Enzybiotics: Enzyme-Based Antibacterials as Therapeutics

  • Dorien Dams
  • Yves BriersEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1148)


Antibiotics have saved millions of lives. However, the overuse and misuse of antibiotics have contributed to a rapid emergence of antibiotic resistance worldwide. In addition, there is an unprecedented void in the development of new antibiotic classes by the pharmaceutical industry since the first introduction of antibiotics. This antibiotic crisis underscores the urgent and increasing necessity of new, innovative antibiotics. Enzybiotics are such a promising class of antibiotics. They are derived from endolysins, bacteriophage-encoded enzymes that degrade the bacterial cell wall of the infected cell at the end of the lytic replication cycle. Enzybiotics are featured by a rapid and unique mode-of-action, a high specificity to kill pathogens, a low probability for bacterial resistance development and a proteinaceous nature. (Engineered) endolysins have been demonstrated to be effective in a variety of animal models to combat both Gram-positive and Gram-negative bacteria and have entered different phases of preclinical and clinical trials. In addition, mycobacteriophage-encoded endolysins have been successfully used to inhibit mycobacteria in vitro. In this chapter we focus on the (pre)clinical progress of enzybiotics as potent therapeutic agent against human pathogenic bacteria.


Endolysin Enzybiotics Clinical trials Animal models Multidrug-resistance 







Antimicrobial peptide


Cell wall binding domain


Cytoplasmic membrane


Coagulase-negative staphylococci




Enzymatically active domain


Ethylenediaminetetraacetic acid




Good laboratory practice


High hydrostatic pressure


Limulus amoebocyte lysate




Mycolic acids


Minimal inhibitory concentration


Methicillin-resistant Staphylococcus aureus


Methicillin-sensitive Staphylococcus aureus


N-acetylmuramic acid


Not applicable


Not defined


Outer membrane


Outer membrane permeabilizing


Post-antibiotic effect


Post-antibiotic sub-MIC effect




Sub-MIC effect


  1. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473:216–220. Scholar
  2. Becker SC, Foster-Frey J, Donovan DM (2008) The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett 287:185–191. Scholar
  3. Becker SC, Roach DR, Chauhan VS et al (2016) Triple-acting lytic enzyme treatment of drug-resistant and intracellular Staphylococcus aureus. Sci Rep 6:25063. Scholar
  4. Brennan PJ (2003) Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis 83:91–97. Scholar
  5. Briers Y, Lavigne R (2015) Breaking barriers: expansion of the use of endolysins as novel antibacterials against gram-negative bacteria. Future Microbiol 10:377–390. Scholar
  6. Briers Y, Volckaert G, Cornelissen A et al (2007) Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL. Mol Microbiol 65:1334–1344. Scholar
  7. Briers Y, Cornelissen A, Aertsen A et al (2008) Analysis of outer membrane permeability of Pseudomonas aeruginosa and bactericidal activity of endolysins KZ144 and EL188 under high hydrostatic pressure. FEMS Microbiol Lett 280:113–119. Scholar
  8. Briers Y, Schmelcher M, Loessner MJ et al (2009) The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144. Biochem Biophys Res Commun 383:187–191. Scholar
  9. Briers Y, Walmagh M, Grymonprez B et al (2014a) Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrob Agents Chemother 58:3774–3784. Scholar
  10. Briers Y, Walmagh M, Van Puyenbroeck V et al (2014b) Engineered endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens. MBio 5:e01379–e01314. Scholar
  11. Callewaert L, Walmagh M, Michiels CW, Lavigne R (2011) Food applications of bacterial cell wall hydrolases. Curr Opin Biotechnol 22:164–171. Scholar
