How Good are Bacteriophages as an Alternative Therapy to Mitigate Biofilms of Nosocomial Infections

Summary

Bacterial biofilms are serious causes of nosocomial and device-related infections. Phages are ubiquitous viruses which infect bacteria and are being extensively explored as alternatives to antibiotics. Phages employ many strategies to better penetrate hard-to-reach bacterial cells in biofilms and have been found to be effective against many resistant nosocomial bacterial strains.

Introduction

Bacteria have a unique protection layer called biofilm constructed by cell aggregation, that facilitate the bacterial immobilization on an extracellular polymeric matrix and form higher-order structures.¹ This extracellular polymeric matrix is comprised of long chain sugars, DNA, and other biological macromolecules.² With the help of such biofilms, bacteria have additional resistant strength against antimicrobial agents like biocides and antibiotics, etc.³ The bacterial cells within the biofilm are protected and tolerant to antibiotics, antiseptic, antimicrobials, and host immune responses.⁴ Although commensal flora also resides in the form of biofilms, especially in sites like the oral cavity, gastrointestinal tract, vagina; this heterogenous community is normally in a homeostatic relationship with the host. Any disturbance in that may lead to the formation of dysbiotic biofilms inhabited largely by opportunistic pathogens and ultimately lead to persistent infections.⁵ Such biofilms of pathogenic bacteria could be present on the surface of various medical instruments and materials too, in addition to the surface of the patient’s tissue.⁶

One of the most prevalent and extensively studied polymicrobial biofilms is oral biofilm. Imbalance and loss of the healthy oral microbial population may lead to various oral diseases. In a previous report, more than 700 bacterial species were observed to be located in the oral cavity⁷ where they continued to survive by constructing biofilm, however excessive synthesis of biofilms on teeth, tongue and other cavity areas may show deregulated immune responses, leading to further human oral diseases,⁸ for example, the dental plaque consisting of Porphyromonas gingivalis and Fusobacterium nucleatum is the crucial agent of gingivitis and periodontitis.⁹ Researchers could not classify many of the oral pathobionts like Porphyromonas gingivalis whether they are bystander microbiota or disease initiators.¹⁰ In addition, to cure oral diseases, alternative treatment processes are required that should target specifically microbes associated with disease in the biofilm without affecting the other beneficial microbiota.¹¹

Orthopedic devices are also one of the most common surfaces colonized by bacteria. Joint replacement is a procedure where prosthetic joint infections due to bacterial biofilms on medical implants can be a frequent complication and may require surgical removal of the implant and prolonged antibiotic therapy.¹² Some of the major pathogens isolated from medical devices and implants are given in Table 1. Similarly, the formation of biofilm on catheters and implants is another major cause of chronic, recurrent, or persistent infections. The urinary bladder usually does not have dangerous levels of bacteria, but when a foreign object like a catheter is put inside, the planktonic bacteria easily get attached to those surfaces forming biofilms. The urease-producing bacteria can form crystalline biofilms, leading to blockage of catheters, urine retention and a medical emergency.¹⁵ Major species involved in crystalline biofilms are Proteus mirabilis, P. vulgaris, Providencia rettgeri.¹⁶ While non-crystallized biofilms are formed by low urease producing bacterial species, namely Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Morganella morganii, Providencia stuartii.¹⁴

Table 1 Most Common Pathogens Isolated from Biofilms Formed Over Medical Prostheses and Devices

It is established that bacteria achieve a good level of resistance to various biocides and antibiotics due to the presence of biofilm. Investigations have suggested that more than 1000 times antibacterial compounds may be required for bacteria within biofilm compared to planktonic forms.¹⁷ Since mechanisms like horizontal gene transfer (HGT) and hypermutability are favoured within biofilms, these structures are now recognized as a reservoir of antibiotic genes.¹⁸ The collective recalcitrance toward antibiotics in a biofilm is majorly dependent on, the developmental stage of biofilm, its EPS composition, the biofilm architecture.¹⁹ These recalcitrant biofilms can also initiate an increase in multidrug resistant (MDR) bacteria. MDR infections are frequently associated with common human pathogens, known as ESKAPE (Enterococcus faecium, S. aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter spp), which are the leading causes of nosocomial infections.²⁰ WHO has declared antimicrobial resistance as one of the top 10 global public health threats facing humanity.²¹ In addition, the economic burden for providing treatment to six MDR ESKAPE pathogens is almost $4.6 billion annually.²² Thus, new therapies acting by totally different mechanisms are highly desired. In WHO’s global action plan on antimicrobial resistance, one of the five objectives is to develop new medicines, diagnostic tools, vaccines, and other interventions.²³ In the present scenario, phage therapy represents a potential non-antibiotic approach worth exploring and current research has re-established phage therapy as an effective antibacterial treatment that works equally well against sensitive as well as resistant bacterial strains.^24,25 Similarly, sufficient evidence is available confirming the potential of phages in biofilm removal under various conditions.^26,27

The aim of the present review is, therefore, to focus on the role of phages as an alternative to antibiotics in the removal of bacterial biofilms, particularly biofilms of common drug resistant pathogens. It primarily summarizes the recent advancements in different phage-therapy strategies, starting from the whole phage to phage cocktail, phage-derived enzymes and engineered phages for mitigating biofilms. Lastly, the potential challenges and future potential of the approach are also highlighted.

Process of Biofilm Generation and Molecular Mechanisms During its Formation

Referring to various investigations, biofilm generation may include three major steps ie, attachment, maturation and dispersion (Figure 1). At the initial stage, attachment can happen in two ways, eg, reversible and irreversible attachment.²⁸ Bacterial cells achieve better tolerance for physical or chemical shear forces due to irreversible attachment. To form a biofilm, motile bacterial cells employ flagella which are crucial for the initial attachment of the cell to the surface, however, O’Toole et al have demonstrated the development of biofilm by non-motile bacteria as well, eg S. aureus lacking flagella.²⁹ During biofilm formation bacterial cells first interact with the surface through their flagella and type IV pili-mediated motilities, further this interaction leads to accumulation and aggregation of cells on the surface and eventually produce microcolonies. Maturation is done through the recruitment of more cells from the same species or other species to the biofilm from the bulk fluid and acquires the “mushroom” or “tower” shape appearance. At this stage, cells are arranged as per their metabolism and aero tolerance state. The bacterial cells within the biofilm secrete more proteins, DNA, polysaccharides for the biofilm scaffold during the maturation time. Figure 2 represents a bacterial biofilm composition, in which the bacterial cells in microcolonies are present as planktonic or embedded centrally in metabolically active or dormant forms. At the final stage, biofilm matures into a mushroom-like structure where bacterial cells achieve their nutrients through channels.²⁹ Various factors have been observed for the dispersed condition of biofilm that may include expanded population, nutrients scarcity, high competition etc. Cell–cell disruptive factors are also present along with the matrix that causes the cell dispersal or detachment from mature biofilms and this may occur simply due to shear stresses. The other passive forces resulting in the breaking of a biofilm can be abrasion with environmental particles, grazing by eukaryotic organisms or sloughing due to frictional forces caused by the flow of ligands.³⁰ During dispersion, the planktonic bacteria are released and spread to other areas, further beginning the formation of new biofilms.³¹

Figure 1 Graphical representation of various steps involved in generation of biofilm by bacterial cells.

Figure 2 Schematic representation of a bacterial biofilm composition. A mature biofilm consists of bacterial cells (2–35%) in close proximity with the help of extracellular matrix, EPS containing exopolysaccharides (40–95%), proteins (1–60%), lipids (1–40%), enzymes, extracellular DNA and RNA (<1%).30 The bacterial cells in microcolonies are present as planktonic or embedded centrally in metabolically active or as dormant form (persister cells).

