human
***task***
the task is to summarize an input biomedical literature in six sentences

***input***
the input is a biomedical literature

***output***
the output is the summary of an input biomedical literature in six sentences

***documentation***

***examples***

input: 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 . 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 . 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 . 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 . although the precise mechanisms of biofilm 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 . in recent years , bacteriophage endolysins ( phage lysins ) have been the focus of research into combatting antibiotic resistance in gram - positive pathogens . these cell wall peptidoglycan hydrolases possess a number of advantages over conventional antibiotics including , rapid lytic activity against bacterial cells , low probability of developing bacterial resistance , and significantly lower chance of disrupting commensal microflora , due to the enzymes ' specificity . our group previously reported the ability of phage k and modified derivatives to prevent biofilm formation and to reduce established biofilm density . 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 modification and abortive infection have been reported in many genera . the 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 after extensive growth of the bacterium in the presence of sublethal levels of enzyme . 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 riskto date a number of phage lysins have been described which demonstrate activity against staphylococci including lysk , lyswmy , 11 lysin , mv - l , lysh5 , lysgh15 , plyv12 , sal - 2 , and sal - 1 . producing tailor - made proteins by combining domains from phage lysins with various other domains has also been investigated . multidomain lysins have previously been examined for control of biofilms namely 11 lysin and sal - 2 . this is the first report of a single domain lysin being used for this purpose . cysteine , histidine dependant amidohydrolase / peptidase chap k ( 18.6 kda ) is a truncated derivative of a phage lysin ( lysk , 54 kda ) from the myoviridae staphylococcal phage k . this endopeptidase specifically cleaves the peptide bond between d - alanine and the first glycine in the pentaglycine cross - bridge of staphylococcal cell wall peptidoglycan . we have previously reported that purified chap k exhibits stronger activity than the original multidomain lysin ( lysk ) and displays rapid lytic activity against a variety of pathogenic staphylococcal species including staphylococcus epidermidis and methicillin - resistant s. aureus ( mrsa ) strains . herewe examine the efficacy of the phage - derived chap k enzyme in eliminating staphylococcus aureus biofilms and demonstrate its efficacy for removal of s. aureus from skin . the strains used in this study were s. aureus strain dpc5246 ( moorepark food research centre , teagasc , fermoy , cork , ireland ) and s. aureus strain xen29 ( caliper lifesciences , uk ) . strain xen29 is a confirmed biofilm producing pleural fluid isolate derived from the parental strain s. aureus atcc 12600 . it has been engineered to emit luminescence when metabolically active . both strains were stored at 80c and routinely grown on tryptic soya agar ( tsa ) , in tryptic soya broth ( tsb ) or tsb supplemented with 1 % d - ( + ) - glucose ( tsbg ) at 37c . chap k is comprised solely of the lytic chap domain of the anti - staphylococcal bacteriophage endolysin , lysk . 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 . highly active chap k ( 18.6 kda ) was purified to 90 % homogeneity by cation exchange chromatography . the protein was then desalted and concentrated using an amicon ultra centrifugal filter ( milipore ) with 10 kda cut - off and subsequently stored at 80c in 25 mm tris ph 7 . the lytic activity of chap k against live planktonic cells of staphylococci including multi - antibiotic resistant strains of clinical origin has been demonstrated previously . a modified static microtitre plate assay , based on previous studies , was used to analyse biofilm formation and treatment with chap k . briefly , overnight ( 1824 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 1.2910 cfu ml . in the biofilm disruption assay ,200 l volumes of the prepared culture were aliquoted into wells of a sterile 96 - well microtitre plate ( sarstedt ) and incubated at 37c for 24 h. after 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 . biofilm containing wells were then treated with 200 l of various concentrations of chap k ( 3.91500 g ml ) in sterile 25 mm tris ph 7 or with 200 l of sterile 25 mm tris ph 7 alone ( control ) , at 37c for 4 h. at the end of treatment all wells were washed again before the plate was inverted and left to dry for 1 h at 60c . the biofilms were then stained with 200 l of 0.5 % crystal violet solution for 15 min . the plate was left to dry , after which , 30 % acetic acid were added to solubilise the stain . the biofilm disrupting ability of chap k was determined by examining the optical density of the wells spectrophotometrically . a 96 - well microtitre plate / peg - lid assay , based on the method used by moskowitz et al . was used to investigate if chap k can completely eliminate a staphylococcal biofilm . 