Abacteriophage (/bækˈtɪrioʊfeɪdʒ/), also known informally as aphage (/ˈfeɪdʒ/), is avirus that infects and replicates withinbacteria. The term is derived from Ancient Greekφαγεῖν (phagein)'to devour' and bacteria. Bacteriophages are composed ofproteins thatencapsulate aDNA orRNAgenome, and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes (e.g.MS2) and as many as hundreds ofgenes. Phages replicate within the bacterium following the injection of their genome into itscytoplasm.
Bacteriophages are among the most common and diverse entities in thebiosphere.[2] Bacteriophages areubiquitous viruses, found wherever bacteria exist.[3] It is estimated there are more than 1031 bacteriophages on the planet, more than every living organism on Earth, including bacteria, combined.[4] Viruses are the most abundant biological entity in the water column of the world's oceans, and the second largest component of biomass afterprokaryotes,[5] where up to 9×108virions per millilitre have been found inmicrobial mats at the surface,[6] and up to 70% ofmarine bacteria may be infected by bacteriophages.[7]
Bacteriophages are known to interact with the immune system both indirectly via bacterial expression of phage-encoded proteins and directly by influencing innate immunity and bacterial clearance.[15] Phage–host interactions are becoming increasingly important areas of research.[16]
Bacteriophage P22, a podovirus by morphology due to its short, non-contractile tailBacteriophage T2, a myovirus due to its contractile tail
Bacterial viruses lack common ancestry and, for that reason, are classified in many unrelated taxa, listed hereafter:[17]
In the realmDuplodnaviria, the classCaudoviricetes contains bacterial viruses. Unlike the other taxa listed here,Caudoviricetes does not exclusively contain bacterial viruses;archaeal viruses are also included in the class.[18] Caudoviruses are also called tailed viruses or head-tail viruses, and they are often sorted into three types based on tail morphology: podoviruses (short tail), myoviruses (long, contractile tail), and siphoviruses (long, non-contractile tail).[19]
Félix d'Hérelle conducted the first clinical application of a bacteriophage
In 1896,Ernest Hanbury Hankin reported that something in the waters of theGanges andYamuna rivers inIndia had a marked antibacterial action againstcholera and it could pass through a very fine porcelainChamberland filter.[34] In 1915,BritishbacteriologistFrederick Twort, superintendent of the Brown Institution of London, discovered a small agent that infected and killed bacteria. He believed the agent must be one of the following:
a virus that grew on and destroyed the bacteria[35]
Twort's research was interrupted by the onset ofWorld War I, as well as a shortage of funding and the discoveries of antibiotics.
Independently,French-CanadianmicrobiologistFélix d'Hérelle, working at thePasteur Institute in Paris, announced on 3 September 1917 that he had discovered "an invisible, antagonistic microbe of thedysentery bacillus". For d'Hérelle, there was no question as to the nature of his discovery: "In a flash I had understood: what caused my clear spots was in fact an invisible microbe... a virus parasitic on bacteria."[36] D'Hérelle called the virus a bacteriophage, a bacterium-eater (from the Greekphagein, meaning "to devour"). He also recorded a dramatic account of a man suffering from dysentery who was restored to good health by the bacteriophages.[37] It was d'Hérelle who conducted much research into bacteriophages and introduced the concept ofphage therapy.[38] In 1919, in Paris, France, d'Hérelle conducted the first clinical application of a bacteriophage, with the first reported use in theUnited States being in 1922.[39]
George Eliava pioneered the use of phages in treating bacterial infections
Phages were discovered to be antibacterial agents and were used in the formerSoviet Republic ofGeorgia (pioneered there byGiorgi Eliava with help from the co-discoverer of bacteriophages,Félix d'Hérelle) during the 1920s and 1930s for treating bacterial infections.
D'Herelle "quickly learned that bacteriophages are found wherever bacteria thrive: in sewers, in rivers that catch waste runoff from pipes, and in the stools of convalescent patients."[41]
They had widespread use, including treatment of soldiers in theRed Army.[42] However, they were abandoned for general use in the West for several reasons:
Antibiotics were discovered and marketed widely. They were easier to make, store, and prescribe.
