Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Infection of cattle by bovine leukemia virus (BLV) causes significant economic losses in terms of milk and meat production in many countries. Because the gut microbiota may be altered by immunomodulation resulting from viral infections, we hypothesized that latent BLV infection would change the gut (i.e., rumen and hindgut) microbiota of infected cattle. In this study, we compared the gut microbiota of 22 uninfected and 29 BLV-infected Holstein-Friesian cows kept on the same farm, by 16S rRNA amplicon sequence analysis of fecal samples. First, we found that the fecal microbial diversity of BLV-infected cows differed slightly from that of uninfected cows. According to differential abundance analysis, some bacterial taxa associated with ruminal fermentation, such as Lachnospiraceae and Veillonellaceae families, were enriched in the fecal microbiota of uninfected cows. Second, the virus propagation ability of BLV strains was examined in vitro, and the correlation of the fecal microbiota with this virus propagation ability was analyzed. Higher virus propagation was shown to lead to less diversity in the microbiota. Differential abundance analysis showed that one bacterial taxon of genus Sanguibacteroides was negatively correlated with the virus propagation ability of BLV strains. Considering these results, BLV infection was speculated to decrease energy production efficiency in the cows via modification of rumen and hindgut microbiota, which partly relies on the virus propagation ability of BLV strains. This may explain the secondary negative effects of BLV infections such as increased susceptibility to other infections and decreased lifetime milk production and reproductive efficiency.

DOI： 10.1016/j.vetmic.2019.108547

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Impetigo is a contagious skin infection predominantly caused by Staphylococcus aureus. Decontamination of S. aureus from the skin is becoming more difficult because of the emergence of antibiotic-resistant strains. Bacteriophage endolysins are less likely to invoke resistance and can eliminate the target bacteria without disturbance of the normal microflora. In this study, we investigated the therapeutic potential of a recombinant endolysin derived from kayvirus S25-3 against staphylococcal impetigo in an experimental setting. First, the recombinant S25-3 endolysin required an incubation period of over 15 minutes to exhibit efficient bactericidal effects against S. aureus. Second, topical application of the recombinant S25-3 endolysin decreased the number of intraepidermal staphylococci and the size of pustules in an experimental mouse model of impetigo. Third, treatment with the recombinant S25-3 endolysin increased the diversity of the skin microbiota in the same mice. Finally, we revealed the genus-specific bacteriolytic effect of recombinant S25-3 endolysin against staphylococci, particularly S. aureus, among human skin commensal bacteria. Therefore, topical treatment with recombinant S25-3 endolysin can be a promising disease management procedure for staphylococcal impetigo by efficient bacteriolysis of S. aureus while improving the cutaneous bacterial microflora.

DOI： 10.3390/v11090769

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

DOI： 10.1007/s00705-017-3513-z

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC  

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S1'' and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13' and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the beta-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the alpha- and beta-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the beta-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the alpha- and b-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13' and S24-1 were structurally similar to the RBPs of phage phi11 of the family Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

DOI： 10.1099/jgv.0.000865

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human-and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci.

DOI： 10.1038/ismej.2014.29

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Nosocomial respiratory infections caused by methicillin-resistant Staphylococcus aureus (MRSA) can progress to lethal systemic infections. Bacteriophage (phage) therapy is expected to be effective against these critical infections. Previously, phage S13' was proposed as a potential therapeutic phage. We here examined phage treatment in a mouse model of lung-derived septicemia using phage S13'. Intraperitoneal phage administration at 6h postinfection reduced the severity of infection and rescued the infected mice. Phage S13' can efficiently lyse hospital-acquired MRSA strains causing pneumonia-associated bacteremia invitro. Thus, phage therapy may be a possible therapeutic intervention in staphylococcal lung-derived septicemia. © 2014 Institut Pasteur.

DOI： 10.1016/j.micinf.2014.02.011

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DOI： 10.1038/509S9a

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BACKGROUND: Streptococcus canis causes deep pyoderma in canines, which raises concerns about the risk of isolates from lesions acquiring an antibiotic-resistant phenotype. It is necessary to identify effective antibiotics and the characteristics of the pathogenic cluster for S. canis-associated deep pyoderma. RESULTS: The signalment, molecular typing, and antibiotic-resistant status of S. canis isolated from deep pyoderma lesions (27 strains) and oral cavities (26 strains) were analyzed. Older dogs tended to have S. canis-associated deep pyoderma (15 of 27 dogs over 10 years old). Veterinarians chose quinolones for 10/16 cases (63%), even though the rate of quinolone-resistant strains of S. canis is 38-59%. Although 70% of the strains showed resistance to three or more antibiotic classes (37/53), 94% (50/53) strains showed sensitivity for penicillins. We also identified β-lactamase activity among penicillin-resistant strains of S. canis. Clonal complex 13 (CC13) was detected only in lesions and formed independent clusters in the phylogenetic tree. One strain of CC13 was resistant to the anti-methicillin-resistant Staphylococcus aureus drugs, vancomycin and linezolid. CONCLUSION: Although antibiotic-resistant strains of S. canis are isolated at a high rate, they can currently be treated with β-lactamase-inhibiting penicillins. CC13 may be a pathogenic cluster with high levels of antibiotics resistance.

DOI： 10.1186/s12917-022-03482-3

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The aim of this study was to investigate the inherent bacteria that contribute to expressing the angiotensin I-converting enzyme (ACE) inhibitory activity and the antioxidant activity of dry-cured meat products without a bacterial starter. Among the ten dry-cured meat product samples, Coppa and Milano salami exhibited high ACE inhibitory activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, and oxygen radical absorbance capacity (ORAC). No consistent trend was observed in the pH values or the total peptide and imidazole dipeptide concentration of the products that exhibited high ACE inhibitory and antioxidant activities in the tested samples. To investigate the bacteria contributing to the ACE inhibitory and antioxidant activities of the product, 16S rRNA sequencing analysis, isolation, and identification of bacteria were performed using not only Coppa and Milano salami but also the Jamon Serrano and Parma prosciutto products that had low functional activities. Results suggest the Lactobacillales order, particularly the species Latilactobacillus sakei and Pediococcus pentosaceus, were the main inherent bacteria in Coppa and Milano salami, respectively, compared with the Jamon Serrano and Parma prosciutto products. Therefore, the inherent lactic acid bacteria in dry-cured meat products without bacterial starter is important for ACE inhibitory and antioxidant activities of the products.

DOI： 10.3390/foods11142123

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Although the biliary system is generally aseptic, gallbladder microbiota has been reported in humans and some animals apart from dogs. We screened and analyzed the bacterial deoxyribonucleic acid in canine gallbladders using bile sampled from 7 healthy dogs and 52 dogs with liver- or gallbladder-associated disease. PCR screening detected bacteria in 17.3% of diseased dogs (9/52) and none in healthy dogs. Microbiota analysis of PCR-positive samples showed that the microbial diversity differed between liver- and gallbladder-associated disease groups. Thus, a specific bacterial community appears to occur at a certain frequency in the bile of diseased dogs.

DOI： 10.1292/jvms.22-0090

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Purpose: Post-cataract surgery bacterial endophthalmitis is a serious postoperative complication, and Enterococcus spp.-induced endophthalmitis reportedly has a particularly poor visual prognosis. This study aimed to demonstrate the prophylactic effect of postoperative intracameral phage administration in Enterococcus faecalis-induced endophthalmitis after cataract surgery in rabbits. Methods: Endophthalmitis was induced in rabbits by injecting E. faecalis into the anterior chamber just after lensectomy while simultaneously administering either phage phiEF24C-P2 or vehicle. Retinal function was evaluated using electroretinography. The number of viable bacteria and myeloperoxidase (MPO) activity in the eye and histopathologic examinations were analyzed 48 hours after infection. Results: In the vehicle-treated group, retinal function at 24 hours after infection was impaired, and the number of viable bacteria and MPO activity in the eye increased 48 hours later. In the phage-administered group, retinal function was maintained; the number of viable bacteria and MPO activity were significantly suppressed. Histopathologic examinations showed disruption of the retinal layers and the presence of numerous E. faecalis in the lens capsule and vitreous cavity in vehicle-treated eyes. In contrast, retinal structures were intact, and no E. faecalis staining was observed in phage-treated eyes. No retinal dysfunction was observed in the group that received phage only without lensectomy; almost no phage was detected in the eyes after 14 days of treatment. Conclusions: Phage administration in the anterior chamber did not cause retinal dysfunction and suppressed postoperative endophthalmitis in rabbits. Translational Relevance: In vivo results of intracameral phage administration suggest that phages are a promising prophylactic candidate for postoperative endophthalmitis.

