Bacteriophages and Lysins in Biofilm Control

Bacteriophages are viruses that specifically infect and kill their bacterial hosts. The growing threat of antimicrobial resistance (AMR) and the paucity of newly developed antibiotics has revived interest in using bacteriophages (phages) to combat AMR. So far no formal proof of effectiveness of phage therapy has been obtained, therefore it is still carried out as compassionate treatment (experimental therapy). Nevertheless, data from animal models and human clinics strongly suggest that phage therapy is safe and has possible beneficial actions (Górski et al. 2019; Hesse and Adhya 2019; McCallin et al. 2019).

Biofilms are communities of microorganisms adhered to both biological and abiotic surfaces. It may be involved in the pathogenesis of chronic diseases, especially infections associated with the use of catheters, drains and implant placement (Akanda et al. 2018; Maszewska et al. 2018; Morris et al. 2019; Taha et al. 2018). Bacterial cells are embedded in a self-produced matrix of extracellular polymeric substances (polysaccharides, proteins, lipids and nucleic acids). These structures are characterized by low susceptibility to antibiotics (Pires et al. 2017b). Increased cell density and physiological changes in a biofilm may be the cause of growing biofilm resistance to antibiotics (Mah and O'Toole 2001). Furthermore, biofilm is characterized by lower metabolic activity, slower growth and greater opportunity for exchange of antibiotic resistance genes, which is also responsible for the induction of antibiotic resistance (Akanda et al. 2018).

Due to the ineffective antibiotic therapy of biofilms, there has been growing interest in phages as a strategy in preventing biofilm formation and elimination. Some studies on the application of phages as an alternative strategy to prevent and control biofilm are encouraging (Pires et al. 2017b). However, the cells located in the deeper layers of biofilm in the absence of oxygen and nutrients show reduced metabolic activity, which affects the lower activity of antibiotics and the lower replication of phages (Pires et al. 2017b; Sillankorva and Azeredo 2014). Within the biofilm, acquisition of phage resistance is also observed (Pires et al. 2017b). The mechanisms by which bacteria acquire resistance to phage are known: prevention of integration of phage DNA into bacterial DNA; degradation of phage DNA by clustered regularly interspaces short palindromic repeats CRISPR/Cas; blocking phage replication, transcription and translation; prevention of phage adsorption by structural modifications of bacterial receptors or by inaccessibility of receptors due to the presence of matrix biofilm.

The phages can be applied as a single phage preparation or a phage cocktail. Phage cocktails increase the spectrum of phage activity and reduce the development of phageresistant variants (Pires et al. 2017b). Phage cocktails have proven to be efficient in preventing biofilm formation and biofilm eradication (Abedon et al. 2017; Abedon 2018). Phages can be used with other antibacterial agents, such as antibiotics, honey and disinfectants, to improve the effectiveness of biofilm elimination (Melo et al. 2019). Synergism of phages with mechanical debridement against biofilms has also been observed (Melo et al. 2019). Due to their ability to penetrate biofilms, bacteriophages may be applied with an antibiotic or as a substitute antibiotic in biofilm treatment (Abedon et al. 2017). Additionally, depolymerases (matrix degrading enzymes encoded by phage) can be used to prevent and disperse biofilm (Abedon et al. 2017; Chan and Abedon 2015; Pires et al. 2017b). Phage-derived lysins are bacteriophage enzymes that cleave peptidoglycan, the main component of the bacterial cell wall of either Gram-positive or Gram-negative bacteria, inducing lysis of a bacterial cell (Borysowski et al. 2011; Fischetti 2018). Recent investigations have indicated that phage-derived lysins can potentially be used as antibacterial agents (Borysowski et al. 2011; Gray et al. 2018; Lood et al. 2015; Sharma et al. 2018). This review is intended to explain the application of phages and lysins or in combination with antibiotics against biofilm formation or eradication as a possible strategy for removing bacterial infections.

