Biocontrol science

Publisher: Nihon Bōkin Bōbai Gakkai

RG Journal Impact: 0.26 *

*This value is calculated using ResearchGate data and is based on average citation counts from work published in this journal. The data used in the calculation may not be exhaustive.

RG Journal impact history

2018 RG Journal impactAvailable summer 2019
2015 RG Journal impact0.26
2014 RG Journal impact1.54
2013 RG Journal impact1.71
2012 RG Journal impact0.77
2011 RG Journal impact0.88
2010 RG Journal impact0.74
2009 RG Journal impact0.83
2008 RG Journal impact0.57
2007 RG Journal impact0.50

RG Journal impact over time

RG Journal impact
RG Journal impact over timeGraph showing a linear path with a yearly representation of impact points of the journal

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Cited half-life5.40
Immediacy index0.23
Eigenfactor0.00
Article influence0.18
WebsiteBiocontrol Science website
Other titlesBiocontrol science
ISSN1342-4815
OCLC37579252
Material typePeriodical
Document typeJournal / Magazine / Newspaper

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  • Malodorants in the human axilla are produced from human biogenic precursors by axillary bacterial enzymes. In the present study, we used pyrosequencing analysis to identify the axillary bacterial microbiota of 13 Japanese male subjects with cumin-like, spicy body odor (C type), and 9 with milky, skin-based body odor (M type). Anaerococcus, Corynebacterium, and Staphylococcus predominated in both C- and M-type subjects, followed by Moraxella and Peptoniphilus. These genera accounted for 96.2-99.9% of the total bacterial population, except in the microbiota of one C-type subject. However, the axillary bacteria in C-type subjects were more abundant than that in M-type subjects. These results suggest that the level of colonization by axillary bacteria is important for the production of malodorants.
  • The four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTMEnterobacter sakazakii agar, CHROMagarTME. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.
  • Recent studies have investigated the efficacy of air-cleaning products against pathogens in the air. A standard method to evaluate the reduction in airborne viruses caused by an air cleaner has been established using a safe bacteriophage instead of pathogenic viruses; the reduction in airborne viruses is determined by counting the number of viable airborne phages by culture, after operating the air cleaner. The reduction in the number of viable airborne phages could be because of "physical decrease" or "inactivation". Therefore, to understand the mechanism of reduction correctly, an analysis is required to distinguish between physical decrease and inactivation. The purpose of this study was to design an analysis to distinguish between the physical decrease and inactivation of viable phi-X174 phages in aerosols. We established a suitable polymerase chain reaction (PCR) system by selecting an appropriate primer-probe set for PCR and validating the sensitivity, linearity, and specificity of the primer-probe set to robustly quantify phi-X174-specific airborne particles. Using this quantitative PCR system and culture assay, we performed a behavior analysis of the phage aerosol in a small chamber (1 m3) at different levels of humidity, as humidity is known to affect the number of viable airborne phages. The results revealed that the reduction in the number of viable airborne phages was caused not only by physical decrease but also by inactivation under particular levels of humidity. Our study could provide an advanced analysis to differentiate between the physical decrease and inactivation of viable airborne phages.
  • Sublethally heat-injured cells of Salmonella in food can recover under favorable conditions, leading to foodborne illness. To elucidate the molecular mechanism of recovery from heat injury, the global changes in gene transcription of Salmonella Typhimurium were investigated in previous study. In this study, the functions of genes involved in phage shock response (viz., phage shock protein (psp) genes), the transcription levels of which were found in previous study to be increased during recovery from heat injury, were investigated in recovering cells. The increase in pspABCDEFG transcription levels during the recovery process was confirmed by qRT-PCR. To understand the role of psp genes in heat injury recovery, a pspA deletion mutant (ΔpspA) and a pspA-overexpressing strain (S. Typhimurium pBAD30/pspA (+) ) were constructed. ΔpspA showed slightly lower viable counts and membrane potential than those of the wild-type strain during recovery. On the other hand, there was no significant difference in the viable counts between S. Typhimurium pBAD30/pspA (+) and the control strains S. Typhimurium pBAD30/pspA (-) and S. Typhimurium pBAD30 (+) during recovery. It would seem that a lack of PspA protein alone somewhat affects the recovery of S. Typhimurium from heat injury, but overexpression of PspA alone is not sufficient to overcome this effect.
  • Fermented vegetables are common part of Cambodian diet. The food safety status for these foods has not been investigated. This study was conducted to evaluate the microbiological hazards that contaminated fermented vegetables. A total of 68 samples of fermented vegetables were purchased randomly from five wet markets in Phnom Penh. The conventional culture methods for microbiological analysis were used. Coliform bacteria (Escherichia coli, Cronobactersakazakii, and Enterobacter spp.), opportunistic non-Entrobacteriaceae, Enterococcus spp., Staphylococcus spp., and Listeria spp. were found in these fermented foods. The highest contamination rate of Enterococcus spp. was 34% of total fermented vegetable samples, followed by Bacillus spp. coliform bacteria and E. coli (31%, 24% and 10%, respectively). The potential foodborne pathogen, C. sakazakii, was identified in one sample. Fermented mixed vegetables showed higher contamination rate of coliform bacteria (50%) than fermented single-type vegetables (13%). The results showed that fermented vegetables sold in wet market are poor in hygiene. The stage in the processing chain where contamination occurred should be identified and basic sanitary practice should be enforced to improve the food safety of fermented vegetables in Cambodia.
