A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses.

Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.

Ecological and functional roles of bacteriophages in contrasting environments: marine, terrestrial and human gut.

While they are the most abundant biological entities on the planet, the role of bacteriophages (phages) in the microbiome remains enigmatic and understudied. With a rise in the number of metagenomics studies and the publication of highly efficient phage mining programmes, we now have extensive data on the genomic and taxonomic diversity of (mainly) DNA bacteriophages in a wide range of environments. In addition, the higher throughput and quality of sequencing is allowing for strain-level reconstructions of phage genomes from metagenomes. These factors will ultimately help us to understand the role these phages play as part of specific microbial communities, enabling the tracking of individual virus genomes through space and time. Using lessons learned from the latest metagenomic studies, we focus on two explicit aspects of the role bacteriophages play within the microbiome, their ecological role in structuring bacterial populations, and their contribution to microbiome functioning by encoding auxiliary metabolism genes.

Isolation and Functional Characterization of Fusobacterium nucleatum Bacteriophage.

Bacteriophages are viruses that specifically lyse bacteria. They have demonstrated potential in applications as antibacterial agents in medicine, agriculture, and environmental remediation. Due to the complex and dynamic nature of the oral microbiome, antibiotic treatment of chronic, polymicrobial oral diseases may lead to dysbiosis. In these diseases, bacteriophages may provide targeted activity against oral bacteria without such disruption to the broader microbial community. In this chapter, we describe the methods for screening samples that may contain bacteriophages against oral pathogenic bacteria, and using the example of FNU1, the bacteriophage we isolated against Fusobacterium nucleatum, describe the process of bacteriophage purification and characterization.

Characterization of Novel Lytic Bacteriophages of Achromobacter marplantensis Isolated from a Pneumonia Patient.

Achromobacter spp. are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis (CF). A. marplatensis, which is closely related to A. xylosoxidans, has been isolated from the lungs of CF patients and other human infections. This article describes the isolation, morphology and characterization of two lytic bacteriophages specific for an A. marplatensis strain isolated from a pneumonia patient. This host strain was the causal agent of hospital acquired pneumonia-the first clinical report of such an occurrence. Full genome sequencing revealed bacteriophage genomes ranging in size from 45901 to 46,328 bp. Transmission electron microscopy revealed that the two bacteriophages AMA1 and AMA2 belonged to the Siphoviridae family. Host range analysis showed that their host range did not extend to A. xylosoxidans. The possibility exists for future testing of such bacteriophages in the control of Achromobacter infections such as those seen in CF and other infections of the lungs. The incidence of antibiotic resistance in this genus highlights the importance of seeking adjuncts and alternatives in CF and other lung infections.

Novel Bacteriophages Capable of Disrupting Biofilms From Clinical Strains of Aeromonas hydrophila.

The increase in global warming has favored growth of a range of opportunistic environmental bacteria and allowed some of these to become more pathogenic to humans. Aeromonas hydrophila is one such organism. Surviving in moist conditions in temperate climates, these bacteria have been associated with a range of diseases in humans, and in systemic infections can cause mortality in up to 46% of cases. Their capacity to form biofilms, carry antibiotic resistance mechanisms, and survive disinfection, has meant that they are not easily treated with traditional methods. Bacteriophage offer a possible alternative approach for controlling their growth. This study is the first to report the isolation and characterization of bacteriophages lytic against clinical strains of A. hydrophila which carry intrinsic antibiotic resistance genes. Functionally, these novel bacteriophages were shown to be capable of disrupting biofilms caused by clinical isolates of A. hydrophila. The potential exists for these to be tested in clinical and environmental settings.

Genomic, morphological and functional characterisation of novel bacteriophage FNU1 capable of disrupting Fusobacterium nucleatum biofilms.

