Involvement of reactive oxygen species in the bactericidal activity of activated carbon fibre supporting silver; Bactericidal activity of ACF(Ag) mediated by ROS

An activated carbon fibre supporting silver (ACF(Ag)) was tested for its antibacterial capacity against Escherichia coli (E. coli). Water that has passed through ACF(Ag) demonstrated strong bactericidal ability. This activity decreased over the time suggesting that generated bactericidal species were short lifespan. Since formation of reactive oxygen species (ROS) might be catalysed by silver impregnated and/or ACF itself, implication of ROS and silver was evaluated by the use of ROS scavengers and a silver ions neutralizing agent. The role of ROS in the E. coli mortality was confirmed by the use of a molecular approach which revealed a strong expression of oxidative stress genes.     

Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate

The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate. The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite.     

Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate

The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate. The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite.     

Effect of hyperbaric oxygen on Vibrio vulnificus and murine infection caused by it

Vibrio vulnificus is a bacterium known to cause fatal necrotizing soft tissue infection in humans. Here, a remarkable therapeutic effect of hyperbaric oxygen (HBO) on V. vulnificus infection provoked by its injection into mouse footpads is described. HBO was shown to be bactericidal to this bacterium in vitro as well as in the infected tissue. The bactericidal activity of HBO was shown to be due to reactive oxygen species (ROS), the efficacy of HBO against V. vulnificus infection being accounted for by the high sensitivity of this bacterium to ROS. Besides being somewhat weak in ROS-inactivating enzyme activities, this bacterium is also unusually sensitive to ultraviolet light and other DNA-damaging agents. It seems likely that the sensitivity of V. vulnificus to HBO is mainly due to its poor ability to repair oxidative damage to DNA. These findings encourage clinical application of HBO against potentially fatal V. vulnificus infection in humans.     

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.     

Hypercapnic hypoxia compromises bactericidal activity of fish anterior kidney cells against opportunistic environmental pathogens

Acute hypoxia can cause massive fish and shellfish mortality. Less clear is the role that chronic sublethal hypoxia might play in aquatic animal health. This study tested whether production of reactive oxygen species (ROS) and bactericidal activity of fish phagocytic cells are suppressed under the conditions of decreased oxygen and pH and increased carbon dioxide which occur in the blood and tissue of animals exposed to sublethal hypoxia. Anterior head kidney (AHK) cells of the mummichog, Fundulus heteroclitus, were exposed in parallel to normoxic (pO2=45 torr, pCO2=3.8 torr, pH=7.6) or hypoxic (pO2=15 torr, pCO2=8.0 torr, pH=7.0) conditions and stimulated with a yeast cell wall extract, zymosan. or live Vibrio parahaemolyticus. Hypercapnic hypoxia suppressed zymosan-stimulated ROS production by 76.0% as measured in the chemiluminescence assay and by 58.5% in the nitroblue tetrazolium (NBT) assay. The low O2, high CO2 and low pH conditions also suppressed superoxide production by 75.0 and 47.3% as measured by the NBT assay at two different challenge ratios of cells:bacteria (1:1 and 1:10, respectively). In addition to its effects on ROS production, hypercapnic hypoxia also reduced bactericidal activity by 23.6 and 72.5% at the 1:1 and 1:10 challenge ratios, respectively. Low oxygen levels alone (pO2=15 torr, pCO2=0.76 torr, pH=7.6) did not significantly compromise the killing activity of cells challenged with equal numbers of V. parahaemolyticus. At the higher 1:10 AHK:bacteria challenge ratio, low oxygen caused a small (26.3%) but significant suppression of bactericidal activity as compared to aerial conditions (pO2=155 torr, pCO2=0.76 torr, pH=7.6). This study demonstrates that while hypoxia alone has detrimental effects on immune function, suppression of phagocytic cell activity is compounded by naturally occurring conditions of hypercapnia and low pH, creating conditions that might be exploited by opportunistic pathogens. These results indicate that the adverse health effects of chronic hypercapnic hypoxia might greatly exceed the effects of low oxygen alone.     

