Light effect and reactive oxygen species in the action of ciprofloxacin on Staphylococcus aureus

Oxygen consumption by Staphylococcus aureus ATCC 29213 sensitive to ciprofloxacin was determined with an oxygen selective electrode. Increase in the O(2) consumption was observed with 0.45 micromL(-1) ciprofloxacin while higher concentrations gave rise to a reduction of O(2) consumption. Resistant S. aureus strain did not show increase of O(2) consumption in presence of ciprofloxacin. Nitro Blue Tetrazolium assay showed that production of reactive oxygen species (ROS) increased intracellularly in sensitive bacteria incubated with this antibiotic. The exposition to UV light (360 nm) augmented the intracellular oxidative stress of S. aureus and provoked increment of ROS in extracellular media. Generation of singlet oxygen O(2) ((1)Delta(g)) in S. aureus was measured by means of oxidation of methionine. The absorbance of methionine was monitored at 215 nm and a clear decrease was detected when sensitive S. aureus was stressed with ciprofloxacin. Sodium azide and 2,5-dimethylfuran were used to reinforce the evidence of O(2) ((1)Delta(g)) generation during oxidative stress. Assays with methionine and 2,5-dimethylfuran demonstrated that resistant S. aureus did not increase the production of O(2) ((1)Delta(g)) in the presence of antibiotic. DNA oxidation was investigated in presence of O(2) ((1)Delta(g)) generated by laser excitation of perinaphthenone and subsequent energy transfer. Deactivation of O(2) ((1)Delta(g)) by reaction with DNA of sensitive and resistant bacteria was observed. According to the results obtained, the effect of ciprofloxacin in S. aureus led to an increment of O(2) ((1)Delta(g)) generating oxidative stress in the bacteria.     

Photoactivated antimicrobial activity of carbon nanotube-porphyrin conjugates

We report the design of antimicrobial nanocomposite films based on conjugates of multiwalled carbon nanotubes (MWNT) and protoporphyrin IX (PPIX) that are highly effective against Staphylococcus aureus (S. aureus) upon irradiation with visible light. S. aureus infections can lead to life-threatening situations, especially when caused by antibiotic-resistant strains. While the light-activated antimicrobial activity of porphyrins against such pathogens is well-known, a facile way to incorporate porphyrins into coatings may lead to their more effective use. To that end, we decided to synthesize and characterize MWNT-PPIX conjugates which combine the biocidal capacity of porphyrins with the mechanical strength of MWNTs. The conjugates could effectively deactivate S. aureus cells in solution upon irradiation with visible light. We also designed large area nanocomposite films comprised of the MWNT-PPIX conjugates that showed potent antimicrobial activity. These MWNT-PPIX conjugates represent a facile strategy for the design of antimicrobial and antifouling coatings.     

Kinetic study of the riboflavin-sensitised photooxygenation of two hydroxyquinolines of biological interest

4-Hydroxyquinoline (4-OHQ) and 8-hydroxyquinoline (8-OHQ), two compounds of interest because of their bioactivity and their structural relation with bioactive products, are effectively photooxygenated when irradiated with visible light in the presence of riboflavin (Rf) (vitamin B2) in solution in air-saturated water-methanol (9:1). Rf behaves as a dye-sensitiser, since both quinolines are transparent to visible light. 8-OHQ degrades about five times faster than 4-OHQ. Kinetic data obtained through time-resolved and stationary detection of Rf-electronically excited states indicate that a superoxide radical anion-mediated mechanism exclusively operates for 4-OHQ, whereas singlet molecular oxygen--mainly--plus superoxide radical anion is the species that reacts with 8-OHQ. The sensitiser Rf, which is known to photodegrade under visible-light aerobic irradiation, is regenerated in the presence of any of the quinolines through an electron transfer process that produces superoxide radical anion. The overall picture indicates that both quinolines act as sacrificial scavengers of the photogenerated oxygen species, thus preventing the photodegradation of Rf.     

Photodynamic destruction of Haemophilus parainfluenzae by endogenously produced porphyrins

Bacterial resistance against antibiotic treatment is becoming an increasing problem in medicine. Therefore methods to destroy microorganisms by other means are being investigated, one of which is photodynamic therapy (PDT).

