The human oral phageome

Oral bacteriophages (or phages), especially periodontal ones, constitute a growing area of interest, but research on oral phages is still in its infancy. Phages are bacterial viruses that may persist as intracellular parasitic deoxyribonucleic acid (DNA) or use bacterial metabolism to replicate and cause bacterial lysis. The microbiomes of saliva, oral mucosa, and dental plaque contain active phage virions, bacterial lysogens (ie, carrying dormant prophages), and bacterial strains containing short fragments of phage DNA. In excess of 2000 oral phages have been confirmed or predicted to infect species of the phyla Actinobacteria (>300 phages), Bacteroidetes (>300 phages), Firmicutes (>1000 phages), Fusobacteria (>200 phages), and Proteobacteria (>700 phages) and three additional phyla (few phages only). This article assesses the current knowledge of the diversity of the oral phage population and the mechanisms by which phages may impact the ecology of oral biofilms. The potential use of phage-based therapy to control major periodontal pathogens is also discussed.

the phage nucleic acid is subsequently injected into the bacterial cell.
The intracellular phage uses the bacterial machinery for genomic replication, and, with a lytic infection, the phage genome is packed into phage particles that are released by a phage-mediated bacterial lysis. Instead of lysing bacterial cells, phages can persist in a latency/ chronic prophage stage by integrating into the bacterial genome or by forming an extrachromosomal, independently replicating plasmid-like episome. 35 Prophages may persist in the lysogenic state or initiate lysis, depending on the energetic/physiologic status of the bacterial host. 36,37 Host starvation (due to lack of energy for phage replication) or nutrient-rich condition (prophage protects expending lysogen from phage attack) 35,38 favor a prophage state. Unfavorable F I G U R E 1 A, Selected phage morphotypes. ds, double-stranded; ss, single-stranded; C, circular; L, linear. DNA, deoxyribonucleic acid. B, Virion morphologies of oral phages. Siphovirus: phages FNU1, ΦAPCM01, transposable phage; Myovirus: Aaphi23-like phage; Podovirus: phage SOCP. Bar represents 100 nm. The electron micrographs (with minor formatting modifications) are licensed but permit reproduction 4,[17][18][19] F I G U R E 2 The replication cycle of a phage. A, Plaques formed by phages on a lawn of the host bacteria. B, Lytic and lysogenic pathways of phage replication. Lytic phages follow a lytic cycle; lysogenic phages can follow either a lytic or lysogenic cycle. DNA, deoxyribonucleic acid host conditions (eg, suboptimal temperature or pH, ultraviolet radiation, presence of DNA-targeting antibiotics, reactive oxygen species, foreign DNA) can trigger a latent phage to enter the lytic cycle and propagate before the death of the bacterial host cell. Phages can also guide lysis-lysogeny decisions by measuring a high abundance of host cells (via quorum sensing) or via phage-encoded arbitrium, an interphage communication peptide. 37,39 Phages are complex macromolecules that exhibit both virulence factors and defense mechanisms against counterattacks by the bacterial host (Figure 3). Recent studies have provided insights into the population genetics of phages, phage defensive mechanisms, and the interplay between phages and bacteria. [40][41][42][43][44][45][46][47][48] The bacterial CRISPR-CRISPR associated (Cas) adaptive immune system aims to inhibit phage infections. Fragments of phage DNA become incorporated into CRISPR memory arrays (protospacers) on the bacterial genome, and RNA probes (spacers) transcribed from these arrays can identify the complementary invading phage DNA and guide antiphage bacterial nucleases. 49 Bacteria-encoded restriction enzymes can cleave unmodified phage DNA but not the methylated bacterial DNA. In response, phages may mutate in the genomic fragments targeted by the bacterial RNA probes, form a nucleus-like compartment barrier, or elaborate proteins inhibitory to the CRISPR-Cas system. [50][51][52][53] Phages may also seek protection by masking phage restriction sites with defensive proteins, by mutation/modification of the restriction sites, by altering the spatial conformation of the bacterial nucleases, or by removing essential enzymatic cofactors. 54 Mutation in bacterial receptors for phage recognition or changes in the expression of bacterial surface structures constitute additional mechanisms of resistance toward phage infection, but phages may attach to another bacterial site or the phage receptor-binding proteins may mutate to fit the mutated bacterial receptor. Bacterial capsule or extracellular matrix, characteristics of sessile growth of biofilm cells, may also hamper the access of phages to receptors on the bacterial surfaces, 55 but some phages can drill through such barriers by means of polymer-degrading hydrolases. 56 Bacteria may release extracellular membrane vesicles that can intercept phages or may block entrance of new phage DNA by superinfection exclusion systems of already resident prophages. 57 Bacterial "defense islands" can provide several additional lines of active defense against phage invasion. 58 Bacteria can also undergo abortive infection to limit phage replication within a bacterial population, which again, however, phages may be able to overcome. The ability of phages to kill bacteria and the bacterial inherent defenses against phage infection and the rapid adaptation of new phage-invading mechanisms and of bacterial countermeasures may be important determinants of the oral/periodontal phageome. A phage infection is traditionally identified by counting areas of clearing (known as plaques) on a lawn of the bacterial host and express the titer as "plaque-forming units" (PFU). Plaques result from successive infections and phage bursts. PFU can be determined by the "spot-titer method" (a phage sample is spotted on a small area where a future bacterial lawn will be formed) or by the "overlaytiter method" (the phage sample is mixed with a bacterial suspension together with an overlaying semisolid layer that allows the formation of plaques within the entire bacterial lawn). Phage sample can also be added to a liquid culture of bacteria, and the phage infection can be estimated as a loss of culture turbidity.

