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.

Bacteriophage isolation. Mouthwash samples were collected from dental practices in Bendigo (Victoria, Australia) and screened for the presence of lytic bacteriophages against F. nucleatum using the enrichment method in BHI broth according to Gill and Hyman 31 . Briefly, 100 µL of F. nucleatum grown previously in broth culture for 48 hours anaerobically was added to 1 mL of sample and 20 mL of broth and incubated anaerobically at 37 °C for seven days. The enrichment was filtered using a 0.20 µm cellulose acetate filter (Microanalytix, Australia) before spotting 10 µL of the filtrate on a freshly prepared lawn of F. nucleatum on 1% agar. The plate was incubated for 48 hours anaerobically at 37 °C. Observed plaques were excised and purified as described previously 32 . To test the host range of the purified bacteriophage, it was also spotted onto cultures of Streptococcus mutans, Porphyromonas gingivalis and C. acnes, which are all found in the oral cavity. electron microscopy. The purified bacteriophage particles were visualised using a JEOL JEM-2100 Transmission Electron Microscope (TEM) using 400-mesh carbon-coated copper grids (ProSciTech, Australia).
The bacteriophage lysate was allowed to adsorb to the grid for 30 seconds before being washed with Milli-Q ® water (Promega, Australia). The adsorbed particles were then negatively stained twice for 30 seconds with 2% [W/V] uranyl acetate (Sigma, Australia). Excess stain on the grids was removed using filter paper before being air dried for 20 minutes. The grid was visualised and images captured with a Gatan Orius SC200D 1 wide-angle camera (Gatan Microscopy Suite and Digital Micrograph Imaging software version 2.3.2.888.0) at 200 kV. Further image analysis was achieved in ImageJ software version 1.8.0_112. DNA extraction. Bacteriophage DNA was extracted from a highly concentrated purified lysate (approximately 10 11 PFU mL −1 ) using the phenol-chloroform method as previously described 32 . All compounds used in the DNA extraction process were obtained from Sigma-Aldrich (Australia) unless stated otherwise. The concentrated bacteriophage stock was treated with 5 mmol L −1 of MgCl 2 and 1.0 µL each of RNase A (Promega, Australia) and DNase I (Promega, Australia) to a final concentration of 10 µg mL −1 . The solution was incubated at room temperature for 30 minutes to digest extraneous DNA or RNA. Polyethylene glycol 8000 (PEG) www.nature.com/scientificreports www.nature.com/scientificreports/ at 10% [W/V] and sodium chloride (NaCl at 1 g L −1 ) were added to the mixture and incubated overnight. The solution was centrifuged at 12000 × g for 5 minutes and pellets resuspended in 50 µL nuclease-free water (Promega, Australia). Bacteriophage proteins were digested by the addition of Proteinase K (50 µg mL −1 ), EDTA (20 mmol L −1 ) and sodium dodecyl sulphate (0.5% (v/v)) and incubating for one hour at 55 °C. An equal volume of phenol-chloroform-isoamyl alcohol (29:28:1) was then added to separate viral DNA from proteins. The mixture was gently vortexed and then centrifuged at 12000 × g for 10 minutes to isolate the aqueous phase. DNA was precipitated by adding an equal volume of isopropanol and incubating overnight at −20 °C. The DNA pellet was collected by centrifugation at 12000 × g for 5 minutes. The DNA pellet was washed in 70% ethanol, air-dried and finally resuspended in 30 µL of nuclease-free water (Promega, Australia).
Whole genome sequencing and in-silico analysis. Nextera ® XT DNA sample preparation kits were used to prepare DNA libraries according to the manufacturer's instructions (Illumina, Australia). The libraries were sequenced on an Illumina MiSeq ® using a MiSeq ® V2 reagent kit (300 cycles) with 150 basepair (bp) paired end reads. Sequence reads were assembled de novo using Geneious software version 11.0.5. Gene prediction was achieved by predicting open reading frames (ORFs) using ATG, GTG, and TTG start codons with a minimum nucleotide length of 50 bp. The ORFs were translated using Geneious and analysed by BLASTP (https://blast. ncbi.nlm.nih.gov/) to ascribe potential function. The genome was further examined for the presence of transfer RNA (tRNA) and transfer-messenger RNA (tmRNA) using ARAGORN 33 and tRNAscan-SE 2.0 34 . Whole genome alignments and phylogenetic tree construction were performed in CLC genomics workbench version 9.5.4 by UPGMA algorithm with 1,000 replicate bootstrapping. The alignment included whole genomes of FNU1 and other bacteriophages specific for oral bacteria obtained from the NCBI Genbank.