The very first step of biofilm formation is attachment to the abiotic surface. This attachment is governed by some physicochemical properties of bacterial surface like hydrophobicity and its composition. There are several factors observed crucial for the attachment and proliferative stages of biofilm generation and pathogens have their own weapons and arsenal for formation of biofilms. Various studies have reported different molecular and genomic mechanisms employed by common pathogens for biofilm formation (Table 2), like uropathogenic E. coli (UPEC) have various adhesins and their receptors on host cells, of which chaperone-usher (CU) fimbrial adhesins are strongly associated with chronic colonization and urosepsis in UTI³² and Type 9 fimbriae in biofilm formation.³⁶ Along with FimH adhesin on Type 1 pili, “curli” amyloid fibers and antigen 43 are also important mediators for attachment and progression of bacteria on abiotic surfaces.⁴⁰ Investigators⁴⁴ have found teichoic acids and the surface protein autolysin associated with abiotic surface attachment in the case of S. aureus, Enterococcus faecalis and Staphylococcus epidermidis. These cell wall anchored (CWA) surface proteins adhesion to a different extracellular matrix including collagen, fibrinogen, fibronectin promotes the formation of biofilms and are being studied in many antibiotic-resistant strains of S. aureus and S. epidermidis, common pathogens forming biofilms in indwelling medical devices.⁴¹ Similarly, researchers have identified the biofilm-associated genes in A. baumannii clinical isolates, which are highly conserved such as csuE and csuA, the proposed tip subunit of the Csu pili,⁵¹ Bap gene for biofilm associated proteins (Bap)⁵² and OmpA for outer membrane protein A (OmpA reported in 81–100% detection).⁵⁵ In contrast to these gram-negative pathogens, the gram-positive ESKAPE member Enterococci though non-motile has also been found to be possessing a number of adhesins, viz. SegA, Acm (in E. faecium), Ace (in E. faecalis), Esp and Ebp, which contribute to biofilm formation and urinary tract infections.⁵⁰ P. aeruginosa employs its Type IV pili and flagella and adhesins and lectins to adhere to epithelial cells in host lungs;⁵⁸ whereas K. pneumoniae has hemagglutinins, Type 1 and Type 3 pili for the purpose.³² After attachment and during proliferation bacteria secretes extracellular matrix that may be composed of various compounds with different chemical properties including exopolysaccharides, proteins, eDNA, and other polymers.

Table 2 Mechanisms of Biofilm Formation Employed by Common Bacterial Pathogens

Finally, biofilm enters the dispersed step which determines its further spread. However, besides the passive dispersal forces as described in the previous section, bacteria also have other signalling methods to become planktonic again. Some of the stressors can be limited availability of nutrients, accumulation of toxic by-products, changes in oxygen level etc.⁶⁶ For example increased iron level in the extracellular environment for UPEC or high carbon and nitrogen levels for P. aeruginosa have been reported for biofilm dispersal. Alteration in gene expression in response to small molecules or autoinducers also promotes dispersal, like c-di-GMP upregulation favors sessility in E. coli and P. aeruginosa.^50,67 The alginate lyase, an EPS hydrolysing enzyme produced by P. aeruginosa helps in bacterial detachment and dispersal. In an extensive review on biofilm dispersal, Guilhen et al have elaborated on multiple strategies shown by bacteria for dispersal as a necessary step which results in a planktonic state with enhanced colonization properties.⁶⁸ In one such example, increased production of rhamnolipids aids in P. aeruginosa dispersal forming new microcolonies and a central void.⁶⁷ In another proof-of-concept study, the in vitro evaluation of P. aeruginosa biofilm dispersal was studied for antimicrobial efficacy. The arabinose-induced biofilm dispersal in the engineered P. aeruginosa strain showed increased efficacy for imipenem and tobramycin.⁶⁹

Biofilm as a Protective Shelter for Bacteria

Biofilms become significant because of characteristic properties and traits, expressed by microbial cells only when in a biofilm. These recalcitrant structures have conferred higher antimicrobial potential to the sessile cells in the biofilm than their planktonic counterparts. For example, cystic fibrosis patients are highly susceptible to chronic infections of P. aeruginosa and A. baumannii, which are frequently identified as biofilm-associated and multidrug-resistant thus highly difficult to treat.⁷⁰ The basis of this biofilm-mediated bacterial antibiotic survival is largely dependent on the developmental stage, the composition of extracellular matrix or EPS and the architecture of biofilm.¹⁹ The EPS in a biofilm comprises a heterogenous mixture of water, polysaccharides, proteins, lipids and glycolipids, extracellular DNA and RNA and ions like Ca²⁺. However, the composition is dynamic and depends on bacterial strain as well as environmental conditions. The EPS protects bacteria by providing nutrients keeping them in close proximity for crosstalk and genetic material exchange and maintaining hydration to protect against desiccation.⁷¹

Many mechanisms, also referred to as “emergent properties” like long-term cell–cell interaction, gradients of pH, nutrients and oxygen, presence of enzymes, persister cells, stress responses and high level of biofilm heterogeneity,⁷² etc are identified. These mechanisms are extensively elaborated in some recent reviews.^19,73,74 Biofilm recalcitrance mechanisms can be a combination of antimicrobial resistance and antimicrobial tolerance and this combination will depend on the type of bacteria, type of antimicrobial agent, age of biofilm, and growth conditions.⁷³ The antimicrobial resistance mechanisms involve horizontal gene transfer (HGT), hypermutation, efflux pumps, quorum sensing,⁷⁵ reduced outer membrane permeability and production of neutralizing enzymes. HGT has been seen at a higher rate in biofilms than in planktonic cells.^76,77 Similarly, a 100 to 1000-fold rise in mutations is reported in a biofilm owing to limited diffusion of antimicrobials due to its architecture.⁷⁸ Another effective mechanism is efflux pumps, which confer higher resistance to bacteria against antimicrobials. Efflux pumps are specialized membrane-bound proteins to expel or throw out different compounds from inside the bacterial cells, thereby reducing the cytoplasmic concentration of antimicrobial compounds below the effective level.⁷⁹ The generation of biofilm starts from initiating and establishing communication by a bacterial cell with other neighboring cells by releasing specific small chemical molecules. This phenomenon which is governed by bacterial population density is known as “Quorum sensing (QS)”.⁷⁵ Blocking of quorum sensing has shown an enhanced susceptibility of biofilm of S. aureus for various classes of antibiotics.⁸⁰ These bacterial cells thus behave in a different way as a community rather than simply a cluster of independent cells.²

In addition, the tolerance mechanisms which enable bacteria to withstand higher concentrations of antimicrobials, include stress-response activation,⁷⁴ hypoxia, initiation of quiescent state, decrease membrane potential, anaerobic metabolism and increased expression of efflux pump. Small colony variants (SCV) in a biofilm give rise to genetic diversity and are correlated with a higher level of persistent and recurrent infection.⁸¹ In a biofilm significant heterogeneity occurs and almost 60% of the cells may show phenotypic variations.⁸² SCVs are slow-growing and dormant cells and the presence of different cell states in the specific zone within a biofilm is dependent on various factors involving the gaseous, as well as nutrient stratifications. Nutrient gradients within a biofilm lead to the formation of dormant “persister cells”.⁷² These metabolically inactive cells present in the matrix have a major role to support the regrowth and resistance of the bacteria within the biofilm and could be the reason behind the drug resistance through biofilm that further led to the revival of the biofilm after treatment.⁸³ Also, the gene expression of bacteria within a biofilm is coordinated by the production and detection of extracellular signal molecules, “anti-inducers”.⁸⁴

Recently, Rasool et al have reported that plastic litter provides space for pathogenic bacteria to form biofilms, leading to the emergence of multidrug resistance.⁸⁵ Sometimes these biofilms are composed of more than one bacterial species too. This multispecies biofilm is more resistant to external stress conditions like the presence of antibiotics or any other antimicrobial agents due to cooperative interactions among them. The existence of this special strength could be associated with an increase in biomass or modified constitution of EPS matrix,^86,87 through which biofilms create a protective environment for resident organisms and is believed to be an important trait for millions of years.⁸⁸ These bacterial reservoirs may lead to noncurable, recurrent or persistent infections at the site or spread as emboli.¹²

Usually, bacteria located on the surface of the biofilms are physiologically different from those located inside the more internal region of the biofilm matrix. The surface bacteria of biofilm are always renewed and help in the initializing primary phage infection.^73,89,90 Whereas bacteria residing in the internal region of the biofilm have restricted entry as well as very limited access to nutrients and oxygen. It is noticeable that bacteria present in the inner matrix of biofilm adapt to environmental anoxia and nutrient limitation leading to lower metabolic rate and a slower rate of cell division.⁹¹ Although it gives them slower growth, it also provides less sensitivity against different antibiotics as well as the bacteriophage infection.⁷³

Impact of Antibiotics in Generation of Antibiotic Resistant Strain and Biofilm

Toward the generation of antibiotic-resistant strains, β-lactamase enzyme that hydrolyses β-lactam antibiotics has been found to be crucial. Studies have been done on enzymes utilizing serine at the active site as well as on those needing divalent Zn ions for hydrolysis. β-Lactamase enzymes comprise more than 2000 unique sequences and some of them have substantial clinical implication such as class A penicillinases, the extended-spectrum β-lactamases (ESBLs), the AmpC cephalosporinases. Therefore, due to this adaptability of β-lactamase enzymes, a new strategy of antimicrobial therapy is urgently needed.⁹² In order to assist researchers, a Beta-Lactamase Database (BLDB) has been established that contains the latest structural and functional information on the β-lactamase superfamily of enzymes and has effective expression on antibiotic resistance. Naas et al has provided a detailed review on functional and structural characteristics of β-lactamase enzymes belonging to various families and subfamilies based on BLDB.⁹³

The different type of antibiotic-resistant genes has been observed in some microbial pathogens belonging to Enterobacteriaceae family and its chromosome constitutes the extended-spectrum-ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) along with the plasmids and transposons as mobile genetic elements. Further various characteristics of MBLs were explained by the Behzadi et al that demonstrated the opportunities in production of effective inhibitors against MBLs.⁶⁴ In some other in vitro works, antibiotics with subminimal inhibitory concentration illustrated its agonist relation with bacterial biofilm formation that represents significant clinical prospects. The induction of biofilm formation can be achieved through the supply of a low dose of antibiotics. But still we need to resolve the intricacies involved in antibiotic-induced biofilm formation and its clinical use as antimicrobial therapy for infection in biomedical devices.⁹⁴

Further, investigators have found out the substantial association among various pathogenic strains of K. pneumoniae linked with pervasiveness of antibiotic resistance and virulent genes.⁹⁵ During this study of 9 months from Dec. 2018 to Aug. 2019, clinical samples were collected from various hospitals located in Tehran and Iran.

