Unrevealing the effects of food-related engineered nanoparticles on the intestinal biofilm

1. Introduction In accordance with the PhD thesis project main goals, this poster reports the main results of the first-year activities concerning: (A1) The selection of a growth medium that can guarantee a constant concentration of dissolved silver nanoparticles (AgNPs). (A2) The effects of different concentrations of AgNPs on bacterial planktonic growth in order to choose sub-lethal concentrations to be later tested on the complex in-vitro model. 2. Materials and Methods Six bacteria from human gut were chosen as representatives of gut microbiome to perform the experiments: Bacteroides thetaiotaomicron DSM 2079, Faecalibacterium prausnizii DSM 17677, Bifidobacterium longum subsp. longum DSM 20219, Lactobacillus acidophilus DSM 20079, Bifidobacterium adolescentis DSM 20083, Escherichia coli MG 1655. Bacillus subtilis subsp. subtilis (natto) ATCC 6051 was used as probiotic model (Charteris et al., 1998; Turroni et al., 2009; Sproule-Willoughby et al., 2010). Tryptic Soy Yeast broth (TSYb) (Sproule-Willoughby et al., 2010), Tryptic Soy Broth (TSB), Luria Bertani (LB) were selected as possible media to perform the project experiments. In order to test the AgNPs stability in the three media, AgNPs (NanoComposix, citrate suspended nanoparticles, 10 nm diameter) were added to 1 mL of sterile TSYb, TSB and LB to reach the final concentration of 50 µg/mL. The media were incubated in anaerobic condition at 37°C. Every two hours, 100 µL of every solution were diluted in 900 µL of MilliQ water to reach the AgNPs concentration of 5 µg/mL, suitable to obtain clear spectra. At each time the absorbance between 200 and 500 nm was measured by the JENWAY 7315 Spectophotometer and spectra were obtained until 24 hours. Control samples without AgNPs for each media were also prepared and spectra were recorded at the same experimental conditions. The expected peak at 390 nm of AgNPs in solution was compared with those of the negative control without nanoparticles. In order to test the ability of AgNPs to affect planktonic growth of E. coli and B. subtilis in anaerobic condition, both bacteria were cultured in pre-reduced TSB medium supplemented with 0 (negative control), 0.01, 0.1, 1 µg/mL of AgNPs. Cultures were obtained in 1.5 mL tubes containing 0.5 mL of TSB inoculated with 5 µl (5 % vol/vol) of an overnight culture (final concentration 105 cells/mL) and grown for 24 hours in anaerobic conditions at 37°C. The negative control was the medium without bacteria incubated at the same conditions. Absorbance at 600 nm (A600) after 24 hours was measured by the Infinite F200 PRO reader (TECAN). Three replicates were performed for each experimental conditions and the experiment was repeated at least two times. To confirm the previous experiment, the bacterial cellular activity with and without AgNPs was evaluated by measuring ATP bioluminescence. The experiment was performed by the Biomass Test Kit (Promicol BV, Nuth, The Netherlands) according to the manufactory instruction and using white 96-well microplates (Greiner Bio-one). The luminescence was measured for 1 s in an Infinite F200 PRO reader (TECAN). A standard curve was used to calculate the pmole of ATP. Four replicates were performed for each experimental conditions and the experiment was repeated at least two times. Analysis of variance (ANOVA) was applied via a software run in MATLAB (Version 7.0, The MathWorksInc, Natick,USA) to statistically evaluate any significant differences among the samples. 3. Results and Discussion The stability of diluted AgNPs at the concentration of 50 µg/mL in TSYb, TSB and LB media was investigated. The TSYb–AgNPs spectrum highlighted a precipitation of the AgNPs immediately after their addition. Indeed, in the TSYb, precipitated AgNPs are even well-visible at the bottom of tube after 6 hours of incubation. The TSB-AgNPs spectrum showed an initial slight decrease of the 390 nm peak that becomes stable after 8 hours of incubation, suggesting a constant solubility of AgNPs in time. Despite the LB-AgNPs spectrum showed a very high 390 nm peak at time 0, indicating the best AgNPs dispersion, there was a relevant progressive decrease of AgNPs peak, indicating a sensible loss of AgNPs solubility. In conclusion, TSB resulted the medium where AgNPs concentration was more stable in time. Thus TSB was chosen as the best medium for the following experiments. The ability of AgNPs to affect both E. coli and B. subtilis planktonic growth in TSB medium was evaluated both measuring the A600 and the ATP cellular content with 0, 0.01, 0.1, 1µg/mL AgNPs after 24 hours. The mean A600 values of the 24 hours cultures showed no significant differences among the samples, suggesting that each of tested concentration of AgNPs does not affect planktonic growth. Moreover, to confirm data obtained by the previous experiment, the toxic potential of AgNPs was evaluated by measuring the intracellular ATP content. Results showed no statistical significant reduction of ATP concentration in both microorganisms at all the AgNPs tested concentrations, in comparison to the control samples. Thus AgNPs did not exert any toxic effect on both bacteria at any of the of the tested concentrations. In conclusion, on the basis of these results, 1 µg/mL AgNPs was chosen as the sub-lethal concentration for all the following experiments, as we demonstrated to be the maximum concentration not affecting both E. coli and B. subtilis growth. 4. 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