Imaging of the Staphylococcus aureus Inactivation Process Induced by a Multigas Plasma Jet.

To identify mechanisms underlying the bacterial inactivation process by atmospheric nonthermal plasma using a unique plasma jet that can generate various gas plasmas, Staphylococcus aureus were irradiated with carbon dioxide plasma, which produces a large amount of singlet oxygens, and nitrogen plasma, which produces a large amount of OH radicals. And damaged areas of plasma-treated bacteria were observed by field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. As a result, bacteria were damaged by both gas plasmas, but the site of damage differed according to gas species. Therefore, it suggests that singlet oxygen generated by carbon dioxide plasma or other reactive species caused by singlet oxygen contributes to the damage of internal structures of bacteria through the cell wall and membrane, and OH radicals generated by nitrogen plasma or other reactive species derived from OH radicals contribute to damage of the cell wall and membrane.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

Cell viability score (CVS) as a good indicator of critical concentration of benzalkonium chloride for toxicity in cultured ocular surface cell lines.

Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60 min using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ≥50% of control. The CVS40/80 was calculated as follows: CVS40/80=(number of measurements for viability values >80%)-(number of measurements for viability values <40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher, %CVS50 and %CVS40/80 became 0 and less than -90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower, %CVS50 became 100. In the case of %CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the %CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.

The reactivity of α-oxoaldehyde with reactive oxygen species in diabetes complications.

The reactions of three α-oxoaldehydes (methylglyoxal, glyoxal, and pyruvic acid) with hydroxyl radicals generated by sonolysis of water were investigated using an electron spin resonance (electron paramagnetic resonance) spin-trapping method, and their reaction kinetics were investigated. It is apparent from our experimental results that methylglyoxal exhibits the highest reactivity of the three α-oxoaldehydes. These α-oxoaldehydes can react with hydroxyl radicals faster than other well-known antioxidants can. The reactivity of hydroxyl radicals is higher than that of hydrogen peroxides.

Microbial resistance in relation to catalase activity to oxidative stress induced by photolysis of hydrogen peroxide.

The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H(2)O(2)) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H(2)O(2) than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H(2)O(2). Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H(2)O(2) than that with weak catalase activity. Kinetic analysis using a modified Lineweaver-Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H(2)O(2). The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H(2)O(2) can be alleviated by decomposition of H(2)O(2) by catalase in microorganisms.

In vitro assessment of the cytotoxicity of six topical antibiotics to four cultured ocular surface cell lines.

To determine the cytotoxicity of antibiotic eyedrops to ocular surface cells using a semi-quantitative method, a range of commercially available antibiotic eyedrops were assessed by using three corneal cell lines and one conjunctival cell line. All antibiotic solutions were free of benzalkonium chloride. Cell viability was determined by the MTT assay and neutral red assay following the exposure of cells to the undiluted, 2- and 10-fold diluted drugs for 10, 30, and 60 min. Toxicity was compared using % cell viability score (%CVS) . The tested eyedrops and values of %CVS50 and %CVS40/80 were Bestron(®) (cefmenoxime, 100, 94) , Panimycin(®) (dibekacin, 86, 58) , Noflo(®) (norfloxacin, 90, 50) , Cravit(®) (levofloxacin, 86, 46) , Tosfulo(®) (tosufloxacin, 57, -3) , and Vigamox(®) (moxifloxacin, 57, -6) . Cell viability markedly increased after dilution. For instance, cell viability assayed by MTT was > 80% for all the measurements in antibiotics diluted 10-fold, and the rate of the measurements showing > 80% cell viability decreased to 43% (31 out of 72 measurements) in the solutions diluted 2-fold. Of the drugs tested, Bestron(®) containing cefmenoxime showed the weakest toxicity. Vigamox(®) containing moxifloxacin and Tosuflo(®) containing tosufloxacin were more toxic when compared with the other antibiotics. CVS was useful for the comparison of the cytotoxicity of the drugs.