DOI： 10.1167/tvst.11.4.2

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Staphylococcus pseudintermedius is one of the major pathogens causing canine skin infection. In canine atopic dermatitis (AD), heterogeneous strains of S. pseudintermedius reside on the affected skin site. Because an increase in specific IgE to this bacterium has been reported, S. pseudintermedius is likely to exacerbate the severity of canine AD. In this study, the IgE reactivities to various S. pseudintermedius strains and the IgE-reactive molecules of S. pseudintermedius were investigated. First, examining the IgE reactivities to eight strains of S. pseudintermedius using 141 sera of AD dogs, strain variation of S. pseudintermedius showed 10-63% of the IgE reactivities. This is different from the expected result based on the concept of Staphylococcus aureus clonality in AD patients. Moreover, according to the western blot analysis, there were more than four proteins reactive to IgE. Subsequently, the analysis of the common IgE-reactive protein at ∼15 kDa confirmed that the DM13-domain-containing protein was reactive in AD dogs, which is not coincident with any S. aureus IgE-reactive molecules. Considering these, S. pseudintermedius is likely to exacerbate AD severity in dogs, slightly different from the case of S. aureus in human AD.

DOI： 10.1093/femsle/fnac019

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Severe adverse reactions in cats after vaccination were examined from 316 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 130 (41%) showed anaphylaxis, and 99 (76%) of the 130 cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis in cats. Bovine serum albumin (BSA) as indicator of purification was detected at high levels in commercially available feline vaccines. BSA might derive from fetal calf serum in culture media. This study provides useful information about anaphylaxis including critical details of the potential clinical signs associated with adverse events to feline vaccination.

DOI： 10.1292/jvms.21-0437

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In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

DOI： 10.1007/s00705-021-05205-9

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As a method of evaluating the effect of inactivaors on allergens while suppressing the effect of inactivator on the assay, we developed new dot-blot method which combines immunostaining and protein detection methods. This method is useful for evaluating whether the inactivator can inactivate allergens rather than removing them from the assay.

DOI： 10.1093/bbb/zbab146

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We investigated the IgE reactivity to crude and purified milk antigens in the sera of 112 dogs with cutaneous adverse food reactions (CAFRs). Of the 112 dogs, 33 (29%) had specific IgE for crude milk antigens. In the dogs with milk-specific IgE, IgE reactivity to casein, bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin, and bovine IgG were 81%, 85%, 39%, 27%, and 35%, respectively. Casein and BSA may be important allergens in dogs with CAFRs. Some canine vaccines contain casein hydrolysate as a stabilizer and the pooled serum with anti-casein IgE showed IgE reactivity to the vaccines containing it. Information about IgE reactivity to casein in dogs with CAFRs could be useful for predicting adverse reactions to the vaccines including casein hydrolysate.

DOI： 10.1292/jvms.21-0162

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Severe adverse reactions after rabies vaccination in dogs were examined from 317 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 109 of the 317 dogs showed anaphylaxis (0.15/100,000 vaccinated dogs), and 71 of the 109 cases of anaphylaxis resulted in death (0.10/100,000 vaccinated dogs). We measured bovine serum albumin (BSA) in four commercially available rabies vaccines and found the levels ranged from 0.1 to 16.6 µg/dose. Our survey showed that the rate of anaphylaxis to rabies vaccines in dogs is rare, although some cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis.

DOI： 10.1292/jvms.21-0090

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We examined several aspects of African hedgehog adenovirus (AhAdv-1) that was isolated from an African pygmy hedgehog, including: replication kinetics of, virus-induced cytopathic effect (CPE), activation status of mitogen-activated protein kinase (MAPK) signaling pathways, and possible roles of these signaling pathways in virus replication and virus-induced CPE in MDCK cells. AhAdv-1 efficiently replicated and induced CPE in infected cells and caused accumulation of cleaved caspase-3 at 24 h post-infection (p.i.), suggesting apoptosis induction. Analysis of several intracellular signal transduction pathways, which are involved in apoptosis, showed activation of p38 MAPK, Akt and ERK1/2 pathways at 3 h p.i., and upregulation of phosphorylated SAPK/JNK at 24 h p.i. Although p38 MAPK inhibitor and SAPK/JNK inhibitor suppressed activation of the respective pathways in infected cells, they did not inhibit virus-induced CPE. Treatment of infected cells with inhibitor of the Akt pathway, the p38 pathway, the SAPK/JNK pathway or the ERK pathway revealed that inhibitors of p38 pathway inhibited viral replication by real-time PCR and TCID50 assay in infected MDCK cells, suggesting that AhAdv-1 uses p38 pathway for multiplication in infected cells.

DOI： 10.1016/j.rvsc.2021.07.016

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Bovine leukemia virus (BLV) infection has spread worldwide causing significant economic losses in the livestock industry. In countries with a high prevalence of BLV, minimizing economic losses is challenging; thus, research into various countermeasures is important for improving BLV control. Because anti-BLV drugs have not been developed, the present study explored a promising chemical compound with anti-BLV activity. Initially, screening of a chemical compound library revealed that violaceoid E (vioE), which is isolated from fungus, showed antiviral activity. Further analysis demonstrated that the antiviral effect of vioE inhibited transcriptional activation of BLV. Cellular thermal shift assay and pulldown assays provided evidence for a direct interaction between vioE and the viral transactivator protein, Tax. These data indicate that interference with Tax-dependent transcription could be a novel target for development of anti-BLV drugs. Therefore, it is suggested that vioE is a novel antiviral compound against BLV.

DOI： 10.1016/j.virol.2021.06.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis.

DOI： 10.3390/microorganisms9020212

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Membrane vesicles (MVs) play biologically important roles in Gram-positive bacteria, and purification is essential for their study. Although high-performance flow cytometry has the capability to quantify and isolate specific small particles, it has not been examined for MV isolation. In this study, we used high-performance flow cytometry to analyze MV from Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, prepared by iodixanol density-gradient ultracentrifugation. Analysis of the quality of MV samples before and after sorting showed that the flow cytometric sorting provided higher purity and uniformity compared to gradient isolation alone. The MV purification method using flow cytometry should prove useful for applications requiring a very high purity of MV samples such as proteomic, metagenomic or lipidomic studies.

DOI： 10.1016/j.resmic.2020.11.003

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Lactic acid bacteria (LAB) are known to promote deterioration of quality in meat products, such as discoloration and slime production. In this study, five types of liquid smoke, considered food additives, were assayed for potential anti-spoilage LABs from meat products. The spoilage LABs isolated from meat products were identified as Lactobacillus plantarum, Enterococcus malodoratus, Streptococcus thermophilus, or Carnobacterium maltaromaticum. The liquid smoke product (LS1), made from mixed wood, was more effective in terms of spoilage LAB isolate activities. It had the highest phenol content along with the most varied component among the tested products. In the model sausages containing 0.5 % LS1, which was incubated at 30 °C for 5 d for accelerating their deterioration, the enumeration of LAB and staphylococci was significantly lower than that of the control. Therefore, LS1 was suggested to be useful for suppressing not only LAB but also the other bacteria related to spoilage of meat products.

DOI： 10.3136/fstr.27.759

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Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

DOI： 10.1016/j.anaerobe.2020.102281

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BACKGROUND: IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS: The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS: The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

DOI： 10.1186/s12917-020-02559-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Bovine leukemia virus (BLV) is a global problem that results in significant economic losses to the livestock industry. We developed three virus strains by inserting the HiBiT reporter tag from NanoLuc luciferase (NLuc) into limited sites within BLV molecular clones. Initial analysis for site selection of the tag insertion revealed a permissible site immediately downstream of the viral envelope gene. Therefore, NLuc activity could be used to measure virus copy numbers in the supernatant and the levels of cell infection. Productivity and growth kinetics of the reporter virus were similar to those of the wild-type strain; therefore, the reporter virus can be used to characterize the replication of chimeric viruses as well as responses to the antiviral drug, amprenavir. Collectively, our results suggest that the BLV reporter virus with a HiBiT tag insertion is a highly versatile system for various purposes such as evaluating virus replication and antiviral drugs.

