Yeshayahu Nitzan

Inactivation of gram-negative bacteria by photosensitized porphyrins.

Abstract— Photosensitization of Escherichia coli and Pseudomonas aeruginosa cells by deuteroporphyrin (DP) is shown to be possible in the presence of the polycationic agent polymyxin nonapeptide (PMNP). Previous studies established complete resistance of Gram‐negative bacteria to the photodynamic effects of porphyrins. The present results show that combined treatment of E. coli or P. aeruginosa cultures with DP and PMNP inhibit cell growth and viability. No antibacterial activity of PMNP alone could be demonstrated and cell viability remained unchanged. Spectroscopically, PMNP was found to bind DP, a mechanism which probably assists its penetration into the cells membranes. Insertion of DP into the cells was monitored by the characteristic fluorescence band of bound DP at 622 nm. Binding times were 5–40 min and the extent of binding increased with decreasing the pH from 8.5 to 6.5. DP binding constants, as well as the concentrations of PMNP which were required for maximal effect on the various Gram‐negative bacteria, were determined fluorometrically. By the treatment of DP, PMNP and light the growth of E. coli and P. aeruginosa cultures was stopped and the viability of the culture was dramatically reduced. Within 60 min of treatment the survival fraction of E. coli culture was 9 × 10–6 that of P. aeruginosa was 5.2 × 10–4. Electron microscopy depicted ultrastructural alterations in the Gram‐negative cells treated by DP and PMNP. The completion of cell division was inhibited and the chromosomal domain was altered markedly.

Understanding the Antibacterial Mechanism of CuO Nanoparticles: Revealing the Route of Induced Oxidative Stress

To date, there is still a lack of definite knowledge regarding the interaction of CuO nanoparticles with bacteria and the possible permeation of the nanoparticles into bacterial cells. This study was aimed at shedding light on the size-dependent (from the microscale down to the small nanoscale) antibacterial activity of CuO. The potent antibacterial activity of CuO nanoparticles was found to be due to ROS-generation by the nanoparticles attached to the bacterial cells, which in turn provoked an enhancement of the intracellular oxidative stress. This paradigm was confirmed by several assays such as lipid peroxidation and reporter strains of oxidative stress. Furthermore, electron microscopy indicated that the small nanoparticles of CuO penetrated the cells. Collectively, the results reported herein may reconcile conflicting concepts in the literature concerning the antibacterial mechanism of CuO nanoparticles, as well as highlight the potential for developing sustainable CuO nanoparticles-based devices for inhibiting bacterial infections.

Antifungal activity of ZnO nanoparticles?the role of ROS mediated cell injury

Metal oxide nanoparticles have marked antibacterial activity. The toxic effect of these nanoparticles, such as those comprised of ZnO, has been found to occur due to an interaction of the nanoparticle surface with water, and to increase with a decrease in particle size. In the present study, we tested the ability of ZnO nanoparticles to affect the viability of the pathogenic yeast, Candida albicans (C. albicans). A concentration-dependent effect of ZnO on the viability of C. albicans was observed. The minimal fungicidal concentration of ZnO was found to be 0.1 mg ml(-1) ZnO; this concentration caused an inhibition of over 95% in the growth of C. albicans. ZnO nanoparticles also inhibited the growth of C. albicans when it was added at the logarithmic phase of growth. Addition of histidine (a quencher of hydroxyl radicals and singlet oxygen) caused reduction in the effect of ZnO on C. albicans depending on its concentration. An almost complete elimination of the antimycotic effect was achieved following addition of 5 mM of histidine. Exciting the ZnO by visible light increased the yeast cell death. The effects of histidine suggest the involvement of reactive oxygen species, including hydroxyl radicals and singlet oxygen, in cell death. In light of the above results it appears that metal oxide nanoparticles may provide a novel family of fungicidal compounds.

Sonochemical coating of paper by microbiocidal silver nanoparticles.

Colloidal silver has gained wide acceptance as an antimicrobial agent, and various substrates coated with nanosilver such as fabrics, plastics, and metal have been shown to develop antimicrobial properties. Here, a simple method to develop coating of colloidal silver on paper using ultrasonic radiation is presented, and the coatings are characterized using X-ray diffraction (XRD), high resolution scanning electron microscope (HRSEM), and thermogravimetry (TGA) measurements. Depending on the variables such as precursor concentrations and ultrasonication time, uniform coatings ranging from 90 to 150 nm in thickness have been achieved. Focused ion beam (FIB) cross section imaging measurements revealed that silver nanoparticles penetrated the paper surface to a depth of more than 1 μm, resulting in highly stable coatings. The coated paper demonstrated antibacterial activity against E. coli and S. aureus, suggesting its potential application as a food packing material for longer shelf life.

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm2, respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization.

FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.