Medical trials of phages were carried out, but a basic lack of understanding of phages raised questions about the validity of these trials.[43]
Publication of research in the Soviet Union was mainly in theRussian orGeorgian languages and for many years was not followed internationally.
The Soviet technology was widely discouraged and in some cases illegal due to thered scare.
The use of phages has continued since the end of theCold War in Russia,[44] Georgia, and elsewhere in Central and Eastern Europe. The first regulated, randomized, double-blindclinical trial was reported in theJournal of Wound Care in June 2009, which evaluated the safety and efficacy of a bacteriophage cocktail to treat infected venous ulcers of the leg in human patients.[45] The FDA approved the study as a Phase I clinical trial. The study's results demonstrated the safety of therapeutic application of bacteriophages, but did not show efficacy. The authors explained that the use of certain chemicals that are part of standard wound care (e.g.lactoferrin or silver) may have interfered with bacteriophage viability.[45] Shortly after that, another controlled clinical trial in Western Europe (treatment of ear infections caused byPseudomonas aeruginosa) was reported in the journalClinical Otolaryngology in August 2009.[46] The study concludes that bacteriophage preparations were safe and effective for treatment of chronic ear infections in humans. Additionally, there have been numerous animal and other experimental clinical trials evaluating the efficacy of bacteriophages for various diseases, such as infected burns and wounds, and cystic fibrosis-associated lung infections, among others.[46] On the other hand, phages ofInoviridae have been shown to complicatebiofilms involved inpneumonia andcystic fibrosis and to shelter the bacteria from drugs meant to eradicate disease, thus promoting persistent infection.[47]
Meanwhile, bacteriophage researchers have been developing engineered viruses to overcomeantibiotic resistance, and engineering the phage genes responsible for coding enzymes that degrade the biofilm matrix, phage structural proteins, and the enzymes responsible forlysis of the bacterial cell wall.[6][7][8] There have been results showing that T4 phages that are small in size and short-tailed can be helpful in detectingE. coli in the human body.[48]
Therapeutic efficacy of a phage cocktail was evaluated in a mouse model with nasal infection of multi-drug-resistant (MDR)A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate compared to those untreated at seven days post-infection.[49]
In 2017, a 68-year-old diabetic patient with necrotizing pancreatitis complicated by a pseudocyst infected with MDRA. baumannii strains was being treated with a cocktail of Azithromycin, Rifampicin, and Colistin for 4 months without results and overall rapidly declining health.
Because discussion had begun of the clinical futility of further treatment, an Emergency Investigational New Drug (eIND) was filed as a last effort to at the very least gain valuable medical data from the situation, and approved, so he was subjected to phage therapy using a percutaneously (PC) injected cocktail containing nine different phages that had been identified as effective against the primary infection strain by rapid isolation and testing techniques (a process which took under a day). This proved effective for a very brief period, although the patient remained unresponsive and his health continued to worsen; soon isolates of a strain ofA. baumannii were being collected from drainage of the cyst that showed resistance to this cocktail, and a second cocktail which was tested to be effective against this new strain was added, this time by intravenous (IV) injection as it had become clear that the infection was more pervasive than originally thought.[50]
Once on the combination of the IV and PC therapy the patient's downward clinical trajectory reversed, and within two days he had awoken from his coma and become responsive. As his immune system began to function he had to be temporarily removed from the cocktail because his fever was spiking to over 104 °F (40 °C), but after two days the phage cocktails were re-introduced at levels he was able to tolerate. The original three-antibiotic cocktail was replaced by minocycline after the bacterial strain was found not to be resistant to this and he rapidly regained full lucidity, although he was not discharged from the hospital until roughly 145 days after phage therapy began. Towards the end of the therapy it was discovered that the bacteria had become resistant to both of the original phage cocktails, but they were continued because they seemed to be preventing minocycline resistance from developing in the bacterial samples collected so were having a useful synergistic effect.[50]
