Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

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, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.


Introduction
Staphylococcal species commonly colonise the skin and mucosal membranes of both humans and animals. ey are a signi�cant causative agent of bovine mastitis in dairy herds [1] 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 [2]. Staphylococcal food poisoning is among the most common food-borne microbial diseases [3] and contamination of food industrial surfaces with staphylococcal species has been demonstrated to be a considerable risk factor [4][5][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 bio�lm-associated infections [7][8][9][10][11].
Bio�lms are multilayered communities of sessile cells protected by an extracellular matrix, which oen adhere to food contact surfaces, damaged tissue and indwelling medical devices [12][13][14]. Once formed, bio�lms may be up to 1,000 times more resistant to antimicrobial agents than planktonic cells alone making them particularly difficult to eliminate [15]. is can ultimately lead to increased risk of persistent infections, as is commonly the case with bovine mastitis [16]. In addition, because of their increased levels of resistance, bio�lm-associated infections can result in a need for explantation of medical devices in human healthcare settings [17,18]. Although the precise mechanisms of bio�lm antibiotic resistance have yet to be fully resolved, failure to successfully treat infections with conventional therapies necessitates the investigation and development of novel treatment strategies [9,18,19].
Our group previously reported the ability of phage K and modi�ed derivatives to prevent bio�lm formation and to reduce established bio�lm density [29]. However, endolysins have a number of advantages over using whole phage as antimicrobial agents. In the case of whole phage, resistance arising from either adsorption inhibition, restriction modi�cation and abortive infection have been reported in many genera [30][31][32]. Bacteriophage and their hosts have coevolved over millions of years. e equilibrium in this relationship has been maintained by continual development of resistance and counter resistance. In contrast, with the use of phage endolysins, there has been no report of bacteria developing resistance to these lytic agents even aer extensive growth of the bacterium in the presence of sublethal levels of enzyme [25,33,34]. In addition, by using phage endolysins instead of whole phage the risk of horizontal gene transfer of virulence genes is avoided. Phage preparations also have the possibility of containing exo-and endotoxins from their respective bacterial host. By overexpressing an endolysin in well-characterised avirulent laboratory strains this risk is circumvented.

2.2.
Production of CHAP . CHAP K is comprised solely of the lytic CHAP domain of the anti-staphylococcal bacteriophage endolysin, LysK [35]. In a previous study by our group, the truncated phage lysin gene was cloned untagged into a pQE60 expression vector (Qiagen) and overexpressed in Escherichia coli (E. coli) XL1-Blue [46]. Highly active CHAP K (18.6 kDa) was puri�ed to >90% homogeneity by cation exchange chromatography. e protein was then desalted and concentrated using an amicon ultra centrifugal �lter (Milipore) with 10 kDa cut-off and subsequently stored at −80 ∘ C in 25 mM Tris pH 7. e lytic activity of CHAP K against live planktonic cells of staphylococci including multi-antibiotic resistant strains of clinical origin has been demonstrated previously [46,53].

Plate Staining Assay.
A modi�ed static microtitre plate assay, based on previous studies [54], was used to analyse bio�lm formation and treatment with CHAP K . Brie�y, overnight (18-24 hr) colonies of S. aureus DPC5246 from a TSA plate were suspended in sterile ringers to an optical density equivalent to 0.5 McFarland standard and subsequently diluted 1 : 100 in TSBg to give a starting inoculum of 1.29 × 10 6 CFU mL −1 . In the bio�lm disruption assay, 200 L volumes of the prepared culture were aliquoted into wells of a sterile 96-well microtitre plate (Sarstedt) and incubated at 37 ∘ C for 24 h. Aer this incubation period, wells were washed three times with 200 L of sterile ringers using a multichannel pipette (Gilson) to remove media and planktonic cells. Bio�lm containing wells were then treated with 200 L of various concentrations of CHAP K (3.91-500 g mL −1 ) in sterile 25 mM Tris pH 7 or with 200 L of sterile 25 mM Tris pH 7 alone (control), at 37 ∘ C for 4 h. At the end of treatment all wells were washed again before the plate was inverted and le to dry for 1 h at 60 ∘ C. e bio�lms were then stained with 200 L of 0.5% crystal violet solution for 15 min. e stain solution was removed and the wells were gently washed as before. e plate was le to dry, aer which, 30% acetic acid were added to solubilise the stain. e bio�lm disrupting ability of CHAP K was determined by examining the optical density of the wells spectrophotometrically.

