New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, , as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4？h. In addition, was able to prevent biofilm formation by this strain. The lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors. 1. Introduction Staphylococcal species commonly colonise the skin and mucosal membranes of both humans and animals. They are a significant causative agent of bovine mastitis in dairy herds  and are also associated with a number of diseases in humans, ranging from a variety of skin conditions to more serious infections such as septicemia . Staphylococcal food poisoning is among the most common food-borne microbial diseases  and contamination of food industrial surfaces with staphylococcal species has been demonstrated to be a considerable risk factor [4–6]. Along with the urgent requirement for novel antibacterials to combat the prevalence of antibiotic/disinfectant resistant staphylococci in food processing, veterinary and healthcare settings, there is an increasing need for effective antimicrobial agents which can prevent and treat staphylococcal biofilm-associated infections [7–11]. Biofilms are multilayered communities of sessile cells protected by an extracellular matrix, which often adhere to food contact surfaces, damaged tissue and indwelling medical devices [12–14]. Once formed, biofilms may be up to 1,000 times more resistant to antimicrobial agents than planktonic cells alone making them particularly difficult to eliminate . This can ultimately lead to increased risk of persistent infections, as is commonly the case with bovine mastitis . In addition, because of their increased levels of resistance, biofilm-associated infections can result in a need for explantation of medical devices in human healthcare settings [17, 18]. Although the precise mechanisms of biofilm antibiotic resistance have yet to be fully resolved, failure to
B. Hole？ková, E. Holoda, M. Fotta, V. Kaliná？ová, J. Gondol', and J. Grolmus, “Occurrence of enterotoxigenic Staphylococcus aureus in food,” Annals of Agriculture and Environmental Medicine, vol. 9, no. 2, pp. 179–182, 2002.
D. Gutierrez, S. Delgado, D. Vazquez-Sanchez, et al., “Incidence of Staphylococcus aureus and analysis of bacterial-associated communities on food industry surfaces,” Applied and Environmental Microbiology, vol. 78, no. 24, pp. 8547–8554, 2012.
J. J. R. Herrera, M. L. Cabo, A. González, I. Pazos, and L. Pastoriza, “Adhesion and detachment kinetics of several strains of Staphylococcus aureus subsp. aureus under three different experimental conditions,” Food Microbiology, vol. 24, no. 6, pp. 585–591, 2007.
E. A. Eady and J. H. Cove, “Staphylococcal resistance revisited: community-acquired methicillin resistant Staphylococcus aureus—an emerging problem for the management of skin and soft tissue infections,” Current Opinion in Infectious Diseases, vol. 16, no. 2, pp. 103–124, 2003.
S. Langsrud, M. S. Sidhu, E. Heir, and A. L. Holck, “Bacterial disinfectant resistance—a challenge for the food industry,” International Biodeterioration and Biodegradation, vol. 51, no. 4, pp. 283–290, 2003.
N. P. O'Grady, M. Alexander, E. P. Dellinger et al., “Guidelines for the prevention of intravascular catheter-related infections,” Infection Control and Hospital Epidemiology, vol. 23, no. 12, pp. 759–769, 2002.
S. C. Marques, J. D. G. O. S. Rezende, L. A. D. F. Alves et al., “Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers,” Brazilian Journal of Microbiology, vol. 38, no. 3, pp. 538–543, 2007.
M. E. Olson, H. Ceri, D. W. Morck, A. G. Buret, and R. R. Read, “Biofilm bacteria: formation and comparative susceptibility to antibiotics,” Canadian Journal of Veterinary Research, vol. 66, no. 2, pp. 86–92, 2002.
L. Rodriguez-Rubio, B. Martinez, D. M. Donovan, A. Rodriguez, and P. García, “Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics,” Critical Reviews in Microbiology, 2012.
P. Szweda, M. Schielmann, R. Kotlowski, G. Gorczyca, M. Zalewska, and S. Milewski, “Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus,” Appl Microbiol Biotechnol, vol. 96, pp. 1157–1174, 2012.
M. Pastagia, C. Euler, P. Chahales, J. Fuentes-Duculan, J. G. Krueger, and V. A. Fischetti, “A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and -sensitive Staphylococcus aureus strains,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 2, pp. 738–744, 2011.
D. Kelly, O. McAuliffe, R. P. Ross, and A. Coffey, “Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives,” Letters in Applied Microbiology, vol. 54, no. 4, pp. 286–291, 2012.
S. O'Flaherty, A. Coffey, W. Meaney, G. F. Fitzgerald, and R. P. Ross, “The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus,” Journal of Bacteriology, vol. 187, no. 20, pp. 7161–7164, 2005.
K. J. Yokoi, N. Kawahigashi, M. Uchida et al., “The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage ？WMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli,” Gene, vol. 351, pp. 97–108, 2005.
P. Sass and G. Bierbaum, “Lytic activity of recombinant bacteriophage φ11 and φ12 endolysins on whole cells and biofilms of Staphylococcus aureus,” Applied and Environmental Microbiology, vol. 73, no. 1, pp. 347–352, 2007.
M. Rashel, J. Uchiyama, T. Ujihara et al., “Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage ？MR11,” Journal of Infectious Diseases, vol. 196, no. 8, pp. 1237–1247, 2007.