Biofilm growth and quantification. Biofilm experiments were conducted anaerobically using BHI broth (Oxoid, Australia) supplemented with 0.5% cysteine, 0.5% haemin and 0.5% glucose in 96 well polystyrene plates (Greiner bio-one, Australia) coated with 0.5% gelatin. F. nucleatum was cultured for 48 hours and 100 µL of approximately 1 × 10 8 CFU mL −1 of F. nucleatum in exponential growth phase was added to each well before an equal volume of broth was added. The inoculated plates were incubated at 37 °C under anaerobic conditions and shaking at 120 rpm (Ratek Medium Orbital shaking incubator) for 4 days with sterile broth replenishment after 48 hours. To each well, 10 µL of bacteriophage FNU1 at 10 11 PFU/mL was added after 4 days of biofilm formation. The biofilms were then incubated for a further 24 hours anaerobically at 37 °C before quantification assays completed as described previously 35 . Briefly, planktonic cells were washed off gently using MilliQ ® deionised water (Merck, Australia) and the attached cells air dried. The attached biomass was then stained with 200 µL of 0.1% crystal violet for 5 minutes, washed using deionised water and air-dried for 5 minutes. An equal volume of ethanol (70%) was added to each well to decolourise the stained attached cells and the absorbance of the crystal-violet stained ethanol was evaluated at a wavelength of 600 nm (OD 600 ) using a FlexStation 3 plate reader (Molecular Devices, United States).

Biofilm viability analysis.
To test for viability, the F. nucleatum biofilm was grown on gelatin-coated microscope slides in the same manner as in the 96 well plates described above. SYBR gold ® and Propidium Iodide (PI) were used to stain nucleic acids of live (membrane intact) and dead (membrane compromised) cells, respectively. Propidium Iodide (3 µL) was added to 100 µL of SYBR gold ® diluted (1:100) in dimethyl sulfoxide (Sigma, Australia). The mixture was applied to the biofilm on slides and incubated for 30 minutes. Excess PI and SYBR gold ® were rinsed off and slides air-dried before mounting with 5 µL Vectorshield ® (Burlingame, USA) and coverslips. The slides were examined using an Olympus Fluoview Fv10i-confocal laser-scanning microscope (Olympus Life Science, Australia) with excitation wavelength at 485 nm. Green emission at fluorescence 535 nm and Red emission at fluorescence 635 nm were measured to indicate live versus dead cells on the slides. statistical analysis. The absorbance values quantifying the biofilms were analysed for normality using the Shapiro Wilk test and the medians compared by a paired T-test. The p-values of less than 0.05 were considered statistically significant. All statistical analysis was performed using the Statistical Package for Social Sciences (SPSS version 25).

Results
Isolation and phenotypic characterisation of F. nucleatum bacteriophage FNU1. One mouthwash sample was found to produce clear plaques on 1% BHI agar. This was not observed on BHI with 1.5% agar. On the less concentrated 1% agar, clear round plaques of approximately 1 mm diameter were seen (Fig. 1A). The host range of FNU1 was restricted to F. nucleatum, and did not extend to other bacteria found in the oral cavity that we tested. TEM revealed a Siphoviridae bacteriophage with an icosahedral head of ≈88 nm in diameter and a long flexible tail terminating in a spike (Fig. 1B). The tail was approximately 310 nm long and ≈10 nm wide with a spike at the end, measuring an average of ≈20 nm long and ≈5 nm wide on the widest section.
Genomic analysis. Bacteriophage DNA extraction and sequencing was performed on three separate occasions. On all occasions, a single contig of 130914 bp was obtained with coverage ranging from 121 to 2400 times. The process was repeated to ensure accuracy and because the sequence generated displayed low homology to any known bacteriophage or other genomes present in the database. The FNU1 bacteriophage genome (NCBI Genbank Accession Number: MK554696) was composed of 178 predicted ORFs of which 30.34% (54/178) had no significant homology to any sequences in the Genbank database and 38.20% (68/178) had some similarity but with E values of less than 1e-4. Of the remaining genome for bacteriophage FNU1, 31.46% (56/178 ORFs) had significant homology to other sequences, and of these, 71.43% (40/56 ORFs) had conserved domains. The overall GC content was 25.0%. The ORFs, their significant matches and E values are shown in Table 1.
