Dottorato in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Titolo della tesi: Transcriptional landscape of green nanocoating with antimicrobial actions

Background: Microbial colonization of surfaces constitutes a dangerous reservoir of pathogens that contribute to spread of infections with repercussions on human health and a heavy economic burden. One of the critical factors in the transmission of infectious diseases is the ability of bacteria to persist on surfaces. Surface disinfection plays a key role in infection prevention and control strategies adopted in response to transmissible disease outbreaks. Typically, antimicrobial agents are chemical compounds, either bioactive or synthesized polymers, used independently or in combination with nanoparticles. Several antimicrobial coatings are available on the market; however, most are based on the leaching of non-environmentally friendly chemicals, such as non-biobased antimicrobial molecules such as antibiotics, phenolic biocides, metal ions and quaternary ammonium compounds. These antimicrobial coatings often raise significant concerns related to antibiotic resistance, complex chemical synthesis, environmental pollution, non-biodegradability, poor performance, toxicity and extremely low sustainability. The optimization of disinfection approaches based on metallic nanoparticles, that can remain longer on surfaces, resist to washing and friction and with good safety environment requisites is a challenge. Aim: The current project aims to explore the antimicrobial properties of an innovative self-disinfecting nanocoating (Cu-SMIN-Ca), based on the antimicrobial action of copper nanoparticles doped with the natural peptide Carvacrol, which is effective against a broad range of viruses and bacteria. In particular, this study investigates the antimicrobial activity of Cu-SMIN-Ca against Escherichia coli (E. coli), exploring the potential mechanisms of action and microbial adaptive responses behind the synergistic effect of copper and Carvacrol. Specifically, genome-wide transcriptional responses were analyzed using the RNA sequencing (RNA-seq) approach to examine the molecular modifications in E. coli after 1 hour of exposure to sublethal and lethal nanoparticle concentrations. Methods: Standard procedures to evaluate minimum inhibition doses to distinct substrate were applied. Total RNA was extracted and libraries for transcriptome investigations (RNA-seq) were prepared for Illumina platform sequencing. For the bioinformatic analysis an ad hoc pipeline was implemented to analyze the sequencing output. Results: The study confirmed that both Carvacrol and Cu-SMIN-Ca, at subinhibitory and inhibitory concentrations, led to the upregulation of genes associated with virulence, DNA damage, and biofilm inhibition. Furthermore, the overexpression of efflux pump-related genes was identified as a potential protective mechanism against the stress induced by Cu-SMIN-Ca nanoparticles and Carvacrol. Additionally, the data indicated that genes commonly regulated at both nanoparticle concentrations were primarily linked to cellular catabolic processes, especially those involved in fatty acid metabolism, purine and pyrimidine biosynthesis, thiamine biosynthesis, and hydrogen metabolism. At the subinhibitory concentration, the modulated genes were mainly related to the catabolism of carboxylic acids and glucose degradation. In contrast, at the inhibitory concentration, modulation of genes associated with cellular respiration and metal ion transport was also observed.