Introduction

There are billions of viruses on the Earth. An estimation of their number reaches an astronomical figure of about 1031 viruses, which is much more than the number of stars in the universe (Weitz and Wilhelm 2013). Most of the viruses play a significant role in the global biogeochemical cycles and thrive by infecting microbes like bacteria, archaea, and microeukaryotes (Suttle 2007). Because of their astounding numbers, and intimate relationship with different microbes, they control both host populations and ecosystem functions (Weitz and Wilhelm 2012). Among the total viral population, bacteriophages, popularly known as phages (Greek “phagein” meaning “to eat”), are the group of viruses that infect and devour bacteria. This potential antibacterial property of phage is unique (Jamalludeen et al. 2009).

Phages are considered to be the most diverse and abundant entity on earth and are thought to exist in every ecosystem (Maranger and Bird 1995; Hendrix 2002; Hanlon 2007; Le Romancer et al. 2007) ranging from extremely hot environments like hot springs, the Sahara, to extremely cold environments like polar inland waters (Breitbart et al. 2004; Glud and Middleboe 2004; Prigent et al. 2005; Suttle 2005; Säwström et al. 2008; Lin et al. 2010). Sea water is among the hugely diverse and most dense natural environments for phages and other viruses; for example, the surface seawater has a concentration of approximately 10 million viruses per milliliter (Breitbart 2012). Similarly, forest floors and agricultural soils usually harbor a phage count of approximately108–109 per gram of soil (dry mass; Williamson et al. 2003; Williamson et al. 2005). It is also reported that to carry out important ecological functions, phages express a variety of auxiliary metabolic genes (Breitbart 2012; Weitz and Wilhelm 2013).

A single phage particle can hunt for a specific bacterium species or a subset of the same species. After infecting a bacterium, phages replicate inside it. Once a bacterial cell is infected, phages multiply exponentially using the cellular machinery of the bacteria including protein-synthesizing and energy-generating systems. However, the method of propagation may either be lytic or lysogenic in nature. The lytic phages cause lysis of the host bacterial cells to release progeny viruses (Young et al. 2000). On the other hand, lysogenic or the temperate phages integrate their nucleic acid (genome) within the host bacterial cell and replicate along with the host, conferring new properties to the host bacteria (Brock et al. 1988). Phages are classified on the basis of certain criteria including its host specificity, morphology, nucleic acid type, mode of infection, morphogenesis, phylogeny, serology, sensitivity to physical and chemical agents, and its environment (Abeles et al. 1984). All bacterial genera, including the cyanobacteria, archaebacterial, and mycoplasmas, are known to be vulnerable to a plethora of phages.

Brief history of bacteriophage

British bacteriologist Ernest Hanbury Hankin, in 1896, first reported antibacterial activity in the waters of the river Ganga and the river Yamuna in India (Hankin 1896: http://icmr.nic.in/buapril02.pdf). The then unidentified antibacterial agent was also noted as filterable and heat labile; it was found to limit the spread of cholera epidemic (Hankin 1896; Van Helvoort 1992; Sulakvelidze et al. 2001). However, how the implications of Hankin’s findings related to the existence of bacteriophages remain dubious in the scientific society.

In 1915, Frederick William Twort FRS (October 22, 1877–March 20, 1950), an English bacteriologist, superintendent of the Brown Institute for Animals and professor of bacteriology at the University of London, while growing viruses in a laboratory condition, found zones of clearance in the form of “transmissible glassy transformation” with micrococcus bacteria. He also concluded that this agent multiplied itself in the process of killing the bacteria (Twort 1915, 1922, 1949).

On the other hand, Félix d’Herelle (April 25, 1873–February 22, 1949), a French-Canadian microbiologist working at the Pasteur Institute in Paris, observed the bacteriophage phenomenon (in the year 1917) on severe hemorrhagic dysentery among French troops stationed at Maisons-Laffitte. He had observed the same phenomenon in 1910 when he was studying microbiology due to an epizootic locust infection in Mexico. d’Herelle prepared bacteria-free filtrates from fecal samples of patients and incubated the filtrate with the bacterium Shigella, isolated from those patients. His primary goal was to develop a vaccine against bacterial dysentery. For observing the growth of the bacteria, an aliquot of the filtrate-bacteria mixture was spread on agar medium and incubated. d’Herelle found the appearance of small, clear zones, which he primarily termed taches, then taches vierges, and later, plaques. This finding was presented in September 1917 and subsequently published (d’Herelle 1917, 1930; Summers 1999; Sulakvelidze et al. 2001). d’Herelle also proposed that the phenomenon was caused by a bacteria parasitizing virus and named “bacteriophage”—phages implying to “eat” or “devour” bacteria. As per the recollection of d’Herelle, the name “bacteriophage” was decided together with his wife Marie, and the word came into existence on 18 October 1916—the day before their youngest daughter’s birthday (d’Herelle 1930; Summers 1999; Sulakvelidze et al. 2001). Thus, the credit of discovery of bacteriophages goes independently to two scientists: F.W. Twort and Félix d’Herelle. Primarily, the scientific society first called it “Twort-d’Herelle phenomenon,” and later, the “bacteriophage phenomenon” (d’Herelle 1949; Duckworth 1976; Sulakvelidze et al. 2001).