Phages have increasingly been used to safen food products and to forestallspoilage bacteria.[51] Since 2006, theUnited States Food and Drug Administration (FDA) andUnited States Department of Agriculture (USDA) have approved several bacteriophage products. LMP-102 (Intralytix) was approved for treating ready-to-eat (RTE) poultry and meat products. In that same year, the FDA approved LISTEX (developed and produced byMicreos) using bacteriophages on cheese to killListeria monocytogenes bacteria, in order to give themgenerally recognized as safe (GRAS) status.[52] In July 2007, the same bacteriophage were approved for use on all food products.[53] In 2011 USDA confirmed that LISTEX is a clean label processing aid and is included in USDA.[54] Research in the field of food safety is continuing to see if lytic phages are a viable option to control other food-borne pathogens in various food products.[55]
Switzerland authorized a phage for use in cheese production in 2016. The European Union has not yet (2025) authorized any.[56]
Bacteriophages, including those specific toEscherichia coli, have been employed as indicators of fecal contamination in water sources. Due to their shared structural and biological characteristics, coliphages can serve as proxies for viral fecal contamination and the presence of pathogenic viruses such as rotavirus, norovirus, and HAV. Research conducted on wastewater treatment systems has revealed significant disparities in the behavior of coliphages compared to fecal coliforms, demonstrating a distinct correlation with the recovery of pathogenic viruses at the treatment's conclusion. Establishing a secure discharge threshold, studies have determined that discharges below 3000 PFU/100 mL are considered safe in terms of limiting the release of pathogenic viruses.[57]
In 2011, the FDA cleared the first bacteriophage-based product for in vitro diagnostic use.[58] The KeyPath MRSA/MSSA Blood Culture Test uses a cocktail of bacteriophage to detectStaphylococcus aureus in positive blood cultures and determinemethicillin resistance or susceptibility. The test returns results in about five hours, compared to two to three days for standard microbial identification and susceptibility test methods. It was the first accelerated antibiotic-susceptibility test approved by the FDA.[59]
Government agencies in the West have for several years been looking toGeorgia and the formerSoviet Union for help with exploiting phages for counteracting bioweapons and toxins, such asanthrax andbotulism.[60] Developments are continuing among research groups in the U.S. Other uses include spray application in horticulture for protecting plants and vegetable produce from decay and the spread of bacterial disease. Other applications for bacteriophages are as biocides for environmental surfaces, e.g., in hospitals, and as preventative treatments for catheters and medical devices before use in clinical settings. The technology for phages to be applied to dry surfaces, e.g., uniforms, curtains, or even sutures for surgery now exists. Clinical trials reported inClinical Otolaryngology[46] show success in veterinary treatment of pet dogs withotitis.
Thesensing of phage-triggered ion cascades (SEPTIC) bacterium sensing and identification method uses the ion emission and its dynamics during phage infection and offers high specificity and speed for detection.[61]
Phage display is a different use of phages involving a library of phages with a variable peptide linked to a surface protein. Each phage genome encodes the variant of the protein displayed on its surface (hence the name), providing a link between the peptide variant and its encoding gene. Variant phages from the library may be selected through their binding affinity to an immobilized molecule (e.g., botulism toxin) to neutralize it. The bound, selected phages can be multiplied by reinfecting a susceptible bacterial strain, thus allowing them to retrieve the peptides encoded in them for further study.[62]
Phage proteins often have antimicrobial activity and may serve as leads forpeptidomimetics, i.e. drugs that mimic peptides.[63]Phage-ligand technology makes use of phage proteins for various applications, such as binding of bacteria and bacterial components (e.g.endotoxin) and lysis of bacteria.[64]
Phages can be used to combat bacterial infections such as blackleg. A line of phage-based products is licensed in the United States, and Georgia has long used agricultural phages. Elsewhere, research and pilot testing are still underway. This is notably the case in Switzerland, where research is being conducted by the Fribourg School of Engineering and Architecture in collaboration with theLausanne University Hospital (CHUV).[66]
Bacteriophages present in the environment can cause cheese to not ferment. In order to avoid this, mixed-strain starter cultures and culture rotation regimes can be used.[67]Genetic engineering of culture microbes – especiallyLactococcus lactis andStreptococcus thermophilus – have been studied for genetic analysis and modification to improvephage resistance. This has especially focused onplasmid andrecombinant chromosomal modifications.[68][51]
Some research has focused on the potential of bacteriophages as antimicrobial against foodborne pathogens and biofilm formation within the dairy industry. As the spread of antibiotic resistance is a main concern within the dairy industry, phages can serve as a promising alternative.[69]
