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

Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test. IMPORTANCE Endolysin is a bacteriophage-derived enzyme that degrades the peptidoglycan in the cell wall of host bacteria; it could be used as an antimicrobial agent for selectively eliminating specific bacterial genera/species. Group B Streptococcus (GBS) causes neonatal infection via vertical transmission; prenatal GBS screening test, in which enrichment culture is followed by bacterial identification, is used to detect the presence of GBS in pregnant women. However, the presence of commensal bacteria such as Enterococcus faecalis in clinical specimens can inhibit GBS growth in the selective enrichment culture, resulting in false-negative result. Here, we demonstrated that the application of originally isolated endolysin in the enrichment culture improved the test accuracy by inhibiting unwanted E. faecalis growth and therefore avoiding false-negative results, not only in experimental settings, but also in tests using vaginal swabs.

DOI： 10.1128/Spectrum.00077-21

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Authorship：Corresponding author   Language：English   Publisher：Elsevier {BV}  

To introduce phage therapy against multidrug-resistant Staphylococcus aureus in Western medicine, the establishment of phage manufacturing, particularly phage propagation, is indispensable. For the propagation of S. aureus phages, knowledge of the effects of phage types, process parameters, and analytical methodologies should be investigated. In this study, S. aureus phage propagations were studied in a flask with a new class of design of experiments, definitive screening design, using S. aureus phages S13' and S25-3 in different taxonomies. Four process parameters, namely, multiplicity of infection, bacterial density at infection, time of harvest, and temperature, were evaluated with the regression models based on the phage concentration data measured using plaque assay and quantitative polymerase chain reaction. As a result, phage propagations measured using plaque assay and quantitative polymerase chain reaction were overall similar to each other in the case of phage S13', while they differed in the case of phage S25-3. These results suggest that the propagation processes need to be developed according to phage type, and the choice of methodologies for phage concentration measurements should be carefully considered.

DOI： 10.1016/j.virusres.2021.198406

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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21H04151/

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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Authorship：Lead author   Language：English  

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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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-24791025/

Language：English   Publisher：7498  

DOI： 10.1038/509S9a

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Other Link： http://www.nature.com/articles/509S9a

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There is so far no available data about how the additive, synergistic, or antagonistic effects of the combined form of alpha-lactalbumin (α-La) and bacteriophages might modulate the cellular milieu of the host-pathogen interface. A co-culture of colonocytes and hepatocytes was stimulated with Pseudomonas aeruginosa PAO1 in the presence of KPP22 phage and incubated for 6 hours in medium alone or medium supplemented with bovine milk-origin α-La. The combination of KPP22 phage and α-La significantly inhibited P.a PAO1-elicited secretion of IL-1β, IL-6, and ICAM-1, which are the mediators and enzymes associated with the inflammatory response to an infectious-inflamed milieu. Cell viability was higher in the P.a PAO1+ KPP22 phage group compared to the P.a PAO1alone group. KPP22 phage and α-La, either alone or in combination, rescued P.a PAO1-induced aberrant PGE1/PGE2 production ratios. The convergence of ingested α-La and phages mitigates pro-inflammatory mediators. α-La leads to an increased sensitivity of opportunistic pathogenic bacteria to phages. Structural, functional, or immunological similarities between ingested α-La and phages play an important role in the mitigation of infection-driven pathobiological processes.

DOI： 10.1016/j.cimid.2024.102271

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Bacteriophage (phage) KHP40 was previously isolated from the supernatant of a culture of Helicobacter pylori KMT83 cells. In this study, we analyzed the infection characteristics of KHP40, phage release pattern from KMT83 cells, and state of KHP40 DNA in KMT83 cells. The findings revealed that KHP40 phage showed varied adsorption efficiencies for different strains, long latent periods, and small burst sizes. Additionally, KHP40 activity was maintained at pH 2.5-12. KHP40 phages were released during the vegetative growth phase of the KMT83 cells. PCR analysis demonstrated that KHP40 DNA was stably maintained in KMT83 clones. Next-generation sequencing analysis revealed the presence of two distinct types of circular double-stranded DNA in H. pylori KMT83 cells. One was an H. pylori-specific DNA consisting of 1,578,403 bp, and the other was a 26,412 bp sequence that represented the episomal form of phage KHP40 DNA. Furthermore, defective KHP40-lysogenic DNA was detected in the H. pylori-specific DNA, the deleted portion of which appeared to have been transferred to another location in the bacterial genome. These findings indicate that KHP40 DNA exists in both episomal and defectively lysogenized states in KMT83 cells, and active phages are produced from KHP40-episomal DNA.

