Abstract: Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization. We observed 26 equipment at our institution for bacterial contamination at least once every 4 weeks for 5 years with another ultrafiltration membrane upstream to prevent bacterial contamination. Bacterial contamination levels were highest and most diverse at the time of the first flush. During subsequent initial cleanng, the contamination level decreased, and bacterial species converged almost exclusively to one genus, namely Methylobacterium spp. During clinical use, the equipment were cleaned and disinfected daily after dialysis, and daily operations and maintenance were performed using aseptic techniques. Although the frequency of bacterial detection decreased annually, the same bacterial genotypes observed at the first flush were isolated even after long time periods and were thought to persist in the equipment possibly by forming biofilm. Pseudomonas aeruginosa was newly detected after the replacement of parts during breakdown maintenance, indicating the need to sterilize replacement parts. Thus, the bioburden should be assessed regularly as part of the management of in-house-produced dialysis fluid.

Abstract: Fatalities caused by pneumonia and underlying diseases from COVID-19 infection show the highest relative frequency among elderly people. Long-term care facilities for elderly people have continued to be the areas most vulnerable to COVID-19. We examined the effectiveness of training for infection control and COVID-19 at elderly care facilities. After sending questionnaires to all long-term elderly care facilities in Ibaraki prefecture, Japan during January 18–29, 2022, we received useful responses from 98 facilities. Using logistic regression, we regressed a dummy variable for outbreak experience to dummy variables representing routine but partial training, routine training for all staff members, long-term care facilities for elderly people, numbers of nurses, and numbers of residents. Outbreak experiences of two types were inferred, as represented by a dummy variable for a COVID-19 outbreak at the facility, and by a dummy variable for outbreak experience at the facility before COVID-19 was found. Multivariate analysis indicated routine training for all staff members as the most effective, in fact the only effective, countermeasure against COVID-19 outbreak.

Abstract: Norovirus (NoV）is a major causative virus of viral gastroenteritis and requires a general disinfection method because it is resistant to common disinfectants such as ethanol and chlorhexidine. This study aimed to find natural extracts as candidates for versatile disinfectant ingredients. The antiviral effect of natural extracts against NoV can be evaluated using the feline calicivirus (FCV）-inactivation test and NoV virus-like particle (NoV-VLP）-binding inhibition test. In this study, screening of natural extracts with anti- NoV effects was performed using these two methods. Of the 63 natural extracts examined, 14 were found to have high FCV-inactivation and NoV-VLP-binding inhibitory effects. In addition, we evaluated the NoV-VLPbinding inhibitory effect of grape seed extract（GSE）containing proanthocyanidins under multiple concentration conditions and treatment times and determined that the binding inhibitory effect of GSE was concentration- and time-dependent. Electron microscopy showed that GSE-treated NoV-VLPs aggregated, distorted, and swelled, suggesting that GSE directly interacts with NoV particles. The results suggest that some natural extracts containing GSE can be used as components of disinfectants against NoV.

Abstract: A new concept of injured population assessment is proposed, in which the size of the injured population in stressed mold spores is evaluated by analyzing the colony formation process on a solid agar medium. In this method, a small paper disc containing mold spores is placed on a subculture agar plate, and the linear increase in the radius of the colony formed by development from the spore is measured over time. Then, the principle of the previously reported growth delay analysis (GDA) method originally using a liquid medium is applied to obtain the integrated viable ratio (IV) of the stressed population from the delay time relative to the growth of the unstressed population. On the other hand, the viable ratio (V) to the initial value as the colony count obtained with the stressed culture is obtained; the difference between the logarithms of V and IV is determined as the log number of the injured population. Applying this analysis method to heated spores of Cladosporium sphaerospermum, we determined the size of the injured population that occurred. This method was considered to be effective as a new method for quantifying injured populations using a solid medium.

Abstract: Considering the lack of detailed research on the antibacterial mechanism of polyoxometalates, we examined the synergistic effect of novel bulky mixed Ti/W hetero-polyoxometalates (K_9.5H_2.5 ［α-Ge₂Ti₄W₂₀O₇₈］・ 29H₂O; αTi4, K₉H₅ ［α-Ge₂Ti₆W₁₈O₇₇］・16H₂O; αTi6, K₂₃H₅［α-Ge₄Ti₁₂W₃₆O₁₅₄］・39H₂O; αTi12, K₉H₅ ［β-Ge₂Ti₆W₁₈O₇₇］・ 45H₂O; βTi6) with the antibiotic oxacillin against vancomycin intermediate-resistant Staphylococcus aureus (VISA) using fractional inhibitory concentration (FIC) index and growth curve in this study. All polyoxometalates used in this study showed remarkable synergistic effects with oxacillin. Its synergistic antibacterial mechanism was examined using reverse transcription PCR (RT-PCR) and penicillin binding protein-2’ (PBP2’) latex agglutination test. The results suggested that these polyoxometalates did not inhibit mecA gene transcription but resulted in PBP2’ protein malfunction. From these results, we concluded that the substances producing resistance in VISA were affected by polyoxometalates depending on their molecular size, facilitating a synergistic antibacterial effect with oxacillin.

