consumption using the formula (Time 0 s − Time 100 s)/100 and then normalized to the c.f.u. count. The percent rate of oxygen consumption percentage was calculated by dividing by the rate of oxygen consumption in the control and multiplying by 100. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test, as described in the figure legends.

ATP assays

HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1. Where indicated, the cells were exposed to 80 µM menadione, 5.5 mM glucose or 4 mM N-acetyl cysteine. The ATP levels of a 100-µl volume of cells were measured after 0.5 or 2 h, as described previously12 using a BacTiter-Glo kit (Promega) according to the manufacturer’s instructions. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test or a one-way ANOVA with Sidak’s multiple comparison test, as described in the figure legends.

Aconitase assays

HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1 and incubated with or without 80 µM menadione, 4 mM N-acetyl cysteine, 5 mM paraquat, 120 mM hydrogen peroxide or 5.5 mM glucose. After 0.5 or 2 h, 2–10 ml of cells were pelleted and resuspended in 200–300 µl assay buffer and lysed using 50 µg ml−1 lysostaphin at 37 °C for 5 min. The samples were pelleted and the supernatant was assayed for aconitase activity using an aconitase assay kit (Abcam) as per the manufacturer’s instructions. The aconitase activity was normalized to the c.f.u. count. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test or one-way ANOVA with either Sidak’s or Dunnett’s multiple comparison test, as described in the figure legends.

Succinate dehydrogenase assays

HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1 and incubated with or without 80 µM menadione, 5 mM paraquat or 120 mM hydrogen peroxide. After 2 h, 2–10 ml cells were pelleted and resuspended in 200 µl SDH sample buffer and lysed with 50 µg ml−1 lysostaphin at 37 °C for 5 min. The samples were pelleted and the supernatant was assayed for succinate dehydrogenase activity using a succinate dehydrogenase activity colorimetric assay kit (Sigma) as per the instructions from Biovision Incorporated. The succinate dehydrogenase activity was normalized to the c.f.u. count. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test or one-way ANOVA with Dunnett’s multiple comparison test, as described in the figure legends.

Isocitrate dehydrogenase assays

HG003 was cultured in MHB to approximately 2 × 108 c.f.u. ml−1 and incubated with or without 80 µM menadione, 5 mM paraquat or 120 mM hydrogen peroxide. After 2 h, 2.4 ml cells were pelleted and resuspended in 400 µl isocitrate dehydrogenase assay buffer and lysed using 50 µg ml−1 lysostaphin at 37 °C for 5 min. The samples were pelleted and the supernatant was assayed for isocitrate dehydrogenase activity using an isocitrate dehydrogenase activity assay kit (Sigma) as per the manufacturer’s instructions. The isocitrate dehydrogenase activity was normalized to the c.f.u. count. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using an unpaired two-tailed Student’s t-test or one-way ANOVA with Sidak’s multiple comparison test, as described in the figure legends.

Strain construction

Transcriptional reporter plasmids were constructed by amplifying the region between −755 and +76 bp of the acnA start codon38, and 222 bp (sdhC) and 209 bp (icd) upstream of the coding regions from HG003 genomic DNA using primers flanked with EcoRI and XbaI sites, and inserted upstream of gfpuvr into the EcoRI and XbaI sites of the plasmid pALC1434 (ref. 39) to yield the transcriptional green-fluorescent-protein (gfp)-reporter plasmids Pacn::gfp, Psdh::gfp and Picd::gfp, respectively. The HG003acnA::erm (acnA) and HG003sdhB::erm (sdhB) strains were constructed by transducing the transposon mutants from the Network on Antimicrobial Resistance in Staphylococcus aureus library40 into HG003.

Transcriptional reporter assays

S. aureus strain HG003 harbouring the gfp promoter plasmids or the promoterless gfp plasmid pALC1434 were cultured to approximately 2 × 108 c.f.u. ml−1 in MHB containing 10 μg ml−1 chloramphenicol. Where indicated, 80 µM menadione was added. After 2 h, 200 μl of each culture was added to the wells of a clear-bottom black-sided 96-well plate and the absorbance (OD600) and GFP fluorescence (emission 528 nm and excitation 485 nm) were measured using a Biotek Synergy H1 microplate reader. The GFP values were divided by the OD600 and then the values obtained for the promoter gfp plasmid were subtracted. The averages and s.d. of three biological replicates are shown (n = 3). Statistical significance was calculated using a two-tailed Student’s t-test, as described in the figure legends.