DOI： 10.1093/femsle/fnae082

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This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2-14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4-8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens.

DOI： 10.3390/antibiotics13090846

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Bacterial and food allergens are associated with immune-mediated food allergies via the gut-skin axis. However, there has been no data on the potential use of phages to rescue this pathological process. A human triple cell co-culture model incorporating colonocytes (T84 cells), macrophages (THP-1 cells), and hepatocytes (Huh7 cells) was established and infected with Pseudomonas aeruginosa PAO1 (P.a PAO1) in the absence or presence of its KPP22 phage in Dulbecco's Modified Eagle's Medium (DMEM), DMEM+ ovalbumin (OVA), or DMEM+β-casein media. The physiological health of cells was verified by assessing cell viability and Transepithelial electrical resistance (TEER) across the T84 monolayer. The immune response of cells was investigated by determining the secretions of IL-1β, IL-8, IL-22, and IL-25. The ability of P.a PAO1 to adhere to and invade T84 cells was evaluated. The addition of either OVA or β-casein potentiated the P.a PAO1-elicited secretion of cytokines. The viability and TEER of the T84 monolayer were lower in the P.a PAO1+OVA group compared to the P.a PAO1 alone and PAO1+β-casein groups. OVA and β-casein significantly increased the adherence and invasion of P.a PAO1 to T84 cells. In the presence of the KPP22 phage, these disruptive effects were abolished. These results imply that: (1) food allergens and bacterial toxic effector molecules exacerbate each other's disruptive effects; (2) food allergen and bacterial signaling at the gut-skin mucosal surface axis depend on a network of bacteria-phage-eukaryotic host interactions; and (3) phages are complementary for the evaluation of pathobiological processes that occur at the interface between bacteria, host cellular milieu, and food antigens because phages intervene in P.a PAO1-, OVA-, and β-casein-derived inflammation.

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The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

DOI： 10.3390/antibiotics13080716

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<jats:title>Abstract</jats:title><jats:p>In dogs, <jats:italic>Porphyromonas gulae</jats:italic> is a major periodontal pathogen with 41-kDa proteins polymerizing to form a filamentous structure called fimbriae or pili, termed FimA. FimA is classified into three genotypes: A, B, and C, and there are combinations of types A, B, C, A/B, A/C, B/C, and A/B/C. Periodontal disease is the most common oral disease in small dogs, but the periodontal disease status and <jats:italic>P. gulae</jats:italic> colonization at each dog age and breed remain unclear. In this study, we stratified 665 small dogs and analyzed the periodontal status and distribution of <jats:italic>P. gulae</jats:italic> with each FimA genotype. Dogs with periodontal disease and FimA genotype tended to increase with age. The dogs with at least one FimA genotype had significantly more severe periodontal disease compared with <jats:italic>P. gulae</jats:italic>-negative dogs (<jats:italic>P</jats:italic> &lt; 0.01). Additionally, periodontal status was significantly associated with specific FimA genotype distribution in Toy Poodles and Chihuahuas (<jats:italic>P</jats:italic> &lt; 0.05), whereas there was no such association in Dachshunds. These results suggest that the onset of periodontal disease and <jats:italic>P. gulae</jats:italic> colonization are related and progress with age. The relationship between periodontal disease and FimA genotype may differ depending on the dog breeds.</jats:p>

DOI： 10.1038/s41598-024-55842-8

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Bacterial infections pose significant global health concerns, necessitating precise and sensitive detection methods. This study introduces a multifunctional probe for bacterial detection. Bifunctional magnetoplasmonic nanoparticles (NPs) serve a dual purpose as carriers for magnetic separation (MS) and enhancers for light scattering of the target bacteria. We achieved exceptional selectivity at the bacterial species level by bioconjugating them with bacteriophages (phages). Optimal phage coverage was carefully determined to enhance the capture efficiency of the target bacterial cells. Additionally, we identified the ideal mixing ratios of the phage probe to bacteria, ensuring efficient MS and high light-scattering intensity. The use of the phage probe enabled the successful separation of captured target bacteria from other bacterial strains in the sample mixture. Bacterial detection was accomplished through dark-field light-scattering imaging, eliminating the need for additional labeling with other probes. This method showed a remarkable sensitivity, achieving a detection limit of ∼102 colony-forming units ml-1. This value is 2 orders of magnitude lower than that achieved using our previously reported technique using monofunctional plasmonic NPs. These phage probes hold promise for healthcare, biotech, and environmental monitoring owing to their adaptability to diverse bacterial species.

DOI： 10.1093/bulcsj/uoad010

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

Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Δ20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Δ15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development.

