Silver nitrate (AgNO₃) (99.9%) and ruthenium red (RR) was purchased from Sigma–Aldrich (United States). AgNPs were provided by nanoComposix (United States) as aqueous suspensions [citrate coated, mass concentration (Ag) 0.02 mg/mL] of the size 10 nm (9.4 ± 1.7 nm, AgNP-10). Cis-Platinum (II) diamine dichloride (cisPt) was purchased from Enzo Life Sciences GmbH (Lörrach, Germany). Nitric acid (HNO₃) was purchased from Merck (Germany). M12 medium and PBS compositions were shown in Supplementary Table S1. For washing purposes 18.2 MΩ⋅cm water (MilliQ, Germany) was used.

Pseudomonas putida KT2440 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). Bacterial standard-growth was performed in M12 medium on a rotary shaker at 30°C and 170 rpm. The growth was monitored by optical density at λ = 600 nm (Spectra max Plus 384 photometer, Molecular Devices, Sunnyvale, CA, United States).

An overnight pre-culture of P. putida KT2440 was incubated in M12 medium with an initial OD₆₀₀ of 0.09 and grown for 72 h (30°C, 170 rpm). Either AgNP-10 (1.29 mg/L) or AgNO₃ (0.19 mg/L) were implemented in the cultivations and chosen concentrations referred to the determined EC₅₀ values from an earlier publication (Guo et al., 2017). Cultivations without silver treatment served as silver-ion negative control while application of AgNO₃ served as silver-ion positive control. Cells were harvested at 0, 12, 48, and 72 h and treated separately according to the mass cytometry staining protocol (see below).

To analyze bacteria on the single cell level at the mass cytometer, a concentration of 5.0 × 10⁵ cells/mL was required for each injection. Therefore, a fast and accurate cell counting method was required and for this a range of linear relationship between cell counts and OD₆₀₀ was exploited. Cell counts were determined by a flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) together with a calibrated suspension of microsphere standard (6.0 μm diameter microspheres at a concentration of 10⁸ beads/mL in Milli-Q water containing 2 mM sodium azide, L34856, Thermo Fisher Scientific, Germany) for accurate cell count measurements. OD₆₀₀ was analyzed by a spectrophotometer. All measurements were done in replicates and shown in Supplementary Figure S1.

To optimize RR staining for P. putida KT2440 populations, a stock solution of 1.3 mM of RR in PBS was prepared and stored at 4°C. Before use the solution was ultra-sonicated (Ultrasonic bath, Merck Eurolab, Darmstadt, Germany) for 10 min. 10⁸ cells were treated with different concentrations of RR (0 to 0.33 μM) for 30 and 60 min. The final staining protocol requires 0.13 μM RR/10⁸ cells/200 μL PBS and a staining time of 30 min at room temperature (RT). Details of staining optimization were shown in Supplementary Figure S2A.

To optimize cisPt staining for P. putida KT2440 populations, a stock solution of 25 mM of cisPt in dimethyl sulfoxide (Sigma–Aldrich, United States) was prepared and stored at -20°C. 10⁸ cells were stained with different concentrations of cisPt (0 to 20 μM) and treated for 1, 5, 10, 30 min. The final staining protocol requires 5 μM cisPt/10⁸ cells/1 mL PBS and a staining time of 10 min at RT. Details of staining optimization and calibration curves generated on the basis of RR and cisPt measurements were shown in Supplementary Figures S2B,C.

The fluorescent dye propidium iodide (PI) was used as dead cell indicator for P. putida KT2440. Staining optimization and calibration were published (Guo et al., 2017). The final protocol requires 2 μM PI/10⁸ cells/1 mL PBS and a staining time of 2 min. For quantification of PI stained dead cells (PI+dead) of RR labeled P. putida KT2440, cells were double stained with RR (0.13 μM, 30 min), followed by PI (2 μM, 2 min) and analyzed by flow cytometry at log scale.

Harvested cells were diluted to 10⁸ cells/1 mL PBS and centrifuged at 3200 × g for 10 min. The pelleted cells were re-suspended in cisPt solution (5 μM, 10 min, RT). Subsequently, the cisPt stained dead cells (cisPt+dead) were washed (3200 × g, 10 min) twice with 3 and 1 mL PBS, respectively, to remove unbound cisPt. Finally, cells were stained with RR (0.13 μM, 30 min, RT), washed twice (3200 × g, 10 min) with 1 mL water to remove salts and unbound RR. For mass cytometry measurement the cell concentration was adjusted to 5.0 × 10⁵ cells/mL in Milli-Q water. Four element calibration beads (Fluidigm, United States) were added 1:10 v/v before acquisition for later normalization (Finck et al., 2013).