DOI： 10.1016/j.virol.2020.07.011

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In this study, dogs with atopic dermatitis were separated into non-food-induced atopic dermatitis (NFIAD) group (n = 15) and food-induced atopic dermatitis (FIAD) group (n = 37) based on an elimination diet test. IgE reactivity for crude Malassezia pachydermatis (M. pachydermatis) and house dust mites (HDM) allergen extracts was investigated in the two groups using fluorometric enzyme-linked immunosorbent assay (ELISA) and intradermal skin test (IDST). Nine (60%) of the 15 dogs in NFIAD group and 6 (16%) of the 37 dogs in FIAD group showed specific IgE for M. pachydermatis (Mann-Whitney U-test, P < 0.01). By immunoblotting analysis, the pooled serum samples from dogs with IgE for M. pachydermatis showed IgE reactivity for 50 kDa protein of M. pachydermatis. Twelve (80%) of the 15 dogs in NFIAD group and 8 (22%) of the 37 dogs in FIAD group showed specific IgE for HDM (Mann-Whitney U-test, P < 0.01). In addition, the dogs in NFIAD group significantly show a positive IDST to M. pachydermatis and HDM extracts compared with the dogs in FIAD group. The results suggest that dogs with NFIAD are at increased risk of becoming sensitized to the normal commensal organism M. pachydermatis compared with dogs with FIAD, perhaps co-sensitization occurred due to an HDM protease antigen's, Der f 1 and/or Der p 1, proteolytic activity related epidermal skin barrier defects. Treatment to limit skin colonization may thus be especially important in NFIAD.

DOI： 10.1016/j.vetimm.2020.110070

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This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

DOI： 10.1007/s00705-020-04577-8

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New hydroquinone derivatives bearing a vinyl alkyne, pestalotioquinols A and B, were isolated from a fungal culture broth of Pestalotiopsis microspora. The structures of these novel compounds were determined by interpretation of spectroscopic data (1D/2D NMR, MS, and IR), and the absolute configuration of the stereogenic center of pestalotioquinol A was assigned using the modified Mosher's method. Nerve growth factor-differentiated neuronal PC12 cells were pretreated with pestalotioquinols A and B and removed from the medium, and then treated with a generator of peroxynitrite (ONOO-), a reactive nitrogen species, to induce cell death. The cytotoxicity of the treated cells was assessed by measuring lactate dehydrogenase leakage. As a result, 1-3 μM pretreatment of pestalotioquinols A and B rescued neuronal PC12 cells from peroxynitrite-induced cytotoxicity and the protective activity was sustained after removing each compound from the medium. These results demonstrate that pestalotioquinol derivatives are a new class of hydroquinones possessing a vinyl alkyne and exhibiting relatively high neuroprotective effects.

DOI： 10.1038/s41429-019-0213-9

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It is important to establish the molecular basis of the high transmissibility of bovine leukemia virus (BLV) to develop new methods of preventing viral transmission. Hence, the aim of this study was to determine whether some strains had transmission advantages. First, we determined the whole BLV genome sequences of all 34 BLV-infected cows from one farm. Phylogenetic analysis divided strains into 26 major and 8 minor strains. The major strains dominantly spread independent of host factor, bovine leucocyte antigen. Further analysis, with molecular clones, associated transmissibility with viral productivity in vitro. In addition, the two groups could be classified by group-specific mutations. The reverse genetic approach demonstrated that a spontaneous mutation at nucleotide 175 of the BLV genome, which is located in the viral promoter region, could alter viral productivity by changing viral transactivation, suggesting that BLV transmissibility is affected by a spontaneous mutation associated with viral productivity.

DOI： 10.1016/j.virol.2019.08.015

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Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 104 cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 107 CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.

DOI： 10.1128/AAC.01088-19

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DOI： 10.1007/978-3-030-26736-0_2

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This study reports the complete genome sequence of an African pygmy hedgehog adenovirus-1 isolate from an African pygmy hedgehog which displayed respiratory symptoms that included nasal discharge, sniffling, coughing, and respiratory distress. The viral genome is 31,764 bp long and shows four deletion sites compared to that of skunk adenovirus-1.

DOI： 10.1128/MRA.00695-19

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To replace molecular biological and immunological methods, biosensors have recently been developed for the rapid and sensitive detection of bacteria. Among a wide variety of biological materials, bacteriophages have received increasing attention as promising alternatives to antibodies in biosensor applications. Thus, we herein present a rapid and highly selective detection method for pathogenic bacteria, which combines dark-field light scattering imaging with a plasmonic biosensor system. The plasmonic biosensor system employs bacteriophages as the biorecognition element and the aggregation-induced light scattering signal of gold nanoparticle-assembled silica nanospheres as a signal transducer. Using Staphylococcus aureus strain SA27 as a model analyte, we demonstrated that the plasmonic biosensor system detects S. aureus in the presence of excess Escherichia coli in a highly selective manner. After the sample and the S. aureus phage S13'-conjugated plasmon scattering probe were mixed, S. aureus detection was completed within 15-20 min with a detection limit of 8 × 104 colony forming units per milliliter.

DOI： 10.1021/acs.analchem.9b02715

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<jats:p>Endophthalmitis due to infection with <jats:italic>Enterococcus</jats:italic> spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant <jats:italic>Enterococcus faecalis</jats:italic> has been increasing, the development of novel therapeutics is urgently required. We have now demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of <jats:italic>E. faecalis</jats:italic>. Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with <jats:italic>E. faecalis</jats:italic>–related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 10<jats:sup>4</jats:sup> cells) into the vitreous. The number of viable bacteria in the eye increased to &gt;1 × 10<jats:sup>7</jats:sup> colony forming units and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of <jats:italic>E. faecalis.</jats:italic></jats:p>

DOI： 10.1128/aac.01088-19

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DOI： 10.1038/s41429-019-0213-9

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Dogs are model animals that can be used to study the gut microbiome. Although the gut microbiome is assumed to be closely related to aging, information pertaining to this relationship in dogs is limited. Here, we examined the association between the canine gut microbiome and age via a bacterial 16S rRNA gene amplicon sequence analysis in a colony of 43 Japanese purebred Shiba Inu dogs. We found that microbial diversity tended to decrease with aging. A differential abundance analysis showed an association of a single specific microbe with aging. The age-related coabundance network analysis showed that two microbial network modules were positively and negatively associated with aging, respectively. These results suggest that the dog gut microbiome is likely to vary with aging.

DOI： 10.1093/femsle/fnz095

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Language：English   Publisher：MOSBY-ELSEVIER  

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Antibiotic-resistant bacteria can cause intractable infections in humans and animals, with damaging effects to health care and economics. Phage therapy is considered a possible alternative to chemotherapy for treating infections, but still requires laborious in vivo experiments before its introduction into society and its further development. Recently, silkworm larvae have been recognized as highly convenient and useful model animals, and an alternative to higher animals. We describe the procedure for experimental phage therapy to treat Staphylococcus aureus infections in silkworm larvae.

DOI： 10.1007/978-1-4939-8940-9_14

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：MDPI  

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, a vaginal-rectal sample is recommended to be enriched, followed by bacterial identification. In some cases, Enterococcus faecalis overgrows in the enrichment culture. Consequently, the identification test yields false-negative results. Bacteriophages (phages) can be used as antimicrobial materials. Here, we explored the feasibility of using phages to minimize false-negative results in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of vaginal E. faecalis strains. The simple coculture of E. faecalis and S. agalactiae was used as an experimental enrichment model. Phage mixture treatment led to suppression of E. faecalis growth and facilitation of S. agalactiae growth. In addition, testing several sets of S. agalactiae and E. faecalis strains, the treatment with phage mixture in the enrichment improved S. agalactiae detection on chromogenic agar. Our results suggest that the phage mixture can be usefully employed in the S. agalactiae culture test to increase test accuracy.