Catalytic sterilization of Escherichia coli K 12 on Ag/Al2O3 surface

Bactericidal action of Al(2)O(3), Ag/Al(2)O(3) and AgCl/Al(2)O(3) on pure culture of Escherichia coli K 12 was studied. Ag/Al(2)O(3) and AgCl/Al(2)O(3) demonstrated a stronger bactericidal activity than Al(2)O(3). The colony-forming ability of E. coli was completely lost in 0.5 min on both of Ag/Al(2)O(3) and AgCl/Al(2)O(3) at room temperature in air. The configuration of the bacteria on the catalyst surface was observed using scanning electron microscopy (SEM). Reactive oxygen species (ROS) play an important role in the expression of the bactericidal activity on the surface of catalysts by assay with O(2)/N(2) bubbling and scavenger for ROS. Furthermore, the formation of CO(2) as an oxidation product could be detected by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and be deduced by total carbon analysis. These results strongly support that the bactericidal process on the surface of Ag/Al(2)O(3) and AgCl/Al(2)O(3) was caused by the catalytic oxidation.     

Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.     

Rosiglitazone Modulates Insulin-Induced Plasma Membrane Area Changes in Single 3T3-L1 Adipocytes

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.     

Anti-inflammatory effects of Melaleuca alternifolia essential oil on human polymorphonuclear neutrophils and monocytes

OBJECTIVE AND DESIGN: The fungicidal and bactericidal actions of the essential oil (EO) of Melaleuca alternifolia seem well established, but their anti-inflammatory and anti-oxidative effects remain unclear. In this study, we investigated in vitro the possible role of whole M. alternifolia EO as a modulator of the oxidative response, i.e. reactive oxygen species (ROS) production, of leukocytes (monocytes and polymorphonuclear neutrophils (PMNs)) in humans. METHODS: Whole blood leukocytes from healthy human volunteers (n = 7), isolated from erythrocytes by haemolytic shock, were incubated for 30 min with M. alternifolia EO (0-0.1%) to determine their ROS production by flow cytometry with or without stimulation of cells. We compared the effects of 3 different stimulating agents acting differently on transductional pathways to stimulate the ROS production: a phorbol ester (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP) and opsonised zymosan (OZ). RESULTS: As attested by the Krüskall-Wallis test, M. alternifolia EO at 0.1% directly stimulated ROS production by PMNs (x 8.7 vs. 0% EO, p < 0.05) and increased the intracellular ROS produced by monocytes. Whichever the stimulating agent used (PMA, fMLP or OZ), M. alternifolia EO decreased the intracellular ROS production at the dilution of 0.1% by PMNs and monocytes, more so with PMNs. CONCLUSION: M. alternifolia EO may be both a direct active mediator of the bactericidal action of the circulating leukocytes and may be efficient in protecting the organism from an excess of ROS, through an anti-oxidant and radical scavenging activity.     

Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers.

The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.     

The blood platelets contribution to innate host defense – What they have learned from their big brothers

Bactericidal effects of blood platelets have been known for more than 120 years, but the underlying mechanisms are largely obscure. Keeping in mind structural and functional analogies of platelets to neutrophils, three different mechanisms are thinkable: Engulfment of pathogens, release of microbicidal proteins, and production of reactive oxygen species (ROS). Here, we focus on the release of ROS and a possible contribution of blood plasma and thrombin to the bactericidal effects. Killing of bacteria was evaluated by DNA fluorescence labeling and electron microscopy. Release of ROS by platelets was measured photometrically by cytochrome C and phenol red/peroxidase assays and was further evaluated by topological methods. We found that (i) platelets produce 1500 times less O₂^– and 4000 times less H₂O₂ compared to neutrophils, (ii) ROS do not affect the killing rates, and (iii) no local enrichment of ROS was detectable. On the other hand, thrombin and plasma proteins with a molecular mass of >100 kDa are essential for bactericidal effects. We suggest that platelets contribute to the innate host defense by providing a catalytical surface for synthesis of thrombin. In the presence of a heat-instable plasma protein, thrombin may generate a strong bactericidal complex, which is only effective in close vicinity to the platelet membrane.     