Biofilm may form on catheters or implants during chronic infections such as urinary tract infections and orthopedic implant-related infections. During treatment of biofilm with single Pseudomonas aeruginosa phage, phage-resistant bacteria may form at 6 h after infection (Pires et al. 2017a). Phage-resistant variants have also been observed in human and animal studies (Oechslin 2018). The appearance of bacterial mutations may be the cause of emergence of phage-resistant mutants (Oechslin 2018). Bacteriophage cocktails delay the appearance of phage-resistant bacteria compared to single phages. Application of bacteriophage cocktails that target different host receptors is recommended to prevent phage-resistance (Bai et al. 2019). Phages with different ranges of lytic activity can be used as a single phage preparation or a phage cocktail to enhance their lytic activity. Phage cocktails improve the lytic effects by extending the phage host range and increasing the number of target pathogens (Oechslin 2018). According to various authors, phage alone or phage cocktails may be applied to prevent bacterial colonization and biofilm formation on such medical devices (Maszewska et al. 2018; Melo et al. 2016; Morris et al. 2019).

Urinary tract infections are the most widespread human infections and about 75% of them are associated with the use of a urinary catheter (Maszewska et al. 2018). Phage cocktails or single phages were used in vitro to against catheter-associated urinary tract infections causing by Proteus mirabilis or P. aeruginosa biofilms created on catheters or on a polystyrene plate (Fu et al. 2010; Maszewska et al. 2018; Melo et al. 2016). Comparison of the effects of a single phage and a phage cocktail is important in the treatment of biofilm (Fu et al. 2010; Maszewska et al. 2018; Melo et al. 2016).

A phage cocktail consisting of two phages against P. mirabilis was used on a dynamic biofilm model simulating a catheter-associated urinary tract infection (Melo et al. 2016). The phage cocktail significantly reduced the number of bacteria after 96 h and 168 h and influenced the lower colonization of bacteria, which were confirmed by fluorescence microscopy and electron microscopy study. Similarly, recent studies in vitro but on a polystyrene plate have demonstrated the positive effect of P. mirabilis phage cocktails on the prevention of biofilm formation and biofilm destruction (Maszewska et al. 2018). After 24 h of action of P. mirabilis three-phage cocktail on 24 h-old biofilm, elimination of P. mirabilis causing catheter-associated urinary tract infections at a similar or slightly greater level compared to phage alone was observed. Additionally, phages included in the cocktail did not inhibit each other's activity (Maszewska et al. 2018).

Studies in vitro compared the application of a single phage and a phage cocktail on biofilm formation of P. aeruginosa on hydrogel-coated catheters (Fu et al. 2010). Two hours prior to the administration of the bacteria, a single phage of P. aeruginosa M4 phage was applied at a dose of 10 log plaque forming units (PFU/mL) on the catheter. Such administration significantly reduced the number of bacteria on 24 h-old biofilm from 6.87 to 4.03 log CFU/cm² (colony-forming unit/cm²). Administration of phage simultaneously with bacterial inoculation significantly reduced the biofilm formation to 4.37 log CFU/cm². The reappearance of bacteria occurred between 24 and 48 h, but additional phage administration at 24 h significantly reduced the growth of biofilm. The pretreatment of 48 h-old biofilm with the P. aeruginosa phage cocktail resulted in a significant reduction of the number of bacteria from 7.13 to 4.13 log CFU/cm², but phage-resistant bacteria were rarely isolated. The potential use of a phage cocktail in reducing the formation of biofilm by clinically important bacteria was suggested (Fu et al. 2010). It is worth noting that application in vitro of a phage cocktail rather than single phages improves the killing of biofilm by increasing lytic activity of phages and preventing the appearance of phage-resistant bacteria (Fu et al. 2010; Maszewska et al. 2018). Recently Malik et al. (2019) have also highlighted the high efficacy of phage cocktails in combating biofilms and the possibility of enhancing their efficacy by combining with antibiotics and depolymerases.

