Cu Nanoparticles Have Different Impacts in Escherichia coli and Lactobacillus brevis than Their Microsized and Ionic Analogues.

Bacterial strains

Two bacterial strains were used in this study: Escherichia coli strain ATCC 25922, a standard strain widely used for antimicrobial disk susceptibility tests, and Lactobacillus brevis strain (Orla-Jensen) ATCC 14869. Chemicals and media components used in these studies are detailed in the Supplemental Materials and Methods section of the Supporting Information . Physiochemical characterization of copper species The copper species tested herein were obtained from a variety of sources as described in Table S1 . Filtered deionized water was used to make stock solution at 20 mg/mL. Dynamic light scattering (DLS, ZetaPALS, Brookhaven Instruments Ltd., UK) was utilized to analyze the average size and size distribution of copper species (50 µg/mL) in water, E. coli media (MMD) and L. brevis media (Lactobacilli MRS broth). The ζ-potential values of each Cu species in aqueous solution were determined using a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Ltd, UK).

Transmission Electron Microscopy

(TEM) was used to determine the primary size and morphology of the particles. To prepare samples for TEM, a drop of each Cu species in purified water was applied to carbon-coated TEM grids and evaporated at room temperature. Images were taken with a JEOL 1200 EX TEM microscope. The percent dissolution of each of the Cu species in water and bacterial media was measured by quantifying dissolved Cu by ICP-OES (ICPE-9000 plasma atomic emission spectrometer, Shimadzu). For the analysis, 1 mg/mL of Cu species was suspended in water or bacterial growth media to yield a final volume of 1 mL for 24 hours and then was centrifuged at 15,000 rpm for 30 minutes to precipitate any remaining particles. The supernatant was collected and transferred to a clean tube for acid digestion. 10 mL of nitric acid (HNO 3 , 65–70%, Trace Metal Grade) was added to the supernatant before incubating in a HotBlock (SC100, Environmental Express) at 80°C for 6 hours. The temperature was then raised to 95°C overnight to evaporate all liquid present in sample. The dried sample was allowed to cool down at room temperature before being dissolved in 2% (v/v) nitric acid at 80°C for 3 hours. The extract was transferred to a 15 mL ICP-OES analysis tube to measure Cu ion concentration. Growth inhibition effects and IC 50 calculation To assess the half-maximal inhibitory concentrations (IC 50 ), a growth inhibition curve was constructed for each Cu species. A 20 mg/mL stock of Cu species was diluted to 10 mg/mL with 2X media (MMD for E. coli and Lactobacilli MRS broth for L. brevis ). Humic acid was added to MMD media as a dispersing agent and was chosen because of its relevance for environmental systems. In this experiment, humic Acid (HA) was added to a final concentration of 0.01 mg/mL and then the resulting mixture were sonicated in water bath (Branson 2510, CT, USA) for 15 minutes at room temperature. 10 mg/mL of each Cu species was then diluted with 1X media supplemented with 0.01 mg/mL HA to a step-wise concentration gradient at 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 mg/mL. 50 µL of NP at each concentration were pipetted into 384-well polystyrene microplates. Nine replicates were performed for each concentration. In a separate plate, 50 µL of a log-phase bacterial culture (OD 600 between 0.5 – 0.7) was pipetted into the 384-well plate and then a plastic 384 pin replicator (Genetix Molecular Devices) was used to inoculate bacteria from this plate to the plate containing the serial dilution of Cu species. Sterility and blank controls (bacterial media with no inoculation) were also included for each concentration. A Biotek Synergy plate reader (BioTek, VT) was used to monitor OD 600 every 30 minute at 37°C for 24 hours. A growth curve was constructed using equation: Growth ( % ) = A Np , B − A Np , A A Bl , B − A Bl , A × 100 In the above equation, A Np,B is the absorbance of the bacterial culture in the presence of each concentration of Cu NPs (average of 9 replicates); A Np,A is the absorbance of the Cu NPs at the respective concentrations which contain no bacteria (average of 3 replicates); A Bl,B is the absorbance of