Rami Pedahzur

Antibacterial activity of silver-killed bacteria: the "zombies" effect

We report a previously unrecognized mechanism for the prolonged action of biocidal agents, which we denote as the zombies effect: biocidally-killed bacteria are capable of killing living bacteria. The concept is demonstrated by first killing Pseudomonas aeruginosa PAO1 with silver nitrate and then challenging, with the dead bacteria, a viable culture of the same bacterium: Efficient antibacterial activity of the killed bacteria is observed. A mechanism is suggested in terms of the action of the dead bacteria as a reservoir of silver, which, due to Le-Chateliers principle, is re-targeted to the living bacteria. Langmuirian behavior, as well as deviations from it, support the proposed mechanism.

Bacterial genotoxicity bioreporters.

Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose‐dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu‐test, has been fully validated and ISO‐ and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial‐based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual genotoxicity testing devices.

Zinc dioxide nanoparticulates: a hydrogen peroxide source at moderate pH.

Solid peroxides are a convenient source of hydrogen peroxide, which once released can be readily converted to active oxygen species or to dissolved dioxygen. A zinc peroxide nanodispersion was synthesized and characterized, and its solubility was determined as a function of pH and temperature. We show that zinc peroxide is much more stable in aqueous solutions compared to calcium and magnesium peroxides and that it retains its peroxide content down to pH 6. At low pH conditions H2O2 release is thermodynamically controlled and its dissolution product, Zn(2+), is highly soluble, and thus, hydrogen peroxide release can be highly predictable. The Gibbs free energy of formation of zinc peroxide was found to be -242.0 ± 0.4 kJ/mol and the enthalpy of formation was -292.1 ± 0.7 kJ/mol, substantially higher than theoretically predicted before. The biocidal activity of zinc peroxide was determined by inactivation studies with Escherichia coli cultures, and the activity trend agrees well with the thermodynamic predictions.

A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Bioactive doped metals: high synergism in the bactericidal activity of chlorhexidine@silver towards wound pathogenic bacteria

The entrapment of chlorhexidine (CH) within metallic silver (CH@Ag) results in a composite material bearing powerful synergetic bactericidal action towards two opportunistic wound pathogenic bacteria, the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus epidermidis. TEM and STEM imaging revealed morphological abnormalities in bacteria exposed to the composite. Release profiles of the active ingredients, CH and ionic silver, from the composite were evaluated by UV spectroscopy and by ICP-MS elemental analysis, and indicated that the strong bactericidal effects were associated with their co-release and presence in the solution. The use of both powder form and pressed form of CH@Ag provides means to control the release kinetics of entrapped CH from the composite and demonstrates the potential application of such composites in wound treatments.

An antibacterial copper composite more bioactive than metallic silver

Although known for its biocidal activity, copper is still not considered as a viable alternative to silver in many of its biocidal applications, mainly because it is generally considered to be a milder antibacterial metal. As copper is much cheaper than silver (1/100), it is potentially more accessible to the health and hygiene needs of third-world countries, to large volume consumer products, and to large-scale agricultural and water treatment needs. Therefore, enhancing the biocidal efficacy of copper is a sought-after goal. We report a method for achieving this goal: by entrapping molecules of the biocidal agent chlorhexidine (CH) within a metallic copper metal powder, using a new materials methodology, the antibacterial efficacy of copper towards two model nosocomial opportunistic bacteria - the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus epidermidis- is enhanced to provide a powerful antibacterial agent exceeding the activity of silver. ICP-MS elemental analysis and UV-spectroscopy indicated that the enhanced bactericidal effects of the synthesized composite, CH@Cu, are associated with the sustained release of both copper ions and CH, giving rise to synergistically enhanced activity.

Electronically Directed Integration of Whole-Cell Biosensors on Bio-Chips

Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.