The pathogenesis of peri-prosthetic joint infections is associated with adhesion of pathogenic bacteria to orthopedic implants and with formation of biofilm (Taha et al. 2018). Staphylococcus aureus and coagulase-negative staphylococci contribute to 50%–60% of peri-prosthetic joint infections (Akanda et al. 2018). The comparison of the use of phages and antibiotics in the removal of S. aureus bacteria is important in the treatment of orthopedic infections (Kaur et al. 2016; Morris et al. 2019). An in vitro study showed that S. aureus phage cocktail composed of five phage preparations reduced the number of bacteria from 6.8 log to 6.2 log colony forming units (CFU, P < 0.01) in biofilms on titanium surfaces, which is of great importance in orthopedic implant-related infections (Morris et al. 2019). No significant reduction of S. aureus biofilm was observed after antibiotic action of 100×MIC (minimum inhibitory concentration) of cefazolin (Morris et al. 2019). In vitro studies comparing the effect of a phage cocktail with an antibiotic in the treatment of a S. aureus biofilm-related orthopedic infection indicated the advantage of using a phage cocktail over an antibiotic (Morris et al. 2019). The study in vivo involved mice with post arthroplasty model of infection with S. aureus successfully treated with phages and linezolid (no mention on the presence of biofilm was supplied) (Kaur et al. 2016). Mice were implanted with a wire coated with phage and/or linezolid into the intra-medullary canal of the femur bone followed by inoculation of methicillin resistant Staphylococcus aureus (MRSA). Maximum bacterial burden in the surrounding joint tissue in mice implanted with the naked and polymer coated wire was achieved on day 5, reaching ~8 log CFU. Mice with phage coated wires indicated control of the tissue bacterial burden with significant reduction of > 3 log on day 5 and 7 and sterile tissue by day 10. A maximum significant decrease in bacterial load of ~4.5 log in the joint tissue from days 5 and 7 and sterile tissue by day 10 was observed in mice implanted with wires coated with phage and linezolid (mice with dual coated wire). Faster resumption of locomotion in this examined group was observed. Studies showed no formation of resistant mutants. Treating orthopedic device related infections with two agents (phage and antibiotic) is a potential approach to control infections caused by methicillin-resistant S. aureus (Kaur et al. 2016). However, in vivo studies have shown the best effectiveness in removing S. aureus bacteria from an orthopedic device, but without forming biofilm using a single phage and an antibiotic simultaneously (Kaur et al. 2016).

Acquired antibiotic resistance genes and biofilm formation are involved in oral diseases such as periodontal and periimplant disease (Khalifa et al. 2016; Pinto et al. 2016; Szafrański et al. 2017). Phages for the oral bacteria Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus spp., Neisseria spp., Streptococcus spp., and Veillonella spp. have been reported (Szafrański et al. 2017). The effect of phages on oral biofilm bacteria was investigated (A. actinomycetemcomitans, E. faecalis and S. mutans) (Szafrański et al. 2017). A. actinomycetemcomitans is involved in periodontitis, infective endocarditis and abscesses. E. faecalis is involved in tooth root canal infections and implant placement. S. mutans is also involved in dental infections such as caries. In all studies, the number of bacteria significantly decreased, by applying phage alone, from 2.3 log to complete bacterial removal. Phage killed 95% of A. actinomycetemcomitans bacteria but did not remove the biofilm matrix. Application of phage against S. mutans and E. faecalis biofilm in vitro reduced the number of bacteria by 5 log. The effectiveness of oral phages was not confirmed in vivo in an animal model of biofilm (Szafrański et al. 2017).