Structure of bacteriophage

Length of phages varies widely and usually ranges from 24 to 200 nm (Mayer 2016). T4 phages are among the largest phages that are approximately 200 nm in length and 80–100 nm wide. Phages having an icosahedral shape or a shape with 20 sides and filaments are also common (http://www.microrao.com/micronotes/bacteriophage.pdf). After the first commercial production of transmission electron microscope (called “Hypermicroscope”) by Siemens and Halske Company (Germany) in 1939, photomicrography of bacteriophages came into the existence. Ruska (1940) and Pfankuch and Kausche (1940), both working in the same company, reported separately in the same journal and on same date showing the first pictures of bacteriophages to the scientific world (Ruska 1940; Pfankuch and Kausche 1940; Ackermann, 2011a, b). On the other hand, in North America, Prebus and Hillier of University of Toronto constructed separately the first electron microscope in 1938, and subsequently, commercialized the same after years of research by Hiller at Radio Corporation of America (RCA, Princeton, NJ; Prebus and Hillier 1939; Haguenau et al. 2003; Ackermann 2011a, b). Luria and Anderson, in 1942, reported different unstained coli and staphylococcal phage particles, which were renamed later as T2 (T-even type; Luria and Anderson 1942; Ackermann 2011a, b). Despite continental difference, huge political unrest (because of World War II) and extreme limitations on scientific exchange, interestingly, both the group of scientists were aware of other progress in the development of this extremely important scientific instrument (Pfankuch and Kausche 1940; Ackermann 2011a, b). Later on, using the “negative contrast” technique in an electron microscope, Brenner and his team first reported a clearer image of bacteriophages in 1959 (Brenner and Horne 1959; Brenner et al. 1959). In subsequent years, Bradley and Kay (1960) and Bradley (1967) elaborated fine structures of phages. The basic features of a typical bacteriophage, in this case the T4 bacteriophage, include a “head” or capsid and a “tail” (Fig. 1).

Fig. 1
figure 1

Schematic representation of a typical structure of a phage emphasizing tail region and contractile ability to insert its DNA into host bacterial cell by penetrating cell wall

Full size image

The head or capsid structure, regardless of its size or shape, is a congregation of one or more protein subunits called protomers. The morphological subunit of a capsid is a capsomere which protects the viral nucleic acid (genome). The tail is a hollow tube through which the nucleic acid passes when the bacteriophage infects a bacterial host cell. Some phages do not possess a tail. The T4 phage has additional structures, namely the base plate and tail fibers attached to the tail that aid the phage in attaching itself to a bacterial cell (Leiman et al. 2003).

Taxonomy of phages

As mentioned earlier, there are billions and billions of phages. Deciphering taxonomic characterization is a challenging task, especially for such nano-sized phage particles. In 1967, phages were first classified by Bradley and were subsequently approved by the International Committee on Taxonomy of Viruses (ICTV) and total 111 phages were listed in classification (Wildy 1971). Bradley’s classification projected six basic morphological types of tailed phages that further, categorized on the basis of morphotypes (contractile tails, long and noncontractile tails, and short tails), small isometric ssDNA viruses, filamentous phages, and small ssRNA phages. The regulating body for the viral taxonomic system (ICTV) characterizes phage particles taking into consideration numerous parameters like host range, physical characteristics (such as structure, capsid size, and shape), type of genomic material (single or double-stranded DNA or RNA), genome size, and resistance to organic solvents (Murphy et al. 1995). More than 96 % of phages are tailed and carries dsDNA as genetic material; however, they may vary in shape like cubic, filamentous, and pleomorphic (Ackermann 2011a, b). Polyphasic taxonomy was revised and emphasis was given on genomic relationship (Thompson et al. 2015). Earlier in 2008, only 18 genera and 36 species were listed among three caudoviral families, Myoviridae, Podoviridae, and Siphoviridae. Subsequently, the order caudovirales expanded and an updated and detailed bacteriophage classification was presented in ICTV Release 2015 (http://www.ictvonline.org/virusTaxonomy.asp; Krupovic et al. 2016). Some alterations have also been suggested by committee, like replacement of word “phage” with “virus” in prokaryotic virus taxon names, omission of infix “like” from prokaryotic virus genus names, discouragement of use of “Phi” and other Greek letters in prokaryotic virus genera, exclusion of hyphens, and encouragement to use of host genus name in replacement of taxon names is being encouraged to avoid confusion related to phage action on specific bacterial host (Krupovic et al. 2016; Pietilä et al. 2016). The overview of the various bacteriophage families has been included in Table 1 compiled from (ICTV 2015 Release; EC 47, London, UK, July 2015 Email ratification 2016 (MSL #30) for ready reference to the readers.