www.nature.com/scientificreports www.nature.com/scientificreports/ Functional genomics predictions were mapped using the Geneious software 11.0.5 ( Fig. 2) with putative structural genes (pink), putative DNA manipulation genes (green), putative regulatory genes (blue), putative lytic genes (red) and hypothetical genes (yellow) marked. Although the majority of genes couldn't be assigned functionality, a pattern of clustering of related genes was observed (Fig. 2). Putative structural genes appeared to be orientated in a clockwise direction while the rest were orientated anticlockwise. The putative structural genes were interspaced with putative lysis and regulatory genes, as well as other genes that have no known functionality or homology located between sites 1 and 40000 bp in the genome map (Fig. 2). The genes in anticlockwise orientation were comprised mostly of putative DNA manipulation genes located between sites 65000 and 130000 bp in the genome map ( Fig. 2) that may be involved in the infection and packaging processes, as well as a cluster of putative lysis genes (located between 45000 and 55000 bp). Putative bacteriophage FNU1 defence against bacterial anti-phage systems. Conserved protein family (Pfam) prediction on BLASTp analysis indicated there were 13 ORFs with predicted Pfam domains that may be involved in bacteriophage FNU1 evading host immunity (Table 2). These included at least three genes each putatively encoding antirepressors, methylation genes and toxin-antitoxin systems. According to the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) database (http://crispr.i2bc.paris-saclay.fr/) 36 , no CRISPR regions were found in the phage FNU1 genome. phylogenetic relatedness with other bacteriophage targeting oral bacteria. The genome of bacteriophage FNU1 showed little homology to any bacteriophage deposited in NCBI Genbank. To understand the relatedness of FNU1 to other bacteriophages, a phylogenetic tree was constructed. Whole genomes of bacteriophages targeting oral bacteria were downloaded from NCBI Genbank and compared with FNU1 as they are found in the same microenvironment. Bacteriophage FNU1 was found to be most closely related to Streptococcus mutans and Streptococcus spp. bacteriophages (Fig. 3). The only F. nucleatum bacteriophage genome in the database is the prophage ΦFunu1, which is phylogenetically distant from FNU1, sharing very little genetic homology. ΦFunu1 branches off earlier in the phylogenetic tree and is most closely related to P1, a bacteriophage for Lactobacillus plantarum and the bacteriophage ΦEf11 for Enterococcus faecalis (Fig. 3).

Effect of bacteriophage FNU1 on F. nucleatum biofilm mass.
To evaluate the potential application of bacteriophage FNU1 in the treatment of gastro-intestinal biofilms, a biofilm model of F. nucleatum was generated as described above. The median [Inter-Quartile Range (IQR)] absorbance at OD 600 of the biofilm without bacteriophage treatment was 2.17 (1.81-2.21). This was significantly higher (p < 0.001) than that following FNU1 bacteriophage treatment for 24 h, where median (IQR) was 0.76 (0.71-0.89), or 35% of the untreated value (Fig. 4). The bacteriophage treated biofilm had a significantly higher absorbance (p < 0.001) compared to the negative control (no bacteria control): median (IQR) absorbance at OD 600 of the negative control was 0.14 (0.14-0.15) (Fig. 4). Subtracting absorbance readings for the negative control (0.14) from both the treated (0.76) and untreated (2.17)  www.nature.com/scientificreports www.nature.com/scientificreports/ biofilms results in untreated biofilms having an OD of 2.03 and treated biofilms having an OD of 0.62. Therefore, FNU1 phage treatment results in a 70% reduction in F. nucleatum biomass.
Viability of the F. nucleatum biofilm after treatment with bacteriophage FNU1. To evaluate the viability of F. nucleatum in the biofilms following treatment with bacteriophage FNU1, live/dead staining using SYBR ® gold and propidium iodide was applied to biofilms formed on microscope slides and imaged using confocal microscopy. The untreated biofilm had predominantly green fluorescent cells, indicating structurally intact membranes. The bacteriophage treated biofilm showed few cells, most of which were red/yellow, indicating structurally compromised membranes, and only very few cells with intact membranes (green) in clumps (Fig. 5).

Discussion
The novel bacteriophage FNU1 genome is over 130 kb in length and displays little homology to other known viral genomes. Because of this, it is difficult to describe any potential synteny between FNU1 and other bacteriophage genomes, although organisational similarity to some of the more abundant bacteriophages found in the human gut exists, where the structural and infection/packaging genes are in opposite orientation to each other 37 . The relatively large genome and structure of the virus, with a capsid of approximately 90 nm diameter and tail of over 300 nm in length, may have contributed to the fact that it was only able to produce clear discernible plaques when the concentration of agar it was grown on was reduced from 1.5% to 1%. The presence of several tRNAs in the FNU1 genome may indicate the requirement for additional translational mechanisms to complement those provided by the host cell. In its phylogeny, bacteriophage FNU1 clusters more closely to several bacteriophages against the oral pathogen S. mutans, and distantly from the only F. nucleatum bacteriophage in the database, the prophage ΦFunu1.