DOI： 10.1371/journal.pone.0310962

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BACKGROUND AND AIMS: Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. MATERIALS AND METHODS: 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. RESULTS: Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1β, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. CONCLUSION: The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.

DOI： 10.2174/0118715303257321231024094904

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<jats:sec>
<jats:title>Background</jats:title>
<jats:p>Methicillin-resistant <jats:italic>Staphylococcus aureus</jats:italic> (MRSA) is listed as a highly prioritized pathogen by the World Health Organization (WHO) to search for effective antimicrobial agents. Previously, we isolated a soil <jats:italic>Brevibacillus</jats:italic> sp. strain SPR19 from a botanical garden, which showed anti-MRSA activity. However, the active substances were still unknown.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Methods</jats:title>
<jats:p>The cell-free supernatant of this bacterium was subjected to salt precipitation, cation exchange, and reversed-phase chromatography. The antimicrobial activity of pure substances was determined by broth microdilution assay. The peptide sequences and secondary structures were characterized by tandem mass spectroscopy and circular dichroism (CD), respectively. The most active anti-MRSA peptide underwent a stability study, and its mechanism was determined through scanning electron microscopy, cell permeability assay, time-killing kinetics, and biofilm inhibition and eradication. Hemolysis was used to evaluate the peptide toxicity.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Results</jats:title>
<jats:p>The pure substances (BrSPR19-P1 to BrSPR19-P5) were identified as new peptides. Their minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) against <jats:italic>S. aureus</jats:italic> and MRSA isolates ranged from 2.00 to 32.00 and 2.00 to 64.00 µg/mL, respectively. The sequence analysis of anti-MRSA peptides revealed a length ranging from 12 to 16 residues accompanied by an amphipathic structure. The physicochemical properties of peptides were predicted such as pI (4.25 to 10.18), net charge at pH 7.4 (−3 to +4), and hydrophobicity (0.12 to 0.96). The CD spectra revealed that all peptides in the water mainly contained random coil structures. The increased proportion of α-helix structure was observed in P2−P5 when incubated with SDS. P2 (NH<jats:sub>2</jats:sub>-MFLVVKVLKYVV-COOH) showed the highest antimicrobial activity and high stability under stressed conditions such as temperatures up to 100 °C, solution of pH 3 to 10, and proteolytic enzymes. P2 disrupted the cell membrane and caused bacteriolysis, in which its action was dependent on the incubation time and peptide concentration. Antibiofilm activity of P2 was determined by which the half-maximal inhibition of biofilm formation was observed at 2.92 and 4.84 µg/mL for <jats:italic>S. aureus</jats:italic> TISTR 517 and MRSA isolate 2468, respectively. Biofilm eradication of tested pathogens was found at the P2 concentration of 128 µg/mL. Furthermore, P2 hemolytic activity was less than 10% at concentrations up to 64 µg/mL, which reflected the hemolysis index thresholds of 32.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Conclusion</jats:title>
<jats:p>Five novel anti-MRSA peptides were identified from SPR19. P2 was the most active peptide and was demonstrated to cause membrane disruption and cell lysis. The P2 activity was dependent on the peptide concentration and exposure time. This peptide had antibiofilm activity against tested pathogens and was compatible with human erythrocytes, supporting its potential use as an anti-MRSA agent in this post-antibiotic era.</jats:p>
</jats:sec>

DOI： 10.7717/peerj.16143

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Language：English   Publisher：The Chemical Society of Japan  

Infectious disease mortality has decreased due to effective drugs and healthcare. However, global health remains threatened by infectious diseases. New methods of rapid and accurate bacterial detection have attracted considerable attention. Fluorescence detection of whole bacterial cells offers high sensitivity, quantitative analysis, and simple operation. A highly fluorescent bioconjugated probe improves sensitivity and selectivity. This study presents a novel, bright fluorescent probe comprising a bacteriophage and a fluorescent nanoemulsion (fNE) as biorecognition and signal transduction elements, respectively. We demonstrate that fluorescence microscopy imaging using the S. aureus-specific phage, S13′-fNE (phage-fNE), detects S. aureus in the presence of E. coli or S. pseudintermedius, another closely related Staphylococci, in a highly selective manner. Furthermore, fNEs with high dye loadings exhibit considerably greater brightness compared to the fluorescent dye alone, making them suitable for sensitive fluorescence imaging. Phage-fNEs can quantitatively detect S. aureus at 104-108 colony-forming units per milliliter (CFU mL-1), with a limit of detection of 8 × 104 CFU mL-1. This result is comparable to the lowest value achieved by microscopic bacterial detection, with no preconcentration or enzymatic signal enhancement methods used. Bioconjugated fNEs open new avenues for highly selective and sensitive fluorescent detection of bacteria.