Virucidal activity of alcohol-based hand rub disinfectants.

We investigated the virucidal activity of commercially available alcohol-based hand rub products against coxsackievirus A7, B5, feline calicivirus F9, and human adenovirus type 3, type 7, type 8 using susceptible cell lines, Vero cells, CRFK cells, and A549 cells. Fifteen tested hand rub products were ethanol (EtOH) for disinfection (Japanese Pharmacopoeia Grade), two EtOH-based products, one povidone iode-containing product, one alkyldiaminoethylglycine hydrochloride-containing product, six benzalkonium chloride (BAK)-containing products, and four chlorohexidine gluconate (CHG)-containing products. Some active ingredients (BAK, benzetonium chloride, and CHG) were diluted with EtOH to make 0.5% and 0.2% solutions. Virus inactivation rates were calculated after contact with each hand rub product for 10 or 60 seconds. Of the hand rub products tested, only the povidone iode-based product showed antiviral activity superior to that of EtOH against all the strains. EtOH solutions of active ingredients (0.2% and 0.5%) also showed decreased antiviral activity. In conclusion, antiviral activity of all the commercially available alcohol-based hand rub products except that containing povidone idode was dependent on their active ingredients. The povidone idode-containing hand rub product kept its effectiveness even after the dilution with EtOH. Although alcohol-based hand rub products are convenient and suitable for the control of some microbes, they are not generally recommended for the control of viral infections.

[Annual incidence of human group A rotavirus G-serotypes detected in a Japanese University Hospital between 2003 and 2007].

Group A rotavirus G-serotyping by polymerase chain reaction (PCR) using university hospital subject samples in September 2003 to August 2004, September 2004 to August 2005, September 2005 to August 2006, and September 2006 to August 2007 showed the most common serotypes G1 and G3, detected in 27 and 33 subjects, compared to 4 subjects in whom serotype G4 was detected. Between 2003 and 2004, serotypes G1 accounted for 50% and G3 for 38%, contrasting with serotype G3 at 79% between 2004 and 2005, serotype G1 at 91% between 2005 and 2006, and serotype G1 and G3 at 37% and 63% between 2006 and 2007, respectively. Serotypes G2 and G9 were not detected at all during any of our time periods. No correlation was seen between subject age and G serotype, although subjects younger than two years old accounted for 73% of subjects. This infection caused combined fever, diarrhea, and vomiting in 48% of subjects but showed no correlation with G serotype. These findings under-score the importance of G-serotyping in understanding rotavirus infection epidemiology at different times and in different locales.

Cell viability of four corneoconjunctival cell lines exposed to five preservatives and a surfactant used for infection control in eyedrops.

The aim of this study was to evaluate the cytotoxicity of six ingredients used in eyedrops with regard to four corneoconjunctival cell lines. Cells were treated with the undiluted solution, and 2-fold, and 10-fold dilutions of each solution for 10, 30, and 60 min and cell viability was measured with the neutral red assay and the MTT assay. The degree of toxicity was based on the cell viability score (CVS). The CVS50 was determined by the number of measurements with a viability ≥ 50% of control. The CVS40/80 was calculated as follows: CVS40/80 = (number of measurements with a viability value >80%) - (number of measurements with a viability value <40%). Results were expressed as % of total measurements (%CVS). The results of each ingredient for %CVS50, and %CVS40/80 were 0.01% benzalkonium chloride (51, -13), 1% boric acid (100, 99), 0.4% methyl paraoxybenzoate (100, 100), 0.4% propyl paraoxybenzoate (100, 100), 1.0% polysorbate 80 (68, 18), and 0.5% chlorobutanol (100, 100). The use of benzalkonium chloride led to apparently low cell viability compared to the other five solutions.

Bactericidal effects and cytotoxicity of new aromatic dialdehyde disinfectants (ortho-phthalaldehyde).