Involvement of cytoplasmic membrane damage in the copper (II)-dependent cytotoxicity of a novel naturally occurring tripyrrole

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC8739 cells were killed by a novel tripyrrole 1, isolated as a red pigment from the Serratia sp. Cell killing was accompanied by a depletion in the potassium pools of the cells due to the damage to the cytoplasmic membrane, without any detectable DNA damage as revealed by the transformed plasmid DNA and phage induction assay. This revealed that the bactericidal activity of compound 1 in the presence of Cu(II) results from membrane damage. Induction of endogenous catalase in the E. coli cells increased their resistance against the combination of compound 1 and Cu(II). Although compound 1 alone generated large amount of reactive oxygen species (ROS), it did not show any cell killing against E. coli in the absence of Cu(II). The Cu(II)-dependent bactericidal activity of compound 1 was suppressed by ethylenediaminetetraacetate, bathocuproine, catalase and superoxide disumutase (SOD), but not by dimethyl sulfoxide. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving compound 1 and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing. Compound 1 alone showed selective bactericidal activity against the gram positive bacterium, Bacillus cereus ATCC 6630, possibly due to its differential cellular transport.     

Antibacterial activity of colloidal copper nanoparticles against Gram-negative (Escherichia coli and Proteus vulgaris) bacteria

Antibacterial activities of as-synthesized nanoparticles have gained attention in past few years due to rapid phylogenesis of pathogens developing multi-drug resistance (MDR). Antibacterial activity of copper nanoparticles (CuNPs) on surrogate pathogenic Gram-negative bacteria Escherichia coli (MTCC no. 739) and Proteus vulgaris (MTCC no. 426) was evaluated under culture conditions. Three sets of colloidal CuNPs were synthesized by chemical reduction method with per batch yield of 0·2, 0·3 and 0·4 g. As-synthesized CuNPs possess identical plasmonic properties and have similar hydrodynamic particle sizes (11–14 nm). Antibacterial activities of CuNPs were evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests, cytoplasmic leakage and reactive oxygen species (ROS) assays. MIC and MBC tests revealed dose dependence bactericidal action. Growth curves of E. coli show faster growth inhibition along with higher cytoplasmic leakage than that of P. vulgaris. This might be because of increased membrane permeability of E. coli. CuNP–microorganism interaction induces oxidative stress generated by ROS. Leakage of cytoplasmic components, loss of membrane permeability and ROS generation are the primary causes of CuNP-induced bacterial cell death. As-synthesized CuNPs exhibiting promising antibacterial activities and could be a promising candidate for novel antibacterial agents.     

Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants

Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.     

Generation of reactive oxygen species by polymorphonuclear leukocytes and its modulation by calcium ions during tumor growth

The generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) and the role of Ca2+ in regulating their activity during Zajdela hepatoma growth in the animal peritoneal cavity were studied. We found a marked increase in the ROS-generating activity of PMNL in circulating blood, the result of increases in both the specific activity of leukocytes and total number of PMNL in circulating blood. The ROS-generating activity of PMNL was substantially activated by Ca2+ ions and a calcium ionophore (ionomycin), but this effect virtually completely disappeared during tumor growth. Perhaps the high ROS-generating activity of PMNL and the lack of the sensitivity to extracellular Ca2+ during tumor growth in the organism are due to an accumulation of intracellular Ca2+ ions.     

Metal Ions,Not Metal-Catalyzed Oxidative Stress,Cause Clay Leachate Antibacterial Activity

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H₂O₂, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1β. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1β was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.