Bacteriophages and Their Dynamics in Biofilm

As one of the most abundant (~10³¹) living species on planet earth, bacteriophages—the viruses infecting bacteria, have acquired a lot of attention from researchers for decades.⁹⁶ They are found both in aquatic and terrestrial ecosystems, thus influence the microbial communities in various ways. There is a huge heterogeneity in terms of their morphology and genomic organization. Different morphology like tailed, icosahedral, filamentous, or pleomorphic have been described; however, the majority (>90%) of identified phages are tailed.⁹⁷ Similarly, phage with single- or double-stranded DNA and single- or double-stranded RNA are identified and documented with genome sizes from as less as 4 kb to much larger of 550 kb.^97–99 Bacteriophages have significant importance because of their specificity to infect one bacterial species, though it is also reported that many phage species may infect one bacterial type, eg more than 25 phage species have been identified which infect E. coli.¹⁰⁰

Having been discovered more than a century ago, phage research has come a long way in terms of understanding not only its biology, genetics or role in ecology and biodiversity but also the huge applications in various fields. Potential applications of bacteriophage in medical and clinical, agriculture, and food industry has been documented, with few acquiring necessary regulatory approvals and are commercially available. Similarly, the ability of phage to affect bacteria in planktonic and within biofilms makes them a potential candidate to be explored more. And when it comes to bacterial biofilms, phage also faces the same fate as other bactericidal modes, it becomes quite hard for them to penetrate biofilm due to its thick polymeric matrix. Usually, every biofilm differs in context, matrix, and physiological heterogeneity within the species and even differs from strain to strain. Before going into further detail, it is important to understand the mechanism of penetration, multiplication, and proliferation of bacteriophage within a biofilm.

Bacterial Resistance Mechanisms vs Phage Penetration into Biofilms

There are many factors that affect the penetration of phage into bacterial biofilm matrix such as the age of biofilm, density of biofilm, its molecular structure as well as the extracellular phage-inactivating enzymes secreted by different bacteria. In a detailed review on bacteria and phage interactions in biofilm, Hansen et al have elaborated on important mechanisms and factors for bacterial protection against phage attack while in a biofilm.¹⁰¹ Major factors which affect phage infection are phage diffusion within the biofilm,¹⁰² presence of spherical bacterial “microcolonies” resulting in poor adsorption of phages in such tightly packed bacterial cells,¹⁰³ presence of EPS and cell debris forming “phage sinks” around high density areas,^104,105 production of amyloid fibers “curli” enhancing matrix density and retaining phages at the outer periphery of biofilms.¹⁰⁶ In addition, the metabolically inactive persister cells inhibit the growth of phage; however, this may delay the process but does not completely inhibit it. Pearl et al in their work have shown that coliphage lambda could infect persister cells and resume its lytic cycle once persisters become metabolically active again.¹⁰⁷ Another factor affecting phage infection is coinfection, wherein many phage particles infect the same host due to clusters of host cells and limited mobility. This also decreases the population of progeny virions.¹⁰⁸

Lastly, QS has also been found to have an important role in biofilm development and phage-resistance by regulating CRISPR-associated (Cas) gene expression, phage receptors and phage inactivating proteases.^109–111 QS alters the population of phage receptors on particular cell surfaces. Its mode of action has been described in E. coli, where, in response to the N-acyl-1-homoserine lactone, a reduction in the number of λ receptors on the bacterial surface is observed, which directly reduces the rate of phage adsorption.¹¹⁰ This mechanism has also been observed in the phages of P. aeruginosa, when a quorum-sensing inhibitor, penicillinic acid was introduced along with the phage dilution, phage efficacy has been exponentially increased in the biofilm matrix.¹¹² Some phages can make use of these autoinducers and can induce lysis, eg, a family of temperate phages of Bacillus subtilis uses a bacterial QS system to initiate lysis-lysogeny cycles.¹¹³ In a recent work by Shah et al, a novel QS-sensing anti-activator protein Aqs1 from Pseudomonas phage DMS3 was characterized to inhibit LasR, the main receptor of QS. The small protein also inactivated the pilus assembly protein PilB which results in inhibition of super infection by phages. This has highlighted the ability of small phage proteins to bind with multiple host proteins and disrupt key biological pathways.¹¹⁴

Conversely, old or aged biofilm matrix is less favourable for bacteriophage penetration, resulting in the limitation of phage therapy, especially during chronic bacterial infection. However, the least metabolically active bacteria inside biofilm matrix, still can produce new virion particles, thus resulting in the infection of freshly divided biofilm matrix. In such cases phage therapy is effective to some extent, depending upon the vulnerability of bacteria to phage virion particles. Usually, more aggressive, and extensive treatment is required in the eradication of biofilm. The density of biofilm is a common indicator of whether the phage dilution can penetrate through the biofilm or not. Since biofilm contains a good water channel inside its matrix, they provide a good mode of penetration through these channels.¹¹⁵

Phage Derived Enzymes as Potential Bactericidal Agents

Of all phage therapy methods, EPS-degrading enzymes against biofilms are gaining a lot of interest. They can be used as an alternative to conventional antibiotics. It is also estimated that the use of phage products is easier and safer. The two major categories of degrading enzymes, found useful in mitigating biofilms, are lysins and depolymerases. Both are described below in separate sections:

Bacteriophage Lysins

Although lytic phages are being reported widely for controlling biofilms; their penetration, propagation, and diffusion rate through EPS matrix are important characteristics that may limit a phage’s use. To overcome this, phage-derived endolysins have been studied for the purpose. Bacteriophage-encoded enzymes, endolysin, are a group of unrelated proteins with a molecular weight range of 15–40 kDa, produced by double-stranded DNA phage at the final stage of the lytic cycle. Lysins are reported to be fast acting bactericidal proteins that will instantly cause hydrolysis of bacterial cell walls, thus helping in the release of phage particles. Vincent Fischetti has identified the endolysin that was used to control several important gram-positive pathogens viz. S. pyogenes, S. aureus, S. pneumoniae, E. faecalis, E. faecium and Group B Streptococci.¹¹⁶ Structurally endolysins against gram-positive bacteria have a cell wall binding (CBD) domain to help in enzyme diffusion and an enzymatically active domain (EAD) towards the N-terminal of lysins. EAD has three distinct catalytic modes of action on the host cell wall (Figure 3) which are (i) glycosidases which cleave β-1,4 glycosidic bonds, (ii) amidases which cleave the link between N-acetylmuramoyl residues and L-alanine amino acid residues in certain cell walls, and (iii) endopeptidases cleaving the peptide bond of stem amino acids.^119–121 In contrast to that, most endolysins of gram-negative bacteria have only EAD and no CBD; but have an individual catalytic domain with terminal charged residues for binding. During the lytic cycle, phages produce holin protein which creates holes in the cytoplasmic membrane of host bacteria and ensures the delivery of lysins to the peptidoglycan layer.¹²² Whereas, for gram-negative hosts, an additional protein, spanin is required for outer membrane degradation.¹²¹

Figure 3 Representation of the natural and engineered endolysins. For natural endolysins, the site of action for three major enzyme classes on bacterial cell wall is illustrated on the upper right-side box; the amidases cleave amide bond between N-acetyl muramoyl residues and L-alanine; the glycosidases target the β-1,4-glycosidic bond between NAM (N-acetylmuramic acid) and NAG (N-acetylglucosamine), whereas endopeptidases act on peptide bridges.117 In case of modified and engineered lysins, different approaches have been tried.118 Some examples of next generation approaches are—chimeric lysins—engineered by shuffling, eg HY-133 a recombinant lysin of N-terminal domain of phage K and cell wall binding domain of lysostaphin used against Staphylococcus aureus biofilms in vascular graft infections; artilysins—is a fusion protein of lysin and outer membrane destabilising peptide thus targeting both gram-positive and gram-negative organisms, eg Art-175; virion associated lysins (VALs) or peptidoglycan hydrolases are tail-associated muralytic enzymes, not containing own cell wall binding domain thus are fused with that of other endolysins, EC300 is one such example to target Enterococcus faecalis; truncated lysins—are modified proteins where cell wall binding domain is removed to increase activity, single-domain truncated enzyme, CHAPk is one such example.