We investigated the bactericidal effects and cytotoxicity of an ortho-phthalaldehyde product in comparison with those of its predecessor glutaraldehyde products. Bactericidal effects ware examined on Mycobacterium terrae, a standard organism used for investigating the bactericidal effect of high-level disinfectants. Cytotoxicity as determined by the MTT assay was examined by using four cell lines. The colony forming test, a method to examine residual toxicity, and the evaporation test, a newly developed method to examine the toxicity of the evaporated ingredients, were performed. Test solutions were 2.25% and 3.5% glutaraldehyde (GA) products and a 0.55% ortho-phthalaldehyde (OPA) product, and glutaraldehyde itself. All the disinfectants showed sufficient bactericidal effects on M. terrae. Meanwhile, the OPA product was less toxic than GA products and GA itself to all the cell lines tested. The colony forming test showed that GA products and GA itself exerted residual cytotoxicity more potently than did the OPA product. The evaporation test showed that GA products and GA itself exerted cytotoxicity via evaporation more potently than did the OPA product. In conclusion, OPA appears to be less cytotoxic than GA even though bactericidal effects were comparable. This may be due to the lower concentration of the active ingredient (ortho-phthalaldehyde) in the OPA product.

Rapid detection of bacteria that produce extended-spectrum ß-lactamase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

OBJECTIVES: Proper use of antibacterial agents is necessary to prevent the spread of drug-resistant bacteria. To support clinicians, laboratories need to rapidly determine bacterial drug susceptibility/resistance. We have established a method to distinguish extended-spectrum ß-lactamase (ESBL)-producing clinical isolates by capturing structural changes in ß-lactam antibiotics using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). METHODS: Clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, classified into ESBL-producing strains and sensitive strains based on the presence or absence of a CTX-M-type gene, were used. Test bacteria were cultured aerobically in solid-phase wells of Eiken DPD1 dry plates at 35°C for 15 min or 30 min with the antibiotics cefotaxime (CTX), cefpodoxime (CPDX) or piperacillin (PIPC). Culture supernatants were then used for analysis with a MALDI Biotyper. RESULTS: Signals derived from non-hydrolyzed products of antibiotics were observed in all strains. In the case of ESBL-producing strains, signals derived from the hydrolysis products of antibiotics were also observed. Since the ratio of signal intensity derived from hydrolysis products divided by the total signal intensity detected was ≥11% for CTX and ≥6% for CPDX and PIPC, all strains were determined to be ESBL-producing bacteria. CONCLUSION: The short incubation time of 15 min suggests that this method can identify ESBL-producing strains much more rapidly than conventional methods.

Photolysis of hydrogen peroxide, an effective disinfection system via hydroxyl radical formation.

The relationship between the amount of hydroxyl radicals generated by photolysis of H(2)O(2) and bactericidal activity was examined. H(2)O(2) (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H(2)O(2) resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 µM hydroxyl radicals would result in reductions of viable counts of >99.99%.

Preserved and unpreserved 12 anti-allergic ophthalmic solutions and ocular surface toxicity: in vitro assessment in four cultured corneal and conjunctival epithelial cell lines.

In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.

Cytotoxicity of topical medications used for infection and inflammation control after cataract surgery in cultured corneal endothelial cells.

Postoperative vision-threatening corneal edema sometimes occurs after eye surgery, and corneal endothelial damage may be caused or exacerbated by drug toxicity. A range of commercially available antibiotic and anti-inflammatory ophthalmic solutions used postoperatively, namely levofloxacin, moxifloxacin, gatifloxacin, cefmenoxime, diclofenac, bromfenac, pranoprofen, betamethasone, and fluoromethorone, were assessed by using human corneal endothelial cells (HCECs). Propylparaoxybenzoate and methylparaoxybenzoate were also examined. Cell survival after 48 h exposure to the drugs was evaluated using the WST assay. Cefmenoxime and betamethasone were the least toxic antibiotic and anti-inflammatory drug, respectively. Cell survival was concentration dependent and increased markedly to ≥ 80% with dilutions of 100-fold or more. Two preservatives seemed to cause minimal cytotoxicity among those tested. Antibiotic cytotoxicity to HCEC was ranked as cefmenoxime < levofloxacin = gatifloxacin < moxifloxacin, while the toxicity of anti-inflammatory drugs was dependent on benzalkonium chloride and polysorbate. These drugs are unlikely to cause HCEC damage at the concentrations used under the usual conditions. Preservatives are essential ingredients in ophthalmic solutions to control postoperative infection and inflammation and we should be aware of their toxicity as well as efficacy.

