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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., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2024 Munetomo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare no conflict of interest.

Atopic dermatitis (AD) is a chronic and relapsing multifactorial inflammatory skin disease that also affects dogs. The oral and gut microbiota are associated with many disorders, including allergy. Few studies have addressed the oral and gut microbiota in dogs, although the skin microbiota has been studied relatively well in these animals. Here, we studied the AD-associated oral and gut microbiota in 16 healthy and 9 AD dogs from a purebred Shiba Inu colony. We found that the diversity of the oral microbiota was significantly different among the dogs, whereas no significant difference was observed in the gut microbiota. Moreover, a differential abundance analysis detected the Family_XIII_AD3011_group (Anaerovoracaceae) in the gut microbiota of AD dogs; however, no bacterial taxa were detected in the oral microbiota. Third, the comparison of the microbial co-occurrence patterns between AD and healthy dogs identified differential networks in which the bacteria in the oral microbiota that were most strongly associated with AD were related to human periodontitis, whereas those in the gut microbiota were related to dysbiosis and gut inflammation. These results suggest that AD can alter the oral and gut microbiota in dogs., (© 2022 Society for Applied Microbiology.)

Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed "dU jumbophages" in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria., (Copyright © 2022. Published by Elsevier B.V.)

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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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.

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.

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.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13' 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 β-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the α- and β-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 β-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the α- and β-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 thefamily 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.

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., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: 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., Importance: Pseudomonas 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (© 2015 The Societies and John Wiley & Sons Australia, Ltd.)

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 6 h postinfection reduced the severity of infection and rescued the infected mice. Phage S13' can efficiently lyse hospital-acquired MRSA strains causing pneumonia-associated bacteremia in vitro. Thus, phage therapy may be a possible therapeutic intervention in staphylococcal lung-derived septicemia., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2014 Yamaguchi et al.)

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., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.)

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

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.