  12. Cassino C (2016) Results of the first in human study of lysin CF-301 evaluating the safety, tolerability and pharmacokinetic 1248 profile in healthy volunteers. In: 26th ECCMID, April 9. Accessed 20 Dec 2017
  13. Catalão MJ, Milho C, Gil F, et al (2011) A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6. PLoS One 6:e20515. doi: 10.1371/journal.pone.0020515PubMedPubMedCentralCrossRefGoogle Scholar
  14. Centres for Disease Control and Prevention (US) (2013) Antibiotic resistance threats in the United States, 2013. Centres for Disease Control and Prevention, US Department of Health and Human ServicesGoogle Scholar
  15. Channabasappa S, Chikkamadaiah R, Durgaiah M, et al (2017) Preclinical studies of anti-staphylococcal ectolysin P128 for potential systemic hypersensitivity and evaluation of efficacy in Staphylococcus aureus bacteremia with renal abscesses in rats. In: ASM Microbe, June. 2017 Rat final.pdf. Accessed 28 Dec 2017Google Scholar
  16. Climo MW, Patron RL, Goldstein BP, Archer GL (1998) Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrob Agents Chemother 42:1355–1360PubMedPubMedCentralCrossRefGoogle Scholar
  17. Czaplewski L, Bax R, Clokie M et al (2016) Alternatives to antibiotica – a pipeline portfolio review. Lancet Infect Dis 16:239–251. Scholar
  18. Defraine V, Schuermans J, Grymonprez B et al (2016) Efficacy of artilysin Art-175 against resistant and persistent Acinetobacter baumannii. Antimicrob Agents Chemother 60:3480–3488. Scholar
  19. D’Herelle F. (1917) Sur un microbe invisible antagoniste des bacilles dysentériques. C. R. Acad. Sci. 165:373–375Google Scholar
  20. Diez-Martinez R, de Paz HD, Bustamante N et al (2013) Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module. Antimicrob Agents Chemother 57:5355–5365. Scholar
  21. Djurkovic S, Loeffler JM, Fischetti VA (2005) Synergistic killing of Streptococcus pneumoniae with the bacteriophage lytic enzyme Cpl-1 and penicillin or gentamicin depends on the level of penicillin resistance. Antimicrob Agents Chemother 49:1225–1228. Scholar
  22. Drilling AJ, Cooksley C, Chan C et al (2016) Fighting sinus-derived Staphylococcus aureus biofilms in vitro with a bacteriophage-derived muralytic enzyme. Int Forum Allergy Rhinol 6:349–355. Scholar
  23. Entenza JM, Loeffler JM, Grandgirard D et al (2005) Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats. Antimicrob Agents Chemother 49:4789–4792. Scholar
  24. Eugster MR, Loessner MJ (2012) Wall teichoic acids restrict access of bacteriophage endolysin Ply118, Ply511, and PlyP40 cell wall binding domains to the Listeria monocytogenes peptidoglycan. J Bacteriol 194:6498–6506. Scholar
  25. Fischetti VA (2005) Bacteriophage lytic enzymes: novel anti-infectives. Trends Microbiol 13:491–496. Scholar
  26. Fischetti VA (2010) Bacteriophage endolysins: a novel anti-infective to control gram-positive pathogens. Antimicrob Agents Chemother 300:357–362. Scholar
  27. George SE, Chikkamadaiah R, Durgaiah M et al (2012) Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128. BMC Res Notes 5:280. Scholar
  28. Gerstmans H, Rodríguez-Rubio L, Lavigne R, Briers Y (2016) From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria. Biochem Soc Trans 44:123–128. Scholar
  29. Gerstmans H, Criel B, Briers Y (2017) Synthetic biology of modular endolysins. Biotechnol Adv. Scholar
  30. Ghahramani P, Khariton T, Jones S, et al (2017) Population pharmacokinetic-pharmacodynamic assessment of cardiac safety endpoints for CF-301, a first-in-class antibacterial lysin. In: ASM Microbe, June 3.
  31. Gil F, Catalao MJ, Moniz-Pereira J et al (2008) The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity. Microbiology 154:1364–1371. Scholar
  32. Gilmer DB, Schmitz JE, Euler CW, Fischetti VA (2013) Novel bacteriophage lysin with broad lytic activity protects against mixed infection by Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 57:2743–2750. doi: 10.1128/AAC.02526-12PubMedPubMedCentralCrossRefGoogle Scholar