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Phage enrichment and expansion Filtration, polyethylene glycerol precipitation, ultracentrifugation, or successive co-incubation cycles with a bacterial propagator strain can be used to concentrate or amplify phages. Phage samples spiked with phage standards help to control the impact of phage enrichment on the phageome. Antibody-based "reverse genomics" technique may be used to enrich specific phages. [72][73][74][75][76] Phage production ("rebooting") Functional virions can be "produced" using cell-deficient propagator cells transformed with phage genomes that are engineered/rebuilt in vitro. 77,78 Prophage detection and induction Phage preservation Expended phage clones are usually stored in stabilizing buffer at 4°C or −80°C. Functional prophages can be stored in bacterial stocks. As phage titers decline over time, virion stability ought to be monitored during long-term storage of phages. 81 Efficiency of plating Plating efficiency is expressed as the phage titer for a specific bacterial strain compared with the maximum titer for the reference phage/host combination. Low efficiency of plating indicates the phage is not fully virulent or productive, or the phage infection is partly controlled by defensive mechanism(s) of the bacterial host. 82 One-step growth curve One-step growth curve can characterize the growth of phages in selected host strains, in terms of eclipse period, latent period, and burst size. Bacterial cells with phages and phage titter (PFU per infected cell) are monitored over time with or without added chloroform, which releases phage particles from intact cells and allows for a distinction between intracellularly functional phages and phages released during cell lysis. Number of infected cells is determined as a decrease in virion numbers due to adsorption to the host. Eclipse period is the time between phage adsorption and production of the first virions. Latent period is the time between adsorption and progeny release. Burst size is defined as the number of progeny phages released during lysis of a single bacterial cell. Low phage multiplicity rate ensures that only one phage infects each target cell. Physical and chemical parameters strongly shape phage-host interaction, and in vitro experiments may not fully mimic the physiological situation. Biofilm, organoid, animal, and ex vivo models may more properly reflect phage-host interaction. 83,84 Host range testing and host prediction A phage host range is defined by the bacterial strains being lysed by the phage. High phage specificity is advantageous for phage therapy because it leaves the commensal flora unaffected. However, a phage that is effective against most strains of a targeted species can be broadly applied, without extensive prior activity testing. Knowledge of phage specificity is also key to understand the effect of phages on microbiome dynamics. Spot-titer or plaque testing is used to determine the host range for a given phage isolate. Host specificity for metagenomically assembled phages (ie, genome sequences reconstructed from reads obtained by high-throughput sequencing of environmental phage DNA) can be predicted based on high DNA similarity between the phage isolate and a known host range. Degree of phage DNA sequence matching bacterial CRISPR spacers can also be used to indicate host range. Phage-host coevolution assessed by guanine-cytosine content, k-mer frequency, frequency of DNA uptake signal sequences, and codon usage may also reveal the most likely host. 4,30,85 Transmission electron microscopy Transmission electron microscopy of negatively stained phage particles is the classic method to study size and morphology of phage particles. Phages are concentrated by ultracentrifugation, adsorbed onto a carbon film, washed, stained with uranyl acetate or phosphotungstate, attached to a copper specimen grid, and examined. 86