Viability Plate Count Assay.
A 96-well microtitre plate/peg-lid assay, based on the method used by Moskowitz et al. [55] was used to investigate if CHAP K can completely eliminate a staphylococcal bio�lm. A peg-lid plate was used in order to ensure that the maximum number of cells were removed from experimental wells with the same efficiency. is method permits removal of the bio�lm matrix by centrifugation prior to plating. Brie�y, an overnight colony (18-24 h) of S. aureus DPC5246 was suspended in sterile International Journal of Microbiology 3 Ringers to an optical density equivalent to 0.5 McFarland standard and subsequently diluted 1 : 100 in TSBg to give a starting inoculum of 1.29 × 10 6 CFU/mL. 200 L of the TSBg cell suspension was transferred to the wells of a 96-well plate. As a negative control for bio�lm formation, 200 L of TSBg was used. A peg lid was added and the plate was incubated statically for 24 h at 37 ∘ C [56]. Aer incubation the peg lid was removed and washed three times by placing it in a 96-well plate containing sterile ringers for 30 sec each time. 200 L of CHAP K at concentrations ranging from 125-1000 g mL −1 (diluted in 25 mM Tris pH7) was added to treatment wells. 200 L 25 mM Tris pH 7 was added to the control wells. e bio�lm peg lid was placed on the antimicrobial challenge plate and incubated for 4 h at 37 ∘ C. Aer incubation the peg lid was washed three times in sterile ringers as before. Finally the lid was placed in a plate containing 200 L sterile ringers in each well and centrifuged at 800 g for 20 mins to remove any bio�lm remaining on the pegs. Serial dilutions were performed on the contents of each well and a viable plate count was performed using Baird Parker Agar supplemented with egg yolk tellurite.

Plate Staining Assay.
In order to investigate the ability of CHAP K to prevent the formation of S. aureus bio�lms on arti�cial surfaces, the staining assay, as described previously for the bio�lm reduction assay, was carried out with the following modi�cation. At the beginning of the assay 100 L of CHAP K , at concentrations ranging from 0.78 to 125.0 g mL −1 , were added to 100 L of TSBg with 1.3 × 10 6 CFU mL −1 of DPC5246 cells, in a sterile 96-well microtitre plate and incubated for 24 h at 37 ∘ C.

Viable Plate Count
Assay. e ability of CHAP K to prevent bio�lm formation was also investigated using a method similar to the viable plate count method described previously with the following changes. At the beginning of the assay 100 L of CHAP K , at concentrations ranging from 0.78 to 125.0 g mL −1 , were added to 100 L of TSBg with 1.3 × 10 6 CFU mL −1 of DPC5246 cells, in a sterile 96-well microtitre plate.

Skin Decolonization Assay.
is study was carried out using a modi�ed version of the spray test of Hoopes et al. [57]. Brie�y, three individual areas, 25 cm 2 in size, were marked out on a section of porcine skin (obtained fresh from an abattoir). Each area was disinfected with 70% isopropyl alcohol wipes and allowed to dry at room temperature for up to 30 min. All three marked areas were then seeded with 100 L of 6.2 × 10 7 CFU mL −1 (2.5 × 10 5 CFU cm −2 ) of S. aureus DPC5246 by pipette, distributed evenly within each area with a sterile plastic spreader (Sarstedt) and allowed to dry for 30 min. CHAP K (200 g mL −1 in sterile H 2 O) was then misted 20 cm above one of the 25 cm 2 areas, in two passes. e two remaining sections served as controls where one 25 cm 2 area was misted with sterile H 2 O and the other was le untreated. e skin was then le to dry at room temperature for 30 min. Sterile cotton tipped swabs (Deltalab sterile swabs, Fisher Scienti�c, Ireland) were moistened in sterile Ringer's solution and used to sample each section of skin by rotating and rubbing the swab, in a zigzag pattern, and repeating at right angles. e tips of each swab were placed in 10 mls of Ringers solution and vigorously mixed using a vortex mixer to dislodge cells. e suspensions were serially diluted and plated on Baird Parker agar supplemented with egg yolk tellurite for enumeration of surviving cells. e work was also similarly done using the bioluminescent producing S. aureus Xen29 strain.

Sta��ylococcal �io�l� �e�uction �sing C�AP
3.1.1. Plate Staining Assay. A strong bio�lm of S. aureus DPC 5246 was routinely formed when the strain was grown in TSB supplemented with 1% D-(+)-glucose for 24 hr at 37 ∘ C. is is represented by the strong staining seen in the untreated well in Figure 1. Solubilising of crystal violet stain and subsequent measurement of OD 590 nm allowed accurate quanti�cation of staining and comparison between control and enzyme-treated wells. e data shown in the bar chart in Figure 1 represents the OD 590 nm of triplicate wells ± standard error. Mature bio�lms (24 h) were treated with enzyme at concentrations ranging from to 3.91-500 g mL −1 , for 4 h at 37 ∘ C. e OD 590 nm data for the bio�lm disruption staining assay demonstrated that at all concentrations tested, CHAP K treatment reduced bio�lm formation relative to the untreated control well (Figure 1). A one-way ANOVA indicated that CHAP K treatment caused a statistically signi�cant change in bio�lm formation ( value < 0.001). It is clear from the graph in Figure 1 that CHAP K successfully disrupted the S. aureus bio�lms in a concentration dependant manner. Visual inspection of the degree and intensity of staining in the CHAP K treated wells compared to untreated bio�lm wells indicated that even at a concentration as low as 3.91 g mL −1 , CHAP K caused a visible reduction in bio�lm mass. At a concentration of 62.5 g mL −1 CHAP K there was little or no visibly detectable staining of the wells (Figure 1).