Because lysins have a narrow antibacterial spectrum range, they have an advantage over broad-spectrum antibiotics and are suitable for drug-resistant pathogenic bacteria without affecting the host microbiome much, thus inhibiting dysbiosis.¹²³ Lysins are much less likely to show resistance, therefore they are seen as a more suitable alternative for antibiotic-resistant pathogens. In some recent reports, the efficacy of endolysin in treatment of biofilms is reviewed.^121,124,125 Table 3 represents the list of endolysins and their use in mitigation of gram-positive and gram-negative bacteria-induced biofilms. Lysins are also thermostable to a certain degree, high ion intolerant bodies and have shown synergistic effects as well with antibiotics. The lysins can exchange catalytic and binding domains efficiently with other lysins, have independent activity, or can fuse with other antimicrobial peptides to develop new lysins, which may have enhanced bactericidal properties. They are also found in phage tail proteins or virion-associated lysins, working as receptor recognition proteins to break the cell wall locally and allowing the transfer of phage genomic material into host bacterial cells.¹⁴⁸ In a recent review on “endolysins in a clinical setting” by Murray et al¹¹⁸ the development and applications of next generation lysins are elaborated. Compared to natural endolysins, these engineered lysins – chimeric lysin, artilysin, virion-associated lysin, truncated lysin will have improved thermostability, better bioavailability, protease-resistance, improved target infections, etc. Along with natural endolysins, the proposed bioengineering modifications for these lysins are also broadly represented in Figure 3, which is adapted from Murray et al.¹¹⁸

Table 3 Major Endolysins, Their Target Pathogens and Potential Applications

Bacteriophage Depolymerases

Depolymerases are the enzymes that bind and digest the polysaccharide components of the host bacteria’s cell wall, thus helping the phage in transferring its genome into the host cell. Depolymerases are divided into two types, hydrolases and lyases. Triacylglycerol lipase is a third type of depolymerase, that acts on the carboxylic ester bonds of triacylglycerols. These enzymes can be further subdivided based on their target polysaccharides which is either capsular polysaccharides, lipopolysaccharides (LPS), O-polysaccharides or exopolysaccharides of biofilms. Such depolymerases are also reported which break proteins or lipids present on the cell wall.¹⁴⁹ Modes of action of depolymerases are very diverse due to the co-evolution of phages with their host bacteria which result in horizontal gene transfer.¹⁵⁰ The enzyme can be found in free rapidly diffusible soluble form or it can be found attached to the phage particle. The soluble form which is released during the lysis of the host,¹⁴⁸ is seen as “halo” or a circular bull’s eye outside the plaque in soft agar gel and is characteristic of capsular hydrolytic activity.¹⁵¹ Many times, phages attacking gram-negative bacteria use tail spikes that specifically bind to a particular capsule site and release depolymerase enzyme which initiates sequential cleavage of polymer bonds.^152,153

The major advantages of depolymerases are their narrow spectrum of activity without causing dysbiosis, can be used against multidrug resistant bacteria, or recombinantly modified to improve activity. Interestingly, such depolymerases have been recognized, which degrade EPS in biofilm thus helping in penetration and targeting resident bacteria. KPO1K1, a depolymerase synthesized by lytic phage can specifically target K. pneumoniae B5055, which is a frequent cause of nosocomial infection. The enzyme decreased the size of the microcolony with subsequent alteration of even old biofilm matrix.¹⁴⁹ Depolymerases can be used as tail-spike proteins (TSP) along with whole virions where biofilms are present as a barrier in the effective delivery of drugs during clinical infections. However, if the phages can pose safety problems in terms of the transfer of genes for encoding toxins and antibiotic resistance, then depolymerase proteins may be employed as more genetically stable, faster, safer, and effective options.

Bacteriophage or Phage-derived Enzymes as Bacterial Biofilm Mitigating Agents—Advantages Offered

Phage studies have given sufficient evidence for the versatile and potential application of phage therapy in the field of medical, agriculture and food processing.¹⁵⁴ Phage therapy is mainly focused on the lytic phages since they completely rupture the bacterial cells. Being highly host-specific, they recognize only one bacterial species or sometimes one particular strain only. As phages are ubiquitous in all-natural environments; it is possible to isolate them against any target bacteria. These “green” alternatives also have a much less environmental impact compared to standard antibiotics.¹⁵⁵ In addition, being viruses for bacteria, phages are unlikely to infect mammalian cells. A number of studies have shown that they have minimum or low inherent toxicity, no major side effects, or localized reactions.^156,157 Another advantage is that phages will have minimum impact on commensal bacterial flora in contrast to broad-spectrum antibiotics.¹⁵⁸

The ability of bacteriophages to penetrate the host cell can be used to treat the bacterial infection such as in the treatment of multidrug resistant bacterial infection or infections due to the development of biofilm around medical devices where it is hard to treat the infection due to the thick biofilm matrix. Using lytic phages also prevents the horizontal gene transfer because these phages lack the integrases and other enzymes which are mainly responsible for the horizontal gene transfer.¹⁵⁹ The specificity of phages to counter any particular bacteria is generally limited to a single strain which also limits the strain bio-spreading. This property of phages can be problematic in the process of treatment of acute infections, where a swift response is required. However, these issues can be countered by using multiple phages at the same time. These mixtures of phages are called phage cocktails. As the different phages in a cocktail target different receptors on host bacteria, studies have reported that such cocktails delay the development of phage-resistant mutants. Moreover, these additional phages also work as puncture devices that lead to the lysis of bacterial cells,¹⁶⁰ though incompatible interactions between phages are also possible in certain conditions.

The applications and potential of phages or phage derived products for biofilm control have been reviewed in some recent works, in which multiple techniques of phage usage, like single phage dilution, multiphage cocktails, a phage derived proteins, a mixture of phages along with different antibiotics as well as genetically modified phages have been described as the mitigating strategy for biofilms.^{90,105,115,123,124,150,161,162} Most of the bacteria given in the WHO priority pathogens list,¹⁶³ have been targeted by phage therapy in various ways (Figure 4). We present here some evidence-based data for all major approaches of phage therapy, namely, monophage, phage cocktail, a cocktail with antibiotics, phage-derived proteins, and genetically modified phages or enzymes, against common opportunistic and drug-resistant pathogens including ESKAPE in clinical settings.

Figure 4 Bacteriophage therapies have shown promising results with WHO priority 1 and 2 pathogens.

Evidence-based Data for Major Pathogens Using Monophage

Conventional phage therapy is using a single phage against the bacterial pathogen and multiple studies have reported successful results in ex vivo and in vivo settings. Some significant examples were oral and buccal biofilms of E. faecalis, F. nucleatum, Streptococcus mutans, or Aggregatibacter actinomycetemcomitans were treated with specific phages and survival of bacteria was studied. For E. faecalis, EFDG-1 phage brought a 5–7 log fold reduction in viable counts in an in vitro model;¹⁶⁴ whereas the specific phage ΦAPMO1 brought complete inhibition of biofilm activity and 5 log₁₀ reduction in viable counts for S. mutans.¹⁶⁵ In another in vitro experiment, biofilms of A. actinomycetemcomitans, commonly associated with aggressive periodontitis and even infective endocarditis, were treated with AabΦO1, AabΦO1-1 which brought down viable count by 2.3 log₁₀ without much affecting amount of biofilm.¹⁰⁹ The synergistic effects of phage with antibiotics have been reported to cover a broader range of periodontopathogens.⁵