| THE OR AL PHAG EOME
Metagenomic profiling of oral biofilms has led to an increased understanding of the potential role of phages in the development, regulation, and treatment of pathogenic microbiomes of the periodontium and other oral sites. 1,2,4,29 However, metagenomic profiling has yielded many new phage genomic sequences that remain to be characterized, and most phages that were phenotyped in the past lack genetic information. 3 The phages reviewed here infect six bacterial Mutagenesis of phages Phage genetics is typically employed in functional phage gene studies. Mutagenesis targets specific phage traits, such as antimicrobial performance or phage range, and the mutants obtained can be evaluated for phenotypic changes. Ultraviolet irradiation or hydroxylamine treatment of virions (substitutes cytosine with thymine) can induce mutations. 104 Genetic engineering of phages Genetic modification of phages aims to improve phage antimicrobial properties, change phage host range, reduce phage immunogenicity, target phages against bacteria harboring specific sequence signatures, detect bacteria, deliver drugs, or create new biomolecules. Phage engineering techniques include homologues recombination, recombination of electroporated DNA, in vivo recombination, CRISPR-Cas-mediated genome engineering, rebuilding/refactoring phage genomes in vitro, wholegenome synthesis, and assembly from synthetic oligonucleotides.

105,106
Heterologous expression of phage enzymes Phage lysins (cell-wall hydrolases) constitute intriguing antibiotic alternatives. Phage lysins have been developed against gram-positive and, more recently, against gram-negative pathogenic bacteria and have been engineered to improve efficiency. 107,108 Antiphage sera Polyclonal antibodies against phage virions are used in downstream immunochemical and biological studies of patient sera. The antiphage activity in sera can be assessed by a neutralization test that estimates the rate of phage inactivation at various incubation times. (Spirochaetia). 114 The oral phyla Synergistetes and Saccharibacteria (formerly known as TM7) have each been associated with a single phage. The oral cavity also hosts phages of pathogenic invaders (eg, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis) and Lactobacillus phages that can be found in environments other than the oral cavity. [115][116][117][118][119][120][121] No phages of the phyla Absconditabacteria (formerly known as SR1), Chlamydiae, or Chloroflexi have been identified in the oral cavity. Figure 4 depicts the distribution of oral phages across host taxons. Figure 5 shows the oral phage diversity at the bacterial phylum level.

| Phages infecting Actinobacteria
Actinomyces and Rothia (both of the order Actinomycetales) and Corynebacterium (order Corynebacteriales) are diverse and ecologically relevant genera of the Actinobacteriia class. 114 Actinomyces spp are primary colonizers of dental plaque and are typically associated with oral health, but they can also cause oral morbidity (eg, caries, endodontic, periodontal and peri-implant diseases) and systemic diseases (eg, orocervicofacial, thoracic, and abdominal/pelvic actinomycosis). 122,123 Newly described Actinomyces species are emerging as opportunistic pathogens in several body sites. 122 Rothia spp can inhabit cariogenic biofilms and produce extracellular levan from sucrose. 124 Corynebacterium spp form long filamentous cells that may provide a scaffold for oral biofilms. 125 Species of the Coriobacteriia class are less studied, but Atopobium spp and Olsenella uli have been linked with periodontal disease. 123,126 The Integrated Microbial Genome/Virus (IMG/VR) database suggests that 241 phages exhibiting high-quality draft genomes have an oral origin and are predicted to infect hosts of the phylum Actinobacteria. 29 However, the IMG/VR database occasionally contains concatenation artefacts, and huge phages in particular need to be validated experimentally. 34 The National Center for Biotechnology Information (NCBI) database describes genomes of two Actinomyces phage isolates, Av-1 and xhp1 ( Figure 4). 127    has been sequenced and reported to effectively kill biofilm cells of Fusobacterium nucleatum. 19 The ecologic significance (a core oral bacterium) and medical relevance (role in colorectal cancer) of F. nucleatum should encourage research on Fusobacterium phages.

