Antibacterial Activity of Chlorhexidine-Killed Bacteria: The Zombie Cell Effect.

We report a biocidal zombie effect of chlorhexidine, a wide-scope biocidal agent commonly used in disinfectant and antiseptic formulations. The zombie effect refers to the ability of dead bacteria killed by a biocidal agent to act as efficient biocidal agents toward a new generation of viable bacteria. The killed bacteria serve as a reservoir for the antibacterial agent incorporated within them; and the new viable population of bacteria acts as a trap of the bioactive agent, shifting the equilibrium of this agent between the reservoir in the dead cells and their aqueous environment. This report is a major generalization of the zombie phenomenon reported previously for silver from the points of view of extending to organic antibacterial agents; extending the effect to both Gram-negative-Pseudomonas aeruginosa PAO1-and Gram positive-Staphylococcus aureus-representative bacteria; showing that the zombie effect is maintained in the second and third generations; showing the effect to operate in an environment of growth media, which extends it to life-supporting environments; and proving that cross-killing is possible, that is, killed S. aureus cells fully inactivated viable P. aeruginosa.

Sustained release from a metal - Analgesics entrapped within biocidal silver.

Matrices for sustained release of drugs have been based on polymers, biomaterials and oxides. The use of the major family of metals as matrices for sustained release is, to the best of our knowledge, unknown. In this context we describe a new family of bio-composites for sustained release of drugs, namely analgesic drugs entrapped within metallic silver. Synthetic methodologies were developed for the preparation of ibuprofen@Ag, naproxen@Ag, tramadol@Ag and bupivacaine@Ag composites. Detailed kinetic analysis of the release of the drugs from within the metal, is provided, demonstrating that metals can indeed serve as reservoirs for drug release. The metal in our case acts not only as a drug releasing source, but also as an antibacterial agent and this property of the composites was studied. Unexpectedly, it was found that the entrapment of the analgesics within silver, dramatically enhances the growth inhibition activity of wild type Pseudomonas aeruginosa, exceeding by far the inhibition activity of the separate components. A mechanism for this interesting observation is provided. The strong antimicrobial activity combined with the analgesic activity open the road for future applications of these materials as dual-purpose components in wound treatment.

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.

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-Chatelier's principle, is re-targeted to the living bacteria. Langmuirian behavior, as well as deviations from it, support the proposed mechanism.

Aqueous stability of alumina and silica perhydrate hydrogels: experiments and computations.

Alumina and silica perhydrate hydrogels were synthesized. Raman spectroscopy and solid (27)Al MAS NMR confirmed alumina perhydrate formation. Thermal and aqueous stability of alumina and silica perhydrates was studied, and they showed exceptionally high stabilities. Alumina perhydrate retained some of the hydrogen peroxide even at 170 °C, higher than any other reported perhydrate, whereas the silica perhydrate lost its hydrogen peroxide content already at 90 °C. The silica perhydrate lost all its peroxide content upon immersion in water, whereas the alumina perhydrate was stable under near-neutral pH conditions. A computational study was conducted in order to glean molecular insight into the observed thermal and aqueous stability of alumina compared to silica perhydrate. Comparison of the hydrogen bond features and the stabilization energies of the hydrate and perhydrate of silica and alumina revealed a higher preference for hydrogen peroxide over water by alumina relative to silica. This is shown to be due to hydrogen peroxide being a better hydrogen donor than water and due to the superior hydrogen accepting propensity of alumina compared to silica.

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.

Microbial genotoxicity bioreporters based on sulA activation.

A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents-hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)-detected 30-60 min after exposure. Detection thresholds were 0.15 µM for MMC, 7.5 µM for nalidixic acid, and approximately 50 µM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.

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.

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.