  • The subtilisin-like serine protease gene TghSS42 was cloned from biocontrol agent Trichoderma ghanense ACCC 30153. Its coding region is 1302 bp in length, encoding 433 aa with a predicted protein molecular weight of 42.5 kDa and pI of 5.53. The accession number of cDNA sequence of TghSS42 gene is KJ740359. Furthermore, the transcription of the TghSS42 gene was all up-regulated under nine different treatments by RT-qPCR analysis, and the highest transcription level of TghSS42 approached 177.29-fold at 4 h under induction with 1% (w/v) Alternaria alternata cell walls, indicating that TghSS42 could be induced by the plant or phytopathogen. Furthermore, Escherichia coli recombinant strain BL21-TghSS42 was constructed. The recombinant protease rTghSS42, with an expected molecular weight of approximately 68.5 kDa (containing 26.0 kDa GST tag), has been successfully expressed and purified from BL21-TghSS42. The purified protease rTghSS42 activity reached a peak of 18.7 U/mL at 4 h following 1.0 mM IPTG induction. The optimal enzyme reaction temperature was 40℃ and the optimal pH was 7.0. The recombinant protease rTghSS42 exerted broad-spectrum antifungal ability against Rhizoctonia solani, Fusarium oxysporum, A. alternata, Sclerotinia sclerotiorum and Cytospora chrysosperma. The inhibition rate of mycelial growth varied between 21.2% and 50.0%.
  • Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infections. The presence of MRSA in a hospital is detrimental to patients and to hospital management. Thus, rapid identification of MRSA is needed. Here, we report on a prospective method to rapidly discriminate of MSSA from MRSA using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) analysis in 160 clinical isolates of S. aureus. The predictive model was tested using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.0% for MSSA and 87.5% for MRSA in a 10-fold cross-validation SVM. In blind test sets, 60 S. aureus isolates (30 MSSA and 30 MRSA) were correctly classified, with identification rates of 93.3% for MSSA and 86.7% for MRSA. The method proposed in this study using the predictive model enables detection of one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.
  • Acanthamoeba is found in seawater, fresh water, and soil and is an opportunistic pathogen that causes a potentially blinding corneal infection known as Acanthamoeba keratitis. The anti-amoeba activity of 9 fatty acid salts (potassium butyrate (C4K), caproate (C6K), caprylate (C8K), caprate (C10K), laurate (C12K), myristate (C14K), oleate (C18:1K), linoleate (C18:2K), and linolenate (C18:3K)) was tested on Acanthamoeba castellanii ATCC 30010 (trophozoites and cysts). Fatty acid salts (350 mM and pH 10.5) were prepared by mixing fatty acids with the appropriate amount of KOH. C8K, C10K, and C12K showed growth reduction of 4 log-units (99.99% suppression) in A. castellanii upon 180 min incubation at 175 mM, whereas the pH-adjusted control solution showed no effect. After the amoeba suspension was mixed with C10K or C12K, cell membrane destruction was observed. The minimum inhibitory concentration of C10K and C12K was also determined to be 2.7 mM. Confirmation tests were conducted using contact lenses to evaluate the effectiveness of C10K and C12K as multi-purpose solutions. Experiments using increasing concentrations showed reduced numbers of living cells in C10K (5.5 mM, 10.9 mM) and in C12K (5.5 mM, 10.9 mM). These results demonstrate the inhibitory activity of C10K and C12K against A. castellanii and indicate their potential as anti-amoeba agents.
  • Differences in the physical properties of individual cells cannot be evaluated with conventional experimental methods that are used to study groups of cells obtained from pure cultures. To examine the differences in the thermal tolerance of individual cells that are genetically identical, a method is needed to measure the thermal energy required to kill single cells. We developed a scanning thermal microscopy (SThM) system and measured the thermal conductivity of various bacterial cells, for example, spore formeing Bacillus genus and non spore-forming bacteria such as Escherichia coli. The thermal conductivity of vegetative cells (0.61 to 0.75 W/m・K) was found to be higher than that of spores (0.29 to 0.45 W/m・K). Furthermore the newly developed method enables us to estimate the thermal energy needed to kill individual cells or spores. We believe that this method can estimate the thermal energy required to achieve the cell for sterilization by heating.
  • The effect of the amount of the proline transporter PutP expression on the mechanism of adaptation of E. coli cells to high salinity was analyzed. The PutP gene derived from the E. coli expression plasmid was introduced into the E. coli cell, and a high PutP expression strain was developed. At 1.2 M NaCl culture condition, the growth of normal E. coli cells was inhibited, whereas high ProP expression cells showed growth under 2.5 M NaCl conditions. The uptake of proline by E. coli as a compatible solute and substrate for metabolization was in good accordance with those seen in cell growth. These data suggested that the amount of the proline transporter PutP expression played an important role in the adaptation of E. coli cells to high saline conditions.
