Gene expression is influenced due to 'nano' and 'ionic' copper in pre-formed Pseudomonas aeruginosa biofilms

Today, researchers across the globe suggest the use of antimicrobial coatings containing copper nanoparticles (CuNPs) complementing the traditional protocols to prevent hospital-acquired infections (HAIs). Since Pseudomonas aeruginosa is one of the commonest opportunistic pathogens, we assessed the anti-biofilm activity of CuNPs in P. aeruginosa MTCC 3541 and compared it with Cu²⁺ (copper sulphate) since the latter continues to be used as an antimicrobial-of-choice in food industries, agriculture and water treatment. In this study, we synthesized and characterized stable poly-acrylic acid (PAA) coated CuNPs with a size of 66-150 nm and zeta potential -13 mV. Pseudomonas aeruginosa MTCC 3541 biofilms were highly resistant to both CuNPs and Cu²⁺ (minimum biofilm inhibitory concentration, MBIC 300 and >600 μg/mL respectively). Scanning electron microscopy revealed alterations in cell morphology upon treatment with CuNPs. A closer analysis of the biofilm-specific gene expression (qRT-PCR) revealed that CuNPs downregulated the genes involved in biofilm matrix formation, motility, efflux, membrane lipoprotein synthesis and DNA replication. Both, CuNPs and Cu²⁺ up regulated copper resistance and biofilm dispersion genes. Copper did not affect the bacterial communication system as evidenced by downregulation of the negative regulator of quorum sensing. The gene expression analysis reveals multiple cellular targets for CuNPs and ionic Cu. The present study highlights the fact that CuNPs affect the membrane functions adversely damaging the cell surface. In pre-formed biofilms, CuNPs were more toxic and displayed distinct responses attributable due to 'nano' and 'ionic' copper. Our findings thus support the use of CuNPs for curbing HAIs.