Gentamicin B

ENMs and reagents. The CPPs obtained ZnO from Meliorum Technologies Inc. (Rochester, NY). TiO₂-P25 (81% anatase and 19% rutile) was purchased from Evonik (Parsippany, NJ); TiO₂-A was provided by P. Biswas (Washington University, St. Louis, MO); and the CPPs prepared the TiO₂-NBs as previously described (Hamilton et al. 2009 (link)). The CPPs obtained the O-MWCNT stock in powder form from Cheap Tubes Inc. (Brattleboro, VT); obtained the P-MWCNT by treating O-MWCNT with dilute acids, chelating agents, and mild conditions to minimize oxidized or damaged tubes; and created F-MWCNT through further acid treatment of P-MWCNT, which introduced carboxyl groups on 5.27% of the carbon backbone (on a per weight basis) (Chen and Mitra 2008 ; Wang et al. 2011 (link)).
The CPPs purchased low-endotoxin bovine serum albumin (BSA) from Gemini Bio-Products (West Sacramento, CA); dipalmitoylphosphatidylcholine, phorbol 12-myristate, 13-acetate (PMA), and lipopolysaccharide (LPS from Escherichia coli 0127:B8) from Sigma-Aldrich (St. Louis, MO); and 1,25-dihydroxy-vitamin D₃ from EMD Millipore (Billerica, MA). The CPPs purchased the cytotoxicity assays CellTiter 96 (MTS assay) and CytoTox 96 [LDH (lactate dehydrogenase) assay] from Promega (Madison, WI).
Preparation of ENMs in cell culture media. The CPPs prepared ENM stock solutions (5 mg/mL) from dry powder using endotoxin-free sterile water and then prepared all ENM suspensions in cell culture media using the stock solutions as needed. Briefly, the CPPs vortexed and then sonicated ENM stock solutions (with the exception of TiO₂-NB, which was stirred to prevent mechanical shear) using a water bath sonicator or cup horn sonicator (depending on laboratory availability) immediately before diluting the solutions into complete cell culture media.
Cell culture and co-incubation with EMN. The CPPs grew all cells at 37°C in a 5% CO₂ atmosphere. RLE-6TN cells, a rat alveolar type II epithelial cell line, from American Type Culture Collection (ATCC; Manassas, VA) were cultured in Ham’s F12 medium (ATCC) supplemented with l-glutamine, bovine pituitary extract (BPE), insulin, insulin growth factor (IGF)-1, transferrin, and epithelial growth factor (EGF), supplemented with 10% fetal bovine serum (FBS). THP-1 cells, a human acute monocytic leukemia cell line (ATCC) were cultured in HEPES-buffered RPMI 1640 supplemented with l-glutamine (Mediatech, Corning, NY), 0.05 mM β-mercaptoethanol, and 10% FBS (PAA Laboratories, Dartmouth, MA). BEAS-2B cells (ATCC) were cultured in bronchial epithelial growth medium (BEGM) obtained from Lonza Inc. (Walkersville, MD) supplemented with BPE, insulin, hydrocortisone, human EGF, epinephrine, triiodothyronine, transferrin, gentamicin/amphotericin-B, and retinoic acid. For the THP-1 differentiation performed in the first series of experiments (phase I), the CPPs pretreated cells with 1.62 µM (1 µg/mL) PMA for 18 hr. However, the CPPs identified excessive cell clumping and cell death during the phase I studies. Therefore, the CPPs alternatively pretreated THP-1 cells with vitamin D₃ at 150 nM overnight and then 5 nM PMA in order to obtain the differentiated macrophage-like cells used during the second series of experiments (phase II). For the IL-1β release, co-culturing THP-1 cells with 10 ng/mL LPS was necessary to initiate transcription of pro-IL-1β. The CPPs initiated aggressive phagocytic activity by adding PMA just before particle exposure.
Before ENM exposure, the CPPs cultured aliquots of 1.5 × 10⁴ cells (for THP-1 cells, 10⁵ cells were seeded into each well of a 96-well plate) in 0.2 mL of the cell culture media in 96-well plates (Costar, Corning, NY) at 37°C for 24 hr. The CPPs freshly prepared all of the ENM suspensions at final concentrations of 10, 25, 50, and 100 µg/mL in the cell culture media. After exposure of the cells to the ENMs for 24 hr at 37°C, the CPPs collected supernatants to measure LDH and IL-1β production then used the remaining cells to test cellular viability by MTS assay.
Physicochemical characterization of ENMs. The CPPs identified the primary particle size and morphology of the ENMs by using a transmission electron microscope (TEM; model 100CX) and a scanning electron microscope (SEM; model JSM-7600F) (both from JEOL Ltd., Tokyo, Japan). In addition, the CPPs characterized the particle hydrodynamic size in H₂O and cell culture media using dynamic light scattering (DLS) (Ji et al. 2010 (link)). The CPPs characterized particle crystallinity and structure using X-ray diffraction measurements and measured particle surface area by Brunauer–Emmett–Teller (BET) surface area analysis. The CPPs performed zeta-potential measurements of the ENM suspensions using a ZetaSizer Nano-ZS instrument (Malvern Instruments, Worcestershire WR, UK). Finally, the CPPs determined the elemental composition of the particles as well as ZnO dissolution rate using inductively coupled plasma mass spectrometry (ICP-MS) (model SCIEX Elan DRCII; PerkinElmer, Norwalk, CT).
Endotoxin analysis of ENMs. CPPs measured the endotoxin content of ENM stock suspensions, as well as dispersions in PBS and tissue culture media, using the colorimetric Limulus amebocyte lysate assay (Lonza Inc.). The LPS content of all ENM suspensions was < 0.3 EU/mL.
Determination of cell viability. The CPPs determined cellular viability using MTS (CellTiter 96) and LDH (CytoTox 96; both from Promega) according to the manufacturer’s protocols. To avoid the interference created by ENMs while measuring formazan absorbance at 490 nm, the CPPs introduced a centrifugation (2000 × g for 10 min) procedure in phase II experiments to collect particles in the wells after incubation with the MTS reagents. CPPs then followed this centrifugation step with a brief mixing and transfer of the supernatant to a new 96-well plate before measuring the formazan absorbance at 490 nm. The CPPs eliminated interference of any residual LDH in FBS by heat-inactivation (70°C water bath for 5 min).
ELISA for IL-1β quantification. The CPPs determined IL-1β production in the THP-1 culture supernatant using a human IL-1β ELISA kit (R&D Systems Human IL-1β DuoSet™; R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
Statistical analysis. The CPPs used the two-way analysis of variance followed by Tukey or Bonferroni correction for multiple comparisons of means for statistical analysis of responses across ENMs and cell lines. In order to define interlaboratory comparisons across two harmonization rounds, the CPPs conducted a meta-analysis of LDH, MTS, and IL-1β assays across eight different laboratories for three cell lines (BEAS-2B, RLE-6TN, and THP-1) exposed to several ENMs (TiO₂-P25, TiO₂-A, TiO₂-NBs, ZnO, O-MWCNT, P-MWCNT, and F-MWCNT). The CPPs combined information within assays and cell lines using a robust two-stage hierarchical model of toxicity. For all quantities of interest, the CPPs obtained Monte Carlo inference by implementing a custom Gibbs sampler in the R computing environment (R Foundation for Statistical Computing, Vienna, Austria). To normalize data, the CPPs subtracted background negative control values (MTS, LDH, and IL-1β) and provided adjustments for positive control values in the case of LDH assays. Details about the statistical model used for analysis are provided in Supplemental Material, p. 8 (http://dx.doi.org/10.1289/ehp.1306561).