  • We compared the TBT-resistant ability of resting cells prepared from isolates that formed colonies on nutrient agar plates containing 100 µM tributyltin (TBT) chloride, such as Photobacterium sp. TKY1, Halomonas sp. TKY2, and Photobacterium sp. NGY1, with those from taxonomically similar type strains. Photobacterium sp. TKY1 showed the highest ability among those three isolates. The number of surviving Photobacterium sp. TKY1 cells was hardly decreased after 1 h of exposure to 100 µM TBTCl, regardless of the number of resting cells in the range from 109.4 to 104.2 CFU mL⁻¹. In such an experimental condition, the maximum number of TBT molecules available to associate with a single cell was estimated to be approximately 6.0 x 1011.8. Resting cells prepared from type strains Photobacterium ganghwense JCM 12487T and P. halotolerans LMG 22194T, which have 16S rDNA sequences highly homologous with those of Photobacterium sp. TKY1, showed sensitivity to TBT, indicating that TBT-resistant marine bacterial species are not closely related in spite of their taxonomic similarity. We also estimated the impact of TBT-resistant bacterial species to indigenous microbial populations of TBT-polluted surface sediments. The number of surviving TBT-sensitive Vibrio natriegens ATCC 14048T cells, 106.2±0.3 CFU mL⁻¹, was reduced to 104.4±0.4 CFU mL⁻¹ when TBT-resistant Photobacterium sp. TKY1 cells, 109.1±0.2 CFU mL⁻¹, coexisted with 109.4±0.2 CFU mL⁻¹ of V. natriegens ATCC 14048T cells in the presence of 100 µM TBTCl. These results indicate that the toxicity of TBT to TBT-sensitive marine bacterial populations might be enhanced when a TBT-resistant marine bacterial species inhabits TBT-polluted surface sediments.
  • The growth and survival of Cronobacter isolates were examined under incubation at different temperatures, and their thermal death behavior was investigated at high temperature conditions of above 50℃. Seventy three strains isolated from fresh vegetables, dried foods and soil were tested: 28 of Cronobacter sakazakii, 5 of C. dublienensis, 27 of C. malonaticus and 13 of C. turicensis. All Cronobacter strains grew and multiplied predominantly at 35 and 44℃ until 16 hours of incubation, but showed poor growth at 15℃, and no growth at 5℃. At 48℃, the bacteria grew slightly during 6 to 8 h-incubation but decreased or were inactivated after 16 h-incubation. The heat resistance of Cronobacter spp. was measured under the conditions of 50, 55, 60, 65 and 70℃. Cronobacter strains survived almost without decrement for 30 min at 50℃, but decreased suddenly and perished completely within 10 to 20 min at 55℃ and within 2 - 5 min at above 60℃. Some food materials should be stored below 5℃ until the preparation, and dried food including powdered infant milk formula should be utilized immediately after reconstitution and preparation.
  • A novel double subculture method, termed DiVSaL (Differential Viabilities between Solid and Liquid media) method, for the enumeration of injured cell population of a microorganism, which occurs after some sublethal to lethal treatment, was proposed. In this method injured cells were enumerated as the differential value between viabilities determined with two different techniques, the conventional plate counting using a solid agar medium and the growth delay analysis using a liquid medium. In the former technique, the viable cell number is obtained as colony forming unit (CFU) formed on an agar medium where sublethally injured cells are as much rescued as possible. In the latter technique, on the other hand,“ the integrated viability” defined by Takano and Tsuchido (1982) is introduced and is calculated from the growth delay of a stressed population, referred to unstressed one. For the growth delay analysis, in this paper, not only the original theoretical model, where the specific growth rate (and therefore the defined G10 value) does not change after the exposure to a stress treatment, but also a novel modified theory, where the parameter changes, is proposed. On the theoretical background, this DiVSaL method as a double subculture method can be used to enumerate the injured cells without selection by addition of some inhibitor or by nutritional shortage.
  • The present study surveyed the occurrence of Cronobacter spp. in dried foods including milk powder, spices and herbs and others, and fresh vegetables commercially available in markets, and ground soil materials for the agriculture. Cronobacter spp. were isolated from 15% of 33 spice and herb samples and 3% of 36 taste foods, and these were C. turicensis, C. malonaticus, C. sakazakii and C. dubliensis. Cronobacter spp. from fresh vegetables were detected in 12% of field vegetables and 13% of hydroponic vegetables. C. turicensis was prevalent in field vegetables, and C. malonaticus was in hydroponic ones. And, Cronobacter spp. in shredded vegetables were detected from 44% of 9 samples, and these were C. dubliensis, C. turicensis and C. sakazakii. Also, Cronobacter spp. in soil from rice field, vegetable field and sandpits were predominantly C. sakazakii and C. malonaticus.
