Biocontrol science最新文献

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^T 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^T, 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^T, which may be used as an index for evaluating the sporulation progress of B. cereus.

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.

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.

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.

The aim of this study was to evaluate bacterial decontamination of boiled young sardine by treatment with violet-blue light followed by cooling storage of the irradiated boiled sardine. Viable cell count in the samples was evaluated after irradiation with four types of violet-blue light-emitting diodes (LEDs; peak wavelength at 405, 412, 421 or 455 nm) and subsequent cooling storage for two days. LED (405 nm) exhibited bactericidal and growth suppression effects. The irradiation gave a 47% bactericidal rate in comparison with no irradiation samples (control) and the two-day storage suppressed the increase in cell counts to 24%, while the rate of increase was 545% for the control. Integrated viability (IV) based on growth delay analysis was estimated after irradiation of four isolates from boiled sardine with 405 nm light. The irradiation caused growth delay against all isolates, resulting in smaller IV values for three isolates compared to those viabilities estimated from colony forming units. Exposure (405 nm) at 432 J/cm² fluence resulted in a decrease in water content, resulting in an increase in salinity of the samples. This study demonstrated the advantages of light emitting a narrow violet region as a non-thermal disinfection technology in the processing and storage of boiled sardines.

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.

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.

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.

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.