  33. Grover N, Paskaleva EE, Mehta KK et al (2014) Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes. Enzym Microb Technol 63:1–6. Scholar
  34. Guo M, Feng C, Ren J et al (2017) A novel antimicrobial endolysin, LysPA26, against Pseudomonas aeruginosa. Front Microbiol 8:293. Scholar
  35. Gutierrez D, Ruas-Madiedo P, Martinez B et al (2014) Effective removal of staphylococcal biofilms by the endolysin LysH5. PLoS One 9:e107307. Scholar
  36. Haddad KH, Schmelcher M, Sabzalipoor H, et al (2017) Recombinant endolysins as potential therapeutics against antibiotic-resistant Staphylococcus aureus: current status of research and novel delivery strategies. Clin Microbiol Rev 31. doi:
  37. Hanlon GW (2007) Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int J Antimicrob Agents 30:118–128. Scholar
  38. Hermoso JA, Monterroso B, Albert A et al (2003) Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11:1239–1249. showArticle InfoPubMedCrossRefGoogle Scholar
  39. Hermoso JA, García JL, García P (2007) Taking aim on bacterial pathogens: from phage therapy to enzybiotics. Curr Opin Microbiol 10:461–472PubMedCrossRefGoogle Scholar
  40. Huang G, Shen X, Gong Y et al (2014) Antibacterial properties of Acinetobacter baumannii phage Abp1 endolysin (PlyAB1). BMC Infect Dis 14:681. Scholar
  41. Jado I, López R, García E et al (2003) Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J Antimicrob Chemother 52:967–973. Scholar
  42. Jandourek A, Boyle J, Cassino C, et al (2017a) Long term immunology results of a phase 1 placebo controlled dose escalating study to examine the safety of CF-301 in human volunteers. In: 27th ECCMID, April 22. Accessed 16 Dec 2017
  43. Jandourek A, Boyle J, Murphy G, Cassino C (2017b) Inflammatory markers in a phase 1 placebo controlled dose escalating study of intravenous doses of CF-301 in human subjects. In: ASM Microbe, June 2. Accessed 16 Dec 2017
  44. Johnsborg O, Håvarstein LS (2009) Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae. FEMS Microbiol Rev 33:627–642PubMedCrossRefGoogle Scholar
  45. Jun SY, Jung GM, Yoon SJ et al (2013) Antibacterial properties of a pre-formulated recombinant phage endolysin, SAL-1. Int J Antimicrob Agents 41:156–161. Scholar
  46. Jun SY, Jung GM, Yoon SJ et al (2014) Preclinical safety evaluation of intravenously administered SAL200 containing the recombinant phage endolysin SAL-1 as a pharmaceutical ingredient. Antimicrob Agents Chemother 58:2084–2088. Scholar
  47. Jun SY, Jung GM, Yoon SJ et al (2016) Pharmacokinetics of the phage endolysin-based candidate drug SAL200 in monkeys and its appropriate intravenous dosing period. Clin Exp Pharmacol Physiol 43:1013–1016. Scholar
  48. Jun SY, Jang IJ, Yoon S et al (2017) Pharmacokinetics and tolerance of the phage endolysin-based candidate drug SAL200 after a single intravenous administration among healthy volunteers. Antimicrob Agents Chemother 61:e02629–e02616. Scholar
  49. Junjappa RP, Desai SN, Roy P et al (2013) Efficacy of anti-staphylococcal protein P128 for the treatment of canine pyoderma: potential applications. Vet Res Commun 37:217–228. Scholar
  50. Lai M-J, Soo P-C, Lin N-T et al (2013) Identification and characterisation of the putative phage-related endolysins through full genome sequence analysis in Acinetobacter baumannii ATCC 17978. Int J Antimicrob Agents 42:141–148. Scholar
  51. Langdon A, Crook N, Dantas G (2016) The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation. Genome Med 8:39. Scholar
  52. Lavigne R, Robben J (2012) Professor Dr. Richard Bruynoghe: a 1951 overview of his bacteriophage research spanning three decades. Bacteriophage 2:1–4. Scholar
  53. Lim J-A, Shin H, Heu S, Ryu S (2014) Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria. J Microbiol Biotechnol 24:803–811. Scholar