DOI： 10.3390/v10100552

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

The combined use of phage and antibiotics can show synergistic antimicrobial effects, so-called phage-antibiotic synergy (PAS). Here, we screened and examined PAS against Pseudomonas aeruginosa in vitro. Testing four different phages infecting P. aeruginosa, phage KPP22 classified within the family Myoviridae genus Pbunavirus showed PAS with the widest range of antibiotics, and showed PAS with anti-Pseudomonas drugs such as piperacillin and ceftazidime. Thus, evidence suggests that the combined use of phage and antibiotics is a promising therapeutic strategy against P. aeruginosa infections, with consideration needed regarding the optimal selection and adequate application timing of these phages and antibiotics.

DOI： 10.1007/s00705-018-3811-0

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Mycobacteriophage archival stocks have been kept for ca. 20-50 years in Japan. In this study, we attempted to recover mycobacteriophages from 50 archival stocks and briefly analyzed the recovered phages. The phages were recovered from 72.2% (13/18) of the lyophilized stocks that had been stored for 47-56 years. Moreover, the analysis of 12 representative recovered phages led to their classification as belonging to the family Siphoviridae, and seven of them were typed by polymerase chain reaction (PCR) targeting the gene that encodes the tape measure protein. Considering these results, lyophilization seems to be suitable for phage archival storage.

DOI： 10.1007/s00705-018-3788-8

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This study aimed to determine the characteristics of the Helicobacter pylori host NY43 strain and its prophage-cured derivative. H. pylori colonizing the human stomach cause many diseases. They show high genetic diversity, allowing the development of mutant strains that can form bacterial communities adapted to specific environmental conditions. Bacteriophage activities are associated with bacterial evolution, including pathogenicity development. Herein, we reported the complete genome sequence and genomic organization of two H. pylori prophages, KHP30 and KHP40; the effects of KHP30 on the behaviours of NY43 are not yet known. We showed that approximately 57 % prophage-cured derivatives spontaneously appeared in the exponential phase during liquid culture, and the biological characteristics of these derivatives differed from those of the host NY43. KHP30 reinfected the cured derivatives, and the curing ratio was influenced by culture conditions. KHP30 was shown to promote the development of a flexible H. pylori community with variable characteristics.

DOI： 10.1099/mic.0.000665

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Using Mycobacterium smegmatis mc2155, 12 siphoviruses were recovered from long-term archival stocks stored in Japan. Their genome sequences were 46.0 to 61.3 kbp with 63 to 68% G+C contents, which allowed them to be categorized within cluster W and subclusters A1, A2, B3, A7, I1, and K4.

DOI： 10.1128/genomeA.00472-18

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DOI： 10.1007/s00705-018-3723-z

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© Springer Science+Business Media LLC 2018. One of the most important factors for successful bacteriophage therapy is, undoubtedly, the isolation of excellent therapeutic candidate bacteriophages. There are only a few reports about active bacteriophages in the fastidious bacteria Helicobacter pylori. In this chapter, we describe a method for isolating and purifying KHP30-like bacteriophages in H. pylori, which have lytic and pseudolysogenic life cycles.

DOI： 10.1007/978-1-4939-7395-8_1

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The nonstructural G4 gene of bovine leukemia virus (BLV) has been thought to function in virus replication. However, the discovery of the AS1 gene on the antisense strand of the G4 gene has affected this interpretation. In this study, we investigated the function of G4 in virus production independent of the AS1 gene using a reverse genetic approach, and briefly examined the association of the G4 protein with Tax, which is also a nonstructural protein that promotes virus replication. First, we constructed a mutant molecular clone of BLV with a nonsense mutation in G4 that had a minimal effect on the AS1 gene. Comparison of the wild-type and mutant molecular clones indicated that the nonsense mutation resulted in a reduction of virus in the culture supernatant and accumulation of viral RNA (vRNA) in cells. Moreover, G4 and Tax expression in cells was shown to synergistically enhance virus production. Therefore, we suggest that G4 enhances virus production through abrogation of vRNA accumulation.

DOI： 10.1016/j.virusres.2017.07.005

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© 2017 Elsevier B.V. The nonstructural G4 gene of bovine leukemia virus (BLV) has been thought to function in virus replication. However, the discovery of the AS1 gene on the antisense strand of the G4 gene has affected this interpretation. In this study, we investigated the function of G4 in virus production independent of the AS1 gene using a reverse genetic approach, and briefly examined the association of the G4 protein with Tax, which is also a nonstructural protein that promotes virus replication. First, we constructed a mutant molecular clone of BLV with a nonsense mutation in G4 that had a minimal effect on the AS1 gene. Comparison of the wild-type and mutant molecular clones indicated that the nonsense mutation resulted in a reduction of virus in the culture supernatant and accumulation of viral RNA (vRNA) in cells. Moreover, G4 and Tax expression in cells was shown to synergistically enhance virus production. Therefore, we suggest that G4 enhances virus production through abrogation of vRNA accumulation.

DOI： 10.1016/j.virusres.2017.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

© 2016 The Authors. Enzootic bovine leucosis is caused by bovine leukemia virus (BLV) infection, which is highly prevalent in several regions of the world and significantly impacts the livestock industry. In BLV infection, the proviral load in the blood reflects disease progression. Although the BLV genome is highly conserved among retroviruses, genetic variation has been reported. However, the relationship between proviral load and genetic variation is poorly understood. In this study, we investigated the changes in proviral load in BLV-infected cattle in Japan and then identified and analysed a BLV strain pvAF967 that had a static proviral load. First, examining the proviral load in the aleukaemic cattle in 2014 and 2015, cow AF967 showed a static proviral load, while the other cows showed significant increases in proviral load. Sequencing the provirus in cow AF967 showed a deletion of 12 nt located in the G4 gene. An in vitro assay system using BLV molecular clone was set up to evaluate viral replication and production. In this in vitro assay, the deletion mutation in the G4 gene resulted in a significant decrease in viral replication and production. In addition, we showed that the deletion mutation did not affect the viral transcriptional activity of Tax protein, which is also important for virus replication. The emergence of strain pvAF967 that showed a static proviral load, combined with other retrovirus evolutionary traits, suggests that some BLV strains may have evolved to be symbiotic with cattle.

DOI： 10.1099/jgv.0.000583

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© 2016 Elsevier B.V. Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

DOI： 10.1016/j.vetimm.2016.06.003

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage ( phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086. The information obtained in this study should be useful for further development of safe and efficient phage therapy.IMPORTANCEPseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage ( phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

DOI： 10.1128/AEM.00090-16

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR, in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.

DOI： 10.1093/femsle/fnw157

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© 2016 The Societies and Wiley Publishing Asia Pty Ltd. Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.

DOI： 10.1111/1348-0421.12347

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Pseudomonas aeruginosa phages belonging to the family Podoviridae are one of the well-characterized phage groups. In this study, a novel P. aeruginosa phage, KPP25, was isolated and characterized. Phage KPP25's morphology was indicative of the family Podoviridae; however, analyses of the whole genome and the virion proteins suggested that it did not belong to any of the known podophage genera. Based on these analyses, phage KPP25 appears to be a novel podophage infecting P. aeruginosa. © 2014 Elsevier B.V.

DOI： 10.1016/j.virusres.2014.04.019

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Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13' (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13', open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses.

DOI： 10.1002/mbo3.166

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© 2014 Sakaguchi et al. Clostridium botulinum strain Osaka05, which has been isolated from an infant patient with botulism in Japan, is the first strain producing botulinum neurotoxin subtype B6. Here, we report the draft genome sequence of C. botulinum Osaka05.

DOI： 10.1128/genomeA.e01010-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Am Soc Microbiol  

© 2014 Yamaguchi et al. Bacteriophage (phage) therapy is expected to become an alternative therapy for Pseudomonas aeruginosa infections. P. aeruginosa phage KPP23 is a newly isolated phage belonging to the family Siphoviridae and may be a therapeutic phage candidate. We report its complete genome, which comprises 62,774 bp of double-stranded DNA containing 95 open reading frames.