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 . briefly , an overnight colony ( 1824 h ) of s. aureus dpc5246 was 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 1.2910 cfu / ml .200 l of the tsbg cell suspension was transferred to the wells of a 96 - well plate . as a negative control for biofilm formationa peg lid was added and the plate was incubated statically for 24 h at 37c . after 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 1251000 g ml ( diluted in 25 mm tris ph7 ) was added to treatment wells .200 l 25 mm tris ph 7 was added to the control wells . the biofilm peg lid was placed on the antimicrobial challenge plate and incubated for 4 h at 37c . after 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 biofilm 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 . in order to investigate the ability of chap k to prevent the formation of s. aureus biofilms on artificial surfaces , the staining assay , as described previously for the biofilm reduction assay , was carried out with the following modification . at the beginning of the assay 100 l of chapk , at concentrations ranging from 0.78 to 125.0 g ml , were added to 100 l of tsbg with 1.310 cfu ml of dpc5246 cells , in a sterile 96 - well microtitre plate and incubated for 24 h at 37c . the ability of chap k to prevent biofilm 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 chapk , at concentrations ranging from 0.78 to 125.0 g ml , were added to 100 l of tsbg with 1.310 cfu ml of dpc5246 cells , in a sterile 96 - well microtitre plate . this study was carried out using a modified version of the spray test of hoopes et al . . briefly , three individual areas , 25 cm 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 6.210 cfu ml ( 2.510 cfu cm ) 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 in sterile h2o ) was then misted 20 cm above one of the 25 cm areas , in two passes . the two remaining sections served as controls where one 25 cm area was misted with sterile h2o and the other was left untreated . sterile cotton tipped swabs ( deltalab sterile swabs , fisher scientific , 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 . the tips of each swab were placed in 10 mls of ringers solution and vigorously mixed using a vortex mixer to dislodge cells . the suspensions were serially diluted and plated on baird parker agar supplemented with egg yolk tellurite for enumeration of surviving cells . a strong biofilm of s. aureus dpc 5246 was routinely formed when the strain was grown in tsb supplemented with 1 % d - ( + ) - glucose for 24 hr at 37c . this is represented by the strong staining seen in the untreated well in figure 1 . solubilising of crystal violet stain and subsequent measurement of od590 nm allowed accurate quantification of staining and comparison between control and enzyme - treated wells . the data shown in the bar chart in figure 1 represents the od590 nm of triplicate wells standard error . mature biofilms ( 24 h ) were treated with enzyme at concentrations ranging from to 3.91500 g ml , for 4 h at 37c . the od590 nm data for the biofilm disruption staining assay demonstrated that at all concentrations tested , chap k treatment reduced biofilm formation relative to the untreated control well ( figure 1 ) . a one - way anova indicated that chap k treatment caused a statistically significant change in biofilm formation ( p value 0.001 ) . it is clear from the graph in figure 1 that chap k successfully disrupted the s. aureus biofilms in a concentration dependant manner . visual inspection of the degree and intensity of staining in the chap k treated wells compared to untreated biofilm wells indicated that even at a concentration as low as 3.91 g ml , chap k caused a visible reduction in biofilm mass . at a concentration of 62.5 g ml chap k there was little or no visibly detectable staining of the wells ( figure 1 ) . the 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 . after treatment with 125 g ml a 2 - log decrease was seen in the number of cells in the biofilm matrix on the pegs . the average plate count from the wells with untreated dpc5246 biofilms was 2.710 cfu ml . the average viable plate count for the wells treated with 125 g ml was 2.210 cfu ml . after treatment with chap k at