Efficient disinfection of Escherichia coli in water by silver loaded alumina

The catalytic inactivation of Escherichia coli (E. coli) in water by silver loaded alumina as catalyst was investigated. Ag/Al₂O₃ and AgCl/Al₂O₃ catalysts exhibited high bactericidal activity at room temperature in water with no need for any light or electrical power input. Dissolved oxygen which can be catalyzed to reactive oxygen species (ROS) was found to be essential for the strong bactericidal activities of the catalysts. Decomposition of the cell wall leading to leakage of the intracellular component and the complete lysis of the whole cell were directly observed by transmission electron microscopy (TEM). The resultant change in cell permeability was confirmed by potassium ion leakage. The different morphological changes between E. coli cells treated with the catalysts and Ag⁺ were also observed. The formation of ROS involved in the bactericidal process by AgCl/Al₂O₃ was confirmed by addition of catalase and OH scavenger. Higher temperature and pH value were found to have positive effect on the bactericidal activity of AgCl/Al₂O₃. All these results indicated that the bactericidal effect of the catalyst was a synergic action of ROS and Ag⁺, not an additive one. A possible mechanism is proposed.     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli

In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag⁺ (in the form of AgNO₃). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.     

Nanocrystal Cu2O-loaded TiO2 nanotube array films as high-performance visible-light bactericidal photocatalyst

In this work, we report the use of a non-toxic nanocrystal Cu₂O-loaded TiO₂ nanotube array (Cu₂O/TNTs) film as high-performance visible-light bactericidal photocatalyst. The samples were characterized by field-emission scanning electron microscopy, X-ray photoelectron spectroscopy, and ultraviolet–visible diffusion reflection spectroscopy. This Cu₂O/TNTs film photocatalyst is capable of complete inactivation of Escherichia coli in 5?×?10⁷ colony-forming units/mL within a record short disinfection time of 20?min under visible-light irradiation. The average bactericidal percentage of the Cu₂O/TNTs for E. coli under visible-light irradiation are 20 times and 6.6 times higher than those of TNTs under the same conditions and Cu₂O/TNTs without light, respectively. This superior bactericidal performance is mainly attributed to the high ability to produce OH radicals by both photogenerated electron and hole of the prepared photocatalyst under visible light. The Cu₂O/TNTs film photocatalyst makes it applicable to broad fields including drinking water disinfection.     

Comparative effects of TiO2-immobilized photocatalytic supporters on the bactericidal efficiency of a novel photoreactor

A novel and highly effective UV-TiO₂ photocatalytic reactor was developed for killing microorganisms, including Escherichia coli. Among tested four types of TiO₂-immobilized photocatalytic supporters (glass bead, muscovite bead, alginate bead, and TiO₂ thin film coated quartz tube), the muscovite bead had a 99.9% percent bactericidal activity within 5 min along with permanent longevity. Adding air bubbles or H₂O₂ (<50 mg l^–1) to the sample solution significantly enhanced the killing activity in that 100% percent of bacterial cells were killed within 3 min.     

Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

Antimicrobial activity of stable silver nanoparticles of a certain size

Conditions for obtaining stable silver nanoparticles smaller than 10 nm were developed using a binary stabilizer polyvinylpyrrolidone/sodium dodecylsulphate in optimal ratio. Optical spectra, morphology and dependence of size of the nanoparticles on the amount of reducing agent were studied. Colloidal solutions of nanosilver showed a high bactericidal activity against strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and fungicidal activity against Candida albicans. The mechanism of action of nanosized silver on microbial cell was examined by laser scanning confocal microscope using fluorescent label. First step of antimicrobial effect on microorganisms was membrane damage and penetration of silver nanoparticles into the cell. Prolonged stability of nanoparticles and their antimicrobial activity over the past two years were showed.     

Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis

Bactericidal actions of the silver ion on Escherichia coli as a model microorganism were studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). EFTEM observations demonstrated that the silver ion readily infiltrates the interior of E. coli, contrary to the early hypothesis that it resides initially in the cell membrane area. Furthermore, 2-DE and MALDI-TOF MS indicated that the expression of a ribosomal subunit protein as well as that of some other enzymes and proteins is affected by the silver ion. The present results demonstrate for the first time that one of the major bactericidal functions of the silver ion is its interaction with the ribosome and the ensuing inhibition in expression of the enzymes and proteins essential to ATP production.     

Novel benzimidazol-2-ylidene carbene precursors and their silver(I) complexes: Potential antimicrobial agents

Novel benzimidazolium salts were synthesized as N-heterocyclic carbene (NHC) precursors, these NHC precursors were metallated with Ag₂O in dichloromethane at room temperature to give novel silver(I)–NHC complexes. Structures of these benzimidazolium salts and silver(I)–NHC complexes were characterized on the basis of elemental analysis, ¹H NMR, ¹³C NMR, IR and LC–MS spectroscopic techniques. A series of benzimidazolium salts and silver(I)–NHC complexes were tested against standard bacterial strains: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and the fungal strains: Candida albicans and Candida tropicalis. The results showed that benzimidazolium salts inhibited the growth of all bacteria and fungi strains and all silver(I)–NHC complexes performed good activities against different microorganisms.     

Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological approach

The development of reliable processes for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research. Reports on the cell-associated biosynthesis of silver nanoparticles using microorganisms have been published, but these methods of synthesis are rather slow. In this paper, we report on the rapid synthesis of metallic nanoparticles of silver using the reduction of aqueous Ag+ ion using the culture supernatants of Klebsiella pneumonia, Escherichia coli, and Enterobacter cloacae (Enterobacteriacae). The synthetic process was quite fast and silver nanoparticles were formed within 5 min of silver ion coming in contact with the cell filtrate. Through a limited screening process involving a number of common microorganisms, we observed that the culture supernatants of different bacteria from Enterobacteriacae were potential candidates for the rapid synthesis of silver nanoparticles; further, we revealed that this method of synthesis requires far less time than previously published biological methods. Our investigation also showed that piperitone can partially inhibit the reduction of Ag⁺ to metallic silver nanoparticles by Enterobacteriacae.     

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.     

Comparative Effects of Anesthetics on the Viability and Integrity of Escherichia coli ML30

Cells of Escherichia coli ML30 in a mineral salts medium were exposed to dichlorodifluoromethane (f-12), cyclopropane, halothane, or Ethrane at concentrations of 1.25, 0.2, 0.04, and 0.008× saturation for times up to 1,200 min, and at temperatures in the range of 2 to 37 C. When any of these anesthetics were applied for 300 min at 1.25× saturation, a substantial decrease in number of survivors occurred. Halothane was most bactericidal, cyclopropane and Ethrane were moderately bactericidal, and f-12 was least bactericidal. At saturation values of less than 1.0, none of the four anesthetics had an appreciable effect on viability of E. coli. Greatest increases in cell permeability occurred when anesthetics were used at saturation values of 1.25, and permeability changes generally decreased as the concentrations of the chemicals were reduced. In many instances, anesthetics in the vapor state caused significant increases in cell permeability but little or no loss of viability. This indicated that a close relationship did not exist between loss of viability and increased permeability. All four anesthetics caused E. coli to lose substantial and similar amounts of compounds absorbing at 260 nm. Release of compounds absorbing at 260 nm generally increased as the saturation value of a given chemical was increased. Halothane, Ethrane, and cyclopropane but not f-12 caused lysis of E. coli ML30. Considering all results, E. coli ML30 was damaged more by halothane or cyclopropane than by f-12 or Ethrane. When f-12 was applied at a saturation value of 1.25, the bactericidal effect on E. coli was much greater at 37 or 22 C than at 12 or 2 C.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F₇₀¹⁰ of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F₇₀¹⁰ = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F₇₀¹⁰ = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Comparative assessment of the bactericidal effect of nanoparticles of copper oxide,silver, and chitosan-silver against Escherichia coli infection in broilers

Escherichia coli infection is considered one of the most economically important multi-systemic diseases in poultry farms. Several nanoparticles such as silver, chitosan, and copper oxide are known to be highly toxic to several microbes. However, there are no data concerning their success against in vivo experimental E. coli infection in broilers. Therefore, the present study was designed to investigate the bactericidal effect of low doses of CuO-NPs (5 mg/kg bwt), Ag-NPs (0.5 mg/kg bwt), and Ch-Ag NPs (0.5 mg/kg bwt) against E. coli experimental infection in broilers. One hundred chicks were divided into five groups as follows: (1) control; (2) E. coli (4 × 10⁸ CFU/ml) challenged; (3) E. coli +CuO-NPs; (4) E. coli +Ag-NPs; (5) E. coli +Ch-Ag NPs. The challenged untreated group, not NPs treated groups, recorded the lowest weight gain as well as the highest bacterial count and lesion score in all examined organs. The highest liver content of silver was observed in Ag-NPs treated group compared with the Ch-Ag NPs treated group. Our results concluded that Ch-Ag NPs not only had the best antibacterial effects but also acted as a growth promoter in broilers without leaving any residues in edible organs. We recommend using Ch-Ag NPs in broiler farms instead of antibiotics or probiotics.     

The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction

Phospholipase A₂ present in a highly purified, potently bactericidal, fraction from rabbit granulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A₂ but also by phospholipase A₁, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30–60 min and at higher concentrations of the fraction.Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains.The addition of 0.04 M Mg²⁺, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg²⁺ in either strain. Less than 5 μg of granulocyte fraction causes loss of viability of from 90 to 99% of $\text{1 × 10}{}^8$ microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.