The CyTOF instrument (Fluidigm Corp, South Francisco, CA, United States) was tuned, calibrated, and cleaned on the daily base according to the manufacturer’s advice. Aqueous bacterial cell suspensions were acquired on a CyTOF instrument version 1 upgraded to the control software of v6.0.626 with a flow rate of 45 μL/min. Argon gas 5.0 was used to generate the plasma and for nebulizing the cell suspension. About 1.5 × 10⁵ cells per sample were subsequently loaded onto a 450 μL sample loop and measured in a dual instrument mode with noise reduction turned on, a cell length range from 1 to 75 and with ‘on the fly’ processing. The initial raw data were processed into standard FCS file format, randomized and normalized by Software Helios version 6.5.358 (Fluidigm Corp, South San Francisco, CA, United States). Data analysis was performed with FlowJo (version 10) (TreeStar, Ashland, OR, United States). Monitoring was done by analyzing natural abundance isotopes of ruthenium (¹⁰²Ru and ¹⁰⁴Ru), platinum (¹⁹⁵Pt) and silver (¹⁰⁷Ag and ¹⁰⁹Ag, 51.8 and 48.2%, respectively). Silver was measured in ionic form and resulting data were related to the ¹⁰⁷Ag isotope.

Cytometric measurements were performed with a BD Influx v7 Sorter USB, (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) equipped with a blue 488-nm Sapphire OPS laser (400 mW, Coherent, Santa Clara, CA, United States). The 488-nm laser light was used for the analysis of the forward scatter (FSC, 488/10), the side scatter (SSC, 488/10, trigger signal), and the PI induced red fluorescence (616/23). The fluidic system was run at 33 psi using a 70-μm nozzle. The sheath fluid consisted of 0.5 × FACSFlow buffer (BD). For the optical calibration of the cytometer in the linear range, 1-μm blue fluorescent FluoSpheres (Molecular Probes, F-8815, Eugene, OR, United States) and 2-μm yellow-green fluorescent FluoSpheres (Thermo Fisher Scientific, F8827, Waltham, MA, United States) were used. For calibration in the log range, 0.5-μm UV Fluoresbrite Microspheres (Polysciences, 18339, Warrington, PA, United States) were applied. Sorting of PI unstained live (PI-live) and PI stained dead (PI+dead) cells into plastic tubes was performed at an event rate of 5.000/sec which corresponded to a sort rate of 700–1.500 cells/sec. The sort mode was 1.0 Drop Pure. To obtain sufficient cell amounts for the following mass cytometry and ICP-MS measurements, up to 3.0 × 10⁶ cells were sorted for either PI-live or PI+dead cells. After sorting cells were pre-processed before CyTOF or ICP-MS analysis: sorted cells were transferred from plastic tubes into glass tubes and centrifuged at 3200 × g for 10 min, supernatant was removed and the cell pellets were stored at -20°C. Before mass cytometry measurements, cell pellets were washed once with 1 mL Milli-Q water by re-suspension in 1 mL Milli-Q water for injection.

For bulk ICP-MS analysis, the sorted PI-live and PI+dead cell pellets were digested by addition of 50 μL HNO₃ (60%, ultrapure) for a period of 15 min. Subsequently the solution was suspended in Milli-Q water dispensed with rhodium (¹⁰³Rh) as internal standard [c (Rh) = 0.75 ng/mL] to a 1 mL solution. The solution was measured with an Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Element XR, Thermo, Germany) equipped with a micro-concentric nebulizer (Micromist-100, Glass Expansion, Australia) consuming 135 μL sample per minute. Silver was measured and quantified in ionic form related to the ¹⁰⁹Ag isotope.

Cytometry by Time of Flight technology substitutes isotopes of transition elements and lanthanides for fluorescent dyes. The cells are directed into a narrow flow to be screened one by one and vaporized. Elements of the cells are atomized, ionized, and subsequently analyzed by a time-of-flight mass spectrometer. The measured signal of natural abundance isotopes of the rare-earth-metals ruthenium and platinum per cell from applied RR and cisPt provided the number of labeled cells and the isotopes’ quantity per cell. RR is a cationic reagent and has been widely used to locate acidic polysaccharide-like material (Fletcher and Floodgate, 1973; Fassel and Edmiston, 1999; Waller et al., 2004) and to visualize numerous ultra-structural details in and outside of cells (Lingens et al., 1985; Chatterjee et al., 2010; Perfumo et al., 2014). Because RR is easy to operate by addressing all cells in a population directly without pre-treatment (instead of, e.g., fixation, necessary for flow cytometric measurements; Günther et al., 2008) and its atomic mass is within the detectable mass range of mass cytometers, RR was used as an all cell indicator for the CyTOF workflow. However, the metal was not used in this context for bacteria before. The second dye used in this study, cisPt, is a readily available platinum based chemotherapeutic agent and reacts with protein nucleophiles, with which it can form covalent platinum–sulfur or sulfhydryl bonds (Fienberg et al., 2012). cispt enters cells with compromised cell membranes, where it non-specifically labels total cellular protein. The reagent can thus be used to discriminate live from dead cells which is well practiced for human cells (Bjornson et al., 2013; Nair et al., 2015). However, applications on bacteria haven’t been tested yet to our knowledge.