DOI： 10.1128/genomeA.00233-14

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Silkworm larva has recently been recognized as an alternative model animal for higher mammals to evaluate the effects of antibiotics. In this study, we examined the efficacy of the bacteriophage (phage) therapy, which harnesses phages as antibacterial agents, against Staphylococcus aureus infections, using the silkworm larval infection model. Two newly isolated staphylococcal phages, S25-3 and S13′, were used as therapeutic phage candidates. They were assigned to two different lytic phage genera, Twort-like and AHJD-like viruses, based on their morphologies and the N-terminal amino acid sequences of the major capsid proteins. Both had a broad host range and strong lytic activity and showed preservative quality. Administration of these phages alone caused no adverse effects in the silkworm larvae. Moreover, the viruses showed life-prolonging effects in the silkworm larval infection model 10 min, 6 h, 12 h, and 24 h following infection. Such phage effects in the silkworm larval model were almost paralleled to the therapeutic efficacies in mouse models. These results suggest that phages S25-3 and S13′ are eligible as therapeutic candidates and that the silkworm larval model is valid for the evaluation of phage therapy as well as mouse models. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

DOI： 10.1111/1574-6968.12220

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Genotyping by pulsed-field gel electrophoresis (PFGE) is recognized as one of the most useful methods for epidemiological studies of drug-resistant Enterococcus faecalis nosocomial outbreaks. As the bacteriolysis procedure is a critical step in PFGE, an accurate and reliable alternative bacteriolytic method to the conventional protocol would be useful. In this study, we examined the applicability of endolysin ORF9, derived from the E. faecalis phage φEF24C, and lantibiotic nisin in sample preparation for PFGE. The results show that the cooperative actions of nisin and ORF9 synergistically lysed E. faecalis, which was then amenable to PFGE analysis. © 2012 Springer-Verlag Berlin Heidelberg and the University of Milan.

DOI： 10.1007/s13213-012-0556-y

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The therapeutic effects of bacteriophage (phage) KPP12 in Pseudomonas aeruginosa keratitis were investigated in mice. Morphological analysis showed that phage KPP12 is a member of the family Myoviridae, morphotype A1, and DNA sequence analysis revealed that phage KPP12 is similar to PB1-like viruses. Analysis of the phage KPP12 genome did not identify any genes related to drug resistance, pathogenicity or lysogenicity, and so phage KPP12 may be a good candidate for therapeutic. KPP12 showed a broad host range for P. aeruginosa strains isolated from clinical ophthalmic infections. Inoculation of the scarified cornea with P. aeruginosa caused severe keratitis and eventual corneal perforation. Subsequent single-dose administration of KPP12 eye-drops significantly improved disease outcome, and preserved the structural integrity and transparency of the infected cornea. KPP12 treatment resulted in the suppression of neutrophil infiltration and greatly enhanced bacterial clearance in the infected cornea. These results indicate that bacteriophage eye-drops may be a novel adjunctive or alternative therapeutic agent for the treatment of infectious keratitis secondary to antibiotic-resistant bacteria. © 2012 Fukuda et al.

DOI： 10.1371/journal.pone.0047742

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Helicobacter pylori causes peptic ulcers and gastric cancer, which lead to significantly higher morbidity in Japan than elsewhere in the world. As bacteriophage (phage) and host bacteria coevolve, the study of H. pylori phages is important to extend understanding of the evolution and pathogenesis of H. pylori. Here we report two complete genome sequences of H. pylori phages KHP30 and KHP40, which were released spontaneously from the most pathogenic East Asian-type isolates from Japanese patients.

DOI： 10.1128/JVI.01767-12

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

The genera SPO1-like and Twort-like viruses in the subfamily Spounavirinae of the family Myoviridae have been newly proposed, with the reorganization of the SPO1-related bacteriophages (phages). A criterion defining these viral genera is the presence/absence of DNA modifications. In this study, liquid chromatography/mass spectrometry showed that phages I center dot EF24C and K of the subfamily Spounavirinae have unmodified DNA, which classifies them as Twort-like viruses. Moreover, in the subfamily Spounavirinae, DNA modification and elimination of a particular DNA sequence were suggested to be the major antirestriction strategies of the SPO1-like and Twort-like viruses, respectively.

DOI： 10.1007/s00705-012-1324-9

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Publishing type：Research paper (scientific journal)   Publisher：The Association for Research in Vision and Ophthalmology  

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Objective: To investigate the effects of tissue stretch and muscarinic receptor stimulation on the spontaneous activity of the urothelium/lamina propria and identify the specific receptor subtype mediating these responses. Methods: Isolated strips of porcine urothelium with lamina propria were set up for in vitro recording of contractile activity. Muscarinic receptor subtype-selective antagonists were used to identify the receptors influencing the contractile rate responses to stretch and stimulation with carbachol. Results: Isolated strips of urothelium with lamina propria developed spontaneous contractions (3.7 cycles/min) that were unaffected by tetrodotoxin, Nω-nitro-l-arginine, or indomethacin. Carbachol (1 μM) increased the spontaneous contractile rate of these tissue strips by 122% ± 27% (P <.001). These responses were significantly depressed in the presence of the M3-selective muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (10-30 nM) but were not affected by the M1-selective antagonist pirenzepine (30-100 nM) or the M2-selective antagonist methoctramine (0.1-1 μM). Stretching of the tissue also caused an increase in the spontaneous contractile rate, and these responses were abolished by atropine (1 μM) and low concentrations of 4-diphenylacetoxy-N-methylpiperidine methiodide (10 nM). Darifenacin, oxybutynin, tolterodine, and solifenacin (1 μM) all significantly depressed the frequency responses to carbachol (1 μM). Conclusion: The urothelium with the lamina propria exhibits a spontaneous contractile activity that is increased during stretch. The mechanism appears to involve endogenous acetylcholine release acting on M3 muscarinic receptors. Anticholinergic drugs used clinically depress the responses of these tissues, and this mechanism might represent an additional site of action for these drugs in the treatment of bladder overactivity. © 2011 Elsevier Inc.

DOI： 10.1016/j.urology.2011.08.039

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Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component. © 2011 Uchiyama et al.

DOI： 10.1371/journal.pone.0026648

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Background: Merkel cell polyomavirus (MCPyV) was first identified in Merkel cell carcinoma (MCC) as a new tumor virus. Studies have also reported differing frequencies of MCPyV detection in other skin cancers in western countries. Objectives: Little is known about geographical differences of MCPyV prevalence in non-MCC tumors. We examined the existence of MCPyV in non-MCC skin cancers including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) in Japanese patients. Study design: Paraffin-embedded tissues of cutaneous SCC (n= 30) and BCC (n= 10) from Japanese patients were tested for the presence of MCPyV by polymerase chain reaction (PCR) with primer sets directed against the genes encoding large-T antigen 3 (LT3) and viral protein 1 (VP1). This was followed by DNA fragment sequencing and immunohistochemistry. Results: PCR analysis targeting the LT3 gene showed that the viral sequences were found in 4 of 30 (13%) SCC cases. Nested PCR detected the VP1 region in four cases. Sequencing analysis of these PCR-amplified fragments showed a close homology to the previously published MCPyV sequences. Immunohistochemistry with the monoclonal antibody to MCPyV LT-antigen showed positive staining in 2 of 4 LT3 PCR-positive cases. On the other hand, our BCC samples were all negative for MCPyV. Conclusion: This study suggested that Japanese cutaneous SCC is infrequently associated with MCPyV. Further worldwide epidemiological surveys are warranted to determine the possible association of MCPyV with pathogenesis of non-MCC skin cancers. © 2010 Elsevier B.V.

DOI： 10.1016/j.jcv.2010.09.013

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Am Soc Microbiol  

In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-L-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn 2+ ions for its activity, contrary to the bioinformatics prediction of a Zn2+ ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-L-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics. Copyright © 2011, American Society for Microbiology.

DOI： 10.1128/AEM.01540-10

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A novel bacteriophage, φMR25, was isolated from a lysogenic Staphylococcus aureus strain by mitomycin C induction. Its biological features were analyzed in comparison with φMR11, which was described previously as a prototype therapeutic phage. φMR25 is morphologically similar to φMR11 (morphotype B1 of family Myoviridae) but has a broader host range than φMR11 on S. aureus strains. φMR25 can also multiply on S. aureus lysogens of φMR11. Its DNA is 44,342 bp in size, is predicted to include 70 open reading frames, and does not contain genes related to toxin or drug resistance. The lysogenic module and most of the putative virion protein genes are completely different from those of φMR11. In spite of their genetic diversity, intraperitoneal administration of φMR25 rescued mice inoculated with a lethal dose of S. aureus, as was the case for φMR11. These results suggest that φMR25 could be another candidate phage to treat S. aureus infection. © Springer-Verlag 2010.

