The characteristics of growth and injury of Bacillus subtilis spores whose heat resistance was reduced by the treatment of high hydrostatic pressure processing (HPP) combined with/without alkaline electrolyzed water (AlEW) were investigated. The delay in the lag phase of growth curve observed when used in combination with AlEW, especially at 50 MPa or less, and was prolonged by about 4 to 6 hours. However, the effects of temperature and time during treatment on the lag phase were not significantly different between solo-HPP and in combination with AlEW. The injury characteristics of the treated bacterial spores were evaluated by using antibiotics (penicillin G, rifampicin and chloramphenicol) supplemented trypticase soy broth. In the chloramphenicol supplemented TSB, although the lag phase of bacterial spores treated by HPP with AlEW was not prolonged as compared with the normal-TSB, the decrease in growth rate during logarithmic phase and increase in maximum growth amount were observed. This result could suggest due to a damage by combined treatment of HPP with AlEW such as the inhibition of protein synthesis. Furthermore, the combined treatment with AlEW increased the catalase activity by 1.45, 1.63 and 4.25 times at 30, 80 and 100 MPa, respectively, as compared with solo-HPP, therefore the combined treatment could cause high oxidative stress on bacterial spores.

Purpureocillium lilacinum has been recently found to contaminate a 20% (200,000 μg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB) . We aimed to elucidate the mechanism underlying the resistance of P. lilacinum to PHMB. First, we induced the PHMB-resistant (IR) strains IFM 67050 (IR) and IFM 65838 (IR) from the type strain P. lilacinum CBS 284.36 via cultivation in a medium containing high concentrations of PHMB. We then analyzed the DNA sequences via Illumina sequencing to evaluate the presence of genetic mutations in IFM 65838 (IR) . Further, we established an IFM 65838 (IR) uridine/uracil auxotrophic strain, and using the orotidine-5'-decarboxylase gene, pyrG as a selection marker, we tried to knockout a mutant gene in IFM 65838 (IR) using the CRISPR-Cas9 genome-editing technique. The growth rates of IFM 67050 (IR) and IFM 65838 (IR) in medium containing PHMB increased, and the minimum inhibitory concentrations (MICs) against PHMB also increased. Based on the DNA sequence analysis, we found a nonsynonymous point mutation in the gene PLI-008146 (G779A) in IFM 67050 (IR) and IFM 65838 (IR) . This point mutation leads to site combinations of splicing changes that cause partial sequences deletion (p.Y251_G281del) in the ΔPLI-008146 locus of IFM 65838 (IR) , and deletion sequences include partial adenosine/AMP deaminase motif (PF00962) orthologous to adenosine deaminase (ADA) (GeneBank: OAQ82383.1) . Furthermore, the mutant gene ΔPLI-008146 was successfully knocked out from the resistanceinduced strain using a novel CRISPR-Cas9 gene transformation method. A considerable reduction in growth rate and MIC against PHMB was observed in the absence of the mutant gene. Therefore, ADA may represent an important resistance factor in PHMB-resistant P. lilacinum.

Bacillus cereus is an important foodborne pathogenic bacterium. Although several B. cereus strains have been isolated from the environment, the differences among these strains with respect to spore formation ability and cell morphology need clarification. In this study, a phylogenetic tree was constructed based on the 16S rRNA gene sequences of nine strains of B. cereus. Spore formation and morphology of these nine strains were compared using both phase-contrast and fluorescence microscopy to create an index of the designated sporulation stages. Additionally, to investigate the efficiency of heat-resistant spore formation. Phylogenetic analysis revealed that five strains (ATCC 14579, NBRC 3457, NBRC 3514, NBRC 3836, and NBRC 13597) clustered together and the remaining four (ATCC 10987, NBRC 3003, NBRC 13494, and NBRC 13690) were genetically distinct from each other. Phase-contrast microscopy revealed significant differences in the sporulation stages among the nine strains. Furthermore, the efficiency of heat-resistant spore formation also differed, even among genetically related strains. In conclusion, a variety of cell morphologies during sporulation were observed among the nine B. cereus strains. We propose a designation of sporulation stages in B. cereus ATCC 14579, which may be used as an index for evaluating the sporulation progress of B. cereus.