  • The antiaflatoxigenic and antifungal activities of essential oils (EOs) of finger root (Boesenbergia rotunda (L.) Mansf.), pine (Pinus pinaster), rosewood (Aniba rosaedora), Siam benzoin (Styrax tonkinensis), Thai moringa (Moringa oleifera), and ylang ylang (Cananga odorata) were tested for Aspergillus parasiticus and Aspergillus flavus in potato dextrose broth. Aflatoxin B1 (AFB1) was extracted from culture using a QuEChERS-based extraction procedure and analyzed with high performance liquid chromatography (HPLC) coupled to a fluorescence detector. EO of pine showed the greatest inhibition of growth and AFB1 production of A. parasiticus, followed by EOs of rosewood, finger root, Siam benzoin, and ylang ylang. EO of finger root gave the best inhibitory effects on A. flavus, followed by EOs of rosewood, pine, ylang ylang, and Siam benzoin. EO of Thai moringa did not show any significant inhibition of aflatoxigenic fungi. The antiaflatoxigenic activities of EOs correlated with their antifungal activities in the dosedependent manner. Comparison of the application of the five selected EOs in peanut pods by direct and vapor exposure indicated that the AFB1 production inhibitory effects of the five EOs by direct exposure were faster and more effective than by vapor exposure. EO of finger root showed the best inhibition of AFB1 production of A. flavus in peanut pods by direct exposure, followed by EOs of pine, rosewood, ylang ylang, and Siam benzoin.
  • Ethylene oxide gas is an agent in the sterilization of medical devices due to its effectiveness and compatibility with most materials. The advantages and disadvantages, as well as its recommended uses, are explored in this review article. The variables and their relevance on process optimization are described, the types of processing cycles are detailed and emphasis is given to the design and validation of the sterilization process.
  • Quantitative antifungal testing methods for textile fabrics under growth-supportive conditions were studied. Fungal growth activities on unfinished textile fabrics and textile fabrics modified with Ag nanoparticles were investigated using the colony counting method and the luminescence method. Morphological changes of the fungi during incubation were investigated by microscopic observation. Comparison of the results indicated that the fungal growth activity values obtained with the colony counting method depended on the morphological state of the fungi on textile fabrics, whereas those obtained with the luminescence method did not. Our findings indicated that unique characteristics of each testing method must be taken into account for the proper evaluation of antifungal activity.
  • The kinetics of the inactivation of Vibrio parahaemolyticus in sodium chlorite (NaClO2) solution was studied in the weakly acidic pH range of 4.0 to 6.5 and at various temperatures. The logarithmic reduction of the survival ratio depended on the concentration-time product, and all the inactivation curves showed a linear reduction phase. The first-order inactivation rate constant (k) increased by approximately twice for every 0.44 unit fall in pH. During the inactivation experiments, no formation of chlorine dioxide occurred. These data indicated that undissociated HClO2 was the active species governing the inactivation of V. parahaemolyticus. It was also shown that the use of weakly acidic NaClO2 solutions containing high concentrations of ionized ClO2⁻ gave slower kinetics of the inactivation, whereas it could achieve the significant reduction of viable cells of more than 4-log. The k value showed an Arrhenius-type temperature dependence in the temperature range of 5 to 40℃. The apparent activation energy for the inactivation of V. parahaemolyticus was estimated to be 43.5 kJ/mol. The k value increased by approximately 1.8 times for every 10℃ rise in temperature.
  • A study on bioremediation of soil contaminated with petroleum sludge was performed using Bacillus pumilus/MVSV3 (Accession number JN089707). In this study, 5 kg of agricultural soil was mixed well with 5% oil sludge and fertilizers containing nitrogen, phosphorus and potassium-(N:P:K). The treatment resulted in 97% removal of total petroleum hydrocarbon( TPH) in 122 d in bacteria mixed contaminated soil when compared to 12% removal of TPH in uninoculated contaminated soil. The population of the microorganism remained stable after introduced into the oil environment. The physical and chemical parameters of the soil mixed with sludge showed variation indicating improvement and the pH level decreased during the experiment period. Elemental analysis and Gas Chromatography-Mass Spectroscopy( GC-MS) analysis revealed the bacterial ability to degrade oil sludge components. Growth experiments with Trigonellafoenumgraecum ( Fenugreek) showed the applicability of bioremediated soil for the production.
  • Hypochlorous acid (HOCl) solution (200 ppm, pH 6) was prepared and evaluated for their stabilities and microbicidal activities. We demonstrated that HOCl is unstable against ultraviolet (UV) light, sunshine, contact with air, and elevated temperature (≧25℃). Furthermore, in the HOCl solution, the presence of excess NH2- or CHO-containing organic compounds such as proteins and carbohydrates, or of inorganic ions such as NO2-, SO3-, PO3-, Fe2+, Cu2+, and CuS, resulted in the rapid consumption of HOCl by oxidation reactions, and significantly decreased the microbicidal activity of the HOCl solution against coliform bacteria and total viable cell count. Thus, production of stable HOCl solution requires formulation in pure water harboring concentrations as low as possible of various compounds and ions, as well as storage in dark and cool conditions (<10℃) to maintain the concentration of HOCl molecules and microbicidal activity.
