Effect of long-term exposure to silver nanoparticles on gut microbiota reshaping the gut

The presentation shows an example of the approaches the doctoral students presented to the committee members after submitting the written proposal exam (NIH grant) and make sure all the writing styles meet the CEMB requirements as well as members of committee after that the doctoral students and members agreed to the oral exam and discussed the proposal. Hint: all the ideas based on the critical thinks that students should develop within the two years after starting the doctoral department program. All doctoral students should be creative with their thinking to initiate the idea or the project to pass the candidacy exam.

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1. Effect of long-term exposure to Silver nanoparticles on gut microbiota reshaping the gut Presented by Suad Algarni Doctoral Student at CEMB program Fall 2017
2. Outline • Introduction • Background • Materials & Methods • Expect results • Conclusion
3. Introduction • Silver Nanoparticles: ²Antimicrobial agent (E174) ²Alginate gel coatings of carrots and asparagus. ²Alternatives to antibiotics. ²Topical wound dressing. ²Food packaging. ²Dietary health supplement. Fröhlich EE et., 2016 Int.J.Mol. Sci U.S. Silver Nanoparticles Market size, by application, 2013-2024 (USD Million)
4. Schematic of antimicrobial activity of AgNPs Ag+ Ag+ Ag+ Fröhlich EE, Fröhlich E. Cytotoxicity of Nanoparticles Contained in Food on Intestinal Cells and the Gut Microbiota. Routledge M, Yan B, eds. International Journal of Molecular Sciences. 2016;17(4):509. doi:10.3390/ijms17040509.
5. What makes silver nanoparticles different in term of toxicity? qSize of particles (>30 nm) qShape (Nano spheres (AgNS),nanocubes (AgNC) qDose (low & high) Javurek, A. B., Suresh, D., Spollen, W. G., Hart, M. L., Hansen, S. A., Ellersieck, M. R., Rosenfeld, C. S. (2017). Gut Dysbiosis and Neurobehavioral Alterations in Rats Exposed to Silver Nanoparticles. Sci Rep, 7(1), 2822. doi:10.1038/s41598-017-02880-0
6. Test material Characterizations of AgNPs: qTransmission Electron Microscopy (TEM) qDynamic Light Scattering (DLS) TEM DLS Williams,et al, 2014
7. Exposure to Silver Nanoparticles Direct Indirect
8. Background:
9. Gut microflora
10. The mucus layers of the small intestine and colon Donaldson, Gregory P., S. Melanie Lee, and Sarkis K. Mazmanian. “Gut Biogeography of the Bacterial Microbiota.” Nature reviews. Microbiology 14.1 (2016): 20–32. Antimicrobial peptides produced by Paneth cells at the base of the crypts secreted immunoglobulin A (sIgA) in both the small intestine and colon; and a steep oxygen gradient that influences which bacteria are capable of surviving close to the epithelial surface. A) The surface of the small intestine is shaped into villi and crypts and is colonized by certain adherent species, including segmented filamentous bacteria (SFB), Lactobacillaceae and Helicobacter spp. B) The colon has two distinct mucus structures: the outer layer is colonized by mucin-degrading bacteria and is characterized by the presence of Bacteroides acidifaciens, Bacteroides fragilis, Bifidobacteriaceae and Akkermansia muciniphila.
11. Gut microhabitats as reservoirs of bacterial diversity Donaldson, Gregory P., S. Melanie Lee, and Sarkis K. Mazmanian. “Gut Biogeography of the Bacterial Microbiota.” Nature reviews. Microbiology 14.1 (2016): 20–32. Crypt + Mucosa Specific niches such as crypts, the inner mucus, and the appendix may be crucial to facilitate immune homeostasis, to protect microbial inhabitants from competitors, and to re-populate the gut following perturbations that alter bacterial community structure or deplete certain species from the lumen. A) A subset of species is able to penetrate the inner mucus layer and enter crypt spaces. B) Environmental challenges such as diet perturbations, abnormalities in gastrointestinal motility, and antibiotic consumption massively alter the lumen community. However, the more stable mucosal environment and crypts protect important bacterial species. C) The crypts and mucosa serve as reservoirs to repopulate the lumen.
12. Gut Dysbiosis q Dysbiosis is an imbalance between beneficial microorganisms like Lactobacillus or Bifidobacterium and harmful microorganisms like Escherichia coli. q Dysbiosis a change in the abundance or diversity of some groups of microorganisms, a change in community composition caused by lifestyle.
13. The problem statement • It is unclear how chronic exposure to AgNPs would affect the gut microbiome and gender influences.
