EFFECTS OF SILVER NANOPARTICLES ON IN VITRO GUT MICROBIAL MODELS AND OTHER ANAEROBIC ENVIRONMENTS

The impacts of AgNPs on both natural and engineered ecosystems is a topic of outstanding importance having socio-economic consequences on medical, industrial and environmental fields. Silver nanoparticles are widely used as antimicrobial agents in consumer products for domestic, environmental, medical, and industrial applications. Release of AgNPs from nanoenabled products has been observed, and the potential impacts of such releases on a wide variety of organisms at many trophic levels have been recognized. However, there has been little exploration of the impact of AgNPs on the microflora associated with living organisms and their environments. For instance, of special interest is the effects on the human microbiota considering the range of consumer goods that could be directly or indirectly ingested. Furthermore, most of the studies concerning toxic effects of AgNPs on biologic systems consider high NPs concentrations, while the effects of real environmental and dietary concentrations are still poorly investigated. Thus, the principal aim of my PhD project was to provide science-based evidence needed to elucidate the effects of sub-lethal concentrations of AgNPs on bacterial ecosystems, with the final goal of creating the scientific know-how to master biological processes and develop leading edge methodologies vital for the nanosafety assessment. In particular, I focused my attention on studying the response of in vitro gut microbial models and other anaerobic ecosystems to acute and chronic AgNPs exposures at vicinity of environmental and human intake concentrations. To this end, three different systems have been investigated: 1) Planktonic cultures of two well characterized bacterial strains (Chapter 3). The aim of this work was to compare the impacts of different sub-lethal AgNPs concentrations on the growth kinetic, adhesion ability, oxidative stress, and phenotypic changes of model bacteria under both aerobic and anaerobic conditions. To gain a mechanistic insight, the experiments were conducted using two differ-ent microbial model systems: (i) a Gram-negative bacterium Escherichia coli representative of human intestinal flora and responsible for infection, and (ii) a Gram-positive bacterium Bacillus subtilis, wide-ly distributed in soil, freshwater, marine environments and used as a probiotic. I also established the minimum AgNPs sub-lethal concentration able to evoke effects on planktonic bacteria. 2) Biofilm cultures of the model bacterium Escherichia coli and their interplays with CaCo2 cells system (Chapter 4). The goal was to investigate the physiological response of a mono-species gut bio-film to chronic and acute exposure to 1 μg/mL AgNPs, and how this physiological response affected the intestinal epithelial cells. To study the interplays among sub-lethal concentrations of AgNPs, the gut biofilm and its host, a simplified experimental lab model system was designed and tested. 3) Human fecal microbiota in combination with the probiotic Bacillus subtilis (Chapter 5). I aimed to explore possible impacts of single and combined treatments of dietary AgNPs and the probiotic Ba-cillus subtilis to the composition, functional performances and microbial metabolites of in-vitro batch fecal fermentation models to mimic the human digestive tract environment. Furthermore, I investigat-ed their potential cytotoxicity and genotoxicity on the human intestinal Caco-2 cell line. These experimental designs were created to investigate microbial ecosystems of increasing complexity, assessing whether sub-lethal concentrations of AgNPs influence microbial physiology and behavior in such settings.