Table 1 The overview of the various phage families compiled from International Committee on Taxonomy of Viruses (ICTV 2015 Release; http://www.ictvonline.org/virusTaxonomy.asp)
Full size table

Mechanism of proliferation

Specific receptors (like lipopolysaccharides, teichoic acids, proteins, and flagella) on the surface of the host bacteria are required for the phage to infect the bacteria. Due to this specificity of the receptor present on the bacterial cell surface, phages can infect specific hosts only. However, in solution, this interaction with the host is a random phenomenon for the phages. Bacterial type (Gram-negative and Gram-positive), growth conditions, and virulence also influence the phage to attach on the host’s surface (Rakhuba et al. 2010). The outer membrane of Gram-negative bacteria has an external lipopolysaccharide (LPS) layer and embedded outer membrane proteins (OMPs) for transport and diffusion of nutrients. These act as phage receptors, and in some infection strategies, they are essential for adsorption of phage particles as well (Bugla-Ploskonska et al. 2007; Rakhuba et al. 2010). In comparison, teichoic acids (peptidoglycan interspersed with acid polysaccharides) present in the Gram-positive bacteria cell wall act as receptors for their corresponding phages (Brown et al. 2013; León and Bastías 2015).

The penetration processes in bacterial cell also vary in different phage groups. In general, myoviridae phage inserts its genetic material into the bacterial cell by using a syringe-like movement of its tail (Fig. 2). After receptor recognition, in a reversible binding mode, the phage particle attaches its base plate with the bacterial surface utilizing the flexing activity of tail fibers. The phage takes sufficient time to make its surface binding irreversible. Thereafter, with the help of ATP, the contraction of its tail takes place, along with insertion of its genetic material. On the contrary, podoviridae phage, which is devoid of the tail part of the myoviridae phage, inserts its genetic material after enzymatically degrading a portion of the bacterial cell membrane using its small, tooth-like tail fibers (Rakhuba et al. 2010; Brown et al. 2013; León and Bastías 2015).

Fig. 2
figure 2

Schematic representation of lytic and lysogenic cycle of phage

Full size image

Phages undergo two possible life cycles: lytic and lysogenic cycles.

Lytic cycle

Phages, which are in their lytic phase, are called virulent phages. During the lytic cycle, lysis (death) of host bacterium occurs as the phage multiplies in great numbers within the host, following which the phage disintegrates and ruptures the host cell for release of new phage particles (Fig. 2).

The lytic cycle initiates with the attachment of the phage on the bacteria with the aid of a complex of proteins (Karlsson et al. 2003). Once the attachment of the viral particle is complete, the genetic material of the bacteriophage is inserted into the bacterial host cell. Upon penetration, the bacterial metabolic machinery is utilized by the phage to create multiple copies of its own genetic material (DNA or RNA). DNA viruses directly transcribe themselves into mRNA (messenger RNA) molecules that are then used to direct the host cell’s ribosomes. In case of RNA viruses (retroviruses), a unique enzyme—reverse transcriptase—transcribes the viral RNA into DNA, and thereafter, follows the path of DNA virus for transcription of the viral RNA. Towards the later stages of this translation, the newly translated proteins are assembled to form the capsid and the tail of the phages that break out of the host cell, bursting its cellular membrane. Each newly formed particle continues to infect new host cells and subsequently proliferates. In few cases, instead of the phage genome, host chromosome gets packed in to the capsid during phage replication and represents an example of horizontal gene transfer within the bacterial population via transduction (Madigan and Martinko 2006). A direct application of phages exhibiting only the lytic cycle is that they can be conveniently employed for tackling the problem of antibiotic-resistant pathogenic bacteria.

Lysogenic cycle

In comparison to the lytic cycle, the lysogenic phase is exhibited particularly by temperate phages (Campbell and Reece 2005), and results in the integration of the viral genetic material with the bacterial genome (called prophage), ensuring continued replication of the viral genetic material without any fatal consequences to the infected host (Inal 2003). However, due to incorporation of viral genetic material into the host, change in the phenotype of the infected bacteria is a common phenomenon. This conversion may induce the pathogenicity of bacteria, which is evident for many common bacterial strains (Brussow et al. 2004; Keen 2012). Prevention of such lysogenic conversion may also be done using hydrogen peroxide by production of a reactive oxygen species, glutathione, and overexpression of transcriptional repressors (Wagner et al. 2001; Liu et al. 2005; Ptashne 2006; Keen 2012).