The life cycle of bacteriophages tends to be either alytic cycle or alysogenic cycle. In addition, some phages display pseudolysogenic behaviors.[15]
Withlytic phages such as theT4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect.[15] Lytic phages are more suitable forphage therapy. Some lytic phages undergo a phenomenon known as lysis inhibition, where completed phage progeny will not immediately lyse out of the cell if extracellular phage concentrations are high. This mechanism is not identical to that of thetemperate phage going dormant and usually is temporary.[70]
In contrast, thelysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known astemperate phages. Their viral genome will integrate with host DNA and replicate along with it, relatively harmlessly, or may even become established as aplasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients, then, theendogenous phages (known asprophages) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is replicated in all offspring of the cell. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is thephage lambda ofE. coli.[71]
Sometimes prophages may provide benefits to the host bacterium while they are dormant by adding new functions to the bacterialgenome, in a phenomenon calledlysogenic conversion. Examples are the conversion of harmless strains ofCorynebacterium diphtheriae orVibrio cholerae by bacteriophages to highly virulent ones that causediphtheria orcholera, respectively.[72][73] Strategies to combat certain bacterial infections by targeting these toxin-encoding prophages have been proposed.[74]
In thiselectron micrograph of bacteriophages attached to a bacterial cell, the viruses are the size and shape of coliphage T1
Bacterial cells are protected by a cell wall ofpolysaccharides, which are important virulence factors protecting bacterial cells against both immune host defenses andantibiotics.[75]To enter a host cell, bacteriophages bind to specific receptors on the surface of bacteria, includinglipopolysaccharides,teichoic acids,proteins, or evenflagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn, determines the phage's host range. Polysaccharide-degrading enzymes are virion-associated proteins that enzymatically degrade the capsular outer layer of their hosts at the initial step of a tightly programmed phage infection process.[citation needed]Host growth conditions also influence the ability of the phage to attach and invade them.[76] As phage virions do not move independently, they must rely on random encounters with the correct receptors when in solution, such as blood, lymphatic circulation, irrigation, soil water, etc.[citation needed]
Myovirus bacteriophages use ahypodermic syringe-like motion to inject their genetic material into the cell. After contacting the appropriate receptor, the tail fibers flex to bring the base plate closer to the surface of the cell. This is known as reversible binding. Once attached completely, irreversible binding is initiated and the tail contracts, possibly with the help ofATP present in the tail,[7] injecting genetic material through the bacterial membrane.[77] The injection is accomplished through a sort of bending motion in the shaft by going to the side, contracting closer to the cell and pushing back up. Podoviruses lack an elongated tail sheath like that of a myovirus, so instead, they use their small, tooth-like tail fibers enzymatically to degrade a portion of the cell membrane before inserting their genetic material.
Within minutes, bacterialribosomes start translating viral mRNA into protein. For RNA-based phages,RNA replicase is synthesized early in the process. Proteins modify the bacterialRNA polymerase so it preferentially transcribes viral mRNA. The host's normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead. These products go on to become part of new virions within the cell, helper proteins that contribute to the assemblage of new virions, or proteins involved in celllysis. In 1972,Walter Fiers (University of Ghent,Belgium) was the first to establish the complete nucleotide sequence of a gene and in 1976, of the viral genome ofbacteriophage MS2.[78] SomedsDNA bacteriophages encode ribosomal proteins, which are thought to modulate protein translation during phage infection.[79]
In the case of theT4 phage, the construction of new virus particles involves the assistance of helper proteins that act catalytically during phagemorphogenesis.[80] The base plates are assembled first, with the tails being built upon them afterward. The head capsids, constructed separately, will spontaneously assemble with the tails. During assembly of thephage T4virion, the morphogenetic proteins encoded by the phagegenes interact with each other in a characteristic sequence. Maintaining an appropriate balance in the amounts of each of these proteins produced during viral infection appears to be critical for normal phage T4morphogenesis.[81] The DNA is packed efficiently within the heads.[82] The whole process takes about 15 minutes.
