Abstract: This study aimed to clarify how the phenolic monoterpene carvacrol and its structural isomer thymol both as essential oil components (EOCs) inhibit the germination of Bacillus subtilis spore. Germination was evaluated by the OD₆₀₀ reduction rate in a growth medium and phosphate buffer containing either l-alanine (l-Ala) system or l-asparagine, d-glucose, d-fructose plus KCl (AGFK) system. The germination of the wild-type spores in the Trypticase Soy broth (TSB) was found to be greatly inhibited by thymol than by carvacrol. Such a difference in the germination inhibition was confirmed by the dipicolinic acid (DPA) release from germinating spores in the AGFK buffer system, but not in the l-Ala system. Similar to the wild-type spores, no difference in the inhibitory activity between the EOCs was also indicated with the gerB, gerK-deletion mutant spores in the l-Ala buffer system and the above substantial difference was also done with the gerA-deleted mutant spores in the AGFK. Fructose was found to release spores from the EOC inhibition and inversely even stimulated. Increased concentrations of glucose and fructose partially suppressed the germination inhibition by carvacrol. The results obtained should contribute to the elucidation of the control effects of these EOCs on bacterial spores in foods.

Abstract: Ultraviolet (UV) -C is widely used to kill bacteria as it damages chromosomal DNA. We analyzed the denaturation of the protein function of Bacillus subtilis spores after UV-C irradiation. Almost all of the B. subtilis spores germinated in Luria-Bertani (LB) liquid medium, but the colony-forming unit (CFU) of the spores on LB agar plates decreased to approximately 1/10³ by 100 mJ/cm² of UV-C irradiation. Some of the spores germinated in LB liquid medium under phase-contrast microscopy, but almost no colonies formed on the LB agar plates after 1 J/cm² of UV-C irradiation. The fluorescence of the green fluorescent protein (GFP) -fused spore proteins, YeeK-GFP, YeeK is a coat protein, decreased following UV-C irradiation of over 1 J/cm², while that of SspA-GFP, SspA is a core protein, decreased following UV-C irradiation of over 2 J/ cm², respectively. These results revealed that UV-C affected on coat proteins more than core proteins. We conclude that 25 to 100 mJ/cm² of UV-C irradiation can cause DNA damage, and more than 1 J/cm² of UV-C irradiation can cause the denaturation of spore proteins involved in germination. Our study would contribute to improve the technology to detect the bacterial spores, especially after UV sterilization.

Abstract: For microbiological management of water quality, it is important to identify bacteria and to understand the community structure. To analyze the community structure during water purification and distribution, we selected a distribution system in which water from other water treatment facilities was not mixed with the target water. Changes in the bacterial community structure during treatment and distribution processes in a slow filtration water treatment facility were analyzed using 16S rRNA gene amplicon sequencing with a portable sequencer MinION. The microbial diversity was reduced by chlorination. The genus level diversity increased during distribution and this diversity was maintained through to the terminal tap water. Yersinia and Aeromonas were dominant in the intake water, and Legionella was dominant in the slow sand filtered water. Chlorination greatly reduced the relative abundance of Yersinia, Aeromonas, and Legionella, and these bacteria were not detected in the terminal tap water. Sphingomonas, Starkeya and Methylobacterium became dominant in the water after chlorination. These bacteria could be used as important indicator bacteria to provide useful information for microbiological control in drinking water distribution systems.

Abstract: To evaluate the synergistic effects of physical and chemical antimicrobial treatments, whether for cidal or static effects, I proposed the extended isobologram (EIBo) analysis method, which is a modification of the isobologram (IBo) analysis commonly used to evaluate synergy between drugs. As the method types for this analysis, the growth delay (GD) assay reported previously by the author was introduced, in addition to the conventional endpoint (EP) assay. The evaluation analysis consists of five stages, analytical procedure establishment, antimicrobial activity assay, dose-effect analysis, IBo analysis, and synergy analysis. In EIBo analysis, the fractional antimicrobial dose (FAD) is introduced for the normalization of the antimicrobial activity of each treatment. For the synergy evaluation, the synergy parameter (SP) is defined as a measure of the degree of the synergistic effect of a combined treatment. This method makes it possible to quantitatively evaluate, predict, and compare various combination treatments as a hurdle technology.