the bacterial culture in blank (no Cu species) media (average of 9 replicates), and A Bl,B is the absorbance of media with no bacteria (average of 3 replicates). The growth inhibition curve was plotted using the program Origin version 9 (OriginLab Corporation) using the category Growth/Sigmoidal, function Logistic. The IC 50 and standard error were calculated for each data set using the same program. In vitro assays In vitro DNA damage assay Purified plasmid pUC19 (Thermo Scientific, catalog #SD0061) was incubated in the presence of 100 mg/L Cu species for 24 hours in purified water. Centrifugation at 15000 rpm for 30 minutes was used to separate plasmid DNA from residual Cu NPs. The supernatant containing the plasmid DNA was collected and loaded into a 1.2% Trisacetate EDTA buffer, TAE agarose gel. Electrophoresis was performed at 5 V/cm for 1 hour and the resulting gel was stained with ethidium bromide for 30 minutes. A Bio-Rad FX™ imaging system was used to image the gel; the band intensities were quantified using QuantityOne™ software. To linearize the pUC19 (first positive control), the restriction enzyme, PstI was incubated with the plasmid at 37° C for 1 hour. To induce random nicking and DNA fragmentation (second positive control), the plasmid was treated with a xenon arc UV-B lamp (Asahi Spectra, LAX-Cute) for 10 minutes at 1000 mJ/cm 2 . For the negative control, pUC19 was loaded into the electrophoresis gel whereas the majority of the plasmid as purchased was in the supercoiled-formed of DNA. Determining cell-associated Cu using sucrose gradient centrifugation and ICP MS E. coli and L. brevis were treated with 0.5 and 1 mg/L of a series of the Cu species. After 24 hours, the cells were washed 2 times with PBS and then sucrose gradient centrifugation was used to separate the cells from residual particulate Cu. To make the sucrose gradient, 0.3, 0.4 and 0.6 g/mL of sucrose (Sigma-Aldrich) were completely dissolved in water and filtered with a 0.22 µm Millipore filter. 44 1.2 mL of each sucrose concentration was carefully layered into a 15 mL Falcon tube and then 1 mL of cell suspension was placed on top of the gradient. The mixture was centrifuged (Eppendorf 5810 R) at 2916×g for 5 minutes at room temperature. After centrifugation, a brown band of cells was clearly visible at the upper part of the gradient and Cu species were visibly precipitated at the bottom of the tube. (See Figure S4 .) 1.2 mL of cells suspension were collected. To determine the total number of bacterial cells, a standard curve between OD 600 and the number of cells was constructed ( Figure S5 , A for E. coli and B for L. brevis ). The total amount of cell-associated Cu was normalized as the Cu content per 10 9 cells for each bacterial species. The number of cells was determined by measuring the OD 600 of 200 µL of this suspension. To determine the amount of cell-associated copper, 1 mL of the suspension was digested with 5 mL of pure HNO 3 for overnight and then was evaporated at 95°C until no liquid remained. 5% HNO 3 was used to resuspend the sample. Cu content was analyzed using ICP-OES (ICPE-9000, Shimadzu). Determining bioavailable, intracellular Cu with biosensor strain of E. coli The amount of bioavailable, intracellular Cu was determined using a genetically engineered E. coli biosensor strain in which bioluminescence specifically responds to Cu cellular bioavailability (MC1061 pSLcueR/pDNPcopAlux, “Cu-inducible strain”). The luminescent strain was constructed as previously described in Bondarenko et al . 17 A colony of the bacteria was inoculated into 3 mL of fresh LB media supplemented with 100 mg/L ampicillin and allowed to grow overnight before being diluted 1:50 using fresh LB media with antibiotic. The culture was allowed to grow to reach log phase (OD 600 of 0.6) before the cells were harvested for the experiment. Growth was conducted at 30 °C, shaking at 200 rpm. 25 µL of the Cu species in MMD supplemented with 0.01 mg/mL humic acid were mixed with twenty-five µL of the biosensor bacteria in a 384-well plate to yield a final bacterial OD 600 of 0.1. The plate was kept at 30 °C for 2 hours to allow for luminescence induction, at which point the luminescence was quantified using a micro-plate reader (SpectraMax MS, Molecular Devices, CA).