The biggest problem in recurrent root canal infections is vancomycin-resistant enterococci (VRE). E. faecalis phages may be used against biofilm in root canal infections (Khalifa et al. 2016). In vitro and ex vivo studies in the removal of oral E. faecalis biofilm have been compared (Khalifa et al. 2015). Single E. faecalis phage was applied in a 2-week-old biofilm in vitro (Khalifa et al. 2015). Phages at a dose of 10⁷ plaque forming units (PFU/well) were incubated with biofilm for one week. E. faecalis phage significantly reduced viable counts by 5 log within 7 days. The effective reduction of E. faecalis biofilm ex vivo in a human root canal model by treatment with E. faecalis phage was achieved. The root canals were contaminated with an E. faecalis suspension with OD₆₀₀ 0.1 and phage-treated group teeth were treated with phage 10⁸ (PFU/mL). In the phage-treated group the number of E. faecalis bacteria was reduced by 7 log after 48 h (Khalifa et al. 2015). Compared in vitro and ex vivo studies of application of single E. faecalis phages in the reduction of oral biofilm indicated better phage efficiency in ex vivo studies (Khalifa et al. 2015).

Synergy is the interaction between two factors, when the combined effect is greater than the sum of individual effects. One strategy for more effective biofilm removal is the use of phages and antibiotics, observing a synergistic effect between these factors. A synergic effect of P. aeruginosa phages and antibiotics against biofilm in vitro was observed (Chaudhry et al. 2017; Henriksen et al. 2019; Issa et al. 2019). Two P. aeruginosa phages killed 48-h-old biofilm grown on plastic surfaces in vitro with better efficacy compared to a single phage (Chaudhry et al. 2017). The synergic effect was observed for simultaneous treatment of 48-h-old biofilm on plastic for the next 48 h with phages and some antibiotics, such as ceftazidime (1×MIC and 8×MIC), ciprofloxacin (1×MIC), but not for gentamicin and colistin. The simultaneous combination of phages and most examined antibiotics (1×MIC) was effective after 12 h of treatment in reducing 8 h-old biofilm grown on layers of epithelial cells. Some antibiotics were more effective at lower doses when combined with phage. Administering phage before some antibiotics may cause better efficacy of killing biofilm than using agents simultaneously. Statistically significant results in killing 48-h-old P. aeruginosa biofilm on plastic surfaces were achieved using gentamicin and tobramycin 24 h after phage application (Chaudhry et al. 2017).

Other studies also confirm the strong synergistic effect of phages and ciprofloxacin at sub-MIC levels when treating P. aeruginosa flow-cell biofilm (Henriksen et al. 2019). In this model, a ~6 log reduction in the abundance of bacterial cells in biofilms was achieved. The synergy of phage and ciprofloxacin was confirmed by further research of elimination of P. aeruginosa biofilm in vitro (Issa et al. 2019). Combination of phages and ciprofloxacin enhanced the reduction of bacterial load by ≥ 50%. The authors suggest an association between biofilm inhibition and smaller plaques of phage formed after high adsorption of phage to bacterial cells. It is worth noting that simultaneous treatment of phages and antibiotics caused a synergistic effect in killing of P. aeruginosa biofilm (Chaudhry et al. 2017; Henriksen et al. 2019; Issa et al. 2019), but application of P. aeruginosa phage before antibiotics improves elimination of biofilm (Chaudhry et al. 2017). Additionally, the combination of phage with an antibiotic in the treatment of biofilm may affect the reduction of an antibiotic dose (Chaudhry et al. 2017).