A recent work by Dakheel et al has reported two novel phages against MRSA biofilms. Two phages, UPMK-1 and UPMK-2 were isolated from lake and sewage water, using 25 biofilm-producing MRSA strains and both were assessed for biofilm degradation in in vitro and in situ settings. Though the biofilm was not completely removed even after 24 h treatment of phages in the microtiter plate assay method, there was an increase in phage titer reflecting the replication and propagation of phages within the biofilm.¹⁶⁶ Another lytic, double-stranded DNA staphylophage, Stau2 was also identified and characterized, which was able to lyse 80% of clinical isolates (164/205) of S. aureus. Complete lysis within three hours of phage treatment was seen in most isolates and 100% protection was reported in experimental mice after lethal infection with S. aureus.¹⁵² Bacteriophages have shown good antimicrobial activity against biofilm-producing A. baumannii strains along with their therapeutic potential against drug-resistant A. baumannii in urinary tract infections.¹⁶⁷

In a recent report by Monto et al, the efficacy of eight specific and novel phages was assessed for their biofilm disruption and susceptibility towards nine MDR E. coli O177 strains on artificially contaminated beef samples. A log reduction in viable E. coli cells was observed for seven days and during which two individual and three phage cocktails showed a reduction of cell counts below the detection limit (1.0 log₁₀ CFU/g); whereas greater efficacy of phage cocktails than monophage was obtained in the destruction of preformed biofilms.¹⁶⁸

Roach et al have used monophage therapy in the treatment of respiratory infection by P. aeruginosa (10 CFU). When this modeled monophage therapy, using PAK_P1 was used in the treatment of acute pneumonia by MDR P. aeruginosa in a mouse model, it provided the experimental basis to reveal that neutrophil-phage synergy is essential for resolution of pneumonia. This immunophage synergy led to the tolerance of these therapeutic phages by lung immune effector cells. It was also explained that phage resistance can emerge but does not result in therapeutic failure in healthy immunocompetent hosts. Treatment mainly fails when the host’s innate immune system fails to eradicate phage-resistant subpopulations.¹⁶⁹

The phage TSK1 of Siphoviridae family has demonstrated a narrow host range against K. pneumoniae, including the MDR strains. TSK1 was able to remove 85–100% biomass in bacterial biofilms of different ages, whereas pretreatment by phage reduced >99% of K. pneumoniae biofilm within 24 h of incubation.¹⁷⁰ In another study by Li et al, a recombinant of T4-like phage, WGqlae is designed by modifying the receptor specificity determinant region of gene 37 and has been found to lyse four additional E. coli hosts. WGqlae has demonstrated significant antimicrobial effects on E. coli DE192 and DE205B strains in planktonic tests, along with improved inhibitory effects on biofilm formation as well as clearance of mature biofilms.¹⁷¹ Similarly, Arumugam et al have identified 94 bacteriophage samples from sewage water against P. aeruginosa. These phages were able to infect 34 MDR P. aeruginosa strains too including hundred other pathogenic strains of P. aeruginosa with variable infectivity patterns, however, improved results were obtained when used in combinations.¹⁷² A novel lytic mycobacteriophage, PDRPxv is characterized by having a short latency period and a large burst size thus making it a promising therapeutic candidate against pathogenic Mycobacterium spp.¹⁷³ The successful removal of P. aeruginosa biofilm by phage therapy in the mouse model of cystic fibrosis is another such example. In this study, an artificial sputum medium biofilm model and biofilm-associated murine model of chronic lung infection were developed to study the efficacy of a novel phage, PELP20 against P. aeruginosa LESB65 strain. After 24 h treatment of biofilm with phage, a 3-log reduction in P. aeruginosa CFU is reported. In addition, in the murine model studies, the novel phage showed complete clearance of bacteria after 48 h postinfection and also from the lungs of mice in established infection of six days.¹⁷⁴

Evidence-based Data for Major Pathogens Using Phage Cocktail

Recent observations favor the in vivo use of phage cocktails against bacterial biofilms, in particular, with the multispecies biofilm. More than monophage therapy, the approach of using two or more phages ie, polyphage therapy is more useful because this gives additional advantages in terms of widening of host range, lesser chances of developing phage-resistant bacteria and targeting two or more pathogens. When more than one phage is used, each will have a different receptor for recognizing and attaching with bacteria. For instance, in a study by Alves et al, they have isolated six novel lytic phages against a wide range of P. aeruginosa clinical isolates. Three DL52, DL60, DL68 were identified to belong to Myoviridae, and the rest DL54, DL62 and DL68 of the Podoviridae family. This phage cocktail was assessed for biofilm-inhibiting activity in static and dynamic biofilm models. The six phages were able to eliminate 95% of biofilm biomass within four hours in the static model. Whereas in the dynamic biofilm model, the majority of the cells (>4 log) were eliminated after 48 h of phage cocktail treatment.¹⁷⁵ In another in vivo study, a six phage cocktail was evaluated for its bactericidal activity in mice model with acute respiratory infection caused by P. aeruginosa. The cocktail of four novel phages (PYO2, DEV, E215, E217) and two previously identified phages (PAK_P1 and PAK_P4) was used against 40 clinical strains of P. aeruginosa, including isolates from Italian cystic fibrosis patients. The phage cocktail cured acute respiratory infection in mice and prevented bacteremia in Galleria mellonella larvae. This was found to be superior in terms of its ability to provide prophylaxis, target MDR or mucoid pseudomonal strains and was able to infect 97% of all P. aeruginosa strains tested.¹⁷⁶ Another promising phage cocktail is EFDG1 and EFLK1, effective against planktonic and biofilm cultures of vancomycin-resistant Enterococci. The two phages together have shown improved activity profile against antibiotic resistant E. faecalis V583 strains and even phage-resistant mutant strain EFDG1r.¹⁶⁴ However, an unknown and uncontrolled orientation of phages in a cocktail can be a potential drawback of this strategy.

A major pathogen in orthopedic and joint implant infections is Staphylococcus, specifically coagulase negative Staphylococcus or methicillin-resistant S. aureus (MRSA) in the majority of the cases. In one such study by Kaur et al, the combination of phage and linezolid was coated on naked wire and was surgically implanted in the intramedullary canal of mouse femur bone; along with hydroxypropyl methylcellulose (HPMC) coated and naked wire in other study groups. This was followed by inoculation of MRSA and results have shown that the combination of phage linezolid has the maximum reduction in adherence of MRSA and related inflammation. No emergence of the resistant mutant was also reported by the authors.¹⁷⁷ In a phase I clinical trial, the safety profile and tolerability of phage cocktail was investigated in patients with recalcitrant chronic rhinosinusitis by S. aureus. Nine patients (median age 45) who received chronic rhinosinusitis due to S. aureus were divided into three cohorts and two intranasal irrigants of phage cocktail AB-SA01 at 3×10⁸ PFU daily for seven days or 14 days or 3×10⁹ PFU for 14 days. All groups completed the trial with no serious adverse effects and AB-SA01 was well-tolerated up to a concentration of 3×10⁹ PFA for 14 days. No changes in body temperature and biochemical parameters were observed in all cohorts.¹⁷⁸

Yilmaz et al have successfully demonstrated the reduction in P. aeruginosa and S. aureus biofilm formation by phage-treatment alone (2.3-fold) and improved results by an antibiotic-phage combination (8.6-fold). They have implanted a plastic intravenous catheter having established biofilm into rat tibial medullary canal and assessed all study group animals after two weeks for implant-associated osteomyelitis.¹⁷⁹ In an MRSA osteomyelitis rabbit model, a cocktail mix of seven phages was tested by Kishor et al. These phages were isolated against clinical MRSA strains and were demonstrated to clear infection in active as well as chronic osteomyelitis within one week.¹⁸⁰ Gibb and Hadjiargyrou, in an extensive review on phage therapy for bone and joint infections, have elaborated on its potential for orthopedic infections and the current status of in vitro, in vivo, and animal studies.¹⁸¹

For crystalline or non-crystalline biofilm-related urinary tract infections (UTIs), single phage or phage cocktails have been studied with promising results. For instance, when a phage cocktail containing three phages, 39APmC32, 65APm2833 and 72APm5211, were tested against 50 uropathogenic P. mirabilis strains related to catheter-associated urinary tract infections, a better biofilm destruction profile (2–3 strains more targeted compared to a single phage) was demonstrated, without inhibiting each other’s activity when used in gel or liquid form to wipe the surface of the catheter.¹⁸² As reported by Fu et al the effectiveness of a single phage against P. aeruginosa was found to be only for 24–48 h, after which the regrowth of biofilm and appearance of resistant strains was observed. However, when a five-phage cocktail was used in the same setting, a 99.9% reduction in biofilm formation and equally significant delay in the emergence of resistant strains was reported.^19,183 Another important catheter-associated organism, P. mirabilis can also progress to complicated UTI infection due to the organism’s unique ability to form crystalline biofilms. In a detailed review on P. mirabilis biofilms associated with catheters, one of the potential documented approaches is phage therapy—either single, cocktail or with antibiotics. The phages against P. mirabilis biofilms were identified from the order Caudovirales, with the majority belonging to Siphoviridae, Podoviridae and Myoviridae lytic phages.¹⁸⁴ In addition, a two-phage cocktail was used for P. mirabilis in a dynamic model simulating a catheter-associated biofilm. A significant reduction in the number of viable bacteria in 96 h after treatment with phage cocktail was demonstrated by electron microscopy.¹⁸⁵ In another study, multidrug resistant P. mirabilis strains were 99.9% reduced by a mixture of five specific phages isolated from sewage water.¹⁸⁶ A cocktail of six specific phages were used with a significant reduction in bacterial count in a mixed biofilm of P. aeruginosa and P. mirabilis.¹⁸⁷