DOI： 10.1007/s00705-010-0623-2

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Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd Oxford, UK  

Increases in multidrug-resistant strains of Serratia marcescens are of great concern in pediatrics, especially in neonatal intensive care units. In the search for bacteriophages to control infectious diseases caused by multidrug-resistant S. marcescens, three phages (KSP20, KSP90, and KSP100) were isolated from environmental water and were characterized morphologically and genetically. KSP20 and KSP90 belonged to morphotype A1 of the family Myoviridae, and KSP100 belonged to morphotype C3 of the family Podoviridae. Analysis of the DNA region coding virion proteins, together with their morphological features, indicated that KSP20, KSP90, and KSP100 were related to the P2-like phage (temperate), T4-type phage (virulent), and phiEco32 phage (virulent), respectively. Based on amino acid sequences of the major capsid protein, KSP90 formed a new branch with a Stenotrophomonas maltophilia phage, Smp14, in the T4-type phage phylogeny. Both Smp14 and phiEco32 have been reported as potential therapeutic phages. These results suggest that KSP90 and KSP100 may be candidate therapeutic phages to control S. marcescens infection. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

DOI： 10.1111/j.1574-6968.2008.01455.x

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Pseudomonas aeruginosa bacteriophage (phage) is one of the most taxonomically and genetically diverse phages. Although phage D3 is one of well-studied P. aeruginosa phages, no D3-related P. aeruginosa phage has been reported. We report a novel P. aeruginosa siphovirus, PAJU2, which is genetically related to but morphology distinct (highly elongated head) from phage D3. A PAJU2 capsid protein, Orf3, is thought to be synthesized as a protein fused to a prohead protease and is autocatalytically cleaved, which may form the head chain mail. Despite such morphological differences, PAJU2 is expected to be a useful genetic reference for phage D3. © 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2008.10.005

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A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1-150] and the lysozyme (aa 401-624) domains but not the linker domain (aa 151-400) caused efficient lysis of S. aureus. Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

DOI： 10.1111/j.1574-6968.2008.01152.x

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We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage φ{symbol}MR11. In silico analysis of the φ{symbol}MR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of φ{symbol}MR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34 bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity. © 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.01.045

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Multidrug-resistant uropathogenic Escherichia coli (UPEC) is increasing gradually on a worldwide scale. We therefore examined the possibility of bacteriophage (phage) therapy for urinary tract infections (UTIs) caused by the UPEC strains as an alternative to chemotherapy. In addition to the well-known T4 phage, KEP10, which was newly isolated, was used as a therapeutic phage candidate. KEP10 showed a broader bacteriolytic spectrum (67%) for UPEC strains than T4 (14%). Morphological and genetic analyses showed that KEP10 resembles phage T4. Phages T4 and KEP10 injected into the peritoneal cavity of mice were distributed immediately to all organs examined and maintained a high titer for at least 24 h. They were stable in the urine of both mice and humans for 24 h at 37°C. Administration of these phages into the peritoneal cavity caused a marked decrease in the mortality of mice inoculated transurethrally with a UPEC strain, whereas most of the control mice died within a few days of bacterial infection. Inoculation with phage alone produced no adverse effects attributable to the phage per se. The present study experimentally demonstrated the therapeutic potential of phage for E. coli-induced UTIs, and T-even-related phages may be suitable candidates with which to treat them. © 2008 Springer-Verlag.

DOI： 10.1007/s00705-007-0031-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P < 0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-alpha, interleukin-1 beta [IL-1 beta], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.

DOI： 10.1128/AAC.00635-06

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We report the successful purification of a cloned lysin encoded by the novel Staphylococcus aureus bacteriophage φMR11. The lysin, designated MV-L, rapidly and completely lysed cells of a number of S. aureus strains tested, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus and a subset of vancomycin-intermediate S. aureus (VISA) in growing conditions. MV-L-mediated killing is specific to S. aureus and not to other species, except for S. simulans. MV-L exerted its staphylocidal effect synergistically with glycopeptide antibiotics against VISA. MV-L efficiently eliminated MRSA that had been artificially inoculated into the nares of mice. The intraperitoneal administration of MV-L also protected mice against MRSA septic death, without any harmful effects. Although MV-L evoked detectable levels of a humoral response in mice, the antibodies did not abolish the bacteriolytic activity. These results indicate that MV-L might be useful as a powerful therapeutic agent against multidrug-resistant S. aureus infections. © 2007 by the Infectious Diseases Society of America. All rights reserved.

DOI： 10.1086/521305

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Language：Japanese   Publisher：日本ペット栄養学会  

DOI： 10.11266/jpan.21.2_S11

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Ozone water is currently utilized for antibacterial and antiviral purposes under the pandemic of COVID-19 due to its strong oxidation effect without safety problems. Therefore, clinical applications of ozone water for cutaneous diseases such as atopic dermatitis (AD) and pyoderma are expected recently. The aim of this project is to apply the ozone water for AD and pyoderma in dogs. We here examined the antibacterial effects of ozone water using staphylococci in in vitro setting, and anti-allergic effects of ozone water in AD mouse model.

We first examined the bactericidal effect of ozone water on resident staphylococci on skin. Staphylococcus aureus, which is an exacerbating factor in human AD, and S. lentus collected from AD mouse model was treated with ozone water and the number of bacteria was determined. Our findings indicated that ozone water showed a significant bactericidal effect against both staphylococcal species . We are currently investigating the efficacy of ozone water against staphylococci isolated from AD and pyoderma dogs. In vivo experiment with AD mouse model is also underway to look for the therapeutic effects of ozone water with impact on inflammatory and itch responses.

DOI： 10.1254/jpssuppl.95.0_1-ss-64

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Periodontal disease, which is majorly caused by Porphyromonas gulae, is extremely common in dogs, and is associated with serious systemic diseases such as kidney failure, heart disease and immune disorder. Ozon water is ozone-gas-dissolved water, which has antimicrobial activity by oxidizing molecules. However, both therapeutic and preventive effects of ozone water for periodontal disease in dogs are still unclear. We herein examined the antibacterial and anti-inflammatory effects of ozone water in vitro using P. gulae. Our results indicated that treatment of ozone water significantly inhibited the growth of P. gulae until 24h post ozone water treatment as compared to purified water treatment. Ozone water also showed the anti-inflammatory effects in P. gulae-infected human gingival epithelial cells (Ca9-22). Secretions of IL-1β and TNFα were significantly suppressed by ozone water treatment compared to the treatment of purified water. As IL-1β and TNFα activate the osteoclast and finally promote bone resorption in the oral cavity, our findings imply the potential therapeutic and preventive effects of ozone water in canine periodontal disease. We are currently investigating the in vivo preventive and therapeutic effects of ozone water in dogs.

DOI： 10.1254/jpssuppl.95.0_2-o-090

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Bacteriophages (phages), or bacterial viruses, are the most abundant and diverse biological entities that impact the global ecosystem. Recent advances in metagenomics have revealed their rampant abundance in the biosphere. A fundamental aspect of bacteriophages that remains unexplored in metagenomic data is the process of recombination as a driving force in evolution that occurs among different viruses within the same bacterial host. Here, we systematically examined signatures of recombination in every gene from 211 species-level viral groups in a recently obtained dataset of the Earth’s virome that contain corresponding information on the host bacterial species. Our study revealed that signatures of recombination are widespread (84%) among the diverse viral groups. We identified 25 recombination-intense viral groups, widely distributed across the viral taxonomy, and present in bacterial species living in the human oral cavity. We also revealed a significant inverse association between the recombination-intense viral groups and Type II restriction endonucleases, that could be effective in reducing recombination among phages in a cell. Furthermore, we identified recombination-intense genes that are significantly enriched for encoding phage morphogenesis proteins. Changes in the viral genomic sequence by recombination may be important to escape cleavage by the host bacterial immune systems.