DOI： 10.1246/bcsj.20230200

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Background

Subclinical infection with bovine leukemia virus (BLV) in cows can cause economic losses in milk and meat production in many countries, as BLV-related negative effects. The volatile fatty acids (VFAs) and microbiota present in the digestive tracts of cows can contribute to cow health. Here, we exploratorily investigated the VFAs and microbiota in the rumen and gut with respect to subclinical BLV infection using cows housed at a single farm.

Results

We analyzed a herd of 38 cows kept at one farm, which included 15 uninfected and 23 BLV-infected cows. First, the analysis of the VFAs in the rumen, gut, and blood revealed an absence of statistically significant differences between the uninfected and BLV-infected groups. Thus, BLV infection did not cause major changes in VFA levels in all tested specimens. Next, we analyzed the rumen and gut microbiota. The analysis of the microbial diversity revealed a modest difference between the uninfected and BLV-infected groups in the gut; by contrast, no differences were observed in the rumen. In addition, the investigation of the bacteria that were predominant in the uninfected and BLV-infected groups via a differential abundance analysis showed that no significant bacteria were present in either of the microbiota. Thus, BLV infection possibly affected the gut microbiota to a small extent. Moreover, bacterial associations were compared between the uninfected and BLV-infected groups. The results of this analysis suggested that BLV infection affected the equilibrium of the bacterial associations in both microbiota, which might be related to the BLV-related negative effects. Thus, BLV infection may negatively affect the equilibrium of bacterial associations in both microbiota.

Conclusions

Subclinical BLV infection is likely to affect the rumen and gut microbiota, which may partly explain the BLV-related negative effects.

DOI： 10.1186/s13213-023-01737-4

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Other Link： https://link.springer.com/article/10.1186/s13213-023-01737-4/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

Currently, ozone water is utilized for antibacterial and antiviral purposes without any reported safety concerns. Therefore, ozone water may have clinical applications in treating staphylococcal-specific cutaneous diseases, such as atopic dermatitis (AD) and pyoderma. This study aimed to verify the bactericidal effects of ozone water at different concentrations (3 and 11 mg/L) against staphylococcal species in vitro, as well as evaluate the anti-inflammatory effects of ozone water in a mouse model of AD and pyoderma. Initially, the bactericidal properties of several concentrations of ozone water were confirmed with Staphylococcus aureus and methicillin-resistant S. pseudintermedius. Both 3 and 11 mg/L of ozone water exhibited a significant bactericidal effect against staphylococci at less than 100 times dilution. We next examined the cellular cytotoxicity and cytokine production (Interleukin (IL)-6 and IL-8) induced by S. pseudintermedius pre-treated with ozone water, and our findings indicated that cytotoxicity and cytokine production induced by staphylococci were significantly inhibited after ozone water pre-treatment. In vivo experiments showed that ozone water-pre-treated S. pseudintermedius significantly inhibited the development of pyoderma in mice; however, limited effects were observed in a therapeutic setting. Interestingly, ozone water at concentrations of 3 and 11 mg/L exhibits dual bactericidal and anti-inflammatory effects in mice with AD. This observation was corroborated by the significant inhibition of cytokine production in interferon-γ/tumor necrosis factor-stimulated human epidermal keratinocyte cells exposed to ozone in vitro. These findings indicate that administering ozone can be a novel therapeutic approach for managing allergic skin diseases, such as AD.

DOI： 10.1016/j.intimp.2023.110920

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Periodontitis is one of the most prevalent infectious diseases in humans and animals. It is a multifactorial disease resulting in attachment loss and tooth loss. Therefore, preventive dentistry, such as daily teeth cleaning or providing dental chews from puppyhood is essential. This study aimed to find an alternative option for preventive dentistry by examining both in vitro and clinically, the antibacterial, antihalitosis, and anti-inflammatory effects of folic acid (FA) in dogs with periodontal disease. The antibacterial and antihalitosis responses of FA were evaluated in vitro using Porphyromonas gulae, a bacterium that plays a significant role in the development of periodontal disease in dogs. Anti-inflammatory responses, such as secretion of IL-1β, IL-6, and IL-8 induced by P. gulae infection in human gingival epithelium have been studied. This study used dogs with P. gulae-associated periodontal diseases and was conducted by providing a dental chew containing 0.13% FA for 28 days. The viability and halitosis production (hydrogen sulfide and methyl mercaptan) of P. gulae was significantly inhibited by FA in a dose and time-dependent manner. IL-1β, IL-6, and IL-8 secretion were also significantly suppressed by FA treatment in a dose-dependent manner. In vitro bactericidal, antihalitosis, and anti-inflammatory effects of FA were confirmed in dogs with P. gulae-associated periodontal disease. One month of oral treatment with 0.13% FA-containing dental chews significantly reduced halitosis as well as P. gulae activity. This study suggests that oral treatment with FA can be a preventive option for periodontal disease in dogs as well as humans.