concentrations of 500 g ml or higher there was complete eradication of the biofilm which corresponded to a 4 - log drop in cfu ml when compared to the untreated control wells . to investigate the capacity of chap k to inhibit the formation of s. aureus biofilms , various concentrations of the enzymewere incubated with strain dpc5246 for 24 h at 37c in a microtitre plate assay . after staining and subsequent solubilisation of stain , od590 nm measurements were recorded and used to assess the ability of chap k to prevent biofilm formation . a one - way anova indicated that chap k treatment caused a statistically significant change in biofilm formation ( p value 0.001 ) . increasing degrees of biofilm prevention were evident in the presence of increasing concentrations of enzyme . at 15.63 g ml a considerable decrease in optical densitya concentration of 31.25 g ml indicated complete prevention as the mean od590 nm value ( 0.14 ) is exactly the same as that of the control medium . to confirm that chap k is able to completely prevent biofilm formationa plate count assay was performed on wells in which s. aureus was grown in tsbg at various concentrations of chap k . growth of bacteria in the presence of 7.8 g ml chap k caused a 2 - log drop in biofilm formation and at 15.63 g ml 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 or higher . the 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 ) were seeded with 2.510 cfu cm of s. aureus strain dpc5246 and misted with 200 g ml solution of chap k for two seconds . water - treated and untreated skin sections had similar cfu values when enumerated after 30 minutes . this contrasted with a significant reduction in cfus on the chap k - treated skin , which received approximately 60 g of enzyme . 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 3.710 cfu ml when treated with water to 1.710 cfu ml when treated with the chapk solution . similar results were achieved when chap k was employed against the bioluminescent strain s. aureus xen29 ( figure 5 ) . biofilm - forming bacterial strains are generally much more difficult to kill than their planktonic counterparts . they survive in sub - optimal environmental conditions , display widespread resistance to antibiotics and disinfectants and often lead to persistent infections such as is commonly seen with bovine mastitis . for example , biofilms can impede liquid flow and heat transfer and lead to increased corrosion rates which can lead to economic losses . this study demonstrates that the phage - derived peptidase , chap k , can completely remove a mature staphylococcal biofilm in under 4 h and can also prevent establishment of a staphylococcal biofilm . in the biofilm contextit is likely that chap k rapidly lyses sessile staphylococcal cells with an efficiency that brings about destabilization of the biofilm matrix leading to their subsequent detachment from solid surfaces . for formation ofa mature staphylococcal biofilms a 24 h incubation period is commonly used . in the results presented in thisstudy the untreated wells in the biofilm prevention assay show that bovine mastitis isolate dpc5246 is capable of forming a mature biofilm of 3.910 cfu / ml in under 24 h. disruption of staphylococcal biofilms by phage lysins has previously been reported by sass and son . 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 more efficiently over expressed in the recombinant e. coli strain compared with the full endolysin ; problems of aggregation , which have been encountered with over - expression 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 ) , the possibility of developing resistance seems unlikely but can not be ruled out . in the earlier studies which reported staphylococcal biofilm disruption by phage lysins , biofilm 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 biofilm forming cells . at a concentration of 31.25 g ml , this 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 biofilm formation and infection . previous studies have shown that coating medical implants with antibacterial agents can be effective in preventing formation of biofilms . 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 biofilm 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 . the robustness of the lysin makes it attractive for commercialisation and utilisation as a decontaminating agent . the two main reservoirs of s. aureus on animals are the skin and mucosal membranes . infection can often originate from commensal flora , especially in veterinary and hospital settings , as is the case with both bovine and human mastitis . the 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 . the 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 after 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 biofilm formation in processing systems .
output:

agent
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 .
***task***
the task is to summarize an input biomedical literature in six sentences

***input***
the input is a biomedical literature

***output***
the output is the summary of an input biomedical literature in six sentences

***documentation***

***examples***

input: 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 . 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 . 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 . 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 . although the precise mechanisms of biofilm 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 . in recent years , bacteriophage endolysins ( phage lysins ) have been the focus of research into combatting antibiotic resistance in gram - positive pathogens . these cell wall peptidoglycan hydrolases possess a number of advantages over conventional antibiotics including , rapid lytic activity against bacterial cells , low probability of developing bacterial resistance , and significantly lower chance of disrupting commensal microflora , due to the enzymes ' specificity . our group previously reported the ability of phage k and modified derivatives to prevent biofilm formation and to reduce established biofilm density . 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 modification and abortive infection have been reported in many genera . the 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 after extensive growth of the bacterium in the presence of sublethal levels of enzyme . 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 riskto date a number of phage lysins have been described which demonstrate activity against staphylococci including lysk , lyswmy , 11 lysin , mv - l , lysh5 , lysgh15 , plyv12 , sal - 2 , and sal - 1 . producing tailor - made proteins by combining domains from phage lysins with various other domains has also been investigated . multidomain lysins have previously been examined for control of biofilms namely 11 lysin and sal - 2 . this is the first report of a single domain lysin being used for this purpose . cysteine , histidine dependant amidohydrolase / peptidase chap k ( 18.6 kda ) is a truncated derivative of a phage lysin ( lysk , 54 kda ) from the myoviridae staphylococcal phage k . this endopeptidase specifically cleaves the peptide bond between d - alanine and the first glycine in the pentaglycine cross - bridge of staphylococcal cell wall peptidoglycan . we have previously reported that purified chap k exhibits stronger activity than the original multidomain lysin ( lysk ) and displays rapid lytic activity against a variety of pathogenic staphylococcal species including staphylococcus epidermidis and methicillin - resistant s. aureus ( mrsa ) strains . herewe examine the efficacy of the phage - derived chap k enzyme in eliminating staphylococcus aureus biofilms and demonstrate its efficacy for removal of s. aureus from skin . the strains used in this study were s. aureus strain dpc5246 ( moorepark food research centre , teagasc , fermoy , cork , ireland ) and s. aureus strain xen29 ( caliper lifesciences , uk ) . strain xen29 is a confirmed biofilm producing pleural fluid isolate derived from the parental strain s. aureus atcc 12600 . it has been engineered to emit luminescence when metabolically active . both strains were stored at 80c and routinely grown on tryptic soya agar ( tsa ) , in tryptic soya broth ( tsb ) or tsb supplemented with 1 % d - ( + ) - glucose ( tsbg ) at 37c . chap k is comprised solely of the lytic chap domain of the anti - staphylococcal bacteriophage endolysin , lysk . 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 . highly active chap k ( 18.6 kda ) was purified to 90 % homogeneity by cation exchange chromatography . the protein was then desalted and concentrated using an amicon ultra centrifugal filter ( milipore ) with 10 kda cut - off and subsequently stored at 80c in 25 mm tris ph 7 . the lytic activity of chap k against live planktonic cells of staphylococci including multi - antibiotic resistant strains of clinical origin has been demonstrated previously . a modified static microtitre plate assay , based on previous studies , was used to analyse biofilm formation and treatment with chap k . briefly , overnight ( 1824 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 1.2910 cfu ml . in the biofilm disruption assay ,200 l volumes of the prepared culture were aliquoted into wells of a sterile 96 - well microtitre plate ( sarstedt ) and incubated at 37c for 24 h. after 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 . biofilm containing wells were then treated with 200 l of various concentrations of chap k ( 3.91500 g ml ) in sterile 25 mm tris ph 7 or with 200 l of sterile 25 mm tris ph 7 alone ( control ) , at 37c for 4 h. at the end of treatment all wells were washed again before the plate was inverted and left to dry for 1 h at 60c . the biofilms were then stained with 200 l of 0.5 % crystal violet solution for 15 min . the plate was left to dry , after which , 30 % acetic acid were added to solubilise the stain . the biofilm disrupting ability of chap k was determined by examining the optical density of the wells spectrophotometrically . a 96 - well microtitre plate / peg - lid assay , based on the method used by moskowitz et al . was used to investigate if chap k can completely eliminate a staphylococcal biofilm . 