Other in vitro studies have indicated better reduction of S. aureus biofilm after pretreatment with phage and then with an antibiotic (Kumaran et al. 2018; Tkhilaishvili et al. 2018). Treatment of biofilms with phage, antibiotics or both simultaneously caused minimal reduction of bacterial cells in biofilm. Application of phage treatment before antibiotics caused significant reduction of viable cells by up to 3 log. This effect was evidently stronger for phage with vancomycin and cefazolin at a lower antibiotic dose (Kumaran et al. 2018). A synergistic effect of simultaneous treatment with S. aureus phage and antibiotics of biofilm in vitro was observed (Tkhilaishvili et al. 2018). The biofilm was evidently eradicated after simultaneous treatment at a sub-eradicating titer of S. aureus phage of 10⁵ PFU/mL and at sub-eradicating concentrations of rifampin (64 μg/ mL) and daptomycin (32 μg/mL). No synergistic effect was achieved for fosfomycin and vancomycin. Pretreatment with S. aureus phage at 10⁵ PFU/mL followed by a sub-eradicating dose of antibiotics improved the synergistic effect in killing biofilm in vitro. Additionally, degradation of the matrix exopolysaccharide of biofilm by S. aureus phage was observed (Tkhilaishvili et al. 2018). It is worth noting that a study on another bacterial species has also demonstrated that an increase of phage from 10⁴ to 10⁷ PFU/mL may reduce the concentration of cefotaxime from 256 to 32 μg/mL in Escherichia coli biofilm eradication in vitro (Ryan et al. 2012). It is worth emphasizing that simultaneous treatment of some S. aureus phage and antibiotics resulted in a synergistic effect (Tkhilaishvili et al. 2018), but pretreatment of S. aureus phage before antibiotics increases elimination of biofilm (Kumaran et al. 2018; Tkhilaishvili et al. 2018).

Studies in vivo indicated that a simultaneous combination of S. aureus or P. aeruginosa phage and antibiotics increases killing of biofilm (Yilmaz et al. 2013), but using an antibiotic prophylactically followed by C. difficile phage treatment increases reduction of bacteria (Nale et al. 2016). A synergistic effect of phage and antibiotics was observed in the treatment of biofilm created by S. aureus and P. aeruginosa in an osteomyelitis model in rats (Yilmaz et al. 2013). An implant-related infection model in rats was treated with phages, antibiotics and a combination of both factors. The MRSA group received Sb-1 phage and teicoplanin while the P. aeruginosa group received PAT14 phage and imipenem, cilastatin and amikacin. A reduction of CFU was observed in each treatment group. MRSA biofilm was significantly eliminated by simultaneous treatment with the antibiotic and phage. A significant reduction of bacteria was observed in all P. aeruginosa biofilm groups. Greater reduction of P. aeruginosa biofilm by simultaneous treatment with the antibiotic and phage was observed (Yilmaz et al. 2013). Therapy of Clostridium difficile infection with antibiotics often causes failure or recurrent infection (Nale et al. 2016). Formation of C. difficile biofilms may contribute to this failure. The C. difficile phage cocktail alone or in combination with vancomycin reduced C. difficile biofilms and prevented colonization in a Galleria mellonella larva model (Nale et al. 2016). Pretreatment of Galleria mellonella larva with vancomycin prophylactically and subsequently with C. difficile phage increases reduction of bacteria (Nale et al. 2016).

Phage-derived endolysins are double-stranded DNA phageencoded peptidoglycan hydrolases. They are produced in bacterial cells infected by phage at the final stage of the lytic cycle and have the ability to kill replicating and nonreplicating bacteria. Endolysins cleave covalent bonds in the peptidoglycan cell wall of Gram-positive bacteria that induce rapid lysis (Borysowski et al. 2006; Borysowski et al. 2011; Fischetti 2018). Gram-negative bacteria are strongly resistant against exogenously added lysins due the occurrence of protective outer membranes (Lood et al. 2015). However, engineered lysins - artilysins created by adding peptides or other proteins improved antibacterial activity of lysins (Yang et al. 2014). Lower activity of endolysin against Gram-negative bacteria can be improved by membrane-destabilizing factors (Lood et al. 2015). Lysins against Gram-negative pathogens were discovered recently (Fischetti 2018). Given the above, lysins constitute an alternative to phages and to antibiotics as potential antibacterial agents. They have the potential to kill antibiotic-resistant bacteria. Lysins have an advantage over broad-spectrum antibiotics, because lysins have a narrow antibacterial range, therefore do not disrupt the normal flora. It has been suggested that lysins cause the low chance of inducing bacterial resistance (Borysowski et al. 2011; Fischetti 2017; Fischetti 2018; Gray et al. 2018; Kropinski 2006). Phage-derived lysins can potentially be used in the elimination of biofilm (Table 1).