  • Most of the ice nucleation activity inhibitor reported so far are compounds processing the hydroxyl group such as the polyphenolic derivative. After examining the anti-ice nucleation activity of the purine base, the highest compound is theophylline, and the activity showed 3.80±0.32℃ at a final concentration of 0.1 mg/ml. We found that the activity of the adenine which was essential to genome information DNA was higher than that of guanine. After examining effect of adenine concentration, high activity showed 9.1±1.2℃ and became approximately constant above 0.1 mg/ml. This active rise is a result of effect of concentration under alkaline condition. Therefore after examining effect of pH on the activity of adenine, this activity rose under an alkaline condition. The active rise predicts that an electric charge of adenine is a factor. Among four kinds of nucleotide of 6 bases, poly-A nucleotide was higher and showed 1.33±0.42℃ at a final concentration of 0.1 mg/ml. This activity of poly-A were proportional to the number of the base. From these results, it was suggested that the poly-A and adenine could be able to be applied to the field to preserve the blood and tissue which differentiated in the generative medicine.
  • Linear alkylbenzene sulfonate (LAS) and polyoxyethylene lauryl ether (POLE) are major surfactants contained in the laundry detergents. In the present study, the antibacterial activities of the surfactants to aquatic microorganisms were compared. When freshwater samples from a small river in Okayama city were treated with each of the surfactants, only LAS showed the significant antibacterial activity. Several strains, which survived after the treatment with 2.0% LAS, were isolated and identified by sequencing of 16S rDNA. All strains were classified into the family Enterobacteriaceae. However, this family was not a major member of the aquatic microflora, suggesting that the bacteria in Enterobacteriaceae have a common property of LAS-resistance in the river water.
  • The aim was to isolate Campylobacter jejuni-specific lytic phages from meats on the market in Japan. These phages were effectively isolated from 13 of 15 (86.7%) retail chicken meat samples (skin and liver) by the enrichment method using Preston Campylobacter Selective Enrichment Broth and 10 host Campylobacter strains. Among the 26 phage isolates, 14 were extracted by means of C. jejuni L26 as a host strain. Phage PHC10 showed the broadest lytic spectrum: active against 67.4% of the 46 C. jejuni strains tested. The other phage isolates showed different lytic spectra. Because phages PHC5, PHC10, PHC19, PHC22, and PHC25 possess an icosahedral head and a contracted tail, they seem to be members of the Myoviridae family. Effects of 19 phage isolates on viability of C. jejuni were investigated. These phages reduced viable counts of C. jejuni by 1-3 log after 6-12 h of incubation at 42℃ as compared to the initial counts. The C. jejuni L26 was found to be suitable as a host because of the wide hosting range. The phages isolated in this study seem to be promising biocontrol agents against C. jejuni in food.
  • The supercooling-facilitating (SCF) activities, that is, the anti-ice nucleation activity of the hot water extracts from five types of processed food refuse was examined. The extract with the highest activity among five hot water extracts was coffee refuse, showing 1.50℃ of SCF activity at a final concentration of 0.1 mg/ml. From the hot water extract of coffee refuse, the coffee refuse extract containing various polyphenols was prepared by the ultrafiltration (less than MWCO 10,000), a solvent fractionation of ethyl acetate. The yield of coffee refuse extract was 0.9% (w/w) from dried coffee refuse. The SCF activity of the coffee refuse extract at a final concentration of 1.0 mg/ml was 4.2℃. HPLC analysis of the coffee refuse extract showed that caffeine and chlorogenic acid, which are major components of coffee, could be found at 173 and 62.3 µg/ml, respectively. However, the SCF activities of both compounds (0.70 and 1.06℃) at a final concentration of 0.1 mg/ml were lower than those of ferulic acid and coumaric acid, respectively at 3.40 and 2.35℃. This is the first report to our knowledge on the SCF activity of caffeine. The SCF activity of caffeine at a final concentration of 1.0 mg/ml was 2.3℃. The specificity of caffeine against various ice nuclei containing calcium oxalate, 9-fluorenon, and ice nucleating bacteria was examined. Caffeine at a final concentration of 1.0 mg/ml could inhibit the ice nucleation activity of calcium oxalate, and Pseudomonas fluorescens KUIN-1 at the same level that of as silver iodide. From these results, it was suggested that the extract could be able to be applied to the field to control the frost damage of the vegetables and that the harvested vegetables might be stored unfrozen even at 0℃ or less.
  • The inactivation of Vibrio parahaemolyticus cells that were unattached or attached to a polyethylene terephthalate (PET) disc in pH-controlled sodium hypochlorite (NaOCl) solutions was studied under turbulent conditions. No significant desorption of attached cells occurred at the free available chlorine( FAC) concentrations from 0.1 to 1.0 mg/l. The number of viable cells was estimated by microbial calorimetry. The logarithmic relative reduction of viable cells was proportional to the product of the FAC concentration and time. In the pH range of 5.6 to 9.3, the firstorder inactivation rate constants for unattached and attached cells increased with decreasing solution pH. It was found that the rate constants for unattached cells were approximately 6 to 7 times higher than those for attached cells at all pH values examined. It was confirmed that attached cells were more resistant to NaOCl solutions than unattached cells even when accessibility of attached cells to HOCI/OCI⁻ was enhanced under turbulent conditions.