In our study, the Gram-negative bacterium P. putida KT2440 was used as a test organism to establish a protocol for RR and cisPt staining in order to detect all cells in a population and to differentiate live from dead cells. The RR stained bacteria were well detectable by the CyTOF and entirely identified within a cell gate plotted via the two most natural abundance ruthenium isotopes ¹⁰²Ru/¹⁰⁴Ru. Protein binding cisPt was used as second cell marker for dead cells and identified by the ¹⁹⁵Pt isotope. For this combined marker group optimized staining conditions were tested with regard to dye concentrations and times of incubation and the trade-off between highest intensity and shortest staining time was chosen for the final workflow (Figure 1A and Supplementary Figure S2B). To verify the reliability of the cisPt marker different ratios of live and dead cells (70% ethanol treated, 20 min, RT) were prepared and measured at CyTOF. In parallel the same ratios were analyzed by using the fluorescent marker PI (binding on nucleic acids) for dead cells and flow cytometry. While flow cytometry mirrored the precise ratios, CyTOF gave lower dead cell counts (Supplementary Figure S2C). Up to now the dye was only applied to cell-wall-free cells such as human cells effectively (Majonis et al., 2011; Fienberg et al., 2012). Bacterial cells, however, have rigid cell walls, subsistent also in dead cells, forming a barrier which must be overcome by cisPt. It can be assumed that the limitation in the quantitative detection of dead cells in a population may be caused by this boundary. Nevertheless, despite the determination of dead cell numbers by cisPt in a population of P. putida KT2440 was lower in comparison to PI staining (Supplementary Figure S2C: k_cisPt = 0.45, k_PI = 0.98), the stable isotopes’ calibration of RR and cisPt proved to be useful cell markers. Standard-grown P. putida KT2440 cells (48 h) were represented in typical 2D-plots for RR staining (Figure 1B). The number of dead cells was 2.7% (Q2) measured by cisPt and mass cytometry (Figure 1C), and 4.1% (R2) measured by PI and flow cytometry (Figure 1D).

Pseudomonas putida KT2440 is known to react on various silver concentrations by changes in growth rates and live/dead cell ratios. The toxicity of silver was recently assumed to be caused by the ions only (Xiu et al., 2012; Giao et al., 2017; Juganson et al., 2017) but we detected an additional particle-related effect (Guo et al., 2017). We assumed that the typical fast aggregation of AgNPs to huge complexes in a nature-like environment within 30 min (Guo et al., 2017) might contribute to a further increase of dead cell counts due to their random attachment to bacterial cell surfaces and their thereby steady release of additional silver ions into those cells.

Thus, mass cytometry was involved in this study to clarify if the treatment of bacteria with either silver ions or AgNP (1) generates different frequencies of cells that contain silver which may cause different live/dead cell ratios and (2) produces disparate quantities of silver per cell suggesting that populations exposed to AgNP might contain cells with a higher silver load. To answer these questions P. putida KT2440 cells were treated with the respective EC₅₀ concentrations of 1.29 mg/L for AgNP-10 and 0.19 mg/L for AgNO₃ as the positive control. As determined before, the released ion concentrations were nearly similar because cell-free AgNP-10 dissolution kinetic curves inferred 0.08 mg/L silver ions released from AgNP-10 (1.29 mg/L) in comparison to 0.12 mg/L silver ions from AgNO₃ (0.19 mg/L) over a time period of 72 h (Guo et al., 2017). Standard-grown P. putida KT2440 without silver treatment served as the negative control. The growth curves (Figure 2, bottom) showed a delayed lag-phase for the AgNP-10 treated bacteria and a slower growth rate (μ = 0.14/h) for the AgNO₃ positive control in comparison to the negative control (μ = 0.18/h). CyTOF technology was used to mark all bacterial cells of those populations and to distinguish live from dead cells. The additional measured silver signal was used to determine the frequency of silver containing cells as well as the quantities of silver per cell via the intensity of the mass cytometric signal for silver (IAg).