DOI： 10.1177/08987564231189650

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media {SA}  

<jats:p>The strong bond between dogs and their owners creates a close association that could result in the transfer of antibiotic-resistant bacteria from canines to humans, potentially leading to the spread of antimicrobial resistance genes. <jats:italic>Pseudomonas aeruginosa</jats:italic>, a common causative agent of persistent ear infections in dogs, is often resistant to multiple antibiotics. Assessing the antimicrobial resistance profile and genotype of <jats:italic>P. aeruginosa</jats:italic> is crucial for the appropriate use of veterinary pharmaceuticals. However, in recent years, few studies have been conducted on this bacterium in Japan. We determined the antimicrobial resistance profile and genotype of <jats:italic>P. aeruginosa</jats:italic> isolated from the ear canal of dogs in Japan in 2020. Analysis of antimicrobial resistance using disk diffusion tests indicated a high frequency of resistance to most antimicrobial agents. Particularly, 29 isolates from the ear canals of the 29 affected dogs (100%) were resistant to cefovecin, cefpodoxime, and florfenicol; however, they were susceptible to cefepime and piperacillin/tazobactam. Only 3.4, 10.3, and 10.3% of the isolates were resistant to ceftazidime, tobramycin, and gentamicin, respectively. Furthermore, upon analyzing the population structure using multilocus sequence typing, a considerably large clonal complex was not observed in the tested isolates. Three isolates, namely ST3881, ST1646, and ST532, were clonally related to the clinically isolated sequence types in Japan (such as ST1831, ST1413, ST1812, and ST1849), which is indicative of dog-to-human transmission. Considering the variation in antibiotic resistance compared to that reported by previous studies and the potential risk of dog-to-human transmission, we believe that the survey for antimicrobial resistance profile and population structure should be continued regularly. However, the prevalence of multidrug-resistant <jats:italic>P. aeruginosa</jats:italic> in dogs in Japan is not a crisis.</jats:p>

DOI： 10.3389/fvets.2023.1074127

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

The deterioration in reproductive performance in association with low fertility leads to significant economic losses on dairy farms. The uterine microbiota has begun to attract attention as a possible cause of unexplained low fertility. We analyzed the uterine microbiota associated with fertility by 16S rRNA gene amplicon sequencing in dairy cows. First, the alpha (Chao1 and Shannon) and beta (unweighted and weighted UniFrac) diversities of 69 cows at four dairy farms that had passed the voluntary waiting period before the first artificial insemination (AI) were analyzed with respect to factors including farm, housing style, feeding management, parity, and AI frequency to conception. Significant differences were observed in the farm, housing style, and feeding management, except parity and AI frequency to conception. The other diversity metrics did not show significant differences in the tested factors. Similar results were obtained for the predicted functional profile. Next, the microbial diversity analysis of 31 cows at a single farm using weighted UniFrac distance matrices revealed a correlation with AI frequency to conception but not with parity. In correlation with AI frequency to conception, the predicted function profile appeared to be slightly modified and a single bacterial taxon, Arcobacter, was detected. The bacterial associations related to fertility were estimated. Considering these, the uterine microbiota in dairy cows can be varied depending on the farm management practices and may become one of the measures for low fertility. IMPORTANCE We examined the uterine microbiota associated with low fertility in dairy cows derived from four commercial farms via a metataxonomic approach using endometrial tissues prior to the first artificial insemination. The present study provided two new insights into the relevance of uterine microbiota with respect to fertility. First, the uterine microbiota varied depending on housing style and feeding management. Next, a subtle change was observed in functional profile analysis: a formation of uterine microbiota was detected to be different in correlation with fertility in one farm studied. Considering these insights, an examination system on bovine uterine microbiota is hopefully established based on continuous research on this topic.

DOI： 10.1128/spectrum.04764-22

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Previous research has demonstrated that Porphyromonas gulae (P. gulae) significantly contributes to the development of periodontal disease in dogs. Porphyromonas gulae is divided into three subtypes according to the 41-kDa filamentous appendage (fimA), defined as types A, B, and C. This study aimed to elucidate the association between fimA type of P. gulae with the number of permanent teeth, reflecting the severity of periodontal disease. Two hundred twenty-five dogs were categorized by P. gulae fimA type as negative, type A dominant, type B dominant, and type C dominant. The stage of periodontal disease in P. gulae-positive dogs increased with age, particularly in type C dominant dogs. Correspondingly, the number of permanent teeth in P. gulae fimA type C-dominant dogs was significantly lower than that of P. gulae-negative dogs, suggesting there is a significant association between fimA type of P. gulae and the number of permanent teeth resulting from the development of periodontal disease.