  • Prediction of competitive microbial growth is becoming important for microbial food safety. There would be two approaches to predict competitive microbial growth with mathematical models. The first approach is the development of a growth model for competitive microbes. Among several candidates for the competition model considered, the combination of the primary growth model of the new logistic (NL) model and the competition model of the Lotka-Vorttera (LV) model showed the best performance in predicting microbial competitive growth in the mixed culture of two species. This system further successfully predicted the growth of three competitive species in mixed culture. The second approach is the application of the secondary model especially for the parameter of the maximum cell population in the primary growth model. The combination of the NL model and a polynomial model for the maximum population successfully predicted Salmonella growth in raw ground beef. This system further successfully predicted Salmonella growth in beef at various initial concentrations and temperatures. The first approach requires microbial growth data in monoculture for analysis. The second approach to the prediction of competitive growth from the viewpoint of microbial food safety would be more suitable for practical application.
  • Food is a basic necessity for human survival, but it is still the vehicle for the transmission of food borne disease. Various studies have examined the roles of spices, herbs, nuts, and semi-dried fruits, making the need for safe and convenient methods of decontamination a necessity. The current study determined the bacterial and fungal loads of 26 spices and herbs, 5 nuts, 10 semi-dried fruits and 5 other foods. Spices, herbs and semi-dried foods demonstrated the highest bacterial and fungal loads with the majority showing over 10⁴ CFU/mL. Nuts and other foods showed growths ranging from 10² to 10⁶ CFU/mL. The current study also attempted to determine the effects of heat and plasma treatment. The log reduction of bacterial growth after heat treatment (maximum: 120 min for 60℃) was between 0.08 to 4.47, and the log reduction after plasma treatment (maximum: 40 min) ranged from 2.37 to 5.75. Spices showed the lowest rates of reduction, whereas the semi-dried and other foods showed moderate to high levels of decrease after heat treatment. The log reduction of fungal growth after heat treatment ranged from 0.27 to 4.40, and log reduction after plasma treatment ranged from 2.15 to 5.91.Furthermore, we validated the sterilization effect of plasma treatment against Bacillus spp. and Staphylococcus spp. by using scanning electron microscopy. Both treatment methods could prove to be advantageous in the agriculture related fields, enhancing the quality of the foods.
  • An identified class of antifreeze, a xylomannan-based thermal hysteresis (TH)-producing glycolipid, has been discovered from diverse taxa, including plants, insects, and amphibians. We isolated xylomannan from the mycelium and fruit body of the basidiomycete Flammulina velutipes using successive hot extraction with water, 2% and 25% aqueous KOH, and gel filtration chromatography. The xylomannan from the fruit body had a recrystallization inhibiting (RI) activity (RI=0.44) at 0.5 mg/mL. The dried weight yield of the fruit body (7.7×10-²%, w/w) was higher than that of the mycelium. Although the purified xylomannan from both soures were composed of mannose and xylose in a 2: 1 molar ratio, the molecular weight of the xylomannan from the mycelium and fruit body was 320,000 and 240,000, respectively. The RI activity of mycelial xylomannan was higher than that from the fruit body (RI=0.57) at 45 μg/mL. Although this RI activity was able to remain constant after exposure to various conditions, we confirmed that the decrease of RI activity was stimulated by the decrease of molecular weight that was caused by heating during the alkaline condition. The survival rate of the CHO cells at -20 C for two days increased to 97% due to the addition of 20 μg/mL of purified xylomannan. This was the first report to indicate that xylomannan from the mycelium of Flammulina velutipes had a high level of ice recrystallization inhibiting activity like antifreeze proteins from plants and had rhe potential to become a new material for cell storage.
  • To prevent nosocomial infections caused by even either Ebola virus or methicillin-resistant Staphylococcus aureus (MRSA), healthcare workers must wear the appropriate protective clothing which can inhibit contact transmission of these pathogens. Therefore, it is necessary to evaluate the performance of protective clothing for penetration resistance against infectious agents. In Japan, some standard methods were established to evaluate the penetration resistance of protective clothing fabric materials under applied pressure. However, these methods only roughly classified the penetration resistance of fabrics, and the detection sensitivity of the methods and the penetration amount with respect to the relationship between blood and the pathogen have not been studied in detail. Moreover, no standard method using bacteria for evaluation is known. Here, to evaluate penetration resistance of protective clothing materials under applied pressure, the detection sensitivity and the leak amount were investigated by using synthetic blood containing bacteriophage phi-X174 or S. aureus. And the volume of leaked synthetic blood and the amount of test microbe penetration were simultaneously quantified. Our results showed that the penetration detection sensitivity achieved using a test microbial culture was higher than that achieved using synthetic blood at invisible leak level pressures. This finding suggested that there is a potential risk of pathogen penetration even when visual leak of contaminated blood through the protective clothing was not observed. Moreover, at visible leak level pressures, it was found that the amount of test microbe penetration varied at least ten-fold among protective clothing materials classified into the same class of penetration resistance. Analysis of the penetration amount revealed a significant correlation between the volume of penetrated synthetic blood and the amount of test microbe penetration, indicating that the leaked volume of synthetic blood could be considered as a latent indicator for infection risk, that the amount of exposure to contaminated blood corresponds to the risk of infection. Our study helped us ascertain, with high sensitivity, the differences among fabric materials with respect to their protective performance, which may facilitate effective selection of protective clothing depending on the risk assessment.