| Phages infecting Proteobacteria
Virtually all Proteobacteria are aerobic/facultative organisms. Oral Deltaproteobacteria are less studied, but one member of the class (Desulfobulbus HMT-041) has been related to periodontal disease. 123 The IMG/VR database includes 254 phages with high-quality draft genomes that have an oral origin and which are predicted to infect hosts of the Proteobacteria phylum ( Figure 4)

| Phages infecting Spirochaetes
Treponema denticola and related species represent the Spirochaetes phylum and are implicated in periodontal disease. The IMG/VR database includes two oral phages with high-quality draft genomes that are predicted to infect Treponema socranskii. A single Treponema phage has been isolated, but its functionality is unknown. 155

Anaerolineae [G-1] bacterium HMT-439 of the Chloroflexi phylum
and Fretibacterium spp of the Synergistetes phylum have been associated with periodontal disease. 123 The IMG/VR database includes two oral phages (including one jumbo phage) with high-quality draft genomes that are predicted to infect Fretibacterium fastidiosum and an unnamed species of the Saccharibacteria phylum.

| Phage diversity patterns across oral niches and time
Oral bacteria tend to be site specialists, 156  The oral microbiomes and related phageomes may be affected by disease, aging, and different food intake. A 60-day study of salivary phage and bacterial communities found most phage populations to be stable and highly personalized. 158 An earlier study by the same research group described transient salivary phage populations but may have overestimated the number of phage genotypes. 5 A 30day study of tongue phages showed a generally stable phage community, although some major phage phylotypes reappeared in cycles that appeared to be unrelated to diet and oral hygiene efforts. 91 The apparent stability of major oral phage groups ensures a continuous phage effect on oral microorganisms.

| Oral phage populations in health and disease
The dental phage population is probably more characteristic of periodontal disease and dental caries than the salivary phage population is, which receives contributions from several different oral surfaces.
However, information on the periodontal phageome is sparse. 159 Subgingival myoviruses (ie, phages representing the Myoviridae fam- Aggregatibacter phages were associated preferentially with advanced periodontitis. 168,169 Finally, oral lysogenic bacteria may be at increased risk of causing systemic infections; for example, an A. actinomycetemcomitans producer strain of Aaphi23-like phages was recovered from an actinomycotic lung lesion. 4 The Streptococcus SM1 prophage of S. mitis encodes a platelet-binding factor that can promote platelet activation and aggregation with risk of causing endocarditis. 170173 A streptococcal satellite prophage carrying the vapE gene was related to S. pneumoniae sepsis in a murine model. 143 Transmission of oral phages can take place between mother and children, family members, couples with intimate contact and people in the same household. 91,179,181 A study of couples demonstrated common features in the tongue phage population. 91 People who are sharing or have shared households in the past also harbor many phages of same sequences. 179 Neither a genetic relationship nor a spousal relationship seem to be required to share oral phages, and even relatively brief contact may lead to transmission. 180 However, transmission of oral phages between mother and infant have been rarely observed, perhaps due to methodological study limitations. 181