Mouse infections

The wild-type C57BL/6J (Jackson, cat. no. 000664) and Ncf1−/− (Jackson, cat. no. 027331) mice were housed in a specific-pathogen-free facility. All of the animal protocols were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill and met the guidelines of the US National Institutes of Health for the humane care of animals. For the mouse infections, 8- to 10-week-old female mice were infected by intravenous injection with approximately 5 × 106 c.f.u. of S. aureus strain HG003 in 100 μl PBS. Where indicated, the mice were treated with tempol (4-hydroxy-TEMPO; Sigma) at 25 mM in the drinking water starting 24 h before infection. Sucrose (4%) was added to the tempol water to increase the volume consumed by the mice. The control groups for the tempol experiments also received drinking water supplemented with 4% sucrose. Rifampicin was dissolved in the vehicle (6.25% dimethylsulfoxide + 12.5% polyethylene glycol 300) at a final concentration of 6.25 mg ml−1. Starting at 24 h.p.i., the mice were treated daily with an intraperitoneal injection of 25 mg kg–1 rifampicin or vehicle. At 24, 48 or 72 h.p.i., the organs were homogenized, serially diluted and plated on BHI agar plates for the enumeration of the bacterial c.f.u. (BHI agar plates containing rifampicin 10 μg ml−1 were used for the enumeration of the rifampicin-resistant mutants). The percentage of rifampicin-tolerant cells was determined by comparing the survivors after rifampicin treatment to the survivors of the vehicle-treated group. At 24 h.p.i., samples of homogenized livers and spleens were treated with a RPMI-based digestion buffer containing 2 mg ml−1 collagenase V (Invitrogen), 3 mM CaCl2, 5 U ml−1 DNase I (Fisher), 5 U ml−1 dispase (Sigma), 5% FBS (Millipore) and 10 mM HEPES for 30 min to quantify the extracellular bacteria. The homogenates were then centrifuged at a low speed (300g) to pellet the host cells. Aliquots of the supernatant from the low-speed centrifugation were serially diluted and plated on BHI agar to quantify the extracellular bacteria. For the infections, n = 9 vehicle- and rifampicin-treated wild-type mice, and n = 10 (vehicle-treated) and 11 (rifampicin-treated) Ncf1-/- mice were used. For the tempol-inhibitor experiments, n = 4 (control) and 5 (tempol) mice for the 24 h.p.i. groups, and n = 10 for vehicle- and rifampicin-treated control and tempol mice in the 48 and 72 h.p.i. groups were used. The mean is indicated by a horizontal line. Statistical significance was calculated using a Kruskal–Wallis one-way ANOVA or Mann–Whitney test, as described in the figure legends. The sample size was chosen based on the number of mice required to reach statistical significance using the Mann–Whitney test in previous experiments using the systemic model of infection. Blinding or randomization was not necessary as all outputs (c.f.u. g−1 tissue) are objective.

Histopathology

The mice were infected as above. The organs were harvested at 48 h.p.i. and fixed in 10% neutral buffered formalin for 72 h and transferred to 70% ethanol. The fixed organs were paraffin embedded and sectioned at 5 µm.

Immunofluorescence microscopy

The sections were deparaffinized and rehydrated with the following washes: 3 × xylene, 3 × 100% ethanol, 2 × 95% ethanol, 1 × 80% ethanol and 1 × water. The slides were boiled for 20 min and cooled to room temperature in citrate buffer (pH 6) to unmask antigens. The samples were blocked with 1.5% normal goat serum in PBS for 40 min. The samples were then incubated overnight with block solution (control), rat monoclonal anti-mouse F4/80 (clone BM8; 1:100; eBiosciences, cat. no. 14-4801-82) or rat monoclonal anti-mouse Ly6G (clone 1A8; 1:100; eBiosciences, cat. no. 16-9668-82) at 4 °C. The samples were washed 3× in PBS and permeabilized with 0.5% Tween 20 in PBS for 30 min to allow the S. aureus antibody to enter the cells. The samples were washed 3× in PBS and incubated for 30 min with 1.5% normal goat serum. Next, the samples were incubated overnight at 4 °C with block solution (control) or anti-S. aureus antibody (1:400; Abcam, ab20920). The samples were washed 3× in PBS and incubated with the secondary antibodies (both at 1:1,000), goat anti-rat conjugated to Alexa 594 (Invitrogen, cat. no. A11007) and goat anti-rabbit conjugated to Alexa 488 (Invitrogen, cat. no. A32731), for 1 h at room temperature. The samples were again washed 3× in PBS and then mounted with 1.5 coverslips over tissue sections using ProLong Diamond with 4,6-diamidino-2-phenylindole (Invitrogen). All of the antibodies were diluted in block solution. Images were obtained on a Zeiss LSM 700 confocal laser scanning microscope using a ×63/1.4 plan apo oil objective lens and Zeiss ZEN 2011 software. DeconvolutionLab2 and ImageJ were used to deconvolve the images. The representative images shown are the maximum-intensity projections of the z-stack.

Rifampicin stability test

We aimed to determine whether rifampicin is inactivated by ROS in our in vitro (Fig. 1c) and tissue culture assays (Fig. 1a). Minimum inhibitory concentration (MIC) assays for rifampicin were conducted using a microdilution method. Approximately 5 × 105 HG003 S. aureus cells in MHB were incubated with various concentrations of rifampicin, either from a freshly prepared stock solution or following extraction from in vitro or tissue culture antibiotic-survival assays as described below. An aliquot of rifampicin-containing cultures (from Fig. 1c) was collected, pelleted to remove the cells and filter sterilized following 19 h of exposure to S. aureus with or without 80 µM menadione. This supernatant was used as the source of rifampicin and underwent MIC testing. Similarly, sterilized rifampicin-containing MEM media from either unstimulated or LPS + IFNγ-stimulated J774 cells incubated with S. aureus for 4 h (Fig. 1a) were used in the MIC assays. The MICs were determined following 24 h incubation at 37 °C. Extended Data Fig. 8 shows that after 19 h in vitro, when all of the killing had occurred, rifampicin activity was reduced by twofold in both the control- and menadione-treated samples. Similarly, supernatants extracted from tissue-culture experiments revealed the rifampicin MIC decreased sixfold in both stimulated and unstimulated J774 macrophages. These data indicate that although rifampicin activity is reduced over the course of our assays, it is not affected by ROS.

Statistical information

The statistical methods and sample sizes (n) are indicated in the Methods for each experiment. Statistical analysis was performed using Excel (Microsoft) or Prism 8 (GraphPad) software. For the in vivo studies, n represents the number of mice per group and statistical significance was determined using the Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison test or the Mann–Whitney test. For the in vitro studies, n represents the number of biological replicates and statistical significance was determined using a one- or two-way ANOVA with Tukey’s, Sidak’s or Dunnett’s multiple comparison test or an unpaired two-tailed Student’s t-test (as indicated in the figure legends). Statistical significance was defined as P < 0.05. The error bars in the figures show the s.d. of the mean.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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