DOI： 10.3389/fvets.2023.1022838

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

BACKGROUND: Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats in Japan due to insufficiently reliable seroepidemiological analysis methods that are easy to use in cats. RESULTS: We developed a protein-A/G-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against SARS-CoV-2 in cats. The assay was standardized using positive rabbit antibodies against SARS-CoV-2. The ELISA results were consistent with those of a conventional anti-feline-immunoglobulin-G (IgG)-based ELISA. To test the protein-A/G-based ELISA, we collected blood samples from 1,969 cats that had been taken to veterinary clinics in Japan from June to July 2020 and determined the presence of anti-SARS-CoV-2 antibodies. Nine cats were found to have SARS-CoV-2 S1-specific IgG, of which 4 had recombinant receptor-binding domain-specific IgG. Of those 9 samples, one showed neutralizing activity. Based on these findings, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats in Japan was 0.05% (1/1,969 samples). This prevalence was consistent with the prevalence of neutralizing antibodies against SARS-CoV-2 in humans in Japan according to research conducted at that time. CONCLUSIONS: Protein-A/G-based ELISA has the potential to be a standardized method for measuring anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan might be linked to that in humans.

DOI： 10.1186/s12917-022-03527-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

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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Language：English   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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Authorship：Corresponding author   Language：English   Publisher：(公社)日本獣医学会  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Association for Research in Vision and Ophthalmology ({ARVO})  

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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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

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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Language：English   Publisher：(公社)日本獣医学会  

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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Language：English   Publisher：(公社)日本農芸化学会  

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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Other Link： https://search.jamas.or.jp/default/link?pub_year=2021&ichushi_jid=J04676&link_issn=&doc_id=20211014220001&doc_link_id=10.1093%2Fbbb%2Fzbab146&url=https%3A%2F%2Fdoi.org%2F10.1093%2Fbbb%2Fzbab146&type=Crossref&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00002_2.gif

Language：English   Publisher：(公社)日本獣医学会  

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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Other Link： https://search.jamas.or.jp/default/link?pub_year=2021&ichushi_jid=J04672&link_issn=&doc_id=20211108410003&doc_link_id=1390007515115443328&url=https%3A%2F%2Fcir.nii.ac.jp%2Fcrid%2F1390007515115443328&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_3.gif

Language：English   Publisher：(公社)日本獣医学会  

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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Other Link： https://search.jamas.or.jp/default/link?pub_year=2021&ichushi_jid=J04672&link_issn=&doc_id=20210908260006&doc_link_id=1390007469757517440&url=https%3A%2F%2Fcir.nii.ac.jp%2Fcrid%2F1390007469757517440&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_3.gif

Language：English   Publishing type：Research paper (scientific journal)  

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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Language：English   Publishing type：Research paper (scientific journal)  

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  

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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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-19K08953/

Language：English   Publishing type：Research paper (scientific journal)  

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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Language：English   Publisher：Nature Publishing Group  

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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Other Link： https://search.jamas.or.jp/default/link?pub_year=2019&ichushi_jid=J04548&link_issn=&doc_id=20191101280001&doc_link_id=10.1038%2Fs41429-019-0213-9&url=https%3A%2F%2Fdoi.org%2F10.1038%2Fs41429-019-0213-9&type=Crossref&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00002_2.gif

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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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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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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18K07127/

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

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.

DOI： 10.1016/j.virusres.2018.06.005

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

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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Language：English   Publisher：Springer  

© 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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DOI： 10.1016/j.xphs.2018.04.012

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

© 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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Authorship：Lead author,　Corresponding author   Language：English   Publisher：John Wiley & Sons Australia, Ltd  

© 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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Other Link： https://search.jamas.or.jp/default/link?pub_year=2016&ichushi_jid=J04575&link_issn=&doc_id=20160613130008&doc_link_id=10.1111%2F1348-0421.12347&url=https%3A%2F%2Fdoi.org%2F10.1111%2F1348-0421.12347&type=Crossref&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00002_2.gif

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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Authorship：Lead author   Language：English   Publisher：WILEY  

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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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/mbo3.166

Publishing type：Research paper (scientific journal)   Publisher：Caister Academic Press  

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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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Language：English   Publisher：SPRINGER  