Ultrafine bubbles (UFBs) are gaining attention in diverse industries as a new type of material with specific physical properties. Bactericidal activity has been reported as one of the unique properties of UFB water; however, the bactericidal activities of UFBs related to the gas type remain unclear. In particular, the bactericidal effect of hydrogen (H) -filled UFB water has not been verified. Therefore, this study aimed to evaluate the bactericidal effects of H- or ozone (O) -filled UFB water using a bacterial suspension test. The results of this study clearly showed that H- or O-filled UFB water had strong bactericidal activity. Exposure of Escherichia coli for 6 h and Staphylococcus aureus for 3 h reduced the survival rate of those bacteria by >90%. This finding suggests that both O gas- and H-filled UFBs are novel environmentally friendly disinfectants that can be employed to avoid the use of chemicals.

Vibrio vulnificus, an opportunistic human pathogen responsible for primary septicemia, initiates pathogenesis by attachment to the intestinal epithelial cells for which the motility by the polar flagellum plays an essential role. The proteomic analysis of outer membrane proteins showed that the treatment with the 1/2 minimum inhibitory concentration (MIC) of polymyxin B (a bacterial antimicrobial peptide) led to the reduced production of flagellin (a major component of the polar flagellum). Furthermore, the bacterial motility was inhibited in the presence of 1/2 MIC of polymyxin B. V. vulnificus has six flagellin genes organized into the flaFBA and flaCDE loci. The flaA was found to be expressed higher than flaC, and its expression was significantly decreased by polymyxin B. As well as polymyxin B, the 1/2 MIC of LL-37 (a human intestinal antimicrobial peptide) reduced the expression of flaA. In addition, among four fragments of LL-37, KI-18 and FK-13 containing FKRIVQRIKDELR could lead to the decreased expression of flaA. Because the motility closely relates to virulence of V. vulnificus, the findings obtained herein indicate that LL-37 may reduce the bacterial virulence through inhibition of the motility via the polar flagellum.

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major burden for health care systems worldwide, and is a threat to our daily lives. Various effective ingredients against SARS-CoV-2 were already reported, however, since products contain various ingredients, it is also important to evaluate the effectiveness of commercially available disinfectants per se. In this study, the virucidal efficacy of forty-eight commercially available products were evaluated according to the standardized suspension method EN 14476 and the following results were obtained: Alcohol-based disinfectants, hand soaps, wet wipes, alkaline cleaners, quaternary ammonium compound sanitizers and oxygen bleach had great virucidal efficacy against SARS-CoV-2. Enveloped viruses such as SARS-CoV-2 are among the most susceptible of pathogens to formulated microbicidal actives and detergents, but as the results of this study showed, it is also necessary to pay attention to the concentration at the time of use and the required contact time.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was approved for medical use in 2011 and is currently used as a rapid, accurate and lowcost technique for bacterial identification. External quality control for medical analysis is monitored using tests of the Japanese Association of Medical Technologists and Prefectural Association of Clinical Laboratory Technologists and through user surveys of reagent and equipment manufacturers. However, external quality control of bacterial typing using MS is not performed. Therefore, we examined procedures for evaluating quality control of bacterial typing using an identification reliability index at 38 facilities.

Legionella pneumophila (L. pneumophila) is responsible for most Legionnaire's disease cases diagnosed worldwide. The species includes 16 serogroups, but most Legionnaire's disease cases (85.7% in Europe, 87.0% in Japan) are caused by L. pneumophila serogroup 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify the L. pneumophila serogroup. In this study, we compared three sample preparation methods that are compatible with MALDI-TOF MS: the direct colony transfer method (DCTM), on-target extraction method (OTEM), and in-tube extraction method (ITEM). The aim was to improve the low identification rates for L. pneumophila, and establish and validate a simple, rapid and robust MALDI-TOF MS-based method for routine use in microbiological laboratories for assignment of L. pneumophila isolates to serogroups and identification of reliable peak biomarkers. Using ITEM, 100.0% (29/29) of hot spring water samples and clinical isolates were correctly identified at the species level. Augmented reference spectra correctly identified all 29 strains at the species level and 29 isolates at the serogroup level, displaying sensitivity, specificity and accuracy of 100.0% for serogroup assignment. MALDI-TOF MS is a relatively inexpensive method for assignment of L. pneumophila serogroups that can serve as a first-line tool for rapid prospective typing.