It has already been shown that a variety of Gram-positive and Gram-negative bacteria can be killed in vitro by PDT using exogenous sensitizers. An alternative method of photosensitizing cells is to stimulate the production of endogenous sensitizers. The purpose of this study was to investigate the bactericidal efficacy of PDT for Haemophilus parainfluenzae with endogenously produced porphyrins, synthesized in the presence of δ-aminolaevulinic acid (δ-ALA). H. parainfluenzae incubated with increasing amounts of δ-ALA showed decreased survival after illumination with 630 nm light. No photodynamic effect on the bacterial viability was found when H. parainfluenzae was grown without added δ-ALA. H. influenzae, grown in the presence of δ-ALA, but not capable of synthesizing porphyrins from δ-ALA, was not affected by PDT. Of the range of incident wavelengths, 617 nm appeared to be the most efficient in killing the bacteria. Spectrophotometry of the bacterial porphyrins demonstrated that the maximum fluorescence occurred at approximately 617 nm, with a much lower peak around 680 nm. We conclude that a substantial killing of H. parainfluenzae by PDT in vitro after endogenous sensitization with δ-ALA can be achieved.     

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.     

Photocatalytic degradation of pathogenic bacteria with AgI/TiO2 under visible light irradiation

The photocatalytic disinfection of pathogenic bacteria in water was investigated systematically with AgI/TiO2 under visible light (lambda > 420 nm) irradiation. The catalyst was found to be highly effective in killing Escherichia coli and Staphylococcus aureus. The adsorbed *OH and hVB+ on the surface of the catalyst were proposed to be the main active oxygen species by study of electron spin resonance and the effect of radical scavengers. The process of destruction of the cell wall and the cell membrane was verified by TEM, potassium ion leakage, lipid peroxidation, and FT-IR measurements. Some products from photocatalytic degradation of bacteria such as aldehydes, ketones, and carboxylic acids were identified by FT-IR spectroscopy. These results suggested that the photocatalytic degradation of the cell structure caused the cell death. The electrostatic force interaction of the bacteria-catalyst significantly affected the efficiency of disinfection on the basis of the E. coli inactivation under different conditions.     

Microbiological Decontamination of Water: Improving the Solar Disinfection Technique (SODIS) with the Use of Nontoxic Vital Dye Methylene Blue

Rational use of water is a major challenge for governments and global organizations, with easy and inexpensive interventions being sought by communities that are not supplied with drinking water. In this context, solar disinfection (SODIS) has shown great efficiency for water disinfection. To speed up the process and improve inactivation, we studied the effects of methylene blue (MB) as a photodynamic agent because of its ability to absorb visible light (red wavelength) and generate singlet oxygen as a reactive species, thereby inactivating bacteria and viruses present in water. In this study, samples of clean mineral water were artificially contaminated with Gram‐positive (Staphylococcus epidermidis or Deinococcus radiodurans) or with Gram‐negative strains (Escherichia coli or Salmonella typhimurium) and exposed to traditional SODIS or to MB‐SODIS. A lethal synergistic effect was observed when cultures were illuminated in the presence of MB. The obtained results indicate that bacterial inactivation can be achieved in a much shorter time when using MB associated with SODIS treatment. Therefore, this technique was able to provide safe water for consumption through the inactivation of microorganisms in general, including pathogens and some strains resistant to the traditional SODIS procedure, thus allowing its use in areas usually less exposed to sunlight.     

In vivo effects of porphyrins on bacterial DNA.

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.     

INHIBITORY EFFECTS OF ETHANOL ON THE PHOTODYNAMIC CELL INACTIVATION AND PETITE INDUCTION BY EUFLAVINE IN YEAST, Saccharomyces cerevisiae

Abstract— Effects of active oxygen scavengers on cell inactivation and petite induction of yeast by the photodynamic action of euflavine were examined. Histidine, sodium azide, 1,3-diphenyl-isobenzofuran and p-carotene, which are singlet oxygen scavengers, inhibited photodynamic cell killing. Histidine and sodium azide inhibited petite induction, too. These results suggested that photobiological effects of euflavine are induced via singlet oxygen-mediated Type II reaction process. In this work, however, we found that ethanol, which is reported to be a hydroxyl radical scavenger, notably inhibited photodynamic cell inactivation and petite induction by euflavine. Inhibition of petite induction was increased with increasing concentration of ethanol. Decrease of absorbance of euflavine by irradiation was also inhibited by the addition of ethanol.
These results suggested that ethanol possibly acts as a singlet oxygen scavenger.     