  54. Loeffler JM, Fischetti VA (2003) Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and-resistant Streptococcus pneumoniae strains. Antimicrob Agents Chemother 47:375–377PubMedPubMedCentralCrossRefGoogle Scholar
  55. Loeffler JM, Djurkovic S, Fischetti VA (2003) Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect Immun 71:6199–6204. https://doi10.1128/iai.71.11.6199-6204.2003PubMedPubMedCentralCrossRefGoogle Scholar
  56. Loessner MJ (2005) Bacteriophage endolysins – current state of research and applications. Curr Opin Microbiol 8:480–487PubMedCrossRefGoogle Scholar
  57. Loessner MJ, Kramer K, Ebel F, Scherer S (2002) C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 44:335–349PubMedCrossRefGoogle Scholar
  58. Lood R, Winer BY, Pelzek AJ et al (2015) Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in a mouse bacteremia model. Antimicrob Agents Chemother 59:1983–1991. Scholar
  59. López R, Garcíia E, García P (2004) Enzymes for anti-infective therapy: phage lysins. Drug Discov Today Ther Strateg 1:469–474CrossRefGoogle Scholar
  60. Lysando (2017) Topical applications in humans. Accessed 14 Jan 2017
  61. Madigan MT, Martinko JM, Dunlap PV, Clark DP (2008) Brock biology of microorganisms 12th edn. Int Microbiol 11:65–73Google Scholar
  62. Nelson D, Loomis L, Fischetti VA (2001) Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Acad Sci U S A 98:4107–4112. Scholar
  63. Nelson DC, Schmelcher M, Rodriguez-Rubio L et al (2012) Endolysins as antimicrobials. Adv Virus Res 83:299. Scholar
  64. Oh J, Schuch R (2017) The sub-MIC effect of lysin CF-301 on Staphylococcus aureus (S. aureus). In: ASM Microbe, June 2. SM+2017+PAE+Final+Version+Poster.pdf. Accessed 20 Dec 2017
  65. Oliveira H, Melo LDR, Santos SB et al (2013) Molecular aspects and comparative genomics of bacteriophage endolysins. J Virol 87:4558–4570. Scholar
  66. Oliveira H, Vilas Boas D, Mesnage S et al (2016) Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-gram-negative bacterial activity. Front Microbiol 7:208. Scholar
  67. Pastagia M, Euler C, Chahales P et al (2011) A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and-sensitive Staphylococcus aureus strains. Antimicrob Agents Chemother 55:738–744. Scholar
  68. Paul VD, Rajagopalan SS, Sundarrajan S et al (2011) A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from phage K and its development into a potent antistaphylococcal protein. BMC Microbiol 11:226. Scholar
  69. Payne K, Sun Q, Sacchettini J, Hatfull GF (2009) Mycobacteriophage lysin B is a novel mycolylarabinogalactan esterase. Mol Microbiol 73:367–381. Scholar
  70. Payne CM, Resch MG, Chen L et al (2013) Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci 110:14646–14651. Scholar
  71. Poonacha N, Nair S, Desai S, et al (2017) Efficient killing of planktonic and biofilm-embedded coagulase-negative staphylococci by bactericidal protein P128. Antimicrob Agents Chemother 61. doi:
  72. Rashel M, Uchiyama J, Ujihara T et al (2007) Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage φMR11. J Infect Dis 196:1237–1247. Scholar
  73. Roach DR, Donovan DM (2015) Antimicrobial bacteriophage-derived proteins and therapeutic applications. Bacteriophage 5:e1062590. Scholar
  74. Rodríguez-Rubio L, Chang W-L, Gutiérrez D et al (2016) “Artilysation” of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci. Sci Rep 6:35382. Scholar
  75. Salmond GPC, Fineran PC (2015) A century of the phage: past, present and future. Nat Rev Microbiol 13:777–786. Scholar
  76. Sandeep K (2006) Bacteriophage precision drug against bacterial infections. Curr Sci 90:631–633Google Scholar
  77. Schirmeier E, Zimmermann P, Hofmann V et al (2018) Inhibitory and bactericidal effect of Artilysin®Art-175 against colistin-resistant mcr-1-positive Escherichia coli isolates. Int J Antimicrob Agents. Scholar