Fusobacterium nucleatum is an important oral bacterium that has been linked to the development of chronic diseases such as periodontitis and colorectal cancer. In periodontal disease, F. nucleatum forms the backbone of the polymicrobial biofilm and in colorectal cancer is implicated in aetiology, metastasis and chemotherapy resistance. The control of this bacteria may be important in assisting treatment of these diseases. With increased rates of antibiotic resistance globally, there is need for development of alternatives such as bacteriophages, which may complement existing therapies. Here we describe the morphology, genomics and functional characteristics of FNU1, a novel bacteriophage lytic against F. nucleatum. Transmission electron microscopy revealed FNU1 to be a large Siphoviridae virus with capsid diameter of 88 nm and tail of approximately 310 nm in length. Its genome was 130914 bp, with six tRNAs, and 8% of its ORFs encoding putative defence genes. FNU1 was able to kill cells within and significantly reduce F. nucleatum biofilm mass. The identification and characterisation of this bacteriophage will enable new possibilities for the treatment and prevention of F. nucleatum associated diseases to be explored.

Isolation and characterization of bacteriophage NTR1 infectious for Nocardia transvalensis and other Nocardia species.

We describe here the isolation and characterization of the bacteriophage, NTR1 from activated sludge. This phage is lytic for Nocardia transvalensis, Nocardia brasiliensis and Nocardia farcinica. NTR1 phage has a genome sequence of 65,275 bp in length, and its closest match is to the Skermania piniformis phage SPI1 sharing over 36% of its genome. The phage belongs to the Siphoviridae family, possessing a long non-contractile tail and icosahedral head. Annotation of the genome reveals 97 putative open reading frames arranged in the characteristic modular organization of Siphoviridae phages and contains a single tRNA-Met gene.

Semi-Solid and Solid Dosage Forms for the Delivery of Phage Therapy to Epithelia.

The delivery of phages to epithelial surfaces for therapeutic outcomes is a realistic proposal, and indeed one which is being currently tested in clinical trials. This paper reviews some of the known research on formulation of phages into semi-solid dosage forms such as creams, ointments and pastes, as well as solid dosage forms such as troches (or lozenges and pastilles) and suppositories/pessaries, for delivery to the epithelia. The efficacy and stability of these phage formulations is discussed, with a focus on selection of optimal semi-solid bases for phage delivery. Issues such as the need for standardisation of techniques for formulation as well as for assessment of efficacy are highlighted. These are important when trying to compare results from a range of experiments and across different delivery bases.

Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca.

AIM: To isolate and characterize bacteriophage lytic for the opportunistic pathogen Klebsiella oxytoca and their formulation into a range of solid dosage forms for in-vitro testing. METHODS AND RESULTS: We report the isolation, genomic and functional characterization of a novel bacteriophage lytic for Klebsiella oxytoca, which does not infect the closely related Klebsiella pneumoniae. This bacteriophage was formulated into suppositories and troches and shown to be released and lyse underlying Klebsiella oxytoca bacteria in an in-vitro model. These bacteriophage formulations were stable for at least 49 days at 4°C. CONCLUSIONS: The successful in-vitro assay of these formulations here suggests that they could potentially be tested in-vivo to determine whether such a therapeutic approach could modulate the gut microbiome, and control Klebsiella oxytoca overgrowth, during antibiotic therapy regimes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriophage specific for Klebsiella oxytoca which can be formulated into solid dosage forms appropriate for potential delivery in testing as a therapy to modulate gut microbiome during antibiotic therapies.

Dynamic interactions between prophages induce lysis in Propionibacterium acnes.

Progress in next-generation sequencing technologies has facilitated investigations into microbial dynamics. An important bacterium in the dairy industry is Propionibacterium freudenreichii, which is exploited to manufacture Swiss cheeses. A healthy culture of these bacteria ensures a consistent cheese with formed 'eyes' and pleasant flavour profile, and the investigation of prophages and their interactions with these bacteria could assist in the maintenance of the standard of this food product. Two bacteriophages, termed PFR1 and PFR2, were chemically induced using mitomycin C from two different dairy strains of P. freudenreichii. Both phages have identical genomes; however, PFR2 was found to contain an insertion sequence, IS204. Host range characterisation showed that PFR1 was able to form plaques on a wild type Propionibacterium acnes strain, whereas PFR2 could not. The lytic plaques observed on P. acnes were a result of PFR1 inducing the lytic cycle of a pseudolysogenic phage in P. acnes. Further investigation revealed that both PFR1 and PFR2 could infect P. acnes but not replicate. This study demonstrates the dynamic interactions between phages, which may alter their lytic capacity under certain conditions. To our knowledge, this is the first report of two phages interacting to kill their host.