Application of phages

Biotechnological relevance

Recently, phages have become an important instrument to biotechnologists. It is being studied for various purposes like drug designing, synthesis of novel protein, delivery of protein and DNA vaccines, detection of pathogenic bacteria, and screening of protein libraries, peptides, or antibodies (Hart et al. 1994; Sperinde et al. 2001; Clark and March 2004; Donnelly et al. 2015; Gao et al. 2015). Further, phages are being used as a substitute to antibiotics for many antibiotic-resistant bacterial strains, biocontrol agents in agriculture, aquaculture, and oil and petroleum industry (Haq et al. 2012). Researchers are also working upon synthesizing a novel peptide by exploiting phage display techniques, gene inclusion, and replication using host machinery (Smith 1985; Sidhu 2000; Haq et al. 2012; Wang et al. 2016). Phage display was first described by Smith (1985) to identify polypeptides with precise bait-binding activity; the technique has evolved with several versatile applications (Paschke 2006; Li and Caberoy 2010). The displayed molecule encoded by the phage genetic information will be expressed on its surface along with the coat protein. Researchers reported using phages like M13, Lambda, and T7 for phage display techniques (Benhar 2001; Willats 2002; Wang et al. 2016). A range of ligands can be prepared by fusing the coat protein gene with different gene-encoding peptides. Such ligands must have capabilities like detecting viable bacteria like Escherichia coli (Wang et al. 2016) and targeting specific pathogen proteins, recognizing specific receptors, and blocking interaction between ligand and receptor (Watson and Eveland 1965; Kodikara et al. 1991; Funatsu et al. 2002; Haq et al. 2012). Furthermore, bacteriophages are being modified as tunable nano-containers for the packaging and delivering of peptides (Kelly et al. 2015). In an interesting study, Dickerson et al. (2005) demonstrated the use of engineered filamentous bacteriophage in cocaine sequestration for blocking the behavioral effects of cocaine in rodent model. Furthermore, phage technology for detecting affinity of antibody to a particular antigen (pathogenic) or phage amplification for detecting pathogenic bacteria, like Mycobacterium tuberculosis, E. coli, Pseudomonas, etc., improvised research in this field (Stewart et al. 1989; Barry et al. 1996; Donnelly et al. 2015). Exploring phage sensitivity and specificity to design phage-based biosensors can be an improved alternative to antibody-based immunoassay techniques (Peltomaa et al. 2015).

Medicine and clinical application

About a century ago, in 1917, the bacteriophage era was started when Félix d’Herelle published a paper demonstrating “un bactériophage obligatoire” (d’Herelle 1917, 1949; Abedon et al. 2011). Bruynoghe and Maisin (1921) initiated bacteriophage therapy by treating patients having staphylococcal infections. “Phage therapy” has become a potential therapeutic option (Duckworth and Gulig 2002; Wright et al. 2009a, b; Sarker et al. 2012). Immense research has been initiated to employ phage therapy as an alternative (Matsuzaki et al. 2003; Quintin et al. 2005; Chatterjee et al. 2015; Cui 2015; Zschach et al. 2015; Abedon 2016; Maszewska et al. 2016). Phage based products were first developed in the commercial laboratory of d’Herelle in Paris. Treatment of various pathogens and antibiotic-resistance bacteria (like Salmonella spp. Clostridium difficile, and diarrheagenic E. coli) has been tried using phage (Mai et al. 2010; Frampton et al. 2012). Phage-based products were first developed in the commercial laboratory of d’Herelle in Paris. The company (later became the successful French company L’Oréal) produced five phage preparations (Bacté-coli-phage, Bacté-rhinophage, Bacté-intesti-phage, Bacté-pyo-phage, and Bacté-staphyphage) (Summers 1999; Sulakvelidze et al. 2001). In 1940s, Eli Lilly and Company (Indianapolis, IN, USA) developed seven phage products for human uses. These products were either in the form of a lysate in broth cultures (e.g., Colo-lysate, Ento-lysate, Neiso-lysate, and Staphylo-lysate) or in the form of a water soluble jelly (e.g., Colo-jel, Ento-jel, and Staphylo-jel) against targeted bacteria like, staphylococci, streptococci, and Escherichia coli. They were used for treating abscesses, septic wounds and vaginitis, mastoid infections, and respiratory tract infections (Sulakvelidze et al. 2001).