| Phage interactions within microbiomes
Phage-induced bacterial lysis may remove pathogenic bacteria and create a niche for previously suppressed low-pathogenic species or may release bacterial components that can contribute to the formation of a protective biofilm matrix. Phages can quickly adapt to environmental changes and apply strong pressure on bacterial populations, especially on bacterial linages that show high fitness and abundance. In in vitro and animal studies, phage-induced lysis of host species has resulted in major changes in the abundance and diversity of bacterial communities. 186,189 As bacteria must adapt to the phage attack to avoid decimation, 190 an oscillatory dynamics may develop between phage and host. 191 Lysogeny protects the host from new phage infection and phage predation. 192 Bacteria may also avoid phage-mediated predation by surface remodeling but face the risk of losing receptors for important biofilm bacterial taxa. Actinomyces strains exposed to phages yielded two different receptor mutants that both lost the ability to coaggregate with streptococci. 193 Phage infection has reduced the expression of Enterococcus genes involved in interspecies interactions. 194 However, phage-induced transfer of antibiotic resistance genes among A. actinomycetemcomitans strains may increase the adaptability of oral biofilms, as implied in an in vitro study. 195 A study on murine fecal phage populations found that antibiotic treatment led to an enrichment of phage-encoded resistance genes and a microbiota with increased resistance. 196 A longterm antibiotic study in humans detected an expansion of genes involved in resistance to numerous antibiotics in fecal phageomes but not in oral phageomes. 197 However, phages can also interfere with transformation and conjugation, and thus negatively affect gene transfer. 44,198,199 Aquatic environmental studies provide another perspective

| PHAG E THER APY AND PHAG E-BA S ED ANTIMICROB IAL S
The clinical use of phages to combat bacterial infections is gain- Pseudomonas phage to be effective and safe against chronic otitis, but recurring infections did occur. 222 A randomized controlled trial on the efficacy and safety of Pseudomonas phages as topical therapy of burn wounds showed phages to be less effective in reducing bacterial loads than the standard of care therapy using sulfadiazine silver. 223 However, the study used low doses of Pseudomonas phages due to phage stability issues, lacked preliminary phagograms (sensitivity testing), recruited a small number of patients, and assessed the therapeutic outcome by semiquantitative methods. 227 Phage safety was confirmed in a study of 13 patients with severe S. aureus infections. 224 Oral coliphages were also found to be safe for treatment of diarrhea in children but were unable to improve outcome, perhaps due to insufficient phage coverage and low phage titers. 225 Phage therapy targeting intestinal E. faecalis in mice was able to attenuate alcoholic liver disease, but a comparable trial has yet to be performed in humans. 230 A recent report describes a 15-year-old patient with cystic fibrosis and bilateral lung transplantation who experienced a life-threatening disseminating infection by antibiotic-resistant Mycobacterium abscessus, which was successfully treated with a genetically engineered threephage cocktail. 226,227 However, the potential release of genetically engineered phages into the environment raises some concerns. 228 Staphylocccocus phages in intracellular compartments of osteoblasts were inactive but were killing bacteria after being released into extracellular compartments. 229 Antimicrobial properties of oral phages and phage enzymes have been studied recently. 3 Fusobacterium phage FNU1 was able to disrupt experimental F. nucleatum biofilms, as assessed by crystal violet staining and confocal microscopy. 19 Haemophilus phiKZ-like oral phages may exert therapeutic activity against pathogens of the Pasteurellacae family. 4,29 The multiple antibiotic-resistant E. faecalis is an organism of major concern in endodontics. A genetically engineered Enterococcus phage ϕEf11-derivative lacked lysogenicity but displayed broadened host range and was able to reduce the level of E. faecalis in human dentin specimens by up to 100-fold. 230 Numerous Streptococcus phages have been identified that eventually may be employed in dental caries prevention and treatment. 3 Phages engineered to express the S. mutans-specific antimicrobial peptide C16G2220 may show anticaries potential. 237 The ClyR lysin was active against cariogenic S. mutans and Streptococcus sobrinus without compromising indigenous S. sanguinis, S. oralis, and S. salivarius. 238,239 High-priority topics in oral phage research also involve identifying phages killing plaque-forming oral Actinobacteria, periodontopathogens, and opportunistic pathogens of nonoral origin.
Oral phage research would benefit significantly from having better-characterized propagator host strains and phage isolates. To sum up, dentistry is at the very beginning of understanding oral phages, and the coming years will undoubtedly uncover some of their secrets. Insights into the phage-bacterium-human cell molecular interaction seem essential to decipher the role of oral phages in health and disease and for producing "intelligent" phages that would fit specific therapeutic purposes.