ALA induced photodynamic effects on Gram positive and negative bacteria

In the present study we examined the production of high amounts of porphyrins upon induction by delta-aminolevulinic acid (ALA) in 9 bacterial strains. This was performed by solely inducing the porphyrin biosynthesis pathway. Four of the strains were Gram positive bacteria and five were Gram negative strains. All strains, except Streptococcus faecalis, produced porphyrins when incubated in PBS with 0.38 mM ALA for 4 h. Excess porphyrin production was excreted to the medium. Gram positive bacteria exhibited fluorescent emission peaks at 622 nm for the endogenous and 617 nm for the excreted porphyrins. Gram negative bacteria exhibited a 630 nm emission peak for the endogenous and a 615 nm emission peak for the excreted extracellular porphyrins. Upon illumination of the ALA induced Staphylococcal strains with 407-420 nm blue light, a decrease of five orders of magnitude was demonstrated with a light dose of 50 J cm(-2). Total eradication of the Staphylococcal strains could be achieved with a 100 J cm(-2) dose, which resulted in a decrease in viability of seven orders of magnitude. The viability of all the induced Gram negative strains and B. cereus decreased by one or two orders of magnitude upon illumination with 50 and 100 J cm(-2), respectively. This difference in the photoinactivation rate was found to be due to the distribution and amounts of the various porphyrins in the bacterial strains. The predominant porphyrin in the Staphylococcal strains was coproporphyrin (68.3-74.6%). In the Gram negative strains there was no predominant porphyrin and the porphyrins found were mostly 5-carboxyporphyrin, uroporphyrin, 7- carboxyporphyrin, coproporphyrin and protoporphyrin. In the B. cereus(Gram positive) strain the predominant porphyrin was uroporphyrin (75.8%). Although the total production of porphyrins in the Gram negative bacteria was higher than in the Staphylococcal strains, the amount of coproporphyrin produced by the latter was twice to three times higher than in the Gram negative strains. The extracellular excreted porphyrins did not contribute to the photoinactivation in any of the tested strains. Significant decreases in the Na(+) and K(+) content were detected in induced S. aureus after illumination while only small changes were observed in E. coli B. The green fluorescent protein within the cytoplasm of induced E. coli strains was only partially disrupted (by 60% only). These results indicate a partial yield of the effects generated by (1)O(2) radicals resulting from the photoinactivation of Gram negative bacteria and a successful generation of the same effects in the Staphylococcal strains.

Eradication of Acinetobacter baumannii by photosensitized agents in vitro

The photodynamic effects of photosensitizers on Acinetobacter baumannii were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrobial agents. The best photodynamic effect was obtained when A. baumannii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 mumoles l-off and polymyxin nonapeptide (PMNP) at a concentration of 200 mumoles l-1. At these concentrations the culture in brain heart infusion (BHI) broth was found to be sterile after l h of treatment. Some inhibition was also obtained under the same conditions with Cd-texaphyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphine (TMPyP) did not exhibit any photodynamic effect on A. baumannii when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivation by the sensitizer alone even in the absence of PMNP. It was found that a high concentration of the proteins present in BHI or in serum prevent TMPyP from acting as a photosensitizer against A. baumannii. Bovine serum albumin at the same high protein concentration prevents Dp (in the presence of PMNP) to act as a photosensitizer. The anionic photosensitizer tetra-sulfonatophenyl porphine (TPPS4) did not show any photodynamic effect in high or low protein media. In this study it was found that despite the high resistance of the Acinetobacter baumannii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPyP in low protein content environments. When the protein concentration is high photoinactivation efficiency depends on the type of protein present in the medium.

ZnO nanoparticle-coated surfaces inhibit bacterial biofilm formation and increase antibiotic susceptibility

Nanotechnology is providing new ways to manipulate the structure and chemistry of surfaces to inhibit bacterial colonization. In this study, we evaluated the ability of glass slides coated with zinc oxide (ZnO) nanoparticles to restrict the biofilm formation of common bacterial pathogens. The generation of hydroxyl radicals, originating from the coated surface, was found to play a key role in antibiofilm activity. Furthermore, we evaluated the ability of the nanoparticle coating to enhance the antibacterial activity of commonly-used antibiotics. The ZnO nanoparticles were synthesized and deposited on the surface of glass slides using a one-step ultrasound irradiation process. Several physico-chemical surface characterization methods were performed to prove the long-term stability and homogenity of the coated films. Collectively, our findings may open a new door for utilizing ZnO nanoparticle films as antibiofilm coating of surfaces, thus providing a versatile platform for a wide range of applications both in medical and industrial settings, all of which are prone to bacterial colonization.

STRUCTURE-ACTIVITY RELATIONSHIP OF PORPHINES FOR PHOTOINACTIVATION OF BACTERIA

Abstract— The antibacterial photodynamic effects of uncharged (o‐tetrahydroxyphenyl porphine [THPP], m‐THPP and p‐THPP), cationic (5,10,15,20‐tetra[4‐N‐methylpyridyllporphine [TMPyP]) and anionic (5,10,15,20‐tetra[4‐sulfonatophenyl porphine] [TPPS4]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane‐disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram‐positive bacteria but only marginally effective on gram‐negative cells, whereas TPPS4 showed no activity on either gram‐positive or gram‐negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram‐positive target cells. For TPPS4, the inactivity toward gram‐positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram‐negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure‐activity relationships of these porphines are affected by their net charge and by aggregation.