Phage lysins that kill S. aureus biofilm in vitro were described for the first time in 2007 (Sass and Bierbaum 2007). After 2 h incubation of S. aureus ϕ11 endolysin with biofilm the reduction of 24 h and 48 h-old S. aureus biofilm was observed. The same efficiency was achieved for killing of S. aureus biofilm cells by endolysin as lysostaphin, but without activity on S. epidermidis biofilm determined by staining of cells with 0.1% safranin. These studies indicated the high specificity of this lysin (Sass and Bierbaum 2007). In vitro studies demonstrated that lysins may eliminate biofilms (Poonacha et al. 2017; Schuch et al. 2017; Sharma et al. 2018; Singh et al. 2014). Lysins can also eliminate biofilms in animal models in endocarditis and catheter-associated infection (Sharma et al. 2018). Lysins could kill persisters that remained after treatment with antibiotics in vitro (Sharma et al. 2018). A positive antibiofilm effect of lysin S. aureus CF-301 was achieved in an ex vivo study in human synovial fluid, which forms a strong antibiotic-resistant biofilm, with the conclusion that this lysin may have a potential role in treating joint infections (Schuch et al. 2017). S. aureus biofilm formed in human synovial fluid was highly susceptible to removal and killing by CF-301 between 4 h and 24 h. Lysin CF-301 removed all biofilm biomass in vitro in the catheter tubing after 4 h and decreased the number of bacteria by > 5 log CFU/mL (Schuch et al. 2017). 24-h-old biofilm formed in vitro on plates was treated for 6 h with S. aureus chimeric lysin P128 (Poonacha et al. 2017). P128 has shown bactericidal activity against S. aureus. However, strong reduction of biofilm cells by 99% was achieved against Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms with 15-31 μg/mL P128, whereas a higher concentration of P128, 62.5 μg/mL, was required to reduce Staphylococcus lugdunensis biofilm. 48-h-old biofilm formed in vitro on catheters was treated for 18 h with P128. 1×MIC (8 μg/mL) of P128 caused > 2 log CFU reduction of S. epidermidis biofilm (Poonacha et al. 2017). S. aureus chimeric lysin Ply187 was tested in vitro for 30 min against S. aureus biofilm grown on glass cover slips (Singh et al. 2014). Ply187 at 1×MIC caused 100% killing of S. aureus biofilm cells (Singh et al. 2014). S. aureus lysins proved to be an efficient new agent in the elimination of staphylococcal biofilm infection in vitro (Poonacha et al. 2017; Schuch et al. 2017; Singh et al. 2014). Melo et al. (2018) described studies of endolysin against S. aureus biofilm, which indicated greater activity of lysin against suspended biofilm cells than intact or scraped biofilms. A biofilm matrix may cause reduction of lysin activity. Moreover, in this study endolysin resistance did not appear in biofilm cells.