  78. Schmelcher M, Shabarova T, Eugster MR et al (2010) Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains. Appl Environ Microbiol 76:5745–5756. Scholar
  79. Schmelcher M, Donovan DM, Loessner MJ (2012a) Bacteriophage endolysins as novel antimicrobials. Future Microbiol 7:1147–1171. Scholar
  80. Schmelcher M, Powell AM, Becker SC et al (2012b) Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands. Appl Environ Microbiol 78:2297–2305. Scholar
  81. Schuch R (2016) Post-antibiotic effects of lysin CF-301 against Staphylococcus aureus in human serum. Accessed 15 Jan 2017
  82. Schuch R, Nelson D, Fischetti VA (2002) A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418:884–889. Scholar
  83. Schuch R, Lee HM, Schneider BC et al (2014) Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia. J Infect Dis 209:1469–1478. Scholar
  84. Shavrina MS, Zimin AA, Molochkov NV et al (2016) In vitro study of the antibacterial effect of the bacteriophage T5 thermostable endolysin on Escherichia coli cells. J Appl Microbiol 121:1282–1290. Scholar
  85. Shen Y, Barros M, Vennemann T et al (2016) A bacteriophage endolysin that eliminates intracellular streptococci. Elife 5:e13152PubMedPubMedCentralCrossRefGoogle Scholar
  86. Sriram B, Channabasappa S, Chikkamadaiah R, et al (2017) Pharmacokinetics and efficacy of ectolysin P128 in a mouse model of systemic Methicillin Resistant Staphylococcus aureus (MRSA) infection. In: ASM Microbe, June. 2017-Mousefinal.pdf. Accessed 18 Dec 2017
  87. Sulakvelidze A, Alavidze Z, Morris JG (2001) Bacteriophage therapy. Antimicrob Agents Chemother 45:649–659. Scholar
  88. Thandar M, Lood R, Winer BY et al (2016) Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 60:2671–2679. Scholar
  89. Thummeepak R, Kitti T, Kunthalert D, Sitthisak S (2016) Enhanced antibacterial activity of Acinetobacter baumannii bacteriophage ØABP-01 endolysin (LysABP-01) in combination with colistin. Front Microbiol 7:1402. Scholar
  90. Totté JEE, van Doorn MB, Pasmans SGMA (2017) Successful treatment of chronic Staphylococcus aureus-related dermatoses with the topical endolysin Staphefekt SA. 100: a report of 3 cases. Case Rep Dermatol 9:19–25. Scholar
  91. Twort FW (1915) An investigation on the nature of ultra-microscopic viruses. Lancet 186:1241–1243CrossRefGoogle Scholar
  92. Vaara M (1993) Outer membrane permeability barrier to azithromycin, clarithromycin, and roxithromycin in gram-negative enteric bacteria. Antimicrob Agents Chemother 37:354–356PubMedPubMedCentralCrossRefGoogle Scholar
  93. Viertel TM, Ritter K, Horz H-P (2014) Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens. J Antimicrob Chemother 69:2326–2336. Scholar
  94. Vipra AA, Desai SN, Roy P et al (2012) Antistaphylococcal activity of bacteriophage derived chimeric protein P128. BMC Microbiol 12:41. Scholar
  95. Walmagh M, Briers Y, Dos Santos SB et al (2012) Characterization of modular bacteriophage endolysins from Myoviridae phages OBP, 201φ2-1 and PVP-SE1. PLoS One 7:e36991. Scholar
  96. Wang IN, Smith DL, Young R (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54:799–825. Scholar
  97. White R, Chiba S, Pang T et al (2011) Holin triggering in real time. Annu Rev Microbiol 108:798–803. Scholar
  98. Witzenrath M, Schmeck B, Doehn JM et al (2009) Systemic use of the endolysin Cpl-1 rescues mice with fatal pneumococcal pneumonia. Crit Care Med 37:642–649. Scholar
  99. Young R (2014) Phage lysis: three steps, three choices, one outcome. J Microbiol 52:243–258. Scholar
  100. Zhang L, Li D, Li X et al (2016) LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation. Sci Rep 6:29344. Scholar

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© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.Laboratory of Applied Biotechnology, Department of BiotechnologyGhent UniversityGhentBelgium

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