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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Other Link： http://link.springer.com/article/10.1007/s13213-012-0556-y/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

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   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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Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23591478/

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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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

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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Language：English   Publishing type：Research paper (scientific journal)  

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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Publishing type：Research paper (scientific journal)   Publisher：The University of Chicago Press  

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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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：家畜感染症学会誌編集委員会  

ウシの低受胎は、生産性の低下に直接的につながることから畜産経営に与える影響は大きい。しかしながら、低受胎の原因は全て解明されているわけではない。細菌叢とは、特定の環境下で生息する様々な細菌の集合体のことをいう。近年、遺伝子解析技術の進歩により、子宮においても細菌叢の存在が明らかにされ、低受胎との関連性が示されはじめている。本報では、ウシの繁殖と子宮内細菌叢の関係について議論する。これまでに、子宮内細菌叢の研究は分娩後の子宮修復の時期と、任意待機期間以降の人工授精が行われる時期の2つに分けて行われてきた。第一に、分娩後の子宮修復の時期においては、子宮環境の変化に伴って子宮内細菌叢は劇的に変化する。周産期の負の栄養状態は、子宮における生体防御能は低下させ、子宮内細菌叢に異常を生じる。そのため、炎症性子宮疾患の発症に応じて子宮内細菌叢は異なり、子宮修復の遅延に関わることが示唆される。また、この分娩後早期の子宮内細菌叢の変化は子宮内膜の遺伝子発現の変化に長期的に関わる。第二に、任意待機期間以降の子宮環境は子宮修復完了とともに安定することから子宮内細菌叢も安定する。繁殖性との関連性を調査した研究では、子宮内細菌叢の違いが人工授精による受胎性と関連することが認められている。以上のことから、子宮内細菌叢はウシの繁殖に関連しており、低受胎の改善のために検査法として利用されることが期待される。(著者抄録)

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Language：Japanese   Publisher：家畜感染症学会誌編集委員会  

ウシの低受胎は、生産性の低下に直接的につながることから畜産経営に与える影響は大きい。しかしながら、低受胎の原因は全て解明されているわけではない。細菌叢とは、特定の環境下で生息する様々な細菌の集合体のことをいう。近年、遺伝子解析技術の進歩により、子宮においても細菌叢の存在が明らかにされ、低受胎との関連性が示されはじめている。本報では、ウシの繁殖と子宮内細菌叢の関係について議論する。これまでに、子宮内細菌叢の研究は分娩後の子宮修復の時期と、任意待機期間以降の人工授精が行われる時期の2つに分けて行われてきた。第一に、分娩後の子宮修復の時期においては、子宮環境の変化に伴って子宮内細菌叢は劇的に変化する。周産期の負の栄養状態は、子宮における生体防御能は低下させ、子宮内細菌叢に異常を生じる。そのため、炎症性子宮疾患の発症に応じて子宮内細菌叢は異なり、子宮修復の遅延に関わることが示唆される。また、この分娩後早期の子宮内細菌叢の変化は子宮内膜の遺伝子発現の変化に長期的に関わる。第二に、任意待機期間以降の子宮環境は子宮修復完了とともに安定することから子宮内細菌叢も安定する。繁殖性との関連性を調査した研究では、子宮内細菌叢の違いが人工授精による受胎性と関連することが認められている。以上のことから、子宮内細菌叢はウシの繁殖に関連しており、低受胎の改善のために検査法として利用されることが期待される。(著者抄録)

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Language：Japanese   Publisher：(公社)北海道獣医師会  

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Language：English   Publisher：(公財)日本眼科学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(公社)北海道獣医師会  

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Language：Japanese   Publisher：(公社)日本化学療法学会  

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Language：Japanese   Publisher：三島海雲記念財団  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：English   Publisher：(一社)日本ヘリコバクター学会  

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Language：Japanese   Publisher：Japanese Pharmacological Society  

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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Language：Japanese   Publisher：Japanese Pharmacological Society  

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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Language：Japanese   Publisher：Japanese Pharmacological Society  

We focus on the ozone water to develop a novel treatment approach for staphylococci related inflammatory skin diseases. In the 95th Annual Meeting of the Japanese Pharmacological Society, we reported the anti-allergic and anti-microbial effect of 3 mg/L ozone water in a mouse model of atopic dermatitis (AD) and staphylococci, however, its effect was far from clinical application. The aim of this study is to evaluate the anti-microbial and anti-inflammatory property of high concentration (11 mg/L) of ozone water to advance our project. Ozone water showed a significant bactericidal effect against Staphylococcus aureus, S. pseudintermedius and S. lentus. Furthermore, S. pseudintermedius induced IL-1β and IL-6 secretion in human epidermal keratinocytes was significantly inhibited by pre-treatment of ozone water. In vivo experiment with a mouse model of AD, significant improvement of AD symptoms and trans epidermal water loss were also found in an ozone water treatment group. Surprisingly, local immune reactions such as type II conventional dendritic cells, effector helper T cells, and IgE-produced B cells are also regulated by ozone water application. Our findings strongly suggest that topical treatment with high concentration of ozone water significantly ameliorated AD symptoms through antibacterial effect toward staphylococci.