FNU1 has almost 8% of its ORFs devoted to putative defence against bacterial anti-bacteriophage systems, with several genes each coding for putative antirepressors, methylation genes to avoid restriction modification and toxin-antitoxin mechanisms that may prevent abortive infections. While some bacteriophages such as Vibrio cholerae ICP1 are known to carry CRISPR sequences which may contribute to their virulence 38 , the FNU1 genome had no such recognisable regions. All the F. nucleatum strains in the CRISPR database (http://crispr.i2bc. paris-saclay.fr/) 36 have one CRISPR with the number of spacers ranging from 3 to 52, (average approximately 25 spacers). Porphyromonas gingivalis, the major etiologic agent associated with chronic periodontitis, and for which no lytic bacteriophage has yet been isolated, has very extensive CRISPR immune mechanisms 39,40 . Each P. gingivalis strain in the database has more than one confirmed CRISPR locus (some with a maximum of five), with the total number of spacers in each bacterial strain ranging from 14 to 136 (http://crispr.i2bc.paris-saclay.fr/). This contrasts with C. acnes strains, where none have a confirmed CRISPR locus, (although some are denoted as having possible CRISPR regions based on the database's algorithms, filtering sequence length, matching repeats and amount of successive repeats) 36 , and against which there are over 80 reported bacteriophages 41 .
In this report, we demonstrate the capacity for FNU1 to disrupt established F. nucleatum biofilms. Our results show the capacity of FNU1 to effectively kill cells within a F. nucleatum biofilm, and although not as complex as polymicrobial biofilms shown to develop during periodontitis 8,29,42 , this work suggests that FNU1 has potential for application in more complex systems. F. nucleatum is one of the late colonisers in oral polymicrobial biofilms. Its capacity to co-aggregate intergenerically with representatives of all oral bacterial species 8 indicates it provides the necessary scaffolding for these communities to grow, develop and flourish synergistically. We have previously shown that diverse bacteriophages are able to be formulated into dosage forms such as lozenges and pastes, and subsequently released to kill underlying bacteria in-vitro 43 . Both of these dosage forms would provide very useful strategies for delivery of bacteriophage such as FNU1 for testing in the treatment of periodontitis and other www.nature.com/scientificreports www.nature.com/scientificreports/ diseases associated with F. nucleatum, as they allow slower "release" of bacteriophage into the oral cavity, and in the case of toothpastes, can be used to gently massage the bacteriophage onto the tooth and gum surface.
Finally, F. nucleatum has recently been described as an oncobacterium, associated with a range of human cancers 4 . The organism has a causal role in tumorigenesis 12 and also confers resistance to chemotherapy 13 . In their animal model, Bullman S. et al. demonstrated that treatment with metronidazole, an antibiotic that their F. nucleatum strains were sensitive to, reduced cancer cell proliferation and tumour growth. However, this approach of using antibiotics to kill Fusobacterium may be unsuitable clinically, as it has been previously demonstrated that perturbation of microbiota by antibiotics leads to reduced efficacy of chemoimmunotherapy for a range of cancers, including colorectal cancer 44,45 . On the other hand, the discovery of FNU1, with capacity to disrupt F. nucleatum biofilms, represents a potentially feasible means of targeted removal of this bacteria for microbiota manipulation in colorectal cancer management. In addition, while it has been suggested that bacteriophages in the gut virome may alter the microbiome such that F. nucleatum is able to overgrow and facilitate neoplasia in Figure 2. FNU1 functional genome map with putative structural genes (pink), putative DNA manipulation genes (green), putative regulatory genes (blue), putative lytic genes (red) and hypothetical genes (yellow) marked. Although majority of genes couldn't be assigned functionality, a pattern of clustering of related genes was observed. Genes in anticlockwise orientation were comprised mostly of putative DNA manipulation genes (between 65000 and 130000 bp and possibly involved in infection & packaging), as well as cluster of putative lysis genes (between 45000 and 55000 bp). www.nature.com/scientificreports www.nature.com/scientificreports/ colon cells 46 , bacteriophages such as FNU1 may assist in overcoming such dysbiosis. That bacteriophages can be successfully formulated into suppositories, as we have previously shown 32 , may assist in delivery.
In conclusion, this work describes the first full genome sequence and functional characterisation of a novel lytic bacteriophage against F. nucleatum, a bacterium associated with periodontitis as well as cancers of the GI tract such as colon cancer. FNU1 is unique in that it shares very little homology with other known bacteriophages.   www.nature.com/scientificreports www.nature.com/scientificreports/ Functionally, FNU1 is capable of breaking down F. nucleatum biofilms and lysing the bacterial cells composing the biofilm. This bacteriophage, then, is able to be tested in more complex oral biofilm assays and could potentially be tested in-vivo to assess capacity to treat periodontitis, as well as possibly assist in colon cancer treatment, following formulation in appropriate dosage forms.