Viable Plate Count Assay.
Puri�ed CHAP K ranging in concentration from 125-1000 g/mL, was used to treat a 24 h staphylococcal bio�lm. e results of the viable plate counts are summarised in the bar chart in Figure 2, where each bar represents the average of triplicate plate counts ± standard error. Aer treatment with 125 g mL −1 a 2-log decrease was seen in the number of cells in the bio�lm matrix on the pegs. e average plate count from the wells with untreated DPC5246 bio�lms was 2.7 × 10 4 CFU mL −1 . e average viable plate count for the wells treated with 125 g mL −1 was 2.2 × 10 2 CFU mL −1 . Aer treatment with CHAP K at concentrations of 500 g mL −1 or higher there was complete eradication of the bio�lm which corresponded to a 4-log drop in CFU mL −1 when compared to the untreated control wells.

Plate Staining Assay.
To investigate the capacity of CHAP K to inhibit the formation of S. aureus bio�lms, various concentrations of the enzyme were incubated with strain DPC5246 for 24 h at 37 ∘ C in a microtitre plate assay. Aer staining and subsequent solubilisation of stain, OD 590 nm measurements were recorded and used to assess the ability of CHAP K to prevent bio�lm formation. is data is presented in Figure 3. A one-way ANOVA indicated that CHAP K treatment caused a statistically signi�cant change in bio�lm formation ( value < 0.001). Increasing degrees of bio�lm prevention were evident in the presence of increasing concentrations of enzyme. At 15.63 g mL −1 a considerable decrease in optical density is seen when compared with the untreated control wells. A concentration of 31.25 g mL −1 indicated complete prevention as the mean OD 590 nm value (0.14) is exactly the same as that of the control medium.

Viable Plate Count Assay.
To con�rm that CHAP K is able to completely prevent bio�lm formation a plate count assay was performed on wells in which S. aureus was grown in TSBg at various concentrations of CHAP K . e bar 24 h staphylococcal bio�lms were grown on peg lids in microtitre plates. e bar chart shows the CFU mL −1 that were retrieved from the pegs aer 4 h treatment with CHAP K , at the concentrations outlined below each bar. e assay was performed in triplicate and each bar is a representation of the mean ± SE. e values indicated above the bars are the mean CFU/mL aer treatment. chart in Figure 4 represents averages of triplicate values ± standard error. Growth of bacteria in the presence of 7.8 g mL −1 CHAP K caused a 2-log drop in bio�lm formation and at 15.63 g mL −1 a 4-log reduction was evident. Complete prevention corresponding to a 6-log drop was achieved when the DPC 5246 was incubated with CHAP K at a concentration of 31.25 g mL −1 or higher.

Removal of S. aureus from Skin
Using CHAP . e potential of CHAP K as a skin decolonization agent was assessed by incorporation of the enzyme into a spray. Sections of porcine skin (25 cm 2 ) were seeded with 2.5 × 10 5 CFU cm −2 of S. aureus strain DPC5246 and misted with 200 g mL −1 solution of CHAP K for two seconds. Water-treated and untreated skin sections had similar CFU values when enumerated aer 30 minutes. is contrasted with a signi�cant reduction in CFUs on the CHAP K -treated skin, which received approximately 60 g of enzyme. CHAP <?brm?>K<?erm?> treatment was found to be sufficient to remove >99% of S. aureus DPC5246 in 30 min when compared with treatment with water, i.e., reduced from 3.7 × 10 3 CFU mL −1 when treated with water to 1.7 × 10 1 CFU mL −1 when treated with the CHAPK solution. Similar results were achieved when CHAP K was employed against the bioluminescent strain S. aureus Xen29 ( Figure 5). Concentrations of enzyme ranging from 7.8−125 g mL −1 were incubated with S. aureus DPC5246 in TSBg at 37 ∘ C for 24 h in a 96 well peg plate. e pegs were washed aer incubation and subsequently any cells remaining adhered to the pegs were removed by centrifugation at 800 rpm for 20 mins. Assays were carried out in triplicate and OD 590 data was expressed as the mean ± SE. e values indicated above the bars are the mean CFU/mL aer treatment.

