In order to improve the photobactericidal activity of ultraviolet-A (UV-A) mediated by reactive oxygen species (ROS), the present study focused on trans-coumaric acid (trans-CA), which is isomerized by UV-A. Generation of ROS was expected during the isomerization of trans-CA. Trans-CA derivatives, in which the carboxyl group was modified with a methyl, n-butyl or phenyl group, thereby changing the interaction with the cellular membrane by quenching the anionic properties of the carboxyl group and changing the UV adsorption properties, were used. The photobactericidal activities of trans-CA derivatives were evaluated by using UV-A light (wavelength 350 to 385 nm). The number of surviving Escherichia coli NBRC12713 was determined by colony-forming assay. Derivative 4c, which was esterified with a phenyl group, reduced survival by more than 5.0-log at a dose of 7.4 J/cm2 and by 3.2-log at a dose of 4.9 J/cm2. This synergistic activity may have been caused by the absorption of photon energy from UV-A, which is attributable to the UV spectrum of 4c. The photobactericidal activity was comparable to that of riboflavin, a known photo-activated agent. Isomerized molecules serve as a promising lead for improving the photobactericidal activity of UV-A by activating molecule-mediated ROS generation.
Eggshells have high bioavailability and can be used as a source of calcium. The main component is CaCO3, which, when heated, is converted to CaO. Seashells are also mainly composed of CaCO3 and were previously found to exhibit antimicrobial activity after being heated. In this study, heated eggshell powder (HESP) was found to have antimicrobial activity against bacterial vegetative cells, fungi and bacterial spores. Parameters, such as the minimum inhibitory concentration, were determined with kinetic analysis using an indirect conductimetric assay. Moreover, HESP was able to kill the Bacillus subtilis spores. There were no significant differences in the activity between HESP, heated scallop-shell powder and pure CaO. The MIC values for HESP against bacteria and fungi were 0.29-0.43 and 1.3-1.5 mg/mL, respectively. Against B. subtilis spores, a reduction of two orders of magnitude of viability was confirmed following 20 min of treatment at 10 mg/mL at 60 ℃. The active oxygen generated from the HESP slurry was examined with chemiluminescence. The intensity of this increased with increasing concentrations of the HESP slurry. This suggests that HESP could be used as a natural antimicrobial agent. Although a high pH is the main contributor to this antimicrobial activity, active oxygen species generated from HESP are likely to be the main antimicrobial agents..
For high-throughput screening of novel cosmetic preservatives, a rapid and simple assay to evaluate the antimicrobial activities should be developed because the conventional agar dilution method is time-consuming and labor-intensive. To address this issue, we evaluated a microbial sensor as a tool for rapid antimicrobial activity testing. The sensor consists of an oxygen electrode and a filter membrane that holds the test microorganisms, Staphylococcus aureus and Candida albicans. The antimicrobial activity of the tested cosmetic preservative was evaluated by measuring the current increases corresponding to the decreases in oxygen consumption in the microbial respiration. The current increases detected by the sensor showed positive correlation to the concentrations of two commercially used preservatives, chlorphenesin and 2-phenoxyethanol. The same tendency was also observed when a model cosmetic product was used as a preservative solvent, indicating the feasibility in practical use. Furthermore, the microbial sensor and microfluidic flow-cell was assembled to achieve sequential measurements. The sensor system presented in this study could be useful in large-scale screening experiments.
A new low-temperature sterilization method to replace the ethylene oxide gas sterilization is needed. Strong bactericidal effects of OH and O2H radicals are well known. The purpose of this study was to evaluate the sterilization effect of wet oxygen ("O2+H2O") plasma in the bubbling method, confirming the effect of humidity. Sterility assurance was confirmed by using a biological indicator (Geobacillus stearothermophilus ATCC7953, Namsa, USA). One hundred and eight samples (105 spores/carrier) were divided into three groups of 36 in each for treatment with a different type of gas (O2, O2+H2O, Air+H2O). Plasma processing was conducted using a plasma ashing apparatus (13.56 MHz, PACK-3®, Y. A. C., Japan) under various gas pressures (13, 25, 50 Pa) and gas flows (50, 100, 200 sccm). Fixed plasma treatment parameters were power at 150 W, temperature of 60℃, treatment time of 10 min. The samples after treatment were incubated in trypticase soy broth at 58℃ for 72 h. The negative culture rate in the "O2+H2O" group was significantly (Mantel-Haenszel procedure, p<0.001) higher than in the other gas groups. It is suggested that the significant sterilization effect of the "O2+H2O" group depends on the bubbling method which is the method of introducing vapor into the chamber. The bubbling method seems able to generate OH and O2H radicals in a stable way.