Microscopy of bacterial cells exposed to Cu species

TEM sample preparation and microscopy

E. coli cells that had been treated with 0.1 mg/mL Cu species for 24 hours were washed 3 times with phosphate-buffered saline (PBS) before being fixed with 2% glutaraldehyde in 0.1 M PBS. Cells were washed with PBS 3 times before treating with osmium tetroxide (OsO 4 ) in PBS for 1 hr. After rinsing 3 times with PBS, the cell pellet was dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol for two hours each). Propylene oxide was used to remove any residual ethanol before the pellet was embedded in Epon resin (Sigma Aldrich). A Reichert-Jung Ultracut E ultramicrotome was used to cut 100 nm thick sections which were placed onto 1GG200 gold grids (Ted Pell Inc.). Sections were stained with uranyl acetate and Reynolds lead citrate, and examined on an FEI T12 transmission electron microscope operated at 80 kV in the Electron Imaging Center for Nanomachines (EICN) at UCLA.

Electron tomography and 3D reconstruction

E. coli cells that had been treated with Cu NPs for 24 hours were washed, dehydrated and embedded in Epon resin as described above. A Reichert-Jung Ultracut E ultramicrotome was used to make 250 nm thick sections from the block, which were placed onto Maxtaform 75/300 Rectangular Mesh copper grids (Ted Pell Inc.). An FEI Tecnai F20 transmission electron microscope was used to capture the tomography tilt series. A Gatan 626 cryo specimen holder was used to collect images in tilt angles ranging from −70° to + 70°, with a 1° increment. The tilt series of 141 images were used to reconstruct 3D volumes using the Etomo tomography processing software in the Imod package (Boulder Laboratory for 3-D Electron Microscopy of Cells). 45 Confocal microscopy E. coli and L. brevis were treated with 50 mg/L of n-FITC-CuO for 24 hours before being washed 3 times with PBS. The bacteria were fixed using 4% paraformaldehyde for 20 minutes at room temperature. After another 3 washes with PBS, the cells were stained with Hoechst 33342, which specifically stains bacterial DNA, yielding blue fluorescence. After 1 hour of incubation with Hoechst dye, the cells were washed 3 times and placed into an 8-well chamber slide (Lab Tek) before being visualized under a confocal microscope (Leica Confocal SP2 1P/FCS).For Hoechst dye, the excitation wavelength used was 358 nm, and E max was monitored at 420–500 nm. For FITC, the excitation wavelength used was 488 nm, and E max was monitored at 520–580 nm. Safe Handling of Nanomaterials Nanoparticles as dry powders were handled in a chemical fume hood or powder enclosure, and manipulated while the researcher was wearing a N95 filer mask. After suspension in aqueous solutions, standard good chemical hygiene practices were employed. Sonication can result in aerolization and thus was only performed on solutions that were in closed containers. More detailed recommendations are available in the Nanotoolkit developed by the California Nanosafety Consortium of Higher Education which is available online at : http://www.cein.ucla.edu/new/p155.php

Safe Handling of Nanomaterials Nanoparticles as dry powders were handled in a chemical fume hood or powder enclosure, and manipulated while the researcher was wearing a N95 filer mask. After suspension in aqueous solutions, standard good chemical hygiene practices were employed. Sonication can result in aerolization and thus was only performed on solutions that were in closed containers. More detailed recommendations are available in the Nanotoolkit developed by the California Nanosafety Consortium of Higher Education which is available online at : http://www.cein.ucla.edu/new/p155.php