  • Structure analysis was performed on the antibiotic-resistance-gene region of conjugative plasmids of four fish farm bacteria. The kanamycin resistance gene, IS26, and tetracycline resistance gene (tetA(D)) were flanked by two IS26s in opposite orientation in Citrobacter sp. TA3 and TA6, and Alteromonas sp. TA55 from fish farm A. IS26-Inner was disrupted with ISRSB101. The chloramphenicol resistance gene, IS26 and tetA (D) were flanked by two IS26s in direct orientation in Salmonella sp. TC67 from farm C. Structures of tetA (D) and IS26 were identical among the four bacteria, but there was no insertion within the IS26-Inner of Salmonella sp. TC67. Horizontal gene transfer between the strains of two different genera in fish farm A was suggested by the structure homologies of mobile genetic elements and antibiotic resistance genes.
  • Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.
  • Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.
  • Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent. The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic has been extensively addressed at conferences and in published documents around the world. More recently, the use of alternative methods for control of the microbiological quality of pharmaceutical products and materials used in pharmaceutical production has been addressed by the compendia in an attempt to facilitate implementation of these technologies by pharmaceutical companies. The author presents some of the rapid method technologies under evaluation or in use by pharmaceutical microbiologists and the current status of the implementation of alternative microbial methods.
  • From May 2014 to February 2015, 319 university students (male, n=173; female n=146) of 18 to 24 years of age who carried mobile phones or computer tablets were selected as subjects. Staphylococcus spp. were detected in 101 of 319 samples (31.7%). In the present study, 11 strains of S. aureus were isolated and identified, not all of which were methicillin-resistant Staphylococcus aureus (MRSA). Overall, 14 species were identified, with 11 strains (10.9%) of S. xylosus being isolated at the highest frequency. Following this were eight strains (7.9%) of S. cohnii and seven strains (6.9%) each of S. capitis and S. haemolyticus. Staphylococcus spp. isolation was performed with bacterial samples obtained from the mobile phones of 22 specific subjects (males, n=12; females, n=10). Staphylococcus spp. isolation was performed on days -1, 7 and 30 of the experiment. Staphylococcus spp. were positively detected one or more times in 12 subjects (54.5%). In one subject (8.3%), all three tests were positive. Furthermore, two tests were positive in three (25.0%). In the eight remaining subjects (66.7%) Staphylococcus spp. were detected only once. For the three abovementioned tests, we investigated the pulsed-field gel electrophoresis (PFGE) patterns of the strains derived from the mobile phone and from the fingers of three subjects in whom the same bacterial species were isolated twice. From the cases with similarities between strains derived from the fingers and the mobile phones and cases, with consistency in the strains derived from the mobile phone at different times, commonality was observed in the strains derived from the fingers and mobile phones along with chronological uniformity in the strains derived from the mobile phones. A total of 101 Staphylococcus spp. strains were isolated from mobile phones. According to drug susceptibility tests, 99 strains (98.0%) were found to have some degree of resistance to drugs (excluding one strain each of S. aureus and S. haemolyticus). Among these, the strain that showed the highest level of drug resistance was one strain (1.0%) of Staphylococcus spp., which showed resistance to nine drugs. The strain that showed the second highest level of drug resistance was one strain (1.0%) of S. caprea, which showed resistance to seven drugs. In this manner, the drug-resistant tendencies of Staphylococcus spp. isolated from mobile phones were observed.
  • Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.
  • The combined effect of several microbial control factors including gas barrier of containers, modified atmosphere packaging, food life extenders and storage temperature was discussed in order to determine the possibility for improving the shelf life for hamburger steak and deepfried chicken, representative ready-made dishes sold at convenience stores in Japan. Multiple measures including cold storage were effective in improving the shelf life of ready-made dishes. It was also suggested that storage tests for ready-made dishes should be conducted at 10℃, a practical temperature, to confirm the storable period, as well as at 15℃, an adequate abuse temperature, to confirm the effects of various microbial control factors. In the present study, the test group 4 (nitrogen + barrier containers + pH modifier) performed most favorably at both temperatures, indicating the efficacy of multiple means including "cold storage" in improving the shelf life (extending the consume-by date) of ready-made dishes. All strains isolated from the tested hamburger steak and deep-fried chicken were common food contaminant bacterial species.