The phage cocktails studied for UPEC are reviewed in detail by Malik et al.¹⁸⁸ In one such example, multidrug resistant E. coli was effectively removed (67% bacteriolytic activity) by T4 and KEP10 phage cocktails in an in vivo study using a mouse model.¹⁸⁹ A three-phage cocktail showed 80% lytic activity when used against a subset of biofilm forming UPEC strains.¹⁹⁰ In an in vivo study on burn wound infection in mice, a combination of five novel phages (Kpn1 – 5) against K. pneumoniae was used and bacterial load, wound contraction and histopathological analysis were done at different periods posttreatment. It was found that in the test group of phage cocktail, the bacterial load in skin tissue was reduced by 3 log CFU/mL on day three. The combination was also reported to give maximum protection against K. pneumoniae B5055 compared to any monophage group.¹⁹¹

Evidence-based Data for Major Pathogens Using Phages in Combination with Antibiotics

Antibiotics and phages work very differently on bacterial infection because phages are usually strain-specific, conversely antibiotics are multispectral drugs that treat a range of bacterial species. Still, phages have an advantage over antibiotics since they are strain-specific and can only target a single strain leaving the rest of the microflora untouched.¹⁹² In addition, the phage can emerge and mutate according to the stress and conditions given, which makes them better than the antibiotics because in the case of antibiotic use bacteria usually develop the resistance resulting in the restriction of antibiotic effect inside biofilms. In combating bacterial resistance, phage-antibiotic therapy has shown better results in multiple reports. It has also shown reduced chances of emergence of double resistance in bacterial pathogens, which means simultaneous resistance against antibiotics and phage is unlikely.¹⁹³ Furthermore, the staggered application of antibiotics and phage cocktail have been found to be more effective in eradicating biofilms. In their latest peer-reviewed paper, Morrisette et al¹⁹⁴ have discussed in detail the interactions between phage and antibiotics and the resultant effects on improved biofilm eradication, reduced bacterial growth, and alterations in bacterial resistance patterns.

The phenomenon of stimulation of host bacterial cell’s production of some virulent phage, after treatment with a sub-lethal concentration of antibiotics, is given the term phage-antibiotic synergy (PAS). It is shown that the production of ϕMFP phage is increased by more than seven-fold in uropathogenic E. coli strain when it is treated with a subinhibitory concentration of cefotaxime. Similar stimulation was seen in T4 like phages with β-lactam and quinolone antibiotics.¹⁹⁵ The idea is to increase the combined bactericidal effect.

A successful study was conducted where the Sb-1 S. aureus phage was given in combination with antibiotics to effectively eradicate the biofilm matrix. In the study, the biofilm of methicillin resistant S. aureus (MRSA) ATCC 43300 was treated with Sb-1 alone or in combination with five antibiotics, namely fosfomycin, rifampin, vancomycin, daptomycin or ciprofloxacin. Pretreatment with Sb-1 followed by subinhibitory concentration of antibiotics resulted in the eradication of MRSA biofilm in a dose-dependent manner. The phage at a concentration of 10⁷ PFU/mL had a direct killing effect on persister cells (5×10⁵ CFU/mL).¹⁹⁶ Combination therapy was also found effective when T4 phage was given in the combination with tobramycin or cefotaxime and resulted in a 99% reduction in antibiotic resistant cells and 39% reduction of phage resistant cells in E. coli biofilms.¹⁹⁷ In another study, a 12-phage cocktail PP1131 in combination with the different antibiotics was studied for its effectiveness in the treatment of experimental endocarditis due to P. aeruginosa. A single dose phage destroyed 7 log PFU within six hours in vitro and the phage resistant mutants which grew after 24 h were effectively removed by phage combined with ciprofloxacin at 2.5× MIC. Whereas, the combination of phage cocktail (≥3 log10 CFU/mL) with ciprofloxacin led to successful treatment of 64% of experimental rats, demonstrating that phage-antibiotic cocktail was highly effective in the treatment of fibrin clots compared to the monophage-therapy with antibiotic or even with the phage cocktail alone.¹⁹⁸ A similar test was conducted for the eradication of the P. aeruginosa biofilm using PB-1 bacteriophage and tobramycin combination which gave 60% and 99% reduction in antibiotic and phage-resistant cells respectively.¹⁹⁷ In another study, the efficacy of Pseudomonas specific phages, NP1 and NP2, isolated from sewage water was tested against P. aeruginosa PA14 and its resistant mutants, PA14Cip-R and PA14Gen-R, which were resistant against ciprofloxacin and gentamicin respectively. The biofilms of P. aeruginosa PA14 were treated in vitro either with phage alone or a combination of two phages and five antibiotics; in which phage NP1 and NP2 in combination with different antibiotics were found to be more effective compared to the monophage treatment or treatment via antibiotic alone. It was also observed that this combined treatment is specifically effective in killing of Pseudomonas biofilms grown on cultured epithelial cell layers.¹⁹⁹

Evidence-based Data for Major Pathogens Using Phage-derived Endolysin Enzyme

Reports of the efficacy of lysins in elimination of biofilms of gram-positive organisms have started appearing since 2007 when Φ11 endolysin was successfully used for reduction of S. aureus biofilm.²⁰⁰ In a detailed review on endolysins as a therapeutic alternative for drug-resistant pathogens, Gondil et al have presented several examples where endolysins have been reported to be effective against fatal infections caused by S. aureus, S. pneumoniae, S. pyogenes, Mycobacterium spp., E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, etc.²⁰¹ Lysins have been found to be effective for catheter-associated biofilm removal. S. aureus lysin CF-301 was found to have a strong antibiofilm effect on human synovial fluid which frequently forms a resistant biofilm during joint infections. The lysin also removed biofilms within one hour from other surfaces such as surgical mesh, catheters and polystyrene. Apart from S. aureus, it was effective against coagulase negative S. aureus, Streptococcus pyogenes, Streptococcus agalactiae as well as effectively killing persister cells of S. aureus. Endolysins showed better activity against suspended S. aureus biofilm compared to intact or scraped biofilms.¹⁸⁵ The activity of CF-301 was much improved by using it with lysostaphin, a cell wall hydrolase.²⁰² Streptococcus uberis, a common cause of bovine mastitis, was effectively killed in vitro by two endolysins PlySs2 and PlySs9 isolated from Streptococcus suis serotype −2 and −9 prophage respectively. Of the two, PlySs9 was reported more potent than PlySs2, with lower MIC and one log higher killing.²⁰³

Though the outer membrane in gram-negative bacteria resists the action of endolysins from outside, many novel lysins have been studied and reported with specific and improved killing actions. P. aeruginosa, cause of nosocomial infections by this gram-negative MDR strains affecting the urinary tract, surgical wound as well as blood infections due to catheters and implants to have been a cause of high morbidity and mortality. PlyPa03 and PlyPa91 are two promising lysins that have shown good bactericidal properties against many clinical and biofilm/embedded strains of P. aeruginosa. PlyP91 has shown efficacy in P. aeruginosa skin infection and PlyPa03 has been reported to be effective in P. aeruginosa pneumonia, on mouse model in vivo studies.¹³⁶ Lys PA26 is a novel endolysin that is used in P. aeruginosa biofilm disruption without the need to add outer membrane permeabilizers. This has also been found to be effective against A. baumannii, K. pneumoniae, and E. coli up to a temperature range of 50°C.¹²⁹