Early studies of bacteriophage T4 (1962–1964) provided an opportunity to gain understanding of virtually all of the genes that are essential for growth of the bacteriophage under laboratory conditions.[83][84] These studies were made possible by the availability of two classes ofconditional lethal mutants.[85] One class of such mutants was referred to asamber mutants.[85] The other class of conditional lethal mutants was referred to astemperature-sensitive mutants[86] Studies of these two classes of mutants led to considerable insight into the functions and interactions of the proteins employed in the machinery ofDNA replication,repair andrecombination, and on how viruses are assembled from protein and nucleic acid components (molecularmorphogenesis).
Phages may be released via cell lysis, by extrusion, or, in a few cases, by budding. Lysis, by tailed phages, is achieved by an enzyme calledendolysin, which attacks and breaks down the cell wallpeptidoglycan. An altogether different phage type, thefilamentous phage, makes the host cell continually secrete new virus particles. Released virions are described as free, and, unless defective, are capable of infecting a new bacterium. Budding is associated with certainMycoplasma phages. In contrast to virion release, phages displaying alysogenic cycle do not kill the host and instead become long-term residents asprophages.[87]
Research in 2017 revealed that the bacteriophage Φ3T makes a short viral protein that signals other bacteriophages to lie dormant instead of killing the host bacterium.[88]Arbitrium is the name given to this protein by the researchers who discovered it.[89][90]
Given the millions of different phages in the environment, phage genomes come in a variety of forms and sizes. RNA phages such asMS2 have the smallest genomes, with only a few kilobases. However, some DNA phages such asT4 may have large genomes with hundreds of genes; the size and shape of thecapsid varies along with the size of the genome.[91] The largest bacteriophage genomes reach a size of 735 kb.[92]
Schematic view of the 44 kbT7 phage genome. Each box is a gene. Numbers indicate genes (or rather open reading frames). The "early", "middle" (DNA replication), and "late" genes (virus structure), roughly represent the time course of gene expression.[93]
Bacteriophage genomes can be highlymosaic, i.e. the genome of many phage species appear to be composed of numerous individual modules. These modules may be found in other phage species in different arrangements.Mycobacteriophages, bacteriophages withmycobacterial hosts, have provided excellent examples of this mosaicism. In these mycobacteriophages, genetic assortment may be the result of repeated instances ofsite-specific recombination andillegitimate recombination (the result of phage genome acquisition of bacterial host genetic sequences).[94] Evolutionary mechanisms shaping the genomes of bacterial viruses vary between different families and depend upon the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the viral life cycle.[95]
Some marineroseobacter phages, also known asroseophages, containdeoxyuridine (dU) instead ofdeoxythymidine (dT) in their genomic DNA. There is some evidence that this unusual component is a mechanism to evade bacterial defense mechanisms such asrestriction endonucleases andCRISPR/Cas systems which evolved to recognize and cleave sequences within invading phages, thereby inactivating them. Other phages have long been known to use unusual nucleotides. In 1963, Takahashi and Marmur identified aBacillus phage that has dU substituting dT in its genome,[96] and in 1977, Kirnos et al. identified acyanophage containing 2-aminoadenine (Z) instead of adenine (A).[97]
The field ofsystems biology investigates the complexnetworks of interactions within an organism, usually using computational tools and modeling.[98] For example, a phage genome that enters into a bacterial host cell may express hundreds of phage proteins which will affect the expression of numerous host genes or the host'smetabolism. All of these complex interactions can be described and simulated in computer models.[98]
For instance, infection ofPseudomonas aeruginosa by the temperate phage PaP3 changed the expression of 38% (2160/5633) of its host's genes. Many of these effects are probably indirect, hence the challenge becomes to identify the direct interactions among bacteria and phage.[99]