After 72 h and in comparison to the negative control AgNP-10 treatment caused an increase of cisPt+dead cells by a factor of about 4 while the AgNO₃ positive control showed a factor of 2 (Figure 2), indicating that the particles caused higher frequencies of dead cells. Further, the frequency of cells that contain silver (Figure 2, dark gray bars) was also higher for the AgNP-10 treatment in comparison to the positive control while cells of the untreated negative control were nearly not loaded with silver (Figure 2). After 72 h AgNP-10 treatment 92% of cisPt+dead cells and 35% of cisPt-live cells were loaded with silver. Instead, the AgNO₃ positive control revealed 48% silver containing cells of the cisPt+dead and only 11% of the cisPt-live cells (Figure 2). The data show that AgNP-10 treatment caused the highest frequencies of dead cells in comparison to both controls after 72 h, and that almost all of the dead cells were loaded with silver. At the beginning of the exponential phase (12 h) where cells thrived at merely low numbers, live cells were still predominant but silver was loaded to any cell independent of its viability state. At longer exposure time the high frequency of silver loaded dead cells was almost consistent between 48 and 72 h. A similar tendency was observed within the larger fraction of surviving silver containing cells, indicating that the absorbed silver (ions) do not cause immediate cell death.

We also determined silver quantities per cell by measuring their IAg values which were different between AgNO₃ positive control and AgNP-10 treated cells (Table 1). Overall, AgNP-10 treatment led to much higher silver quantities per cell in comparison to the positive control. Our data also suggest that the AgNP-aggregates, formed asap from AgNP-10, were not present at all or at least in their entirety due to the stringent washing procedures before CyTOF measurement otherwise we would expect an elevated silver signal in the 1D-plot of the silver channel (Supplementary Figure S3). But the silver ions released earlier by AgNP-aggregates and then bound by the cells were obviously responsible for the higher silver contents in AgNP-10 treated cells. In addition, for AgNP-10 treated cells we found nearly identical IAg values for both cisPt-live and cisPt+dead cells when cells grew exponentially (12 h: 18.3 ± 0.6 vs. 17.1 ± 1.1, Table 1) which was different for early and late stationary cells where the IAg values were higher for the cisPt+dead cells (e.g., for 72 h: 1.6 ± 0.1 vs. 10.3 ± 0.6). For AgNO₃ treated cells the IAg values per cell were generally much lower and increased finally to only 3.0 ± 0.3 after 72 h for cisPt+dead cells. Thus, silver quantities per cell were higher under AgNP-10 treatment with the highest IAg values determined for cells from exponential phase independent if they were dead or alive.

The data of both the calculated frequencies in cell numbers and the silver quantities per cell follow the same trend. AgNP-10 treatment cause higher frequencies of dead cells, higher frequencies of silver affected cells and higher per-cell silver quantities whereby live and dead cells load silver in equal quantities and this preferably during exponential growth.

Independent from this, the population showed heterogeneous cell states with respect to silver quantities which might simply be caused by chemical equilibria of available free silver ions. Although we were not able to distinguish a silver signal related to cell-attached AgNP-aggregates or to distinguish aggregate-dissolved silver ions from free silver ions with CyTOF technology we assume that by a fast process an equilibrium is established and leads to the silver ions’ equal distribution among cells. When the equilibrium changes in favor of the cells, because of increasing cell numbers during growth, these mostly new live cells were found free of silver. This observation was made also with AgNO₃ treated cells. It can also be assumed that a continuous uptake of silver ions by the cells may influence the equilibrium between AgNP-aggregate bound and unbound silver ions in the medium and cause further dissolution of silver ions from the aggregates (Yue et al., 2017).

The equal silver quantities in both live and dead exponential grown cells were verified by sorting identical numbers (3 × 10⁶ cells each) of PI unstained/PI stained cells, from which an aliquot of cells of each sample were injected into the mass cytometer and 10⁶ cells of each sample was used for ICP-MS measurements. As before, the cells were treated by AgNP-10 at EC₅₀ dosage of 1.29 mg/L. From CyTOF analysis we found analogous silver quantities per cell among PI-live and PI+dead cells with IAg = 21.6 and IAg = 17.9, respectively. The equal silver quantities per cell were by tendency confirmed by the ICP-MS results with 1.5 fg and 1.1 fg silver per PI-live and PI+dead cells, respectively. Overall, the equal silver quantity distribution among live and dead cells suggested that random AgNP-aggregate attachment is not only affecting and toxic to an appended cell but also to every other cell in its vicinity.