Electrolyzed water is a novel disinfectant that is widely used in the food industry. We conducted an experimental model-based study to determine the effectiveness of neutral electrolyzed water (NEW) for the daily nozzle cleaning of artificially contaminated tankless and tank-type bidet toilet seats. The toilet seats were designed to automatically self-clean the spray nozzles using tap water or NEW after each use or at specified intervals. The numbers of Pseudomonas aeruginosa and Escherichia coli microorganisms in the spray water were measured twice or thrice per week. A Kruskal-Wallis test was used to compare the bacterial count in the spray water of various cleaning (NEW) and control (tap water) conditions. The number of bacteria was significantly lower in NEW conditions with and without periodic nozzle cleaning functions than in tap water conditions for both tank-type and tankless bidet toilet seats. Microorganisms were detected only on the surface area around the opening for ejecting spray water and not in the internal piping at the spray nozzle tip. These findings demonstrate that NEW has superior decontamination efficacy over tap water when used as a cleaning agent for the spray nozzles of warm-water bidet toilet seats.

The fungal biocontrol agents, Cladosporium sp. have great economic importance on account of their beneficial effects on the integrated pest management (IPM) program. The different species of this genus can control different arthropod pests like sweetpotato whitefly, sugarcane woolly aphid, two-spotted mite, cotton aphid, cotton leaf worm, red spider mite, armyworm; and different plant diseases like- rice blast, wheat stripe rust, chrysanthemum white rust, etc. Chemical pesticides are a common practice by the user to protect their crops from these pests. But the intensive use of chemical pesticides has harmful impacts on human health and ecosystem functioning, and they also reduce plant protection sustainability. Sustainable plant protection could be achieved through the reduction of chemical pesticides, resulting in minimal impact on the environment without compromising crop yields. This review was written based on biocontrol methods using Cladosporium sp. which is an alternative option for pest management. Continued research concerning the commercialization of these biocontrol agents as biopesticides may contribute to sustainable plant protection.

The genus Fusarium causes a wide range of infections in human, animals and herbs. The purpose of this research was to investigate and identify the native strains of Bacillus subtilis playing an inhibitory role against Fusarium oxysporum by producing surfactin. B. subtilis was isolated from the soil of various parks in Tehran-Iran, and identified by biochemical tests. Growth inhibition zone, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of B. subtilis were determined. After purification of surfactin, quantitative and qualitative analysis of surfactin conducted using high performance liquid chromatography (HPLC) . Finally, two selected native strains with the highest production rate of surfactin identified using PCR for 16S rRNA and phylogenetic tree was drawn. Sixty strains of B. subtilis were isolated from soil, after identification through phenotypical and biochemical tests, the antagonistic activity of 27 different strains against F. oxysporum by Agar well diffusion assay determined and the highest inhibition zone was 13.66 mm. Six strains showing the best inhibitory effect, were isolated and their metabolite were purified by methanol. MIC and MFC values of different strains were in the range of 0.5-1.6 and 1.6-2.6 mg/mL. Using HPLC, the purified surfactin content in B. subtilis was about 56.7 - 131.9 μg/mL. Based on the curves of the chromatogram, the preferred strains with the highest production of surfactin, by molecular identification, displayed high similarity to B. subtilis. We got a maximum amount of yellow and transparent surfactin from native strains. Furthermore, the selected bacteria can be good candidates for biological control of fungal pathogens.

Extended-spectrum β-lactamases (ESBLs) are produced mainly by gram-negative bacteria in Enterobacteriaceae. One of the major types of ESBLs is sulfhydryl variable (SHV) -type ESBL. Herein, we attempted to develop a simple and rapid method for the detection of the ESBL bla gene by loop-mediated isothermal amplification (LAMP) . The five-primer set designed could amplify bla gene at an isothermal temperature of 65℃. The detection limit of the LAMP method with the LF loop primer was 1 copy/tube, which was 10,000-fold more sensitive than that of the conventional PCR. The LAMP assay could also detect the direct amplification of bla gene from a single river water sample in Tokyo. The LAMP method has great potential for applications in hospital, soil and water environment, food, and livestock.