However, due to exponential growth of antibiotic-based drug companies and pharmaceutical giant companies, phage products became less popular (Alisky et al. 1998; Sulakvelidze and Kutter 2005). In favor of therapeutic uses of phages, recent reports on the application of phage cocktails for open septic wounds and burn injuries have contributed encouraging results. Phage cocktail containing 82 phages against Pseudomonas aeruginosa and 8 phages against Staphylococcus aureus was successfully applied on eight patients (Merabishvili et al. 2009; Wright et al., 2009a, b; Nilsson 2014). Recently, Pherecydes Pharma, a biotechnology company in France, have announced the phase I/II single-blind multicenter clinical trials of its bacteriophage based product ‘Phagoburn’ (http://www.pherecydes-pharma.com/phagoburn-clinical-study.html; Reardon 2014; Ravat et al. 2015; Servick 2016). Phagoburn contains bacteriophage cocktail targeting pathogenic strain of Escherichia coli and Pseudomonas aeruginosa and its infection in serious burn patients. The Phagoburn is a collaborative European project (www.phagoburn.eu) coordinated by French Ministry of Defense and being conducted in eleven major burns units in France, Switzerland and Belgium along with eight civilian hospitals. Phage therapy is important alternative to overcome critical limitations of antibiotic therapy due to emergence of bacterial resistance, like, in the case of Clostridium difficile infection (CDI). CDI is responsible for inducing the dysbiosis, which has extremely high recurrence rates. As per reports, treatment of CDI using current antibiotics has become more and more ineffective (Sangster et al. 2014). Further, prolonged use of antibiotics can also harm beneficial gut flora causing discomfort to the patients. Phage therapy against C. difficile involves specifically targeting the causative agent, sparing the other bacterial organisms of the human gut (Sangster et al. 2014, 2015). Similarly, treatment of nosocomial infections (common hospital-acquired infections) caused by Pseudomonas aeruginosa is a huge therapeutic challenge currently. High rate of morbidity and mortality is connected with the infection, along with a greater possibility of drug resistance in the bacteria during the course of therapy. With such limited scope for developing new drugs, scientists have also found considerable success in alternative treatment options including phage-based approaches (Viertel et al. 2014; Chatterjee et al. 2015). Babalova et al. (1968) used Shigella phages to treat bacterial dysentery and successfully controlled dysentery in patients. Till date, inadequate references are available on human clinical trials conducted and published; the reported cases are phase I studies and mostly from European Medicines Agency (EMA) or United States Food and Drug Administration (FDA) jurisdictions (Bruttin and Brüssow 2005; Merabishvili et al. 2009; Rhoads et al. 2009). In 2009, phase I clinical trial of bacteriophage cocktail (Biophage-PA) targeting three pathogenic strains of S. aureus, P. aeruginosa, and E. coli targeting venous ulcers was approved US FDA (Rhoads et al. 2009). Similarly, a randomized, double-blind, placebo-controlled phase I/II clinical trial approved by UK Medicines and Healthcare products Regulatory Agency (MHRA) and the Central Office for Research Ethics Committees (COREC) ethical review process was performed to treat chronic otitis against multidrug resistant bacteria P. aeruginosa and found lower bacterial count and significant improvement with a single input dose of 600,000 bacteriophages (Wright et al. 2009a, b). Bacteriophage application trials have shown that PFU count of 102–103 plaque forming unit (PFU) is adequate to counter 106–109 CFU per milliliter proliferation threshold of bacteria in vivo (Marza et al. 2006; Wright et al. 2009a, b; Parracho et al. 2012). However, the reported trials with bacteriophages have been performed with 105 and 109 PFU of individual bacteriophages (Bruttin and Brüssow 2005; Merabishvili et al. 2009; Rhoads et al. 2009; Wright et al. 2009a, b). Development of phage bioderm is another important area of application in clinical sector. It is a therapeutic autodegrading, non-toxic, biopolymer complex containing phages that help in healing of wounds and burns, osteomyelitis, and periodontal diseases (Mathur et al. 2003). Studies reported 20–95 % lysis of clinical isolates of Serratia marcescens using specific phage strains and phage type (Alisky et al. 1998). However, there are certain limitations on the application of phage bioderm, like reduction in phage activity due to development of antibodies against phages, induction of toxin genes, and fast discharge of bacterial endotoxins due to the lytic effect of phage (Mathur et al. 2003).

Biosensor development

IUPAC defines a biosensor as “a device that uses specific biochemical reactions mediated by isolated enzymes, immune-systems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals” (Nagel et al. 1992; Hwang 2014). A biosensor is typically composed of bio-based recognition and transducer components, and electronic systems (signal amplification, processing, and display). Potential applications of biosensors are diverse and numerous, from defense security to pharmaceutical science to environmental monitoring and assessment (Kirsch et al. 2013). Biosensors have a number of advantages like sensitivity, specificity, speed and accuracy of detection, easy sample preparation, cost-effectiveness, etc. (Singh et al. 2013; Hwang 2014; Rackus et al. 2015). Like that of many other biomaterials, unique biological, geometrical, and mechanical characters of bacteriophages can be exploited for bacterial identification, pathogen detection, and biocontrol (Hwang 2014).