14. Central hypothesis • Hypothesis: • 1) A specific size of silver nanoparticles (10 nm) could alter the gut microbiome composition after long-term AgNP exposure in a gender-specific. • 2) Use of a fecal microbiota transplant could restore the gut microbiota composition of exposed mice.
15. Aim 1 • To examine the long-term exposure to silver nanoparticles have gender-specific effects on the mouse gut microbiome composition. AgNPs chronic exposure Change the gut microbial composition Gender specific? Male Female
16. Materials & Methods • Experimental design:
17. Materials & Methods • Synthesis silver nanoparticles and prepare dosage: • A colloidal silver nanoparticles • 2 ml, 5mM AgNO3 heat in the boiling water, add 5 drop of 1% sodium citrate and continue heat to form silver nanoparticles (Yellow). 10 nm
18. Materials & Methods • Bacterial DNA extraction
19. Materials & Methods • PCR amplification and sequencing Illumina ,Miseq-16S.PDF
20. Materials & Methods 16srRNA fast Q-file Quality control Trimmed and filtered OTU Figure 2. Ecological representations of microbial communities Morgan,et al,2012
21. Microbiome data analysis Beta diversity analysis reveals that diet, gender, and stress significantly alter the composition of the gut microbiota. (a) Principle coordinate analysis (PCoA) based on Bray-Curtis dissimilarity of all OTUs from all the groups on Day 0, 81 and 136. (b) Clustering of gut microbiota based on distance between different groups calculated by MANOVA testing of the first 20 principal coordinates (accounting for 80% of total variation). (c) Comparison of the Bray-Curtis distance between NCD and HFD at each time point. (d) Comparison of the Bray-Curtis distance between females and males at each time point. All data is showed as mean ± SE For (c and d) the tests of significance were performed using a two-sided Student’s t-test with Bonferroni- correction Bridgewater LC, Zhang C, Wu Y, et al. Gender-based differences in host behavior and gut microbiota composition in response to high fat diet and stress in a mouse model. Scientific Reports. 2017;7:10776. doi:10.1038/s41598-017-11069-4.
22. Materials & Methods Ø (PCoA) based on Bray- Curtis distance MANOVA Random forest Mean decrease in accuracy greater than 0.002 Pull the key OTU Bridgewater LC, Zhang C, Wu Y, et al. Gender-based differences in host behavior and gut microbiota composition in response to high fat diet and stress in a mouse model. Scientific Reports. 2017;7:10776. doi:10.1038/s41598-017-11069-4.
23. Expected Results Key phylotypes of gut microbiota showed differences between female and male mice Bridgewater LC, Zhang C, Wu Y, et al. Gender-based differences in host behavior and gut microbiota composition in response to high fat diet and stress in a mouse model. Scientific Reports. 2017;7:10776. doi:10.1038/s41598-017-11069-4. Key phylotypes were identified as those OTUs with a value of discrimination mean decrease in accuracy greater than 0.002 in Random Forest models of female and male mice on Day 0. The heat map shows the abundance (log 2 transformation) of OTUs.
24. Fecal Microbiota Transplant • Fecal microbiota transplant is efficacious therapy for reshaping the composition of the gut microbiota. • FMTs become acritical tool to limit the spread of antibiotics resistance. Manichanh, C., Reeder, J., Gibert, P., Varela, E., Llopis, M., Antolin, M., . . . Guarner, F. (2010). Reshaping the gut microbiome with bacterial transplantation and antibiotic intake. Genome Res, 20(10), 1411-1419. doi:10.1101/gr.107987.110
25. Aim2 • Examine whether fecal transplants reshape the bacterial community of each GI tract region after AgNPs exposure.
26. Materials & Methods • Study design:
27. Materials & Methods o Prepare fecal microbiota transplant dose: o The cecum of healthy control donor will be dissected along its length and the materials are transferred to a sterile tube and re-suspended in 40 volumes of pure water. o The 200 ul of this suspension will be given to each exposed recipient via oral gavage, o Selected based on the phylotype bacterial richness. o 200 species-level phylotypes of the beneficial bacteria
28. The dynamics of OTUs significantly correlated with FMT after AgNPs exposure OTU-14 OTU-16 OTU-6 AgNPs FMT AgNPs+FMT Day 140 Day 30 Significantly negatively or positively correlate with FMT treatment on days 30.
29. Conclusion • Long term expousre of Ag NPs & Gender-specific phynotype experiment: ² Silver nanoparticles alter the microbiome composition and treated female mice is more significant than in male mice. • Each GI tract in response to FMT experiment: ² One or more OTUs in the family level could significantly negatively or positively correlate after FMT treatment on days 30.