The remote bactericidal effect of TiO photocatalyst, i.e., the bactericidal effect away from the photocatalyst, was successfully achieved using a humidified airflow. The TiO photocatalyst used was anatase-type TiO nanoparticles (NPs) annealed with a low-temperature O plasma. For comparison, anatase-type TiO NPs annealed in the air were used. The bacteria, Bacillus subtilis, were placed away from the TiO NPs. The plasma-assisted-annealed TiO NPs significantly inactivated 99% of the bacterial cells in 5 h, whereas the pristine and air-annealed TiO NPs inactivated 88-90% of the bacterial cells. The remote bactericidal effect of plasmaassisted-annealed TiO NPs would be attributed to a larger amount of HO molecules traveled by the airflow from the TiO NPs. The molecules were generated by chemically reacting more photoexcited carriers on the TiO surface with HO and O in the airflow. These photoexcited carriers originated from more oxygen-based species adsorbed and more oxygen vacancies introduced on the TiO surface by the plasma-assisted-annealing.

The aims of this study were to determine the impact of storage practice and mold types on mold growth and aflatoxin B (AFB) concentration in corn residue from local seed corn plants, the main roughage source of dairy farms in the northern region in Thailand. A total of 223 samples from 2 types of corn residue - dried and wet - were collected. Mold contamination was determined by spread plate technique, and aflatoxin B (AFB) quantification was performed by a commercial enzyme-linked immunosorbent assay. Multivariate linear models were created to determine factors associated with fungal quantity and AFB concentration. Results showed that the presence of Cladosporium spp. in the samples was associated with a lower risk of AFB1 contamination (P<0.05). In addition, appropriate storage practices, e.g. keeping feeds under a roof and using floor canvas under feed piles, gave lower risk of mold contamination and decreasing AFB contamination.

To assess injury in bacterial spore populations exposed to lethal stress, we proposed a theoretical basis for applying the DiVSaL method, which has already been reported for general microorganisms as a double subculture method. We constructed a mathematical model in which both injuries to the germination system and the spore body were taken into the theory. In this theory, we reasonably assumed that the viable and germinable spore count is constant before the subsequent vegetative growth and that the delay of germination and outgrowth can be included in the concept of λ injury previously reported as the growth-independent injury. By introducing these assumptions, the double subculture method can be considered to apply to spores as well. As examples of the application of this theory, the growth delays of Bacillus subtilis spores treated with heat and UV irradiation were analyzed and the numbers of injured spores were evaluated. Based on the results obtained, heat is indicated to have a higher injury generation ability than UV irradiation. The applicability of the DiVSaL method as a tool for food preservation and sanitation designs is presented.

This study determined the prevalence of Staphylococcus aureus in food of animal origin, investigated its antimicrobial susceptibility profiles and antimicrobial-resistant genes encoding resistance to methicillin (mecA), penicillin (blaZ), and vancomycin (vanA). Two hundred and sixty food samples, including raw retail milk, meat, and meat products, were obtained from local retail shops in Mansoura city, Egypt. The overall prevalence of S. aureus in the total examined food samples was 32.69% (85/260). Methicillin-resistant S. aureus (MRSA) was identified in 11.15% (29/260) of the tested food samples. S. aureus indicated a high resistance to nalidixic acid, penicillin, ampicillin, cefuroxime, trimethoprim/sulfamethoxazole, and azithromycin. The multiple antibiotic resistance (MAR) rate was 89.4% of the total S. aureus isolates, and MARindex ranges from 0.05-0.64. Genotypically, mecA and blaZ genes were identified in a percentage of 34.11% and 82.35%, respectively, while no isolates harbored the vanA gene. The presence of MAR S. aureus particularly, MRSA in food samples, is of great concern and represents a possible threat to the community. Therefore, the study's findings highlight the importance of establishing vigilant food safety practices for food handlers to inhibit the transmission of S. aureus through the food chain to reduce public health risks.

It is necessary to know the appropriate bacterial culture temperature and stock temperature to carry out high-precision preservative efficacy tests. In this study, we investigated whether the temperature conditions within the culture or stock affect the survivability or growth activity of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. We found that the cold stocking condition affected the survivability of P. aeruginosa, but not that of E. coli and S. aureus. Furthermore, stocking temperature affects the growth activity of E. coli, P. aeruginosa and S. aureus, and the effect on P. aeruginosa was greater than that on the others. However, the attenuating effect on P. aeruginosa was reversed by culturing into fresh medium for up to six weeks after stocking. These results suggest that cold stress during stocking may affect the growth activity of P. aeruginosa. The results of this study should provide knowledge for the precise evaluation of preservative efficacy in cosmetics.