It is simpler to develop biosensor surfaces by surface adsorption of phages, though it may give inconsistent results due to unstable immobilization densities (Balasubramanian et al. 2007; Lakshmanan et al. 2007). However, chemically anchoring (cysteamine-modified and glutaraldehyde-activated gold substrate) phages on a detection platform display a consistent improvement in the phage density and detection (Cademartiri et al. 2010; Singh et al. 2013). For chemically functionalizing phage-based biosensors, selection (biopanning) purity of the phage suspension is an important criterion which should be devoid of various other bio-contaminating agents like other carbohydrates, proteins, and lipids (Naidoo et al. 2012). However, genetically modified or engineered phages are more appropriate to develop bio-probes than intact wild-type phages. Being biologically active, wild-type phages upon infection lyse the host bacterium that may lead to reduction of signal on a biosensor platform (Singh et al. 2010, 2013).

Recently, utilizing unique ability of phages to display peptides or proteins on their surface, called “phage display,” is becoming a powerful tool to screen diversity of targets like proteins, carbohydrates, small molecules, or an entire cell. For the phage display technology, lambda, f1, M13, fd, T4, and T7 phages have most widely been used (Smith 1985; Atias et al. 2008; Singh et al. 2013). This emerging technology can revolutionize diagnostics by creating molecules that are otherwise unavailable via conventional approaches. Researchers have successfully expressed cellular proteins and peptides, antibody (or its fragments), and antigen molecule on the surface of phages for developing pathogen detection biosensors, molecular imaging, and gene delivery (Petrenko 2008; Pande et al. 2010; Singh et al. 2013; Hwang 2014; Chuang et al. 2015).

Agriculture and food safety

Phage application in agriculture and food material is an upcoming area of development. Salmonella, Campylobacter, Listeria, and E. coli are common bacterial contaminants in food borne-related infections (DuPont 2007). Bacterial infection in crops is a grave problem that reduces the yields (Allen et al. 2011). High resistivity to multiple antibiotics was reported by a number of researchers in the agricultural field (Price et al. 2012; Zhu et al. 2013; Micallef et al. 2013; Popowska et al. 2012). To evade infection, a number of reports of phage application on various crops (like tomato, citrus, and onion) are available (Huff et al. 2005; Lang et al. 2007; Balogh et al. 2008; Jones et al. 2014). Phage products like AgriPhage™, Omnilytics are being used at a large scale for protecting crops from a number of bacterial diseases. Similarly, other phage products like LISTEX (for Listeria sp.; http://www.listex.eu/product/), Listshield™, and Intralytix were approved by the FDA (USA), for treating food products before they are being marketed (Meaden and Koskella 2013). Phages have been exploited to treat colibacillosis and prevent food-borne pathogens (Huff et al. 2005; Sharma et al. 2009). Phage therapy is applicable in restricting bacterial contamination on a variety of food materials like chicken flesh, meat, fruits, and vegetables (Flaherty et al. 2000; Goode et al. 2003; Allwood et al. 2004; Toro et al. 2005; Higgins et al. 2005; Fiorentin et al. 2005; Ravensdale et al. 2007; Sharma et al. 2009; Guenther et al. 2009; Marcó et al. 2012). Further, antimicrobial packaging against the activity of Listeria monocytogenes for cantaloupes and ready to eat meat and E. coli for alfalfa seeds and sprouts are available in the market (Lone et al. 2016).

Aquaculture industries

Aquaculture industries often undergo economic losses chiefly due to uncontrolled microbial diseases that threaten their development and sustainability (Almeida et al. 2009, Silva et al. 2014). Fish-based products are becoming more popular throughout the world as a cheap source of protein. For the last three decades, considerable growth of aquaculture and related industries has taken place. On the other hand, pathogenic bacteria like Flavobacterium psychrophilum, Photobacterium damselae, Vibrio anguillarum, Vibrio vulnificus, Aeromonas hydrophila, and Aeromonas salmonicida have become more prominent, causing heavy loss and/or diseases in humans due to consumption of contaminated aquaculture products (Subasinghe et al. 2001; Nakai and Park 2002; Flegel 2006; Saksida et al. 2006; FAO Report 2015). Mostly in marine and estuarine fisheries and occasionally in freshwater fisheries, vibriosis is a common disease that causes significant mortality in fish (up to 100 % mortality in larvae). This is also responsible for disease outbreaks in fisheries units. Bacterial genera Vibrio (with its various species like V. vulnificus, V. anguillarum, V. parahaemolyticus, V. alginolyticus, and V. salmonicida) and Photobacterium damselae are the causative agents of vibriosis (Noorlis et al. 2011; Higuera et al. 2013; Martínez-Díaz and Hipólito-Morales 2013; Silva et al. 2014). As antibiotic treatments have exhibited reduced effectiveness against multidrug-resistant bacteria, treatments with phages have proven efficient in case of V. anguillarum infection in fish larvae (Silva et al. 2014). However, the success of aquaculture phage therapy depends mainly on two factors: number of phages produced by each host cell after host lysis and on latent period for fresh infection to a new host by new phage particles. Ideally, for phage therapy in aquatic condition, maintaining abundant phage number and short period for fresh infection is required, which is a challenge for the days to come (Abedon et al. 2001; Crothers-Stomps et al. 2010; Silva et al. 2014). Table 2 has summarized the phage applicability and trials performed in recent era of antibiotic resistance bacteria.