Synergy between lysins and antibiotics in treating MRSA biofilms has been observed (Sharma et al. 2018). It is particularly important to explain the importance of using lysins and antibiotics in the treatment of biofilm (Chopra et al. 2015; Schuch et al. 2017) and the order of administration of these factors (Chopra et al. 2015). An in vitro study of treating S. aureus biofilm with endolysin and an antibiotic was described (Chopra et al. 2015). Simultaneous treatment of MRSA biofilm with endolysin MR-10 and minocycline (a broad range tetracycline) at a dose of 4 μg/mL overnight showed no significant decrease of old biofilm from day 4 to day 7. In a sequential treatment of biofilm, endolysin MR-10 was used for 6 h and next overnight with minocycline at a dose of 4 μg/mL. Compared to simultaneous treatment, a significant decrease of bacterial cells of young biofilm until day 3 was observed. The reduction of old biofilm was achieved to some extent. The use of an antibiotic after endolysin had limited action on mature biofilm cells. Probably the reason for such a result is the lack of metabolically active cells in old biofilm. Endolysins kill rapidly growing cells and non-dividing cells regardless of the metabolic status of cells. They can better penetrate deeper layers of biofilm than antibiotics. A significant decrease of S. aureus bacteria in other sequentially treated MRSA biofilm with minocycline and endolysin MR-10 was observed. Minocycline at a dose of 4 μg/mL was applied for 3 h and next endolysin MR-10 overnight was used in MRSA biofilm. Such a sequential combination with tetracycline and endolysin MR-10 efficiently eliminated younger and older biofilm, so using an antibiotic before application of lysin significantly increases the elimination of even a mature biofilm (Chopra et al. 2015). Potent antibiofilm S. aureus activity of lysin CF-301 in vitro was examined (Schuch et al. 2017). In addition, we can compare the effect of lysine or antibiotics on biofilm treatment. Biofilm was formed in vitro on polystyrene, glass, surgical mesh and catheters. The effect of the action of CF-301 on biofilm was examined by staining with methylene blue and quantitative estimation of biofilm bacteria. Rapid elimination of biofilm that formed on polystyrene, glass and PVC (catheter tubing) by CF-301 was observed. Biofilms of MRSA were grown for 3 days in the catheter lumen. Addition of CF-301 at MIC (32 μg/mL) to 0.019 MIC (0.32 μg/mL) removed all biofilm biomass within 4 h and reduction of bacteria was > 5 log CFU/mL. Daptomycin had a weak effect on biomass biofilm from 50009 MIC (5 mg/mL) to MIC (1 μg/mL) and a decrease of bacteria by 1–3 log CFU/mL. CF-301 eradicated all S. aureus biofilm on catheters after only 1 h and killed all released bacteria by 6 h (Schuch et al. 2017). Evidently, the advantage of lysin CF-301 over the antibiotic to kill biofilm was demonstrated (Schuch et al. 2017). The effect of treatment with lysin combined with other factors (lysozyme, proteinase K and lysostaphin) on biofilm reduction has also been investigated (Schuch et al. 2017; Simmons et al. 2012). The application of the lysin PlyLM against Listeria monocytogenes biofilm was examined in vitro, and was found to effectively reduce monolayer biofilm similarly to lysozyme and proteinase K, as was examined in crystal violet staining intensity (Simmons et al. 2012). A synergistic effect in the elimination of biofilm was achieved for lysin and these two factors (Simmons et al. 2012). A combined treatment of biofilm with lysin S. aureus CF-301 and the cell wall hydrolase lysostaphin had a synergistic effect (Schuch et al. 2017).

The limited progress of research on genetically engineered lysins for Gram-negative bacteria necessitates more studies performed in vitro and in vivo. Acinetobacter baumannii is a Gram-negative bacterium considered to be a common nosocomial pathogen. The studies in vitro and in vivo of biofilm treatment by A. baumannii lysin were compared (Lood et al. 2015). Treatment of biofilm growing on catheters with A. baumannii lysin PlyF307 significantly reduced biofilm A. baumannii in vitro and in vivo (Lood et al. 2015). in vitro lysin PlyF307 reduced by approximately 1.6 log the number of bacteria in a 3-day-old biofilm on a catheter after 2 h of treatment with lysin. In vivo catheter sections were implanted with a 2-day-old A. baumannii biofilm subcutaneously in the backs of mice. After 24 h, two doses of 1 mg PlyF307 were administered subcutaneously at the site of the implant for 4 h. After 3 h the estimation of bacterial viability was performed. 1 mg of PlyF307 caused a 2 log reduction of bacterial viability in biofilm. A stronger reduction of biofilm cells by lysin (2 log vs 1.6 log) was observed in an in vivo study compared to an in vitro study. Additionally, mice were infected with 10⁸ CFU of A. baumannii intraperitoneally (i.p.) and 2 h later received a single dose of 1 mg of lysin PlyF307. The organs were strongly infected by 2 h, suggesting development of systemic infection. PlyF307 rescued 50% of mice from a lethal dose of A. baumannii (Lood et al. 2015).