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筆者らはHelicobacter pyloriに感染する新規バクテリオファージ(ファージ)KHP30を分離・解析し、そのユニークな性状・ゲノムを明らかにした。近年、KHP30に遺伝的に類似した3種のKHP30様ファージが報告されている。本稿では、ファージKHP30の特徴を概説し、KHP30様ファージゲノムに関して解説する。(著者抄録)

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筆者らはHelicobacter pyloriに感染する新規バクテリオファージ(ファージ)KHP30を分離・解析し、そのユニークな性状・ゲノムを明らかにした。近年、KHP30に遺伝的に類似した3種のKHP30様ファージが報告されている。本稿では、ファージKHP30の特徴を概説し、KHP30様ファージゲノムに関して解説する。(著者抄録)

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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-26461504/

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To research the detailed mechanism of the lubrication process using the thermal effusivity sensor, the relationships of the lubrication progress with the pattern of powder flow, the rotation speed and the filling level were investigated. The thermal effusivity profile was studied as a function of the number of rotations at various rotation speeds. It was observed that at lower rotation speeds, the profiles of the lubrication progress were almost the same, regardless of the rotation speed. In this region, the highest speed was defined as the critical rotation speed (CRS), which was found to be one of the important factors. The CRS had close relations with avalanche flow in the blender. The first and the second phases were observed in the lubrication process. The first phase was influenced by the CRS and the filling level in the blender. The second phase was influenced by the rotation speed. The mechanism of two-phase process was proposed as a macro progression of the dispersion of the lubricant (first phase) and micro progression of the coating of the powder particles with lubricant (second phase). The accurate monitoring by the thermal effusivity sensor was able to help a better understanding in the lubrication process.

DOI： 10.3109/03639045.2014.935394

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The principles of thermal effusivity are applied to an understanding of the detailed mechanisms of the lubrication process in a rotating mixer. The relationships and impact of the lubrication process by the pattern of powder flow, the filling level, and the rotating mixer size were investigated. Thermal effusivity profiles of the lubrication process, as obtained, indicate that lubrication is a two-phase process. The intersection point of the first and second phases (IPFS) is influenced by changing the filling level, thus changing the resulting number of avalanche flows created. The slope of the second phase (SSP) is influenced by the relationship between the number and the length of avalanche flows. Understanding this difference between the first and second phases is important to successfully evaluate the impact of proposed changes in the lubrication process. From this knowledge, a predictive model of the lubrication profile can be generated to allow an evaluation of proposed changes to the lubrication process. This model allows estimation of the lubrication profile at different filling levels and in different rotating mixer sizes. In this study, the actual lubrication profile almost coincides with the model predicted lubrication profile. Based on these findings, it is assumed that lubrication profiles at a commercial scale can be predicted from data generated at the laboratory scale. Further, it is assumed that changes in the filling level can also be estimated from the laboratory or current data.

DOI： 10.1248/cpb.c14-00634

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The genomes of Staphylococcus phages S25-3 and S25-4 (family Myoviridae, genus Twort-like viruses) were sequenced and analyzed from an evolutionary perspective. The genome-based phylogeny and genome analyses of phages S25-3 and S25-4 showed that they had diverged evolutionarily from the majority of this viral genus based on the presence of mobile genetic elements, i.e., a putative transposase and the homing endonuclease I-MsaI. These results suggest that genetic elements such as transposases and homing endonucleases are likely to be involved with the evolution of some Twort-like phages, including phages S25-3 and S25-4. © 2013 Springer-Verlag Berlin Heidelberg and the University of Milan.

DOI： 10.1007/s13213-013-0762-2

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Language：Japanese   Publisher：小峰工業出版  

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In the process design of tablet manufacturing, understanding and control of the lubrication process is important from various viewpoints. A detailed analysis of thermal effusivity data in the lubrication process was conducted in this study. In addition, we evaluated the risk and benefit in the lubrication process by a detailed investigation. It was found that monitoring of thermal effusivity detected mainly the physical change of bulk density, which was changed by dispersal of the lubricant and the coating powder particle by the lubricant. The monitoring of thermal effusivity was almost the monitoring of bulk density, thermal effusivity could have a high correlation with tablet hardness. Moreover, as thermal effusivity sensor could detect not only the change of the conventional bulk density but also the fractional change of thermal conductivity and thermal capacity, two-phase progress of lubrication process could be revealed. However, each contribution of density, thermal conductivity, or heat capacity to thermal effusivity has the risk of fluctuation by formulation. After carefully considering the change factor with the risk to be changed by formulation, thermal effusivity sensor can be a useful tool for monitoring as process analytical technology, estimating tablet hardness and investigating the detailed mechanism of the lubrication process.

DOI： 10.3109/03639045.2013.795581

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Differences between laboratory and commercial tablet presses are frequently observed during scale-up of tableting process. These scale-up issues result from the differences in total compression time that is the sum of consolidation and dwell times. When a lubricated blend is compressed into tablets, the tablet thickness produced by the commercial tablet press is often thicker than that by a laboratory tablet press. A new punch shape design, designated as shape adjusted for scale-up (SAS), was developed and used to demonstrate the ability to replicate scale-up issues in commercial-scale tableting processes. It was found that the consolidation time can be slightly shortened by changing the vertical curvature of the conventional punch head rim. However, this approach is not enough to replicate the consolidation time. A secondary two-stage SAS punch design and an embossed punch head was designed to replicate the consolidation and dwell times on a laboratory tablet press to match those of a commercial tablet press. The resulting tablet thickness using this second SAS punch on a laboratory tablet press was thicker than when using a conventional punch in the same laboratory tablet press. The secondary SAS punches are more useful tools for replicating and understanding potential scale-up issues. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

DOI： 10.1002/jps.23989

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The applicability of porous spherical silica (PSS) was evaluated as core particles for pharmaceutical products by comparing it with commercial core particles such as mannitol (NP-108), sucrose and microcrystalline cellulose spheres. We investigated the physical properties of core particles, such as particle size distribution, flow properties, crushing strength, plastic limit, drying rate, hygroscopic property and aggregation degree. It was found that PSS was a core particle of small particle size, low friability, high water adsorption capacity, rapid drying rate and lower occurrence of particle aggregation, although wettability is a factor to be carefully considered. The aggregation and taste-masking ability using PSS and NP-108 as core particles were evaluated at a fluidized-bed coating process. The functional coating under the excess spray rate shows different aggregation trends and dissolution profiles between PSS and NP-108; thereby, exhibiting the formation of uniform coating under the excess spray rate in the case of PSS. This expands the range of the acceptable spray feed rates to coat fine particles, and indicates the possibility of decreasing the coating time. The results obtained in this study suggested that the core particle, which has a property like that of PSS, was useful in overcoming such disadvantages as large particle size, which feels gritty in oral cavity; particle aggregation; and the long coating time of the particle coating process. These results will enable the practical fine particle coating method by increasing the range of optimum coating conditions and decreasing the coating time in fluidized bed technology. © 2014 Informa Healthcare USA, Inc.

DOI： 10.3109/03639045.2013.807277

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Scale-up issues in the tableting process, such as capping, sticking, or differences in tablet thickness, are often observed at the commercial production scale. A new shaped punch, named the size adjusted for scale-up (SAS) punch, was created to estimate scale-up issues seen between laboratory scale and commercial scale tableting processes. The SAS punch's head shape was designed to replicate the total compression time of a laboratory tableting machine to that of a commercial tableting machine. Three different lubricated blends were compressed into tablets using a laboratory tableting machine equipped with SAS punches, and any differences in tablet thickness or capping phenomenon were observed. It was found that the new shaped punch could be used to replicate scale-up issues observed in the commercial tableting machine. The SAS punch was shown to be a useful tool to estimate scale-up issues in the tableting process.

DOI： 10.1002/jps.23781

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© 2014 Sakaguchi et al. Clostridium botulinum strain Osaka05, which has been isolated from an infant patient with botulism in Japan, is the first strain producing botulinum neurotoxin subtype B6. Here, we report the draft genome sequence of C. botulinum Osaka05.

