Variations in Copper during a 72h-exposure hydroponic experiment in presence of Gram- bacteria

The aim of this experiment was to determine if the development of resistance to antibiotics can be driven by the concentration and speciation of copper (Cu). Experimental setup was designed to investigate two hypotheses for which two strains of Gram- bacteria have been selected: - Do Cu enhance antibiotic resistance (AR) in resistant bacteria? Resistant strain: Bioluminescent Pseudomonas aeruginosa PAO1 (Xen41, Tetracycline resistant) - Do Cu induce AR in sensitive bacteria? Sensitive strain: Pseudomonas aeruginosa PAO1 (Wild Type) A 72h exposure hydroponic experiment has been carried out. The growth media was selected to provide optimum free Cu concentration in solution by limiting the complexation of Cu by the constituents in the medium. The description of the Growth minimal media (MES buffered minimal medium) can be found elsewhere (Rathnayake, I. V. N.; Megharaj, M.; Krishnamurti, G. S. R.; Bolan, N. S.; Naidu, R., Heavy metal toxicity to bacteria – Are the existing growth media accurate enough to determine heavy metal toxicity? Chemosphere. 2013, 90, (3), 1195-1200.) This media was spiked with three Cu concentrations: Cu=MIC/10, Cu=MIC and Cu=MIC*10 (MIC= minimal inhibitory concentration). For the studied strains, MIC was determined to be Cu2+=10E-6 mol.L-1 or Cu=3.7E-06 mol.L-1. Samples (bacteria + solution) have been collected after 0, 24, 48 and 72 hours: *After extraction of DNA and RNA, Whole Genome Sequencing (WGS) and RNA sequencing (RNAseq) were analyzed to identify the development of resistance *Solutions were analyzed for total Cu concentration, Cu concentration of solution filtered at 3 kDa and ion Cu2+ concentration.