Some of them have been studied in vivo too in animal models. Lood et al have for the first time shown the in vivo efficacy of gram-negative lysin Ply307, against multidrug resistant A. baumannii. Twenty-one distinct lysins were identified from prophages induced from 13 A. baumannii strains. Ply307 brought >5 log unit decrease in A. baumannii isolates, also significantly reducing planktonic and biofilm organisms in vitro as well as in vivo. The efficacy of PlyF307 was tested against A. baumannii biofilms formed on PVC catheters. The Scanning Electron Microscopy has shown that much of the EPS of biofilm on these catheters were destroyed.¹²⁷ LysAB2 lysin isolated from A. baumannii ΦAB2 showed broad bactericidal activity against MDR A. baumannii, E. coli as well as S. aureus. The antimicrobial peptides from the C-terminal of LysAB2 were also found to be bactericidal for A. baumannii both in vitro and in vivo. Some other A. baumannii specific lysins are lysABP-01, PlyAB1, Ply6A3, ABgp46 which have shown promising results in vitro against several MDR A. baumannii strains.¹²⁵ The phage lysin ABgp46 was additionally active against P. aeruginosa and Salmonella enterica; whereas Ply6A3 lysin showed a 70% bactericidal action against MDR A. baumannii and protected the experimental mouse model from a lethal A. baumannii septic challenge.¹³¹

Other gram-negative pathogens which have been targeted by lysins or their recombinant improved versions are E. coli, K. pneumoniae, Burkholderia cenocepacia, and Salmonella enterica. Lysin KP27 against drug resistant K. pneumoniae, EndoT5, and Lysep3 lysins coliphages against E. coli are few such examples that have been shown to be active even in the presence of different outer membrane factors. The recombinant version of Lysep3 fused with D8 domain has shown improved activity.²⁰⁴ Lastly, “amurins” which are a group of phage-encoded antimicrobial peptides, have shown remarkable bactericidal activity against gram-negative pathogens as well as for the removal of biofilm embedded bacteria. In a proof-of-concept study, the peptide App2-MI amurin was tested Stenotrophomonas maltophilia biofilms on hemodialysis catheters. The amurin was found to be effective at a concentration of 1 µg/mL in eradicating the biofilm.¹²⁵ Table 3 gives a list of endolysins identified from different phages and targeted against common gram-positive and gram-negative pathogenic bacteria.

Evidence-based Data for Major Pathogens Using Phage-derived Depolymerase Enzyme

Among some of the significant examples for this enzyme is depolymerase Dpo7, derived from vB_SepiS-phiPLA7 phage, which has shown the reduction of Staphylococcus spp. biofilm biomass by 53–85% in 67% of the bacterial strains tested in a dose-dependent; but time-independent response.²⁰⁵ Another test based on the biofilm disruptive activity of depolymerase is Dpo42, derived from phage vB_EcoM_ECOO78. Its antibiofilm activity was tested against multiple bacteria like E. coli where it showed dose-dependent biofilm prevention activity.¹²⁹ Depolymerase Dpo1, isolated from the phage Petty, was found to be effective in removing biofilms formed by Acinetobacter strains. The enzyme was able to reduce EPS viscosity, thus reducing the virulence of tested strains; however, it was not very effective in destroying Acinetobacter biofilms as only 20% reduction in that was observed.²⁰⁶ IME180 is another phage carrying a functionally active depolymerase gene, against P. aeruginosa biofilm. The purified enzyme inhibited bacterial biofilm formation and reduced preformed biofilm biomass.²⁰⁷ In a study by Olsen et al, lysin LysK and depolymerase DA7 have been tested in combination against S. aureus biofilms in static as well as in dynamic models, where they showed synergistic behavior, dropping the number of viable cells in biofilm.²⁰⁸ Another depolymerase enzyme Dep42, specific for K. pneumoniae is identified in bacteriophage SH-KP152226 of Podoviridae family. Dep42 was demonstrated to cause lysis of capsular K. pneumoniae. In biofilms, the depolymerase caused degradation of EPS causing the removal of attached cells. This may prove useful in combining it with other antimicrobial agents to effectively remove biofilms.¹³¹

The constitution of biofilm, whether single or multispecies also affects the depolymerase effectiveness in removing biofilms. As mixed bacterial communities are present mostly in naturally occurring biofilms than a single species, the use of a single depolymerase may have limited effect. The different types of EPS in such mixed communities may limit the penetration of antibiofilm agents through biofilms or may even trap phage in biofilm matrix or reduce their multiplication due to the presence of metabolically inactive cells or reduced availability of receptors or inhibited depolymerase activity.²⁰⁹ In addition, there can be huge heterogeneity in the polysaccharide content of EPS layers of bacterial biofilms; thus, a depolymerase that is effective for breaking cell wall polysaccharides may not be effective for degrading EPS layers. This may limit the range of polysaccharides that can be targeted by a specific depolymerase, even the cell surface polysaccharide of closely related bacterial hosts may not be recognized.²¹⁰ Thus, a combination therapy using two or more agents has also been studied with promising results. In this approach, a phage derived enzyme depolymerase is mixed with different active antimicrobial agents like antibiotics, phages, lysins, detergents, chemicals or natural compounds to improve the efficacy. For example, Dpo42 mixed with polymyxin and KPO1K2 depolymerase mixed with gentamycin have given much improved results in reducing K. pneumoniae bacterial biofilms.¹³¹ Similarly, a combination of depolymerase with honey and EC3a phage with depolymerase activity against E.coli biofilm¹⁵³ have more efficient antibiofilm activity than phage or its enzyme alone. Another strategy is to combine alginate lyase with antibiotics for targeting P. aeruginosa in biofilms. The mucoid strains of P. aeruginosa frequently produce alginate which resists antibiotic penetration thus presents a problem in managing drug-resistant and opportunistic P. aeruginosa. The effective killing of P. aeruginosa in biofilms by combining antibiotics with alginate lyase as an adjuvant has been reported. This effect of alginate lyase in dispersing through biofilm was independent of antibiotic action.²¹¹ Many more such examples can be obtained in the review on bacteriophage depolymerases by Topka-Bielecka et al.³⁰

Evidence-based Data for Major Pathogens Using Genetically Modified Phage or Phage-derived Enzymes

Phages can be genetically modified to increase their host range and also to increase their survival in the biofilm matrix, eg a modified T7 E. coli phage was designed to express intracellularly a hydrolase, which is released during infection to the extracellular matrix, enhancing the biofilm degradation. This test showed 99% eradication of E. coli biofilm.²¹² Bacteriophage K1F-GFP is another engineered phage, using CRISPR/Cas selection method, is targeted specifically against E. coli K1, a common nosocomial pathogen for urinary tract infections. The engineered fluorescent phage, K1F-GFP, and E. coli EV36-RFP enter cells via phagocytosis, phage efficiently kills intracellular E. coli from T24 human urinary bladder epithelial cells, and finally, both are degraded by phagocytosis and xenophagy.²¹³ In a study done by Tinoco et al, a lysogenic _Ef11 E. faecalis phage was genetically modified to eliminate all the genes related to lysogeny, eliminating transduction problem and achieving a noteworthy drop in the population of E. faecalis biofilms, wherein vancomycin-resistant as well as sensitive strains were used.¹⁵⁹

One of the most important characters of genetically modified phages is their ability to infect multiple hosts. For example, a T7 coliphage was modified by the insertion of a sequence for 1080. It is a short peptide with a broad spectrum antibiofilm effect, making it a broad spectrum T7 phage.²¹⁴ Temperate phages may be of such interest for delivering programmable DNA nucleases associated with CRISPR to reverse antibiotic resistance. This system can selectively annihilate plasmids that confer antibiotic resistance.²¹⁵

In addition, lysins have also been engineered to modify their target specificity. As an example, chimeric lysin Cs12, obtained by fusion of the catalytic domain of Cp1-7 lysozyme to the CW-7 repeats of the LySMP lysin from a Staphylococcus spp. have increased the target specificity. It was designed to remove Staphylococcus spp. biofilm in vitro and validated in vivo with a zebrafish infection model.²¹⁶ Another chimeric lysin P128 showed bactericidal activity against S. aureus and 99% reduction effects on S. epidermidis and Staphylococcus haemolyticus biofilms.²¹⁷ The removal of biofilms in endocarditis and catheter associated infections is reported from an animal model study by this novel recombinant chimeric ectolysin, P128 which has potential antistaphylococcal activity.²¹⁸ In the study, BALB/c mice were injected with a single dose of P128. The ectolysin exerted a quick bactericidal effect and inhibited the fatal MRSA and VRSA invasion in the test group.²¹⁹ P128 ectolysin has also shown potent antibiofilm activity on planktonic and biofilm embedded cells. The effect was seen for both resistant and sensitive isolates of S. aureus and also in synergy with standard-of-care antibiotics.²²⁰ P128 has also completed a Phase II trial for safety and efficacy in healthy volunteers and patients (https://clinicaltrials.gov/ct2/show/NCT01746654).