The bactericidal activity of a deuteroporphyrin-hemin mixture on gram-positive bacteria. A microbiological and spectroscopic study

The combined antibacterial activity of various porphyrins with hemin on Gram-positive bacteria was studied. Protoporphyrin, hematoporphyrin derivative and deuteroporphyrin show only a marginal inhibitory effect in the dark. However, hemin has a strong cytotoxic effect which is independent of illumination and is equally strong in the dark. The disadvantage of hemin treatment is that it is temporary. In this study, we have demonstrated that a combination of deuteroporphyrin and hemin has a unique cytotoxic activity on Staphylococcus aureus, Streptococcus faecalis and Bacillus cereus. The effect of the combined compound is stronger than that of the separate constituents, and is as strong in the dark as in the light. Only 0.005% of the initial S. aureus population survive after a 2 h treatment. Absorption and fluorescence spectra of hemin-deuteroporphyrin mixtures in water and liposomes suggest the formation of a species with spectroscopic properties which are different from those of the two constituents.     

Production of oxygen by electronically induced dissociations in ice

A solid-state chemical model is given for the production of O2 by electronic excitation of ice, a process that occurs on icy bodies in the outer solar system. Based on a review of the relevant available laboratory data, we propose that a trapped oxygen atom-water complex is the principal precursor for the formation of molecular oxygen in low-temperature ice at low fluences. Oxygen formation then occurs through direct excitation of this complex or by its reaction with a freshly produced, nonthermal O from an another excitation event. We describe a model for the latter process that includes competition with precursor destruction and the effect of sample structure. This allows us to put the ultraviolet photon, low-energy electron, and fast-ion experiments on a common footing for the first time. The formation of the trapped oxygen atom precursor is favored by the preferential loss of molecular hydrogen and is quenched by reactions with mobile H. The presence of impurity scavengers can limit the trapping of O, leading to the formation of oxygen-rich molecules in ice. Rate equations that include these reactions are given and integrated to obtain an analytic approximation for describing the experimental results on the production and loss of molecular oxygen from ice samples. In the proposed model, the loss rate varies, roughly, inversely with solid-state defect density at low temperatures, leading to a yield that increases with increasing temperature as observed. Cross sections obtained from fits of the model to laboratory data are evaluated in light of the proposed solid-state chemistry.     

Photoinactivation Sensitivity of Staphylococcus carnosus to Visible-light Irradiation as a Function of Wavelength

Inactivation properties of visible light are of increasing interest due to multiple possible fields of application concerning antibacterial treatment. For violet wavelengths, the generation of reactive oxygen species by porphyrins is accepted as underlying mechanism. However, there is still little knowledge about photosensitizers at blue wavelengths. While flavins were named as possible candidates, there is still no experimental evidence. This study investigates the photoinactivation sensitivity of Staphylococcus carnosus to selected wavelengths between 390 and 500 nm in 10- to 25-nm intervals. Absorption and fluorescence measurements in bacterial lysates confirmed inactivation findings. By means of a mathematical calculation in MATLAB^®, a fit of different photosensitizer absorption spectra to the measured action spectrum was determined to gain knowledge about the extent to which specific photosensitizers are involved. The most effective wavelength for S. carnosus at 415 nm could be explained by the involvement of zinc protoporphyrin IX. Between 450 and 470 nm, inactivation results indicated a broad plateau, statistically distinguishable from 440 and 480 nm. This observation points to flavins as responsible photosensitizers, which furthermore seem to be involved at violet wavelengths. A spectral scan of sensitivities might generally be an advantageous approach for examining irradiation impact.     

Mechanism of visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum

Phototoxicity of visible light laser on the porphyrin-producing bacteria, Porphyromonas gingivalis, in the absence of photosensitizers and under aerobic conditions was shown in previous studies. Recently, we found that the noncoherent visible light sources at wavelengths of 400-500 nm, commonly used in restorative dentistry, induced a phototoxic effect on P. gingivalis, as well as on Fusobacterium nucleatum, and to a lesser extent on the Streptococci sp. To elucidate the mechanism of this phototoxic effect, P. gingivalis and F. nucleatum were exposed to light (1) under aerobic and anaerobic environments and (2) in the presence of scavengers of reactive oxygen species (ROS). Phototoxic effect was not observed when the bacteria were exposed to light under anaerobic conditions. Dimethyl thiourea, a hydroxyl radical scavenger, was effective in reducing phototoxicity (P 相似文献   

C-N-S-TiO2光催化剂的制备、表征及其性能

以钛酸四丁酯为钛源、功能生物小分子胱氨酸为掺杂剂,采用溶胶-凝胶法同步合成了C-N-S-TiO2光催化剂,利用XRD、XPS、FT-IR和DRS等测试技术对样品的结构及物化性能进行了表征。XRD和DRS分析表明,共掺杂抑制了TiO2晶粒的生长,提高了晶相转变温度,且C-N-S-TiO2样品的吸收带边明显"红移",光吸收范围一直延长至800 nm左右。XPS分析结果显示,C-N-S-TiO2样品表面产生了杂质能级,C、S元素分别取代部分晶格Ti4+以CO23-和S6+形式存在;而N峰呈宽化状态,以O—Ti—N和Ti—O—N键存在,且样品表面羟基含量明显增加。以罗丹明B染料为模型污染物,考察了该催化剂的可见光催化活性。结果表明,与P25 TiO2比较,C-N-S-TiO2光催化剂活性得到改进,C-N-S-TiO2光催化剂在470～800 nm波长下辐射120 min后对罗丹明B的降解率可高达83%。     