Moreover, comparing the activity of A. baumannii lysin PlyF307 and two peptide derivatives against biofilm in vitro has shown higher activity of peptide derivatives than lysin (Lood et al. 2015; Thandar et al. 2016). C-terminal amino acids 10⁸ to 138 of PlyF307 lysin named P307. Derivative peptid P307SQ-8C was engineered from P307 to improved activity (Thandar et al. 2016). P307SQ-8C was obtained by fusing eight amino acids (SQSRESQC) to the C terminus of P307. A. baumannii biofilms in vitro in PVC catheters were treated with two peptide derivatives P307 and P307_SQ-8C for 2 h and 24 h (Thandar et al. 2016). Biofilms treated with P307 and P307_SQ-8C after 2 h were reduced respectively by ~3 and 4 log of CFU/mL. An additional decrease by 1.3 log of CFU/mL was observed with P307 and no further decrease with P307_SQ-8C after 24 h (Thandar et al. 2016). Lysin PlyF307 caused reduction of biofilm cells in vitro on a catheter by 1.6 log after 2 h (Lood et al. 2015).

Activity of P. aeruginosa lysin LysPA26 against biofilm in vitro was similar to the activity of A. baumannii lysin PlyF307 in vitro (Lood et al. 2015; Guo et al. 2017). 48-hold P. aeruginosa biofilm formed in vitro on a polystyrene plate showed a reduction by 1–2 log of the number of bacteria after 2 h treatment with lysin LysPA26 (Guo et al. 2017). Interestingly, P. aeruginosa lysin LysPA26 could destroy other Gram-negative bacteria (Klebsiella pneumoniae, A. baumannii, E. coli), but does not affect Grampositive bacteria (Guo et al. 2017).

Based on the recent reports, potential application of phage-derived lysins for Gram-positive and Gram-negative bacteria as new agents against biofilm-related infections is suggested.

Based on recent discoveries, the potential application of phages as well as phage-derived lysins in the prevention and elimination of biofilm-related infections is a promising therapeutic option. It is important to emphasize the use of a phage cocktail rather than single phages as a positive factor increasing the killing of biofilm and reducing the development of phage-resistant bacteria especially in biofilmrelated urinary tract infections. ex vivo studies of treatment of oral biofilm with a single phage demonstrated a greater reduction of biofilm than in vitro studies. Combining phages with antibiotics improves antibiofilm properties. Some antibiotics were more effective at lower doses when combined with phage. The sequence of use of antibiotics and phages in killing biofilms may be important. Application of phages before antibiotics increases elimination of biofilm. Interestingly, an advantage of applying a phage cocktail over an antibiotic in killing biofilm-related orthopedic infection was observed. Stronger killing of biofilm cells by lysin against Gram-negative bacteria was observed in an in vivo study compared to an in vitro study. Combining lysins with antibiotics increases biofilm elimination. Pretreating biofilm with an antibiotic and then with lysin improves elimination of biofilm, but other studies have shown an advantage of using other lysin over an antibiotic against biofilm. Further research of biofilm elimination by different antimicrobial factors is required, especially an in vivo biofilm model.

This work was supported by the statutory funds from the Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences.

A Górski and B Weber-Da ˛browska are co-inventors of patents owned by the Hirszfeld Institute of Immunology and Experimental Therapy and covering phage preparations. M Łusiak-Szelachowska declares that she has no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.