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 . briefly , an overnight colony ( 1824 h ) of s. aureus dpc5246 was 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 1.2910 cfu / ml .200 l of the tsbg cell suspension was transferred to the wells of a 96 - well plate . as a negative control for biofilm formationa peg lid was added and the plate was incubated statically for 24 h at 37c . after 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 1251000 g ml ( diluted in 25 mm tris ph7 ) was added to treatment wells .200 l 25 mm tris ph 7 was added to the control wells . the biofilm peg lid was placed on the antimicrobial challenge plate and incubated for 4 h at 37c . after 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 biofilm 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 . in order to investigate the ability of chap k to prevent the formation of s. aureus biofilms on artificial surfaces , the staining assay , as described previously for the biofilm reduction assay , was carried out with the following modification . at the beginning of the assay 100 l of chapk , at concentrations ranging from 0.78 to 125.0 g ml , were added to 100 l of tsbg with 1.310 cfu ml of dpc5246 cells , in a sterile 96 - well microtitre plate and incubated for 24 h at 37c . the ability of chap k to prevent biofilm 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 chapk , at concentrations ranging from 0.78 to 125.0 g ml , were added to 100 l of tsbg with 1.310 cfu ml of dpc5246 cells , in a sterile 96 - well microtitre plate . this study was carried out using a modified version of the spray test of hoopes et al . . briefly , three individual areas , 25 cm 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 6.210 cfu ml ( 2.510 cfu cm ) 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 in sterile h2o ) was then misted 20 cm above one of the 25 cm areas , in two passes . the two remaining sections served as controls where one 25 cm area was misted with sterile h2o and the other was left untreated . sterile cotton tipped swabs ( deltalab sterile swabs , fisher scientific , 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 . the tips of each swab were placed in 10 mls of ringers solution and vigorously mixed using a vortex mixer to dislodge cells . the suspensions were serially diluted and plated on baird parker agar supplemented with egg yolk tellurite for enumeration of surviving cells . a strong biofilm of s. aureus dpc 5246 was routinely formed when the strain was grown in tsb supplemented with 1 % d - ( + ) - glucose for 24 hr at 37c . this is represented by the strong staining seen in the untreated well in figure 1 . solubilising of crystal violet stain and subsequent measurement of od590 nm allowed accurate quantification of staining and comparison between control and enzyme - treated wells . the data shown in the bar chart in figure 1 represents the od590 nm of triplicate wells standard error . mature biofilms ( 24 h ) were treated with enzyme at concentrations ranging from to 3.91500 g ml , for 4 h at 37c . the od590 nm data for the biofilm disruption staining assay demonstrated that at all concentrations tested , chap k treatment reduced biofilm formation relative to the untreated control well ( figure 1 ) . a one - way anova indicated that chap k treatment caused a statistically significant change in biofilm formation ( p value 0.001 ) . it is clear from the graph in figure 1 that chap k successfully disrupted the s. aureus biofilms in a concentration dependant manner . visual inspection of the degree and intensity of staining in the chap k treated wells compared to untreated biofilm wells indicated that even at a concentration as low as 3.91 g ml , chap k caused a visible reduction in biofilm mass . at a concentration of 62.5 g ml chap k there was little or no visibly detectable staining of the wells ( figure 1 ) . the 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 . after treatment with 125 g ml a 2 - log decrease was seen in the number of cells in the biofilm matrix on the pegs . the average plate count from the wells with untreated dpc5246 biofilms was 2.710 cfu ml . the average viable plate count for the wells treated with 125 g ml was 2.210 cfu ml . after treatment with chap k at concentrations