  • The aim of this study was to investigate the effect of low-pressure plasma treatment on seed disinfection and the possible mechanisms underlying this effect. Seed-borne disease refers to plant diseases that are transmitted by seeds; seed disinfection is an important technique for prevention of such diseases. In this study, the effectiveness of low-pressure plasma treatment in the inactivation of the seed-borne plant pathogenic bacterium, Xanthomonas campestris, inoculated on cruciferous seeds, was evaluated. The highest inactivation effect was observed when the treatment voltage and argon gas flow rate were 5.5 kV and 0.5 L/min, respectively. The viable cell number of X. campestris was 6.6 log cfu/seed before plasma treatment, and decreased by 3.9 log after 5 min of treatment and by 6.6 log after 40 min. Ethidium monoazide treatment and quantitative real-time PCR results indicated that both the cell membrane and target DNA region were damaged following 5 min of plasma treatment. Although both heat and ozone were generated during the plasma treatment, the contribution of both factors to the inactivation of X. campestris was small by itself in our low-pressure plasma system. Overall, we have shown that our low-pressure plasma system has great applicability to controlling plant pathogenic bacterium contamination of seeds.
  • The characteristics of 11 strains of Stx1-producing and Stx2-non-producing STEC O103:H2 were analyzed to investigate the differences in virulence in a single serotype of Shiga toxin (Stx) -producing Escherichia coli (STEC). Differences in the cell-adhesion activity to Caco-2 cells were observed among the strains. The activity of the one strain, isolated from a patient with hemolytic uremic syndrome was 4-20-fold higher than those of the other strains. Although the strains with high cell-adhesion activity showed high expressions of eae, espB, espD, and tir in the locus of enterocyte effacement related with cell-adhesion, those were not specific for this strain. In addition, the Stx1 production level of the strain was not particularly high. It was indicated that the high adhesion activity might be a potential factor to associate serious symptom.
  • RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 107 cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 102 – 104 copy numbers per ng of the RNA preparation, and the strains produced 104 copies including ones which had the high vacuolation activities of HEp-2 cells.
  • This review discusses the application of several sorts of non-equilibrium gas plasma discharges for sterilization and disinfection treatments against spores or bioburden on/in the healthcare products or biological indicators. The basic properties of electrical discharges are briefly reviewed and thereafter the paper discusses the interactions of gas plasma with several sorts of biological systems such as bacteria, bacterial spores, endotoxins, lipid A and normal and abnormal prion proteins.
  • 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 agarBP according to ISO 6888-1, Mannitol salt agar with egg yolk MSEY , and 3M PetrifilmTM STX 3M-STX in 100 artificially contaminated 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.
  • 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 ("OO2+HO2O") 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 seem). Fixed plasma treatment parameters were power at 150 W, temperature of 60°C, treatment time of 10 min. The samples after treatment were incubated in trypticase soy broth at 58C for 72 h. The negative culture rate in the "O2+H2O" group was significantly (Mantel-Haenszel procedure, p<0.00l) 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.
  • 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.
  • 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.
  • 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.
  • Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.
  • Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.
  • Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent. The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic has been extensively addressed at conferences and in published documents around the world. More recently, the use of alternative methods for control of the microbiological quality of pharmaceutical products and materials used in pharmaceutical production has been addressed by the compendia in an attempt to facilitate implementation of these technologies by pharmaceutical companies. The author presents some of the rapid method technologies under evaluation or in use by pharmaceutical microbiologists and the current status of the implementation of alternative microbial methods.
  • From May 2014 to February 2015, 319 university students (male, n=173; female n=146) of 18 to 24 years of age who carried mobile phones or computer tablets were selected as subjects. Staphylococcus spp. were detected in 101 of 319 samples (31.7%). In the present study, 11 strains of S. aureus were isolated and identified, not all of which were methicillin-resistant Staphylococcus aureus (MRSA). Overall, 14 species were identified, with 11 strains (10.9%) of S. xylosus being isolated at the highest frequency. Following this were eight strains (7.9%) of S. cohnii and seven strains (6.9%) each of S. capitis and S. haemolyticus. Staphylococcus spp. isolation was performed with bacterial samples obtained from the mobile phones of 22 specific subjects (males, n=12; females, n=10). Staphylococcus spp. isolation was performed on days -1, 7 and 30 of the experiment. Staphylococcus spp. were positively detected one or more times in 12 subjects (54.5%). In one subject (8.3%), all three tests were positive. Furthermore, two tests were positive in three (25.0%). In the eight remaining subjects (66.7%) Staphylococcus spp. were detected only once. For the three abovementioned tests, we investigated the pulsed-field gel electrophoresis (PFGE) patterns of the strains derived from the mobile phone and from the fingers of three subjects in whom the same bacterial species were isolated twice. From the cases with similarities between strains derived from the fingers and the mobile phones and cases, with consistency in the strains derived from the mobile phone at different times, commonality was observed in the strains derived from the fingers and mobile phones along with chronological uniformity in the strains derived from the mobile phones. A total of 101 Staphylococcus spp. strains were isolated from mobile phones. According to drug susceptibility tests, 99 strains (98.0%) were found to have some degree of resistance to drugs (excluding one strain each of S. aureus and S. haemolyticus). Among these, the strain that showed the highest level of drug resistance was one strain (1.0%) of Staphylococcus spp., which showed resistance to nine drugs. The strain that showed the second highest level of drug resistance was one strain (1.0%) of S. caprea, which showed resistance to seven drugs. In this manner, the drug-resistant tendencies of Staphylococcus spp. isolated from mobile phones were observed.
  • Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.

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