Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Transparent polycarbonate coated with CeO2 nanozymes repel Pseudomonas aeruginosa PA14 biofilms.

Highly transparent CeO2/polycarbonate surfaces were fabricated that prevent adhesion, proliferation, and the spread of bacteria. CeO2 nanoparticles with diameters of 10-15 nm and lengths of 100-200 nm for this application were prepared by oxidizing aqueous dispersions of Ce(OH)3 with H2O2 in the presence of nitrilotriacetic acid (NTA) as the capping agent. The surface-functionalized water-dispersible CeO2 nanorods showed high catalytic activity in the halogenation reactions, which makes them highly efficient functional mimics of haloperoxidases. These enzymes are used in nature to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). Bacteria-repellent CeO2/polycarbonate plates were prepared by dip-coating plasma-treated polycarbonate plates in aqueous CeO2 particle dispersions. The quasi-enzymatic activity of the CeO2 coating was demonstrated using phenol red enzyme assays. The monolayer coating of CeO2 nanorods (1.6 µg cm-2) and the bacteria repellent properties were demonstrated by atomic force microscopy, biofilm assays, and fluorescence measurements. The engineered polymer surfaces have the ability to repel biofilms as green antimicrobials on plastics, where H2O2 is present in humid environments such as automotive parts, greenhouses, or plastic containers for rainwater.

Nanocomposite antimicrobials prevent bacterial growth through the enzyme-like activity of Bi-doped cerium dioxide (Ce1-xBixO2-δ).

Preventing bacterial adhesion on materials surfaces is an important problem in marine, industrial, medical and environmental fields and a topic of major medical and societal importance. A defense strategy of marine organisms against bacterial colonization relies on the biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidases, a class of metal-dependent enzymes, whose activity can be emulated by ceria nanoparticles. The enzyme-like activity of ceria was enhanced by a factor of 3 through bismuth substitution (Ce1-xBixO2-δ). The solubility of Bi3+ in CeO2 is confined to the range 0 < x < 0.25 under quasi-hydrothermal conditions. The Bi3+ cations are located close to the nanoparticle surface because their ionic radii are larger than those of the tetravalent Ce4+ ions. The synthesis of Ce1-xBixO2-δ (0 < x < 0.25) nanoparticles was upscaled to yields of ∼50 g. The halogenation activity of Ce1-xBixO2-δ was demonstrated with phenol red assays. The maximum activity for x ≈ 0.2 is related to the interplay of the ζ-potential of surface-engineered Ce1-xBixO2-δ nanoparticles and their BET surface area. Ce0.80Bi0.20O1.9 nanoparticles with optimized activity were incorporated in polyethersulfone beads, which are typical constituents of water filter membrane supports. Although Ce1-xBixO2-δ nanoparticles are not bactericidal on their own, naked Ce1-xBixO2-δ nanoparticles and polyethersulfone/Ce1-xBixO2-δ nanocomposites showed a strongly reduced bacterial coverage. We attribute the decreased adhesion of the Gram-negative soil bacterium Pseudomonas aeruginosa and of Phaeobacter gallaeciensis, a primary bacterial colonizer in marine biofilms, to the formation of halogenated signaling compounds. No biocides are needed, H2O2 (formed in daylight) and halide are the only substrates required. The haloperoxidase-like activity of Ce1-xBixO2-δ may be a promising starting point for the development of environmentally friendly, "green" nanocomposites, when the use of conventional biocides is prohibited.

Functional Enzyme Mimics for Oxidative Halogenation Reactions that Combat Biofilm Formation.

Transition-metal oxide nanoparticles and molecular coordination compounds are highlighted as functional mimics of halogenating enzymes. These enzymes are involved in halometabolite biosynthesis. Their activity is based upon the formation of hypohalous acids from halides and hydrogen peroxide or oxygen, which form bioactive secondary metabolites of microbial origin with strong antibacterial and antifungal activities in follow-up reactions. Therefore, enzyme mimics and halogenating enzymes may be valuable tools to combat biofilm formation. Here, halogenating enzyme models are briefly described, enzyme mimics are classified according to their catalytic functions, and current knowledge about the settlement chemistry and adhesion of fouling organisms is summarized. Enzyme mimics with the highest potential are showcased. They may find application in antifouling coatings, indoor and outdoor paints, polymer membranes for water desalination, or in aquacultures, but also on surfaces for food packaging, door handles, hand rails, push buttons, keyboards, and other elements made of plastic where biofilms are present. The use of natural compounds, formed in situ with nontoxic and abundant metal oxide enzyme mimics, represents a novel and efficient "green" strategy to emulate and utilize a natural defense system for preventing bacterial colonization and biofilm growth.

Haloperoxidase Mimicry by CeO2-x Nanorods Combats Biofouling.

CeO2-x nanorods are functional mimics of natural haloperoxidases. They catalyze the oxidative bromination of phenol red to bromophenol blue and of natural signaling molecules involved in bacterial quorum sensing. Laboratory and field tests with paint formulations containing 2 wt% of CeO2-x nanorods show a reduction in biofouling comparable to Cu2 O, the most typical biocidal pigment.