of 500 g ml or higher there was complete eradication of the biofilm which corresponded to a 4 - log drop in cfu ml when compared to the untreated control wells . to investigate the capacity of chap k to inhibit the formation of s. aureus biofilms , various concentrations of the enzymewere incubated with strain dpc5246 for 24 h at 37c in a microtitre plate assay . after staining and subsequent solubilisation of stain , od590 nm measurements were recorded and used to assess the ability of chap k to prevent biofilm formation . a one - way anova indicated that chap k treatment caused a statistically significant change in biofilm formation ( p value 0.001 ) . increasing degrees of biofilm prevention were evident in the presence of increasing concentrations of enzyme . at 15.63 g ml a considerable decrease in optical densitya concentration of 31.25 g ml indicated complete prevention as the mean od590 nm value ( 0.14 ) is exactly the same as that of the control medium . to confirm that chap k is able to completely prevent biofilm formationa plate count assay was performed on wells in which s. aureus was grown in tsbg at various concentrations of chap k . growth of bacteria in the presence of 7.8 g ml chap k caused a 2 - log drop in biofilm formation and at 15.63 g ml 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 or higher . the 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 ) were seeded with 2.510 cfu cm of s. aureus strain dpc5246 and misted with 200 g ml solution of chap k for two seconds . water - treated and untreated skin sections had similar cfu values when enumerated after 30 minutes . this contrasted with a significant reduction in cfus on the chap k - treated skin , which received approximately 60 g of enzyme . 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 3.710 cfu ml when treated with water to 1.710 cfu ml when treated with the chapk solution . similar results were achieved when chap k was employed against the bioluminescent strain s. aureus xen29 ( figure 5 ) . biofilm - forming bacterial strains are generally much more difficult to kill than their planktonic counterparts . they survive in sub - optimal environmental conditions , display widespread resistance to antibiotics and disinfectants and often lead to persistent infections such as is commonly seen with bovine mastitis . for example , biofilms can impede liquid flow and heat transfer and lead to increased corrosion rates which can lead to economic losses . this study demonstrates that the phage - derived peptidase , chap k , can completely remove a mature staphylococcal biofilm in under 4 h and can also prevent establishment of a staphylococcal biofilm . in the biofilm contextit is likely that chap k rapidly lyses sessile staphylococcal cells with an efficiency that brings about destabilization of the biofilm matrix leading to their subsequent detachment from solid surfaces . for formation ofa mature staphylococcal biofilms a 24 h incubation period is commonly used . in the results presented in thisstudy the untreated wells in the biofilm prevention assay show that bovine mastitis isolate dpc5246 is capable of forming a mature biofilm of 3.910 cfu / ml in under 24 h. disruption of staphylococcal biofilms by phage lysins has previously been reported by sass and son . 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 more efficiently over expressed in the recombinant e. coli strain compared with the full endolysin ; problems of aggregation , which have been encountered with over - expression 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 ) , the possibility of developing resistance seems unlikely but can not be ruled out . in the earlier studies which reported staphylococcal biofilm disruption by phage lysins , biofilm 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 biofilm forming cells . at a concentration of 31.25 g ml , this 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 biofilm formation and infection . previous studies have shown that coating medical implants with antibacterial agents can be effective in preventing formation of biofilms . 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 biofilm 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 . the robustness of the lysin makes it attractive for commercialisation and utilisation as a decontaminating agent . the two main reservoirs of s. aureus on animals are the skin and mucosal membranes . infection can often originate from commensal flora , especially in veterinary and hospital settings , as is the case with both bovine and human mastitis . the 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 . the 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 after 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 biofilm formation in processing systems .
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