Table 2 Representing information on phage therapy trials and its applications in various fields
Full size table

Wastewater phages and their application in effluent water decontamination

Common wastewater treatment involves technology based on chemical precipitation, ion exchange, sedimentation, or coagulation-flocculation (Otte and Jacob 2006). These techniques are energy and maintenance intensive and require high-infrastructure facilities. Researchers across the world are trying to harness phage-based techniques in wastewater treatments to improve quality of effluent and sludge either released into the environment or reused (Fu and Wang 2011). This treatment has the potential to manage the problems of environmental wastewater processes like: reduction in pathogenic bacteria, foaming in activated sludge plants, digestibility, and water ability of sludge, and reduction in competition between functionally important bacterial populations and nuisance bacteria (Withey et al. 2005; Mulani et al. 2015). This is a highly potential area of research and development especially in a country like India; there is potential here for application of appropriate, affordable, sustainable, and eco-friendly approaches. Phage-mediated bactericidal effects in any wastewater treatment process have many controlling factors that lead to treatment performance. Wastewater is rich in microbial diversity that contains a wide variety of microbes and bacteriophages, among which, somatic coliphages are the most abundant. These coliphages are virulent, in nature, generally following lytic cycle for their reproduction. These phages belong to the families Myoviridae, Siphoviridae, Podoviridae, and Microviridae that mainly target the member of Enterobacteriaceae family especially E. coli (Hayes 1968). Coliphages find favorable environment in wastewater. However, their abundance depends upon factors like optimal condition and density of the host bacteria, pH, temperature, cations like calcium and magnesium, organic matter concentration, etc. (Grabow 1990; Grabow et al. 1980, 1998). For instance, fecal coliform bacteria closely related to E. coli, especially Klebsiella species, are heterotrophic and favor certain water environments for its multiplication and in turn support the multiplication of somatic coliphages (Grabow et al. 2000). It has been reported that phage titer against Salmonella typhi in both laboratory media and sewage was found to be 109–1010 per milliliter (Goyal et al. 1980). Successful phage replication requires a minimum of 104 host bacteria per milliliter. According to Goyal et al. (1987), phages under nutrient-limited conditions were found concentrated on the solid surface instead of flowing water. Coliphages also inhabit inside the gut of humans and other warm blooded animals, multiply within it, and are excreted along with fecal matter. Different pieces of information are available on phages in sewage. A number of studies have reported density of somatic coliphages to be 106–108 per milliliter (Bell 1976; Ignazitto et al., 1980; Havelaar and Hogeboom 1984; Havelaar et al. 1984; Tartera et al. 1989; Grabow et al. 1993; Grabow et al., 2000).

Interestingly, some members of coliphages showed similarity with human viruses like enteroviruses such as polio viruses (Family Picornaviridae) and F-RNA coliphages (Family Leviviridae) (Kamiko and Ohgaki 1993). Both viral particles consist of an icosahedral capsids (about 25 nm diameter) and a single strand (ss)-RNA genome. Due to these similarities, coliphages are considered to be valuable models or surrogates for human enteric viruses (Grabow 2001), which are also excreted along with fecal matter (Grabow et al. 1995). Although coliphages are shed regularly along with fecal matter, human enteric viruses are excreted generally during infection. Greater number of adenoviruses than enteroviruses have been constantly present in raw sewage around the world (Irving and Smith 1981; Hurst et al. 1988; Krikelis et al. 1985a, b), and according to Hurst et al. 1988, 80 % of infectious adenoviruses shed in the feces and present in raw sewage may be enteric adenoviruses. Recently, human fecal contamination was indicated using potential source indicators such as bacteriophages against Bacteroides (GA-17, GB-124, and ARABA 84) as well as sorbitol-fermenting Bifidobacteria (Wicki et al. 2015). Furthermore, according to Haramoto et al. (2015), coliphages belonging to the genogroup, GI F-RNA, may be used as a suitable indicator of virus reduction during wastewater treatment.

Impediments of phage application: interplay between phage and bacterial resistance

There is a tug of war between phage and bacterial infection and immunity. The selective pressure among these two entities is unique. Bacteria develop resistance to phage; subsequently, phages improve their antiviral mechanism and vice versa. This interplay between phage and bacteria is an interesting phenomenon; elevated mutation rates is a normal event for bacteria like Pseudomonas fluorescens when they are subjected to coevolve in the invitro systems (Buckling and Rainey 2002). Again, Pal et al. (2007) showed that in laboratory conditions, coevolution of P. fluorescens with its bacteriophage help in faster bacterial mutation rates. They have also found that 25 % of the P. fluorescens populations (approx. 200 bacterial generations) coevolving with the lytic phages had evolved 10 to 100-fold increase in mutation rates (Pal et al. 2007). For lytic bacteriophages, reciprocal selection for bacterial resistance and phage infectivity is essential. And this bacteria-phage interface generates more beneficial resistance mutations to the bacteria (Gomez and Buckling 2013). However, natural populations of the bacteria-phage coevolving in their natural environment (as for example, soil) may not follow the same mutation pattern as, in nature, several other factors may influence the selection rates for mutation. Innumerable microbial species including natural microbial and viral community (NMC) as a whole and other physico-chemical factors possibly will influence the rate of mutation (Gomez and Buckling 2013).