In another work by Singh et al, Plt187AN-KSH3b, a chimeric phage endolysin derived from Ply187 was evaluated for its therapeutic potential against ocular infections, specifically S. aureus endophthalmitis in a mouse model. They have reported strong antimicrobial activity of the chimeric endolysin against methicillin-sensitive S. aureus and MRSA, both without any resistance development.²²¹ A novel chimeric lysin, ClyV, is constructed against S. agalactiae by fusing the EAD domain of PlyGBS lysin with the CBD domain of PlyV12 lysin. The engineered lysin has shown better bactericidal activity than the parental enzyme and a single intraperitoneal dose of 0.1 mg of ClyV was able to provide 100% protection against S. agalactiae infection in experimental mouse models, thus demonstrating the potential role of this chimeric lysin against antibiotic resistant beta hemolytic streptococcal infections.²²² The engineered LysH5 lysin was studied for its potential to kill S. aureus persister cells with 100% efficacy at a minimum concentration of 0.15 µM.²⁰⁵

A recombinant version of CF-301, Exebacase a potent antistaphylococcal lysin with many improved features of lower resistance and synergy with conventional antibiotics, is the first direct lytic agent (DLA) clearing phase 2 clinical trial. In this clinical study done on 121 patients with S. aureus bloodstream infection or endocarditis, a single dose of exebacase given with standard antibiotics has caused a 43% point higher clinical response rate in the MRSA subgroup. This is the first proof of concept study for the therapeutic potential of exebacase for MRSA caused bloodstream infections and endocarditis.²²³ Watson et al have reported synergistic activity of exebacase with 12 broad range antibiotics.²²⁴

Many examples of recombinantly designed lysins with improved activity against gram-negative bacteria are also being reported. For example, Art-175 lysin is a fusion protein that contains lysin fused to an outer membrane destabilizing peptide and is active against both gram-negative and gram-positive bacteria.¹³⁵ It has also shown the independent activity against the biofilms of multidrug resistant P. aeruginosa and caused the osmotic lysis in bacterial metabolism. This provides evidence that even at low metabolic rates the lysins can disrupt persistent bacteria within the biofilm.²²⁵ Lysocins are bioengineered lysin-bacteriocin fusion molecule which can translocate through the outer membrane of P. aeruginosa. This proof-of-concept study by Haselpoth et al has shown efficient peptidoglycan cleavage, the log-fold killing of P. aeruginosa, effective biofilm disruption, and no toxicity, thus outperforming standard-of-care antibiotics.²²⁶

Challenges in Phage Therapy and Future of Phage Research

Bacteriophages have been studied for a long time to treat infections in humans, however, bacteria also have different strategies to prevent infection of phages, namely, blocking surface receptors, alter mechanism, secrete extracellular polymeric capsule, quorum sensing, secretion of phage inactivating enzymes, presence of EPS matrix, etc. Thus, phage-bacteria interactions in a biofilm must be understood completely to get successful outcomes. Factors such as the metabolic activity of bacteria in biofilm, the density of biofilm, old or aged biofilm are some important aspects that govern whether bacteriophage can penetrate the biofilm or not and thus must be considered to achieve greater efficacy. In a recent work by Simmons et al, the coexistence of phage resistant and phage susceptible bacteria and dynamics of phage resistance in a biofilm is demonstrated, using resistant and susceptible E. coli strains. In these in vitro and simulation studies on the spatial structure of a biofilm, they have reported that at initial stages when phage is introduced, and phage-resistant bacteria are rare, large interaction of phages with susceptible cells leads to killing of host cells. Space thus created is occupied by resistant cell clusters; whereas when resistant bacteria are common at the time of addition of phage, they make a barrier for the phages to reach a susceptible host, and diffusion is impeded. That is how the susceptible cells are shielded by resistant cells. This barrier effect of resistant cells is an important factor to be considered for phage therapy efficacy and for it to be successful, the biofilm architecture must be disrupted for exposing target bacteria to phages.¹⁰²

Moreover, in a multispecies biofilm matrix, the comparative health depends upon the amplification of phage within the biofilm matrix as well as on the availability of nutrients and oxygen to different bacterial species. The study by Harcombe and Bull suggested that multiple species composition of biofilm can influence the proliferation, penetration of phage inside biofilm matrix thus affecting the phage therapy.²²⁷ Usually, lab studies are focused on the single species biofilm matrices where it is hard to determine the success of bacteriophage infection inside the biofilm matrix. This leads to a constant debate on the usefulness of phages in targeting the bacterial population. Even though phages have been reported to be safe and no side effects have been documented, still it is hypothesized that phages should be administered locally at the site of infection and not systemically.²²⁸ There are almost no studies that have compared local vs systemic administration of phages or quantification of anti-phage antibiotics in biofilm infections. Therefore, for their effective clinical applications, extensive in vivo studies should be done for assessing the response of plasma proteins and components of the host immune system.³⁰ Figure 5 reproduced with permission from Hassan et al depicts the friend and foe roles of bacteriophages, in which as bacteriophriend they have potential in phage therapy and biofilm removal, whereas bacteriophage as foe can be a vehicle for transfer of antibiotic resistance and other virulent factors.⁹⁹

Figure 5 Bacteriophage—Friend vs Foe. The impact of bacteriophages with respect to antimicrobial resistance are summarized, depicting the beneficial applications on the left and the potential risks on right. Notes: Reproduced from Hassan AY, Lin JT, Ricker N, Anany H. The age of phage: Friend or foe in the new dawn of therapeutic and biocontrol applications? Pharmaceuticals. 2021;14(3):1–36. doi:10.3390/ph14030199.99 Copyright: © 2021 by the Her Majesty the Queen in Right of Canada as represented by the Minister of Agriculture and Agri-Food Canada; Licensee MDPI, Basel, Switzerland. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Although phages are highly versatile and stable in storage²²⁹ and have been tried in all forms, such as liquids, creams or impregnated solids via different administration routes like oral, intramuscular, intravenous, topical, or superficial;¹⁵⁷ therapeutic limitations of bioavailability, rapid clearance or non-targeted delivery have been reported. To overcome that, encapsulation of phage or endolysins into a suitable drug delivery system is an alternate approach that is being extensively explored.²³⁰ Lastly, though several clinical trials have been documented until now for chronic wound infections²³¹ or infectious complications of surgical wounds, burn infections and infected ulcers;²³² none of them were able to support completely the in vitro and in vivo studies. Górski et al have summarized some of the recent clinical trials and critically analyzed the reasons for the failure of these studies which were mainly due to insufficient phage coverage or overgrowth of other bacteria. According to them, the major focus points, which must be considered before initiating a clinical trial, should be quality (taking a well-characterized phage), titer of phage preparation (>10⁶/mL of phage titer is recommended) and patient’s antibody response during phage therapy.²³³

Conclusion

With the constant rise of incidences of hospital-acquired and community-acquired infections by common pathogens; the need for alternate therapies is increasing day by day. Phage therapy is gaining attention in clinical medicine as we are getting more and more insight into phage mechanisms. For example, in a recent report, Yan et al have demonstrated incorporation of A. baumannii specific phage into a hydrogel, which has shown a 90% reduction in microbial survival after four hours of the treatment in an ex vivo wound infection model using pig skin.²³⁴ A Staphylococcal bacteriophage, (monophage Sb-1) has been used to treat foot ulcers in patients with osteomyelitis, as well as PYO bacteriophage, a complex preparation designed to treat wound infection is also available commercially in the market.²³⁵ Another potential approach is phage-loaded biocompatible nanofibers for wound dressings.²³⁶ Similarly, some phage-based compounds to treat biofilms, have also emerged in the market and they have shown favorable results. In a recent work, two commercially available formulations of phage namely monophage Sb-1 and PYO (a polyphage) were evaluated for their eradication potential on in vitro biofilms of MRSA and also in a G. mellonella model of MRSA systemic infection for in vivo experiments. The co-incubation with the highest phage titers (10⁷ PFU/mL) was effective in the eradication of MRSA biofilm within seven days, along with preventing MRSA infection and increasing survival rate in G. mellonella larvae.²³⁷

Due to their specificity and narrow spectrum activity, bacteriophages provide an attractive alternative to overcome the increasing antimicrobial resistance,²³⁸ but not many promising results are coming out for their role in mitigating biofilms of medically important microbes. This may be due to the fact that most of the studies for bacteriophage or their derived proteins are done in vitro which have helped us to understand their role and ultimately their application in static environments, but a better understanding of phage dynamics, phage proteins, phage biology, and ecology is needed. Combining laboratory experimental data with newer approaches like metagenomics (especially “viromics”), empirical studies, bioengineering, mathematical models²³⁹ and computational studies have been helpful. However, it is important that we must have a robust clinical trial design and understanding of mechanisms involving co-evolution of bacteria and phage.