Exploring the Interplay between Nutrients, Bacteriophages, and Bacterial Lipases in Host- and Bacteria-mediated Pathogenesis.

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-1ß, 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.

Phylogenic analysis of new viral cluster of large phages with unusual DNA genomes containing uracil in place of thymine in gene-sharing network, using phages S6 and PBS1 and relevant uncultured phages derived from sewage metagenomics.

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.

Intracameral Bacteriophage Injection as Postoperative Prophylaxis for Enterococcus faecalis-Induced Endophthalmitis After Cataract Surgery in Rabbits.

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.

Acid-stable capsid structure of Helicobacter pylori bacteriophage KHP30 by single-particle cryoelectron microscopy.

The acid-stable capsid structures of Helicobacter pylori phages KHP30 and KHP40 are solved at 2.7 and 3.0 Å resolutions by cryoelectron microscopy, respectively. The capsids have icosahedral T = 9 symmetry and consist of each 540 copies of 2 structural proteins, a major capsid protein, and a cement protein. The major capsid proteins form 12 pentagonal capsomeres occupying icosahedral vertexes and 80 hexagonal capsomeres located at icosahedral faces and edges. The major capsid protein has a unique protruding loop extending to the neighboring subunit that stabilizes hexagonal capsomeres. Furthermore, the capsid is decorated with trimeric cement proteins with a jelly roll motif. The cement protein trimer sits on the quasi-three-fold axis formed by three major capsid protein capsomeres, thereby enhancing the particle stability by connecting these capsomeres. Sequence and structure comparisons between the related Helicobacter pylori phages suggest a possible mechanism of phage adaptation to the human gastric environment.

Complete Genome Sequence of Helicobacter pylori Strain 3401, a Suitable Host for Bacteriophages KHP30 and KHP40.

Helicobacter pylori 3401, isolated from a patient with duodenal ulcers in Japan, is susceptible to the bacteriophages KHP30 and KHP40. In this study, we report the complete genome sequence of H. pylori 3401. This study may lead to the establishment of phage therapy against H. pylori infection.

The Efficacy of Phage Therapy in a Murine Model of Pseudomonas aeruginosa Pneumonia and Sepsis.

The emergence of multi-drug resistant Pseudomonas aeruginosa necessitates the search for treatment options other than antibiotic use. The use of bacteriophages is currently being considered as an alternative to antibiotics for the treatment of bacterial infections. A number of bacteriophages were introduced to treat pneumonia in past reports. However, there are still lack of knowledge regarding the dosages, application time, mechanism and safety of phage therapy against P. aeruginosa pneumonia. We used the bacteriophage KPP10 against P. aeruginosa strain D4-induced pneumonia mouse models and observed their outcomes in comparison to control models. We found that the nasal inhalation of highly concentrated KPP10 (MOI = 80) significantly improved survival rate in pneumonia models (P < 0.01). The number of viable bacteria in both lungs and in serum were significantly decreased (P < 0.01) in phage-treated mice in comparison to the control mice. Pathological examination showed that phage-treated group had significantly reduced bleeding, inflammatory cell infiltration, and mucus secretion in lung interstitium. We also measured inflammatory cytokine levels in the serum and lung homogenates of mice. In phage-treated models, serum TNFα, IL-1ß, and IFN-γ levels were significantly lower (P < 0.05, P < 0.01, and P < 0.05, respectively) than those in the control models. In the lung homogenate, the mean IL-1ß level in phage-treated models was significantly lower (P < 0.05) than that of the control group. We confirmed the presence of phage in blood and lungs, and evaluated the safety of bacteriophage use in living models since bacteriophage mediated bacterial lysis arise concern of endotoxic shock. The study results suggest that phage therapy can potentially be used in treating lung infections caused by Pseudomonas aeruginosa.

In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of Enterococcus faecalis Endophthalmitis.

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.

Purification of membrane vesicles from Gram-positive bacteria using flow cytometry, after iodixanol density-gradient ultracentrifugation.

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.

Characterization of two newly isolated Staphylococcus aureus bacteriophages from Japan belonging to the genus Silviavirus.

Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.

Therapeutic Effects of Intravitreously Administered Bacteriophage in a Mouse Model of Endophthalmitis Caused by Vancomycin-Sensitive or -Resistant Enterococcus faecalis.

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.

Therapeutic Potential of an Endolysin Derived from Kayvirus S25-3 for Staphylococcal Impetigo.

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.

Use of a Silkworm Larva Model in Phage Therapy Experiments.

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.

Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening.

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.

Genome Sequences of 12 Mycobacteriophages Recovered from Archival Stocks in Japan.

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.

Polymorphisms in the Helicobacter pylori NY43 strain and its prophage-cured derivatives.

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.

Recovery of mycobacteriophages from archival stocks stored for approximately 50 years in Japan.

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.

Piperacillin and ceftazidime produce the strongest synergistic phage-antibiotic effect in Pseudomonas aeruginosa.

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.

Isolation of Bacteriophages for Fastidious Bacteria.

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.

Prevalence and Genetic Variability of Human Polyomaviruses 6 and 7 in Healthy Skin Among Asymptomatic Individuals.

Background: Despite the pathogenetic potential of human polyomavirus 6 (HPyV6) and human polyomavirus 7 (HPyV7), they have been found in the normal skin of healthy individuals. However, little is known about the prevalence, infection levels, and geographical variations of these polyomaviruses in the skin. Methods: Using skin swabs from 470 participants aged 2-98 years, we estimated the prevalence of copy numbers of HPyV6 and HPyV7 with respect to age and ethnicity. Phylogenetic analyses were conducted based on viral sequences obtained from Asian and white populations. Results: This study provides the first analyses of the age-specific prevalence and levels of HPyV6 and HPyV7 infections in normal skin. Comparisons of age groups revealed that the prevalence and viral loads were significantly higher in elderly persons. Phylogenetic analyses demonstrated the existence of Asian/Japanese-specific strains genetically distinct from strains prevalent in the skin of the white population studied. Conclusions: This large study suggests that HPyV6 and HPyV7 infections in the skin are highly prevalent in elderly adults. Further research is warranted to understand whether persistent infection with high viral loads in the skin could be a risk factor for the development of HPyV6- and HPyV7-associated skin disorders.