Temperature is one of the important parameters regulating the expression of virulence factors in bacteria. The global regulator, a histone-like nucleoid structuring protein (H-NS), is known to play a crucial role in this regulation. In the present study, we first clarified the role of H-NS in the temperature-dependent regulation of virulence factor production in Vibrio vulnificus, including that of the cytolytic toxin (V. vulnificus hemolysin: VVH) and the proteolytic enzyme (V. vulnificus protease: VVP). The expression of hns itself was subjected to temperature regulation, where hns was expressed more at 26℃ than at 37℃. VVH production and the expression of its gene vvhA were increased by disruption of the hns gene. H-NS appeared to affect the vvhA expression by the well-documented transcriptional silencing mechanism. On the other hand, hns disruption resulted in the reduction of VVP production and the expression of its gene vvpE. H-NS was suggested to positively regulate vvpE expression through the increase in the level of the rpoS mRNA. Moreover, H-NS was found to contribute to the survival of V. vulnificus in stressful environments. When compared to the wild type strain, the hns mutant exhibited reduced survival rates when subjected to acidic pH, hyperosmotic and oxidative stress.
Protamine is an arginine-rich polycationic protein extracted from sperm cells of vertebrates including fishes such as salmon. The purpose of this study was to investigate the suppressive effects of protamine on the growth of oral pathogens for possible usage in dental materials. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the microdilution method. Twelve strains of oral viridans streptococci, Actinomyces naeslundii, Actinomyces odontolyticus, Enterococcus faecalis, Lactobacillus acidophilus, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Candida albicans were suppressed by protamine. MIC and MBC values were between 0.009～20 mg/mL and 0.019～80 mg/mL, respectively. The bactericidal activities of protamine against susceptible bacterial species were dependent on the concentration of protamine and incubation time. Based on the results of this study, protamine would be a useful compound for the development of antimicrobial agents against oral pathogens in dental materials.
The growth of black mold (Aspergillus brasiliensis) in black-colored samples such as hair color and mascara was measured with an automatic count system based on time-lapse shadow image analysis (TSIA). A. brasiliensis suspended in a lecithin and polysorbate (LP) solution of each sample (hair color or mascara) was spread on a potato dextrose agar medium plate containing LP. The background image darkness of the agar plate could be adjusted to attain accurate colony counts. 95 colonies in hair color and 22 colonies in mascara could be automatically determined at 48 h. The accuracy of the colony counts could be confirmed from the timelapse image data. In contrast, conventional visual counting at a specified time could not determine the number of colonies or led to false colony counts.
An outbreak of Escherichia coli O157:H7 occurred due to the consumption of sweet dumplings in Japan. We examined the survival of E. coli O157:H7 inoculated into several types of sweet dumplings to evaluate the progress of the residual contaminating pathogens after the production or packing processes. For all 4 types of tested typical sweet dumplings, no significant reduction in the viable cell counts of inoculated E. coli O157:H7 (3 log CFU/g) was observed during storage at -20℃ and 5℃ for 5 weeks. Approximately 1 log CFU/g of reduction was observed after storage for 5 weeks at 10℃ and 15℃, which corresponded to the growth of the naturally contaminating fungi. Similar results were obtained when we used several types of commercially distributed sweet dumplings. It is of vital importance to prevent the cross contamination of sweet dumplings after the heating process in order to reduce the risk of foodborne illness.
We previously developed a method for evaluating the heat resistance of microorganisms by measuring the transition temperature at which the coefficient of linear expansion of a cell changes. Here, we performed heat resistance measurements using a scanning probe microscope with a nano thermal analysis system. The microorganisms studied included six strains of the genus Bacillus or related genera, one strain each of the thermophilic obligate anaerobic bacterial genera Thermoanaerobacter and Moorella, two strains of heat-resistant mold, two strains of non-sporulating bacteria, and one strain of yeast. Both vegetative cells and spores were evaluated. The transition temperature at which the coefficient of linear expansion due to heating changed from a positive value to a negative value correlated strongly with the heat resistance of the microorganism as estimated from the D value. The microorganisms with greater heat resistance exhibited higher transition temperatures. There was also a strong negative correlation between the coefficient of linear expansion and heat resistance in bacteria and yeast, such that microorganisms with greater heat resistance showed lower coefficients of linear expansion. These findings suggest that our method could be useful for evaluating the heat resistance of microorganisms.
Sanita-kunTM SA for Staphylococcus aureus (SkSA), a novel dry sheet quantitative culture system, was evaluated. When the inclusivity and exclusivity of SkSA were assessed using 121 microorganisms including 47 S. aureus strains, the tested S. aureus strains formed blue-colored colonies on the SkSA and all the other microbes failed to grow. The SkSA was then compared with Baird-Parker agar (BP) according to ISO 6888-1, Mannitol salt agar with egg yolk (MSEY), and 3M PetrifilmTM STX (3M-STX) in 100 artificially contami nated food samples. The correlation coefficients between SkSA and BP, SkSA and MSEY, and SkSA and 3M-STX were 0.971, 0.989 and 0.996, respectively. Our results demonstrated that SkSA is a suitable alternative for the enumeration of S. aureus in foods.