Abstract: Shredded cabbage treated with either tap water or electrolyzed water was stored in an active modified atmosphere packaging (MAP) of 10% CO₂ for 5 d at 10℃, 7 d at 5℃, and 8 d at 1℃ to evaluate the occurrence and viability of sublethally injured coliform bacteria. The CO₂ and O₂ concentrations in the packages approached an equilibrium of 10% CO₂ and 10% O₂ during storage at all temperatures tested. Coliforms in shredded cabbage increased during storage at all three temperatures, with the increase being greater at 10℃. Sublethal injury at 65% to 69% for the coliforms was detected only on cabbage samples treated with electrolyzed water and stored at 5℃ for 4 and 7 d. Enterobacter cloacae was one of the injured species of coliform bacteria in shredded cabbage. Shredded cabbage was inoculated with chlorine-injured Escherichia coli O157:H7 and stored at 5℃ for 6 d in an active MAP of 10% CO₂. Counts of E. coli O157:H7 remained almost constant during storage, and injured E. coli O157:H7 ranging from 50% to 70% were found on shredded cabbage throughout the storage period. These results indicate that sublethally injured indicator and pathogenic bacteria would be found on fresh-cut cabbage in the realistic MAP storage at 5℃.

Abstract: Previous studies in our lab have shown that peptidoglycan (PG) enhances the photocatalytic bactericidal effect. Therefore, in this study, we focused on the PG-constituting components. The PG-constituting components were added to Mesoplasma florum with no cell wall, respectively, and their effects on photocatalytic sterilization were investigated. The PG-constituting components used were amino sugars, amino acids, L-Alanine-D-Glutamic Acid (L-Ala-D-Glu) dipeptide of diaminopimelic acid (DAP）-type crosslinking peptide, and Lysine (Lys）-type crosslinking peptide. We compared the survival rates of M. florum cells and PG-constituting components-added M. florum cells after 3 h of photocatalytic reaction. Consequently, the survival rates of the cells that were added N-acetylglucosamine (GlcNAc）, DAP, and L-Ala- D-Glu dipeptide were significantly lower than those of only the cells. Furthermore, the amounts of hydrogen peroxide (H₂O₂) generated by the photocatalytic reaction under the presence of these components were determined. The results showed that DAP, L-Ala- D-Glu dipeptide, and PG of DAP-type significantly increased the amount of H₂O₂ produced. From the above results, it is suggested that the presence of DAP and L-Ala- D-Glu dipeptide in the photocatalytic reaction boosts the production of H₂O₂ and enhances the bactericidal effect and that GlcNAc might produce reactive oxygen species other than H₂O₂.

Abstract: Clavibacter michiganensis, a gram-positive actinomycete, is a major seed-borne tomato pathogen. We investigated the inactivation efficacy of low-pressure plasma treatment against C. michiganensis inoculated on tomato seeds by placing them on a mesh sheet above the bottom dielectric glass plate. The 2- and 5-minute plasma treatment reduced C. michiganensis populations on the tomato seeds by 0.8 and 1.8 log cfu/seed, respectively. The reduction rates were similar to those of C. michiganensis on shirona (cruciferous) seeds, which have different shapes and surface structures. In contrast, the inactivation of C. michiganensis cells using plasma was more difficult than that of X. campestris cells. Additionally, it was found that placing seeds on a mesh sheet laid on the dielectric glass plate was remarkably effective in inactivating the pathogens on tomato seeds. Since the tomato seeds were susceptible to damage from plasma treatment, methods to reduce its damage need to be investigated.

Abstract: For clinical diagnosis of enterohemorrhagic Escherichia coli (EHEC）, it needs to capture viable EHEC cells from stool sample in the view of medical fee points. However, there is no comprehensive solution for the detection of viable EHEC cells since there are wide variety of serotype and susceptibility against potassium tellurite which is commonly used for selective agent in selective medium for EHEC. In these background, EHEC Clear-HT System (EHEC-CHT）, a novel effective chromogenic medium system for screening comprehensive viable EHEC, was developed. When EHEC-CHT was assessed using 128 microbes including 49 clinical isolated EHEC strains, EHEC-CHT detected all 49 EHEC strains as typical blue-colored colony regardless of both serotype and susceptibility to potassium tellurite. EHEC-CHT was compared with Japanese commercially available tellurite-based EHEC selective media using 107 clinical patient stool samples. EHEC-CHT showed higher detection ratio than conventional tellurite-based selective media compared, and 7% improvement at least in detection ratio in this study.

Abstract: The inactivation of Vibrio parahaemolyticus cells attached to a polyethylene terephthalate (PET) disc in a sodium chlorite (NaClO₂) solution was kinetically studied in a weakly acidic pH range of 4.0 – 6.5. The logarithmic reduction in the survival ratio depended on the concentration-time product. All inactivation curves showed a linear reduction phase, and the reduction in viable cells was greater than 4-log. No significant desorption of attached cells was observed during the inactivation treatment. The first-order inactivation rate constant (k) increased by approximately 4.5-fold for every 1.0 unit fall in pH. At all pH values, the k values calculated for the attached cells were approximately half of those for the unattached cells. These findings indicate that a weakly acidic NaClO₂ solution is effective in inactivating bacteria attached to hard surfaces.

Abstract: Many gram-negative pathogens can activate virulence factors under the control of N-acylhomoserine lactone (AHL）-mediated quorum sensing. AHL-degrading enzymes have been investigated for their application in disease control. Trichoderma is a genus of fungi inhabiting various types of soil and are widely used as biocontrol agents for plant pathogens. When the AHL-degrading activity of 33 strains belonging to Trichoderma species was investigated, most strains can degrade AHL. AHL lactonase catalyzes AHL ring opening by hydrolyzing lactone. Two model strains, Trichoderma atroviride MAFF 242473 and MAFF 242475, degrade AHL using their AHL lactonase activity and rapidly metabolize ring-opening AHL. Moreover, co-inoculation with MAFF 242473 and MAFF 242475 effectively inhibited AHL production by the plant pathogens, Pantoea ananatis and Pectobacterium carotovorum subsp. carotovorum. Our study suggested that Trichoderma might be an effective biocontrol agent to inhibit the expression of virulence factors via AHL-mediated quorum sensing.