Eighty-seven strains of Aspergillus section Restricti were isolated from five storage rooms (50 strains) and 21 houses (37 strains) between 2014 and 2020. Eleven species were identified based on their morphological characteristics and molecular phylogeny using the rRNA internal transcribed spacer (ITS) region, calmodulin (CaM), β-tubulin (benA), and RNA polymerase II second largest subunit (RPB2) sequences. A. penicillioides, which was known to cause the deterioration of cultural assets, was isolated at high frequency (73%) from the surfaces of 11 cultural assets in the storage rooms; A. clavatophorus and A. magnivesiculatus, which are closely related to A. penicillioides, were also isolated frequently (45 and 64%, respectively). Five species [A. clavatophorus (42.8%), A. penicillioides (42.8%), A. magnivesiculatus (14.3%), A. reticulatus (28.6%), and A. vitricola (28.6%)] were isolated from dust on the carpets in seven houses. Five species [A. clavatophorus (33.3%), A. penicillioides (55.5%), A. magnivesiculatus (44.4%), A. restrictus (44.4%), and A. gracilis (11.1%)] were isolated from dust on the bedding in nine houses. Using the taxonomic system described by Sklenář et al. (2017), five species (A. clavatophorus, A. magnivesiculatus, A. hordei, A. reticulatus, and A. glabripes) previously identified as A. penicillioides were confirmed as new to Japan.

Bidet-toilets are widely used in households and public spaces in Japan. The effect of nozzle cleanliness on spray water quality of warm-water bidet toilet seats is unknown. We conducted a long-term experimental model-based study (2015-2016) and two survey-based studies (A: 2018-2019 and B: 2020-2021) at a university campus. Survey A measured the effect of nozzle cleansing on total viable count (TVC) and heterotrophic plate counts (HPCs) in spray water. Survey B measured the total organic carbon, residual chlorine concentration, TVC, and HPC of different fractions of sprayed water. We found no upstream migration of microbes from the nozzle to water tanks. While daily cleaning of the nozzle surface could reduce bacterial count in spray water, continuous discharge of water washed away bacterial contamination. Fecal indicator bacteria were in traces in spray water, indicating proper maintenance of these toilet seats.

The mechanism of thermal death of mold conidia has not been understood in detail. The purpose of this study is to analyze the death kinetics of heated conidia of Cladosporium sphaerospermum and to ascertain the expectant cell injury responsible for the death. The death of the dormant (resting) conidia of Cladosporium sphaerospermum was examined at temperatures of between 43 and 54℃ with the conventional colony count method. The death reaction apparently followed the first order kinetics, but the Arrhenius plot of the death rate constant demonstrated seemingly a break. The linearity at temperatures higher than that at the break was lost at lower temperatures, suggesting the involvement of an unusual mechanism in the latter temperatures. In the cell morphology, we observed with quinacrine staining the vacuole rupture at a lower temperature but not at a high temperature. Interestingly, the vacuole rupture by low-temperature heating was found to correlate with the viability loss. Furthermore, active protease originally locating in vacuoles was detected in the cytoplasm of the conidia after heated at a low temperature. The results obtained suggest the involvement of potent autophagic cell death induced by low temperature heating of C. sphaerospermum conidia.

The statistical correlation between the number of oral streptococci and the results of ATP bioluminescence assay was examined and compared with the results from Streptococcus plate counts and an oral bacteria quantification system. Because a significant correlation was found between ATP (RLU) and the number of bacteria in the oral bacteria quantification system for all seven types of oral streptococci examined, ATP would reflect a conditions of oral hygiene. However, using this assay, it was observed it may be difficult to correctly evaluate bacteria that form aggregates. Furthermore, even a small number of bacteria (below 10 CFU/mL) , which cannot be measured by the oral bacteria quantification system, could be estimated by using ATP bioluminescence assay. It was suggested that this assay could be used for quantitative evaluation of the effect of oral cleaning.