Minimum inhibitory concentrations (MICs) of MTC, PEN, and antibiotics against selected P. aeruginosa strains were determined in 96-well microplates as previously described [22 (link)]. The effect of combinations of MTC with PEN or gentamicin against selected P. aeruginosa strains was carried out by checkerboard assays in 96-well microplates. Briefly, plates were prepared by making doubling dilutions of MTC in MHB followed by subsequent addition of PEN or gentamicin. Wells were then inoculated with 1.0 × 10⁶ cfu/mL of P. aeruginosa cells and microplates incubated at 37 °C. After 24 h, each well was scored for visible growth and fractional inhibitory concentration index (FICI) values were calculated for each combination tested. The FICI value was calculated using the equation FICI = Ac/MICA + Bc/MICB, where Ac is the concentration of compound A when combined with compound B; MICA is the MIC of compound A alone; Bc is the concentration of compound B in combination with compound A; and MICB is the MIC of compound B alone. Synergy was defined at the point at which the FICI was ≤0.5. An additive effect was defined if the FICI was >1 and antagonism if it was ≥ 4 [23 ]. Selected P. aeruginosa strains were tested in duplicate.

Overnight cultures of genetically modified thymidine-auxotrophic B. thetaiotaomicron strains were diluted 1:100 in fresh TYG medium with thymidine, and E. coli S17-1 λ pir strains bearing plasmids with the thyA gene and an erythromycin resistance gene were diluted 1:100 in fresh in LB medium with carbenicillin. The cultures were grown anaerobically for B. thetaiotaomicron and aerobically for E. coli at 37 °C to an optical density of 0.4–0.9 at 600 nm, then harvested by centrifugation at 3200 × g for 10 min, washed in PBS twice, and resuspended in the fresh TYG medium with or without thymidine. The optical density of the cell suspension was adjusted to 16 with the medium, and B. thetaiotaomicron and E. coli were mixed at equal volumes. Then, 50 to 100 μL of the mixtures were transferred onto a 0.45 μm filter disc placed on TYG agar plates, with or without thymidine, and incubated anaerobically at 37 °C overnight. To evaluate conjugation efficiency, cells were washed off the filter and resuspended in BHIS medium with gentamicin and thymidine or in TYG medium with gentamicin. The cell suspension was plated on BHIS agar plates with gentamicin, thymidine, and erythromycin or on TYG agar plates with gentamicin. Total CFU/mL of viable cells after conjugation were determined by plating the cell suspension on BHIS agar plates containing gentamicin and thymidine or TYG agar plates containing gentamicin and thymidine. The colonies were counted manually 4 days after culturing.
DNA was purified from transconjugants grown on BHIS agar plates with gentamicin, thymidine, and erythromycin after conjugation, using the DNeasy Blood & Tissue Kits (Qiagen). PCR was performed with primers thyA-F and thyA-R to detect the thyA gene. Primers named qPCR-EmR-F and qPCR-EmR-R were used for the EmR gene (Supplementary Table 6). Whole genome sequencing was also performed with the purified DNA to check for the integration of the plasmid bearing the intact thyA gene.