Bacterial resistance towards phage, therefore, may be due to several mechanisms. These include modification of phage attachment or adsorption sites (receptors) and CRISPR sequence-based adaptive immunity (Hyman and Abedon 2010). Alteration of phage adsorption sites/receptors is the most common phenomenon by which bacteria evade phage infection and become resistant to phage. However, phages can also adapt themselves to recognize these new modified receptors. Synthesis of exopolysaccharide (EPS) or masking proteins (like protein A of S. aureus) to mask the phage receptor is another strategy of bacteria. Again, phages conquer the barrier by cleaving the EPS layer using polysaccharide lyase or a polysaccharide hydrolase (Labrie et al. 2010, Örmälä and Jalasvuori 2013).

Further, self/non-self discrimination in prokaryotes is ubiquitous and provided by restriction-modification (RM) systems that defend hosts from exogenous DNA (Pleška et al. 2016). Bacterial system has the ability to recognize and modify the phage DNA. In many instances, the phage DNA is cleaved by the restriction endonucleases on entering into the bacterial cell through RM system and protects bacterial cell from foreign phage DNA attack (Pleška et al. 2016).

It has also been reported that phages adopt anti-restriction strategy to avoid recognition by endonuclease enzyme. For example, T4 phage escapes of restriction endonuclease attack as it contains hydroxymethylcytosine (HMC) instead of cytosine. However, some bacteria, again, modified their system to specially recognize hydroxymethylcytosine (HMC) and shatter phage DNA (Bickle and Kruger 1993; Borgaro and Zhu 2013). Yet again, anti-restriction strategy in Staphylococcus phage K is interesting where no 5′ GATC-3′ cleavage site is present; hence, DNA is protected from restriction (Kruger et al. 1988; Tock and Dryden 2005, Bryson et al. 2015).

CRISPER-Cas mechanism or Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (cas) genes present a novel example of acquired resistance against viruses in prokaryotes. This mechanism is widely present in genome of bacteria and archaeal population and CRISPER-Cas loci were first described in 1987 in E. coli (Ishino et al. 1987). CRISPER is composed of 21–48 bp direct repeats separated by (26–72 bp) non-repetitive spacers, and at 5′ edge, this loci is flanked by a number of cas genes (4–20 in number) in bacterial strains. The variation in cas gene and spacer make this system more unique and robust (Marraffini 2015). Main function of CRISPER-Cas is to provide immunity against foreign DNA including phage genomic DNA or plasmid DNA (Makarova et al. 2006; Bolotin et al. 2005; Haft et al. 2005; Mojica et al. 2005; Pourcel et al. 2005; Marraffini and Sontheimer 2008). Further, as for example of antiphage activity of CRISPER-Cas mechanism in Streptococcus thermophilus, exposure to virulent phage gave rise to the phage-resistant mutants due to insertion of additional 30 bp spacer similar to protospacer of infecting phage (Barrangou et al. 2007). The event of acquiring immunity against phage can be explained in following steps like adaptation or spacer attainment, transcription of acquired spacer (small CRISPER RNAs (crRNAs), on recurrent phage attack this crRNAs form a complex with Cas protein), and immunity against phage (crRNAs-Cas complex direct nuclease to trace and chop the invading phage DNA; Marraffini 2015).

Conclusive remarks

Phages are omni-present, ubiquitous, and important part of nature. Being antibacterial and natural predators of bacteria, the role of phage can be extensive as nano-cleaner of ecosystem. Detailed classification of bacteriophages may be beneficial to mankind in the screening of therapeutic phages against a number of diseases and economically important phages help in industries and its preservation. High-throughput sequencing like metagenomics study of environmental samples can provide information of novel bacteriophages or new bacteriophage species. Various biological, environmental, medical, and pharmaceutical applications related to the use of phage are becoming more and more attractive. Further, employability of phage for sustainable wastewater treatment is a challenging approach. Labeled phages are used in situ bacterial detection using phage typing of clinical bacterial strains. Although discovered a century ago, their use in various fields are still restrictive. Comprehensive studies on basic molecular biology, host range increment, and genetics processes of these tiny antibacterial agents are required which will help them consider novel beneficial agent for bactericidal activities, even against multidrug-resistant bacteria.