生物分子辅助低成本制备Zn0.9Cd0.1S固溶体及其高效可见光光催化制氢

如何提高光催化制氢量子产率是太阳能分解水制氢研究的重点和焦点. Zn-Cd-S固溶体因具有窄的带隙宽度及合适的导带和价带位置而显示了广阔的应用前景. 然而, 两方面的问题限制了其规模化应用: (1)往往需负载Pt, Pd, Ru和Rh等贵金属助催化剂才能获得可观的光催化性能; (2)传统合成技术通常采用硫代乙酰胺、硫脲及硫化钠等昂贵且有毒的化学试剂作硫源. 与上述硫源相比, 生物小分子L-胱氨酸分子中含有-COOH、-NH2及-SH基团, 这些基团易于与金属阳离子配位, 因此能够有效调控硫源释放S2-的速度, 硫化物的形貌、尺寸以及取向能够灵活地得到调控. 另外, 在强碱或强酸性介质中, L-胱氨酸具有良好的水溶性, 因此材料的合成可选择在水介质中, 这对光催化过程是非常关键的, 有利于改善材料在光催化反应过程中的稳定性. 基于此, 本文以经济环保的生物小分子作硫源, 制备了高效、稳定且有可见光响应的纳米硫化物光催化体系, 旨在发展环境友好、条件温和、成本低廉、操作简单和易于工业化生产的绿色制备技术, .以L-胱氨酸为硫源和结构导向剂, 采用水热合成技术在温和条件下制备了立方相结构的Zn-Cd-S固溶体光催化剂, 采用XRD, TEM, HRTEM, XPS, UV-vis及N2吸附等手段表征了其结构和形貌. 结果表明, 随Zn含量增加, 其带隙在2.11-3.19 eV间连续可调. 在可见光(λ > 420 nm)照射、无助催化剂和Na2S/Na2SO3水溶液为牺牲剂的条件下研究了其光催化制氢的性能. 其中Zn0.9Cd0.1S具有最佳的光催化活性, 其产氢速率约为4.4 mmol h -1g -1(无助催化剂, 远高于CdS), 且显示优良的稳定性及抗光腐蚀能力. 通过经验公式计算得出了其能带结构示意图, 结果表明, ZnxCd1-xS固溶体的导带和价带的位置随着Zn含量的增加而向更负的导带和更正的价带移动. 固溶体导带电位更负促进更有效的氢产生, 电位价带更正导致电荷更容易发生转移. Zn0.9Cd0.1S高的光催化活性可能归因于中等的导带边缘和最合适的带隙. 最后利用光电流及交流阻抗阐明了其光生电子-空穴对的分离及迁移机理. 与CdS相比, Zn-Cd-S固溶体的形成促进了光生载流子在界面间的传输, 抑制了其快速复合, 从而大幅度改善了光催化活性及稳定性. 该硫化物纳米晶的绿色制备技术期望可推广到其它硫化物可见光光催化体系.     

Sporicidal Effects of High‐Intensity 405 nm Visible Light on Endospore‐Forming Bacteria

Resistance of bacterial endospores to treatments, including biocides, heat and radiation is a persistent problem. This study investigates the susceptibility of Bacillus and Clostridium endospores to 405 nm visible light, wavelengths which have been shown to induce inactivation of vegetative bacterial cells. Suspensions of B. cereus endospores were exposed to high‐intensity 405 nm light generated from a light‐emitting diode array and results demonstrate the induction of a sporicidal effect. Up to a 4‐log₁₀ CFU mL^?1 reduction in spore population was achieved after exposure to a dose of 1.73 kJ cm^?2. Similar inactivation kinetics were demonstrated with B. subtilis, B. megaterium and C. difficile endospores. The doses required for inactivation of endospores were significantly higher than those required for inactivation of B. cereus and C. difficile vegetative cells, where ca 4‐log₁₀ CFU mL^?1 reductions were achieved after exposure to doses of 108 and 48 J cm^?2, respectively. The significant increase in dose required for inactivation of endospores compared with vegetative cells is unsurprising due to the notorious resilience of these microbial structures. However, the demonstration that visible light of 405 nm can induce a bactericidal effect against endospores is significant, and could have potential for incorporation into decontamination methods for the removal of bacterial contamination including endospores.     