DOI： 10.1128/genomeA.e01010-13

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尾部のないファージの分離率はわずか約4%とまれである。そのなかに球形ファージを含むさまざまな分類がある。われわれはHelicobacter pylori(H.pylori)が自然放出している二つの球形ファージ(KHP30とKHP40)を発見した。脂質を含むparticleは線状の約26kbp dsDNAで頭部直径約70nmであった。菌体内ではゲノムにintegrateされておらず、おそらくepisomal formで存在するpseudolysogenyで、burst sizeは約13、宿主域は約50%であった。ゲノムおよび性状解析の結果、従来の球形ファージfamily(CorticoviridaeやTectiviridae)には分類できない新規な球形ファージと考えた。(著者抄録)

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Other Link： http://search.jamas.or.jp/link/ui/2014038434

Authorship：Lead author   Language：English   Publisher：AMER SOC MICROBIOLOGY  

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：シーエムシー出版  

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Genotyping by pulsed-field gel electrophoresis (PFGE) is recognized as one of the most useful methods for epidemiological studies of drug-resistant Enterococcus faecalis nosocomial outbreaks. As the bacteriolysis procedure is a critical step in PFGE, an accurate and reliable alternative bacteriolytic method to the conventional protocol would be useful. In this study, we examined the applicability of endolysin ORF9, derived from the E. faecalis phage φEF24C, and lantibiotic nisin in sample preparation for PFGE. The results show that the cooperative actions of nisin and ORF9 synergistically lysed E. faecalis, which was then amenable to PFGE analysis. © 2012 Springer-Verlag Berlin Heidelberg and the University of Milan.

DOI： 10.1007/s13213-012-0556-y

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Bacteriophage (phage) KPP10 has been used in experimental phage therapies directed against P. aeruginosa infections. To examine the eligibility of phage KPP10 as a therapeutic phage, its genome was analyzed. The genomic DNA was shown to be 88,322 bp long, with 158 open reading frames (ORFs), and three tRNA genes were predicted. No ORF-encoded pathogenicity or lysogenization factor was predicted. A comparative genomic analysis revealed that phage KPP10, together with phage PAK_P3, can be grouped as a new type of lytic phage infecting P. aeruginosa. Phage KPP10 is considered to be suitable for therapeutic purposes because it is a lytic phage without ORF-encoded pathogenicity or a lysogenization factors.

DOI： 10.1007/s00705-011-1210-x

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The genera SPO1-like and Twort-like viruses in the subfamily Spounavirinae of the family Myoviridae have been newly proposed, with the reorganization of the SPO1-related bacteriophages (phages). A criterion defining these viral genera is the presence/absence of DNA modifications. In this study, liquid chromatography/mass spectrometry showed that phages I center dot EF24C and K of the subfamily Spounavirinae have unmodified DNA, which classifies them as Twort-like viruses. Moreover, in the subfamily Spounavirinae, DNA modification and elimination of a particular DNA sequence were suggested to be the major antirestriction strategies of the SPO1-like and Twort-like viruses, respectively.

DOI： 10.1007/s00705-012-1324-9

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OBJECTIVE To investigate the effects of tissue stretch and muscarinic receptor stimulation on the spontaneous activity of the urothelium/ lamina propria and identify the specific receptor subtype mediating these responses.
METHODS Isolated strips of porcine urothelium with lamina propria were set up for in vitro recording of contractile activity. Muscarinic receptor subtype-selective antagonists were used to identify the receptors influencing the contractile rate responses to stretch and stimulation with carbachol.
RESULTS Isolated strips of urothelium with lamina propria developed spontaneous contractions (3.7 cycles/min) that were unaffected by tetrodotoxin, N omega-nitro-L-arginine, or indomethacin. Carbachol (1 mu M) increased the spontaneous contractile rate of these tissue strips by 122% +/- 27% (P &lt; .001). These responses were significantly depressed in the presence of the M3-selective muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (10-30 nM) but were not affected by the M1-selective antagonist pirenzepine (30-100 nM) or the M2-selective antagonist methoctramine (0.1-1 mu M). Stretching of the tissue also caused an increase in the spontaneous contractile rate, and these responses were abolished by atropine (1 mu M) and low concentrations of 4-diphenylacetoxy-N-methylpiperidine methiodide (10 nM). Darifenacin, oxybutynin, tolterodine, and solifenacin (1 mu M) all significantly depressed the frequency responses to carbachol (1 mu M).
CONCLUSION The urothelium with the lamina propria exhibits a spontaneous contractile activity that is increased during stretch. The mechanism appears to involve endogenous acetylcholine release acting on M3 muscarinic receptors. Anticholinergic drugs used clinically depress the responses of these tissues, and this mechanism might represent an additional site of action for these drugs in the treatment of bladder overactivity. UROLOGY 78: 1442. e9-1442. e15, 2011. (C) 2011 Elsevier Inc.

DOI： 10.1016/j.urology.2011.08.039

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A novel bacteriophage, phi MR25, was isolated from a lysogenic Staphylococcus aureus strain by mitomycin C induction. Its biological features were analyzed in comparison with phi MR11, which was described previously as a prototype therapeutic phage. phi MR25 is morphologically similar to phi MR11 (morphotype B1 of family Myoviridae) but has a broader host range than phi MR11 on S. aureus strains. phi MR25 can also multiply on S. aureus lysogens of phi MR11. Its DNA is 44,342 bp in size, is predicted to include 70 open reading frames, and does not contain genes related to toxin or drug resistance. The lysogenic module and most of the putative virion protein genes are completely different from those of phi MR11. In spite of their genetic diversity, intraperitoneal administration of phi MR25 rescued mice inoculated with a lethal dose of S. aureus, as was the case for phi MR11. These results suggest that phi MR25 could be another candidate phage to treat S. aureus infection.

DOI： 10.1007/s00705-010-0623-2

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Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model. © 2009 The Societies and Blackwell Publishing Asia Pty Ltd.

DOI： 10.1111/j.1348-0421.2009.00125.x

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A tailed bacteriophage, phi MR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1-150] and the lysozyme (aa 401-624) domains but not the linker domain (aa 151-400) caused efficient lysis of S. aureus. Immunoelectron microscopy localized gp61 to the tail tip of the phi MR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of phi MR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with phi MR11 was also lysed by both proteins. Staphylococcus aureus strains on which phi MR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.

DOI： 10.1111/j.1574-6968.2008.01152.x

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Along with the increasing threat of nosocomial infections by vancomycin-resistant Enterococcus faecalis, bacteriophage (phage) therapy has been expected as an alternative therapy against infectious disease. Although genome information and proof of applicability are prerequisites for a modern therapeutic phage, E. faecalis phage has not been analyzed in terms of these aspects. Previously, we reported a novel virulent phage, phi EF24C, and its biology indicated its therapeutic potential against E. faecalis infection. In this study, the phi EF24C genome was analyzed and the in vivo therapeutic applicability of phi EF24C was also briefly assessed. Its complete genome (142,072 bp) was predicted to have 221 open reading frames (ORFs) and five tRNA genes. In our functional analysis of the ORFs by use of a public database, no proteins undesirable in phage therapy, such as pathogenic and integration-related proteins, were predicted. The noncompetitive directions of replication and transcription and the host-adapted translation of the phage were deduced bioinformatically. Its genomic features indicated that phi EF24C is a member of the SPO1-like phage genus and especially that it has a close relationship to the Listeria phage P100, which is authorized for prophylactic use. Thus, these bioinformatics analyses rationalized the therapeutic eligibility of phi EF24C. Moreover, the in vivo therapeutic potential of phi EF24C, which was effective at a low concentration and was not affected by host sensitivity to the phage, was proven by use of sepsis BALB/c mouse models. Furthermore, no change in mouse lethality was observed under either single or repeated phage exposures. Although further study is required, phi EF24C can be a promising therapeutic phage against E. faecalis infections.

DOI： 10.1128/AEM.02371-07

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[仮説]ファージ療法は、欧米諸国では抗生物質の実用化に伴い一旦放棄されたが、旧ソ連諸国ポーランドでは現在に至るまで使用されている。ファージ療法の導入により、現在の化学療法の弱点(耐性菌、菌交代症の問題など)を補足することは可能であろうか。[新知見の要点]欧米の多くの研究者によってよく計画された動物実験により、ファージ療法(活性ファージおよびライシンの使用)の有効性・安全性を示すデータが蓄積されつつある。[将来の可能性、問題点]今後、各菌種に対応可能な実用的ファージバンクの構築が必要である。(著者抄録)

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Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage phi EF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phi EF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that phi EF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phi EF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c. 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that phi EF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phi EF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.

DOI： 10.1111/j.1574-6968.2007.00996.x