Several attempts have been made to mapprotein–protein interactions among phage and their host. For instance, bacteriophage lambda was found to interact with its host,E. coli, by dozens of interactions. Again, the significance of many of these interactions remains unclear, but these studies suggest that there most likely are several key interactions and many indirect interactions whose role remains uncharacterized.[100]
Bacteriophages are a major threat to bacteria and prokaryotes have evolved numerous mechanisms to block infection (host resistance) or to block the replication of bacteriophages within host cells (anti-phage defense). Some examples include
Retrons and the anti-toxin system encoded by them.[101]
TheThoeris defense system is known to deploy a unique strategy for bacterial antiphage resistance viaNAD+ degradation.[102]
TheHailong anti-phage defense system consists of a two-gene operon encoding a transmembrane ion channel effector (HalA) and anucleotidyltransferase (NTase, HalB). An infecting phage can activate HalA, triggeringmembrane depolarization and thus cell death. Although the infected cell is likely going to die, this protects the bacterial population from further spread.[103]
Temperate phages are bacteriophages that integrate their genetic material into the host as extrachromosomal episomes or as aprophage during alysogenic cycle.[104][105][106] Some temperate phages can confer fitness advantages to their host in numerous ways, including giving antibiotic resistance through the transfer or introduction of antibiotic resistance genes (ARGs),[105][107] protecting hosts from phagocytosis,[108][109] protecting hosts from secondary infection through superinfection exclusion,[110][111][112] enhancing host pathogenicity,[104][113] or enhancing bacterial metabolism or growth.[114][115][116][117] Bacteriophage–host symbiosis may benefit bacteria by providing selective advantages while passively replicating the phage genome.[118]
Metagenomics has allowed the in-water detection of bacteriophages that was not possible previously.[119]
Also, bacteriophages have been used inhydrological tracing and modelling inriver systems, especially where surface water andgroundwater interactions occur. The use of phages is preferred to the more conventionaldye marker because they are significantly less absorbed when passing through ground waters and they are readily detected at very low concentrations.[120] Non-polluted water may contain approximately 2×108 bacteriophages per ml.[121]
Bacteriophages are thought to contribute extensively tohorizontal gene transfer in natural environments, principally viatransduction, but also viatransformation.[122] Metagenomics-based studies also have revealed thatviromes from a variety of environments harbor antibiotic-resistance genes, including those that could confermultidrug resistance.[123]
Recent findings have mapped the complex and intertwined arsenal of anti-phage defense tools in environmental bacteria.[124]
Although phages do not infect humans, there are countless phage particles in the human body, given the extensivehuman microbiome. One's phage population has been called the humanphageome, including the "healthy gut phageome" (HGP) and the "diseased human phageome" (DHP).[125] The active phageome of a healthy human (i.e., actively replicating as opposed to nonreplicating, integratedprophage) has been estimated to comprise dozens to thousands of different viruses.[126]There is evidence that bacteriophages and bacteria interact in thehuman gut microbiome both antagonistically and beneficially.[127]
Preliminary studies have indicated that common bacteriophages are found in 62% of healthy individuals on average, while their prevalence was reduced by 42% and 54% on average in patients withulcerative colitis (UC) andCrohn's disease (CD).[125] Abundance of phages may also decline in the elderly.[127]
The most common phages in the human intestine, found worldwide, arecrAssphages. CrAssphages are transmitted from mother to child soon after birth, and there is some evidence suggesting that they may be transmitted locally. Each person develops their own unique crAssphage clusters. CrAss-like phages also may be present inprimates besides humans.[127]
Among the countless phages, only a few have been studied in detail, including some historically important phage that were discovered in the early days of microbial genetics. These, especially the T-phage, helped to discover important principles of gene structure and function.
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