Porphyrin-cellulose nanocrystals: a photobactericidal material that exhibits broad spectrum antimicrobial activity

Towards our overall objectives of developing potent antimicrobial materials to combat the escalating threat to human health posed by the transmission of surface-adhering pathogenic bacteria, we have investigated the photobactericidal activity of cellulose nanocrystals that have been modified with a porphyrin-derived photosensitizer (PS). The ability of these previously synthesized porphyrin-cellulose-nanocrystals (CNC-Por (1)) to mediate bacterial photodynamic inactivation was investigated as a function of bacterial strain, incubation time and illumination time. Despite forming an insoluble suspension, CNC-Por (1) showed excellent efficacy toward the photodynamic inactivation of Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii (MDRAB) and methicillin-resistant Staphylococcus aureus (MRSA), with the best results achieving 5-6 log units reduction in colony forming units (CFUs) upon illumination with visible light (400-700 nm; 118 J cm(-2)). CNC-Por (1) mediated the inactivation of Pseudomonas aeruginosa, although at reduced activity (2-3 log units reduction). Confocal laser scanning microscopy of CNC-Por (1) after incubation with A. baumannii or S. aureus suggested a lack of internalization of the PS. Research into alternative materials such as CNC-Por (1) may lead to their application in hospitals and healthcare-related industries wherein novel materials with the capability of reducing the rates of transmission of a wide range of bacteria, particularly antibiotic resistant strains, are desired.     

PHOTO ACTIVATION OF ISOFLAVONOID PHYTOALEXINS: INVOLVEMENT OF FREE RADICALS

Abstract— Upon irradiation with ultraviolet light the isoflavonoid phytoalexins phaseollin, 3,6a, 9-trihydroxypterocarpan, glyceollin, tuberosin and pisatin, but not medicarpin, brought about inactivation ofglucose–6-phosphate dehydrogenase in an in vitro assay system. Photoinactivation of the enzyme by photoactivated pisatin in air-saturated solutions was hardly affected by singlet oxygen quenchers such as NaN₃, bovine serum albumin, histidine or methionine. Neither addition of the hydroxyl radical scavengers mannitol, Na-benzoate and ethanol nor the presence of catalase or supcroxide dismutase protected the enzyme against photoinactivation, suggesting that OH, H₂O₂ and O₂ are not the reactive oxygen species involved. However, the free radical scavenger S-(2-amino-ethyl)isothiouronium bromide hydrobromide (AET) protected the enzyme against inactivation by photoactivated pisatin. Direct evidence for the generation of free radicals was obtained by ESR measurements of solutions of phaseollin, pisatin and medicarpin in hexane irradiated with ultraviolet light in the presence or absence of O₂. Phaseollin produced the most stable free radicals, whereas medicarpin hardly gave rise to free radical formation; pisatin took a somewhat intermediate position by producing a strong ESR signal which, however, decayed rather quickly. Photodegradation of all phytoalexins, except for medicarpin, was accompanied with loss of fungitoxicity, as shown in thin-layer chromatographic bioassays, and formation of new products.
These results indicate free radical formation as the causative process for photoinactivation of enzymes by photoactivated isoflavonoid phytoalexins.     

Sensitivity of Staphylococcus aureus strains to broadband visible light

The phototoxic effect of illumination with broadband visible light on the viability of two Staphylococcus aureus strains was examined in the present study. A difference in the light sensitivity of the two strains was found. Illumination of the tested strains with a fluence rate of 180 J cm⁻² caused a reduction of up to 99.8% in the colony count of one of the strains (the "sensitive" strain). Illumination of the other strain (the "resistant" strain) resulted in a 55.5% reduction in viability. Proliferation of both strains was observed at low fluence rates of light. The phototoxic effect was found to be dependent on oxy radical production. The light-sensitive strain produced higher amounts of hydroxyl and superoxide radicals than the "resistant" strain. Adaptation to oxidative stress was exhibited only by the "resistant" strain. The "sensitive" strain produced ten times more endogenous porphyrins and secreted almost nine times more porphyrins than the resistant strain. Furthermore, the "resistant" strain produced twice as many carotenoids that protect the strain from illumination than the "sensitive" strain. These results indicate that high intensities of visible light cause bacterial photoeradication, a reaction which may assist wound healing by killing the infecting bacteria. On the other hand, low intensities of white light were found to enhance bacterial proliferation and thus prolong wound infection.