J. M. Obeso, B. Martínez, A. Rodríguez, and P. García, “Lytic activity of the recombinant staphylococcal bacteriophage ΦH5 endolysin active against Staphylococcus aureus in milk,” International Journal of Food Microbiology, vol. 128, no. 2, pp. 212–218, 2008.
J. Gu, W. Xu, L. Lei et al., “LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection,” Journal of Clinical Microbiology, vol. 49, no. 1, pp. 111–117, 2011.
P. Yoong, R. Schuch, D. Nelson, and V. A. Fischetti, “Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium,” Journal of Bacteriology, vol. 186, no. 14, pp. 4808–4812, 2004.
J. S. Son, S. J. Lee, S. Y. Jun et al., “Antibacterial and biofilm removal activity of a podoviridae Staphylococcus aureus bacteriophage SAP-2 and a derived recombinant cell-wall-degrading enzyme,” Applied Microbiology and Biotechnology, vol. 86, no. 5, pp. 1439–1449, 2010.
S. Y. Jun, G. M. Jung, J. S. Son, S. J. Yoon, Y. J. Choi, and S. H. Kang, “Comparison of the antibacterial properties of phage endolysins SAL-1 and LysK,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 4, pp. 1764–1767, 2011.
M. Schmelcher, A. M. Powell, S. C. Becker, M. J. Camp, and D. M. Donovan, “Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands,” Applied and Environmental Microbiology, vol. 78, pp. 2297–2305, 2012.
E. A. Idelevich, C. von Eiff, A. W. Friedrich, et al., “In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 9, pp. 4416–4419, 2011.
M. Horgan, G. O'Flynn, J. Garry et al., “Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci,” Applied and Environmental Microbiology, vol. 75, no. 3, pp. 872–874, 2009.
S. C. Becker, S. Dong, J. R. Baker, J. Foster-Frey, D. G. Pritchard, and D. M. Donovan, “LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells,” FEMS Microbiology Letters, vol. 294, no. 1, pp. 52–60, 2009.
S. O'Flaherty, A. Coffey, W. J. Meaney, G. F. Fitzgerald, and R. P. Ross, “Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk,” Letters in Applied Microbiology, vol. 41, no. 3, pp. 274–279, 2005.
J. L. Kadurugamuwa, L. V. Sin, J. Yu et al., “Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 10, pp. 3130–3137, 2003.
J. L. Kadurugamuwa, L. V. Sin, J. Yu, K. P. Francis, T. F. Purchio, and P. R. Contag, “Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 6, pp. 2283–2287, 2004.
J. A. Wu, C. Kusuma, J. J. Mond, and J. F. Kokai-Kun, “Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 11, pp. 3407–3414, 2003.
S. M. Moskowitz, J. M. Foster, J. Emerson, and J. L. Burns, “Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis,” Journal of Clinical Microbiology, vol. 42, no. 5, pp. 1915–1922, 2004.
H. Ceri, M. E. Olson, C. Stremick, R. R. Read, D. Morck, and A. Buret, “The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms,” Journal of Clinical Microbiology, vol. 37, no. 6, pp. 1771–1776, 1999.
J. T. Hoopes, C. J. Stark, H. A. Kim, D. J. Sussman, D. M. Donovan, and D. C. Nelson, “Use of a bacteriophage lysin, PlyC, as an enzyme disinfectant against Streptococcus equi,” Applied and Environmental Microbiology, vol. 75, no. 5, pp. 1388–1394, 2009.
é. Hell, C. G. Giske, A. Nelson, U. R？mling, and G. Marchini, “Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis,” Letters in Applied Microbiology, vol. 50, no. 2, pp. 211–215, 2010.
S. Furukawa, Y. Akiyoshi, M. Komoriya, H. Ogihara, and Y. Morinaga, “Removing Staphylococcus aureus and Escherichia coli biofilms on stainless steel by cleaning-in-place (CIP) cleaning agents,” Food Control, vol. 21, no. 5, pp. 669–672, 2010.
K. Bruellhoff, J. Fiedler, M. Moller, J. Groll, and R. E. Brenner, “Surface coating strategies to prevent biofilm formation on implant surfaces,” The International Journal of Artificial Organs, vol. 33, pp. 646–653, 2010.
J. van Heerden, M. Turner, D. Hoffmann, and J. Moolman, “Antimicrobial coating agents: can biofilm formation on a breast implant be prevented?” Journal of Plastic, Reconstructive and Aesthetic Surgery, vol. 62, no. 5, pp. 610–617, 2009.
N. M. Bernthal, A. I. Stavrakis, F. Billi et al., “A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings,” PloS ONE, vol. 5, no. 9, Article ID e12580, 2010.
J. Q. Gong, L. Lin, T. Lin et al., “Skin colonization by Staphylococcus aureus in patients with eczema and atopic dermatitis and relevant combined topical therapy: a double-blind multicentre randomized controlled trial,” British Journal of Dermatology, vol. 155, no. 4, pp. 680–687, 2006.
J. Kluytmans, A. van Belkum, and H. Verbrugh, “Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks,” Clinical Microbiology Reviews, vol. 10, no. 3, pp. 505–520, 1997.