DOI： 10.1254/jpssuppl.96.0_2-b-p-170

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Language：Japanese   Publisher：Japanese Pharmacological Society  

We are currently developing Lactobacillus reuteri isolated from healthy dogs as a probiotic supplement for canine atopic dermatitis (AD). In this study, we examined the anti-allergic effects of live L. reuteri and killed L. reuteri strain mixture (i.e., strains M01, M11, M40, and M41) using a hapten-induced-AD mouse model. First, when UV-killed L. reuteri (overgrowth) was orally administered to AD mice, cutaneous barrier function evaluated by the trans epidermal water loss was maintained as normal in the L. reuteri treatment group, compared with the control group. Immune reactions including lymphocyte proliferation in auricular lymph nodes and total IgE levels in serum are significantly regulated by treatment of killed L. reuteri. In contrast, L. reuteri treatment improve neither AD score nor skin thickness. Second, the anti-AD effects were examined using 4 individual strains of live L. reuteri (i.e., M01, M11, M40, or M41). The symptoms of AD and immune responses such as lymphocyte proliferation and total IgE levels are significantly reduced by L. reuteri M11 administration compared with the vehicle control and other strains. Currently, the interaction with macrophages and regulatory T cells is being studied.

DOI： 10.1254/jpssuppl.96.0_2-b-p-173

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Regulation of the gut microbiome using probiotics such as Lactobacillus spp. is recently focused on as one of the preventive measures for allergy. We originally isolated Lactobacillus sp. (L. AZABU) from the healthy dog guts. We here evaluated the anti-allergic effects of L. AZABU using hapten-induced atopic dermatitis (AD) and asthma mouse models. First, daily oral administration of live L. AZABU (2×10⁸ CFU/ml) during the experimental period significantly prevented the development of AD symptoms, skin thickness, and trans-epidermal water loss in the AD mouse model. Significant decreased number of IgE-positive B cells in auricular lymph node was also observed in L. AZABU treatment group, indicating allergen-specific immunoreaction was regulated by L. AZABU. Group 2 innate lymphoid cells, which play a pivotal role in non-allergen-specific immunoreaction, also significantly suppressed by oral administration of live L. AZABU. Next, the anti-allergic effects of live L. AZABU was simultaneously demonstrated in the asthma mouse model, including significant decrease of inflammatory pathological change of lung, and both allergen-specific and non-specific immunoreaction in hilar lymph node and lung tissue. Our findings suggested that live L. AZABU has anti-allergic properties although the mechanism of action is still being investigated.

DOI： 10.1254/jpssuppl.96.0_2-b-p-172

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(一社)日本アレルギー学会  

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Language：Japanese   Publisher：(一社)日本アレルギー学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本内科学会-関東地方会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：神奈川県医師会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(一社)日本アレルギー学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(一社)日本アレルギー学会  

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Language：English   Publisher：(一社)日本アレルギー学会  

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Language：English   Publisher：(一社)日本アレルギー学会  

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Language：Japanese   Publisher：(公社)日本分析化学会  

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Language：Japanese   Publisher：神奈川県医師会  

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Language：Japanese   Publisher：神奈川県医師会  

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Language：Japanese   Publisher：神奈川県医師会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(公社)日本分析化学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2019197623

Language：Japanese   Publisher：麻布大学  

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Other Link： http://search.jamas.or.jp/link/ui/2020013120

Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：プラスチックス・エージ  

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Language：Japanese   Publisher：(一社)日本臨床微生物学会  

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Language：Japanese  

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Language：Japanese  

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.

DOI： 10.1038/s41598-018-29272-2

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Language：Japanese  

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Language：Japanese   Publisher：(株)インターズー  

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Language：Japanese   Publisher：(一社)日本ペット栄養学会  

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Language：Japanese   Publisher：インターズー  

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Other Link： http://search.jamas.or.jp/link/ui/2018300752

Language：Japanese   Publisher：(株)インターズー  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：Japanese Society of Pet Animal Nutrition  

DOI： 10.11266/jpan.21.3_142

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Language：Japanese  

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