Fungicidal action of hydroxyl radicals generated by ultrasound in water.

It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection.

[Cytotoxicity of antiglaucoma ophthalmic solutions for human corneal endothelial cells].

PURPOSE: The cytotoxicity of a range of commercial antiglaucoma ophthalmic solutions was assessed in human corneal endothelial cells using in vitro techniques. METHODS: Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, latanoplast, unoprostone, levobunolol, bunazosine, betaxolol, nipradiol, dorzolamide, brinzolamide, and pilocarpine were assessed. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 hours exposure. RESULTS: Survival was lower in prostagrandines and in medications containing benzalkonium. It increased to more than 85% after dilution of 1000-fold or more dilution. CONCLUSIONS: Antiglaucoma ophthalmic solutions have corneal endothelial toxicity. The toxicity significantly decreases after dilution of 1000-fold or more dilution and toxicity seems to be due mostly to benzalkonium chloride.

Antiviral activity of proanthocyanidin against feline calicivirus used as a surrogate for noroviruses, and coxsackievirus used as a representative enteric virus.

Proanthocyanidin, which consists of (+) catechin, (-) epicatechin and their gallates (15%), (-) epicatechin gallate-dimers, -trimers, and -tetramers (80%), and (-) epicatechin gallate-pentamers, -hexamers, and -heptamers (5%), was evaluated for its antiviral activity against feline calicivirus F9 strain (FCV/F9), which is thought to be a surrogate for noroviruses, and coxsackievirus A7 strain (Cox.A7), which was selected as a representative enteric virus. To achieve a viral inactivation rate of 99% or greater after contact for 10 sec., at least 1 mg/ml and 10 mg/ml of proanthocyanidin were required against FCV/F9 and Cox.A7, respectively. Although the antiviral mechanism of proanthocyanidin is not clear at present, proanthocyanidin may be an effective disinfectant against enteroviruses such as noroviruses.

Protective effects of blue light-blocking shades on phototoxicity in human ocular surface cells.

OBJECTIVE: Blue light hazards for retina and ocular surface have been repeatedly described and many protective methods are introduced for retina; however, no study has been conducted on ocular surface protection. The purpose of this in vitro study was to examine phototoxicity and shade protection after blue light irradiation in primary human cells of corneal surface origin. METHODS AND ANALYSIS: Primary human cells of corneal surface origin were obtained from eye bank eyes. After blue light irradiation (405 nm) of these cells for 3 min, and a further 24 hours' incubation, surviving viable cells were assessed by the methyl thiazolyl tetrazolium assay. Simultaneously, cell viability was determined in wells covered by ultraviolet and blue light shades. RESULTS: Under subconfluent conditions, viable cells decreased by around 50% after blue light irradiation, compared with control cells without irradiation. The blue light phototoxicity was not blocked by the control shade, but the ultraviolet-blocking and blue light-blocking shades protected the cells from phototoxicity, producing a 30%-40% reduction (ultraviolet) and 15%-30% reduction (blue light) in viable cells. CONCLUSION: These results indicate that blue light injures ocular surface cells and the cells are protected from damage by a shade. We recommend blue light protection to maintain ocular health, especially in high-risk populations, such as people with dry eye, contact lens users, the malnourished and the elderly.

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

PURPOSE: The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. METHODS: Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. RESULTS: HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. CONCLUSIONS: The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.