Discussion
Bio�lms are recognised as a signi�cant problem in the food industry. Bio�lm-forming bacterial strains are generally much more difficult to kill than their planktonic counterparts. ey survive in sub-optimal environmental conditions, display widespread resistance to antibiotics and disinfectants and oen lead to persistent infections such as is commonly seen with bovine mastitis [16]. Bio�lms may also interfere with various processes in food technology and engineering. For example, bio�lms can impede liquid �ow and heat transfer and lead to increased corrosion rates which can lead to economic losses [58].
is study demonstrates that the phage-derived peptidase, CHAP K , can completely remove a mature staphylococcal bio�lm in under 4 h and can also prevent establishment of a staphylococcal bio�lm. In the bio�lm context it is likely that CHAP K rapidly lyses sessile staphylococcal cells with an efficiency that brings about destabilization of the bio�lm matrix leading to their subsequent detachment from solid surfaces. For formation of a mature staphylococcal bio�lms a 24 h incubation period is commonly used [37,56,59,60]. In the results presented in this study the untreated wells in the bio�lm prevention assay show that bovine mastitis isolate DPC5246 is capable of forming a mature bio�lm of 3.9 × 10 6 CFU/mL in under 24 h.
Disruption of staphylococcal bio�lms by phage lysins has previously been reported by Sass and Son [37,42]. While these studies involved the complete endolysin protein with multiple domains, CHAP K is a truncated form of a natural endolysin (Lys K). It contains one lytic domain and only 33% of the original protein. Due to its lower molecular weight, CHAP K is predicted to have a lower chance of inducing a humoral immune response [46]. Also, because it is smaller CHAP K is more efficiently over expressed in the recombinant E. coli strain compared with the full endolysin; problems of aggregation, which have been encountered with overexpression of cow udders full endolysins are greatly reduced. Lysins with just one catalytic domain, such as CHAP K , may run the risk of being more susceptible to development of host resistance, than a protein with multiple catalytic domains. However, because of the position at which CHAP K cleaves the peptidoglycan (between the characteristic S. aureus pentaglycine bridge and the D-alanine of the tetrapeptide crossink [47]), the possibility of developing resistance seems unlikely but cannot be ruled out. In the earlier studies which reported staphylococcal bio�lm disruption by phage lysins, bio�lm staining was the sole method used to estimate the efficacy of the enzymatic treatment. Our study combines the staining approach with viable plate counting in order to more accurately represent the effect of the lysin on bio�lm forming cells.
At a concentration of 31.25 g mL −1 , CHAP K completely prevented the formation of S. aureus bio�lms. is result demonstrates the potential of applying CHAP K as a spray for decontamination of food contact surfaces or of cow udders as a preventative measure for bovine mastitisCHAP K . could also be employed as a coating agent on medical implants such as catheters to prevent the adherence of staphylococci and subsequent bio�lm formation and infection. Previous studies have shown that coating medical implants with antibacterial agents can be effective in preventing formation of bio�lms [61][62][63]. Previous work by our group demonstrated that CHAP K is also effective as a biocidal agent against several pathogenic species of Staphylococcus including the  well known bio�lm former S. epidermidis and all known clonal types of MRSA, and thus can also be considered as a useful antimicrobial agent for prevention or treatment of infections caused by these species. A previous publication by our group on the characterisation of CHAP K demonstrated that the lysin is active over a broad range of temperatures and pH and was not seen to be susceptible to degradation by multiple freeze thawing steps [48]. e robustness of the lysin makes it attractive for commercialisation and utilisation as a decontaminating agent. e two main reservoirs of S. aureus on animals are the skin and mucosal membranes. Infection can oen originate from commensal �ora, especially in veterinary and hospital settings, as is the case with both bovine and human mastitis [64,65]. e present study demonstrated the potential of CHAP K as a decolonisation agent for the removal of S. aureus from the surface of mammalian skin. When applied as a spray, CHAP K eliminated 99% of S. aureus DPC5246 from skin in 30 min. e results of the experiment suggested that CHAP K could be included in bovine teat-dip solution for reduction of mastitis causing staphylococci on the udder prior to and aer milking in dairy farms. In addition, treatment of human skin with CHAP K prior to surgery may help prevent serious nosocomial infections.
In conclusion, our data demonstrates the potential of a novel but natural anti-staphylococcal agent to prevent economically important veterinary infections, nosocomial staphylococcal infections and also reduce bio�lm formation in processing systems.

Authors' Contribution
M. Fenton and R. Keary contributed equally to this work.