Bacteriophage formulated into a range of semisolid and solid dosage forms maintain lytic capacity against isolated cutaneous and opportunistic oral bacteria.

BACKGROUND: Resistance of bacteria to antimicrobial agents is of grave concern. Further research into the development of bacteriophage as therapeutic agents against bacterial infections may help alleviate this problem. OBJECTIVES: To formulate bacteriophage into a range of semisolid and solid dosage forms and investigate the capacity of these preparations to kill bacteria under laboratory conditions. METHODS: Bacteriophage suspensions were incorporated into dosage forms such as creams, ointments, pastes, pessaries and troches. These were applied to bacterial lawns in order to ascertain lytic capacity. Stability of these formulations containing phage was tested under various storage conditions. KEY FINDINGS: A range of creams and ointments were able to support phage lytic activity against Propionibacterium acnes. Assessment of the stability of these formulations showed that storage at 4 °C in light-protected containers resulted in optimal phage viability after 90 days. Pessaries/suppositories and troches were able to support phage lytic activity against Rhodococcus equi. CONCLUSIONS: We report here the in-vitro testing of semisolid and solid formulations of bacteriophage lytic against a range of bacteria known to contribute to infections of the epithelia. This study provides a basis for the future formulation of diverse phage against a range of bacteria that infect epithelial tissues.

Locating and Activating Molecular 'Time Bombs': Induction of Mycolata Prophages.

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences.

The Formulation of Bacteriophage in a Semi Solid Preparation for Control of Propionibacterium acnes Growth.

AIMS: To isolate and characterise phage which could lyse P. acnes and to formulate the phage into a delivery form for potential application in topical treatment of acne infection. METHODS AND RESULTS: Using standard phage isolation techniques, ten phage capable of lysing P. acnes were isolated from human skin microflora. Their genomes showed high homology to previously reported P. acnes phage. These phage were formulated into cetomacrogol cream aqueous at a concentration of 2.5x108 PFU per gram, and shown to lyse underlying P. acnes cells grown as lawn cultures. These phage formulations remained active for at least 90 days when stored at four degrees Celsius in a light protected container. CONCLUSIONS: P. acnes phage formulated into cetomacrogol cream aqueous will lyse surrounding and underlying P. acnes bacteria, and are effective for at least 90 days if stored appropriately. SIGNIFICANCE AND IMPACT OF THE STUDY: There are few reports of phage formulation into semi solid preparations for application as phage therapy. The formulation method described here could potentially be applied topically to treat human acne infections. The potential exists for this model to be extended to other phage applied to treat other bacterial skin infections.

The short-term influence of elevated intraocular pressure on axial length.

PURPOSE: To investigate the influence of a short period of elevated IOP (induced through the wearing of standard swimming goggles) upon axial length. METHODS: Forty young adult subjects (20 myopes and 20 emmetropes, mean age 22 ± 2 years) had their IOP and axial length measured before, during and after a 3 min period of swimming goggle wear. IOP was measured using a non-contact tonometer, and axial length with an optical biometer based upon the principle of partial coherence interferometry. RESULTS: A 3-min period of swimming goggle wear was found to be associated with a significant increase in IOP (mean change 3.7 ± 2.6 mmHg, p < 0.0001) and a significant axial elongation of the eye (mean change in axial length of 18 ± 12 µm, p < 0.0001). Both IOP and axial length were found to return to baseline levels upon removal of the swimming goggles. A significant positive association was found between the changes in IOP and axial length (r(2) = 0.37, p < 0.0001). There were no significant differences between the emmetropic and myopic subjects in terms of their magnitude of change in axial length and IOP during swimming goggle wear. CONCLUSIONS: A small magnitude of elevation in IOP, induced through mechanical means and imposed for a short period of time was associated with a small but statistically significant axial elongation of the eye.