staphylococcus aureus is a major common cause of skin and soft tissue infections , although the majority of serious infections occur in the health care setting and in hospitals ( 1 ) . treatment of severe s. aureus infections can be puzzling and mortality rate remains between 20 - 25% ( 2 ) . over the past 40 years , methicillin resistant s. aureus had become widespread in worldwide hospitals ( 3 ) . increasing incidence of antibiotic resistance poses a serious risk to human health ( 4 ) . approximately 70 different extra - cellular proteins were detected in strain n315 using a proteomics approach ( 6 ) but some expected proteins were not detected e.g. nuclease . many of these exoproteins have been shown to play a direct role in the pathogenicity of the organism or to enhance virulence ( 5 - 8 ) . silver sulphadiazine ( agsd ) is very effective for prevention and treatment of infection in burn wounds which is widely used ( 9 - 11 ) . as it is likely that at least some areas of treated wounds will be exposed to low concentrations we have determined the effects of sub - inhibitory concentrations on toxin synthesis and in previous reports we showed that the production of toxic shock syndrome toxin-1 and exoproteases were affected by sub - inhibitory concentrations of silver sulphadiazine ( 12 , 13 ) . silver sulphadiazine ( agsd ) increased levels of tsst-1 in strains t4 and rn4282 ( termed responder strains ) but no changes in tsst-1 were seen in strain t1 ( termed a non - responder strain ) . agsd also repressed metalloprotease production in strain t4 and stimulated thiol - protease so that the overall protease activity remained the same . there were no effects on proteolytic activities in strain t1 . in order to further define the effects of agsd in this study we investigated the effects of agsd on extracellular protein production in a number of responder and non - responder strains isolated from burn wounds . bacterial strains and culture conditions 
  eleven strains of s. aureus were used for this study ( table 1 ) . t1 ( fri1189s ) was originally isolated from a case of menstrual toxic shock syndrome ( mtss ) and supplied by m. s. bergdoll ; t4 was from a burned patient with confirmed non - menstrual toxic shock syndrome ( nmtss , bury and rochdale healthcare trust , uk ) , rn4282 was obtained from t.j . foster , university of dublin , ireland and 8325 - 4 from r. novick , new york university of medicine , usa . all other strains were isolated from burn wounds ( 12 ) . s. aureus rn4282 , t4 , b6 , b23 , b45 and b54 were responders , t1 , b3 and b35 were non - responders , 8325 - 4 had not been tested and b10 did not produce tsst-1 and was included as a control . all strains were confirmed as s. aureus by performing gram stain , catalase , oxidase and staphaurex ( murex biotech limited , kent , uk ) tests . strains of staphylococcus aureus used in this study and their characteristics key : nmtss ( non menstrual toxic shock syndrome ) , mtss ( menstrual toxic shock syndrome ) , sapi ( staphylococcus aureus pathogenicity island ) , tsst-1(toxic shock syndrome toxin-1 ) , r ( responder ) , nr ( non - responder ) , nt ( not tested ) . kinetics of exoprotein production in sub - inhibitory concentrations of silver sulphadiazine 
  overnight broth cultures of s. aureus of agsd and incubated at 37 c as before . flasks were removed at 2 , 4 , 8 , 24 , 48 and 72 h , turbidities were determined using a unicam sp600 spectrophotometer at 550 nm using distilled water as a blank . cultures with optical densities above 0.7 were diluted 1:10 with distilled water . the remainder was centrifuged for 20 min at 1500 x g and 4 c and the supernatant fluids removed and stored at 20 c . preparation of protein 
  ice - cold propanol-2 ( 100 ml ) was added to 100 ml of culture supernatant fluid in a 250 ml centrifuge tube , kept on ice for 30 min , and centrifuged at 10,000 rpm for 15 min . the pellet was collected and washed with 50% ( v / v ) of propanol-2 in electrophoresis buffer and centrifuged again at 10,000 rpm for 15min.the pellet was collected and dissolved in 10 ml of electrophoresis buffer ( eb ; 10 mm tris - hcl , 1 mm edta , both sigma - aldrich , uk , ph 8) . the sample was placed in a dialysis tube ( medical international ltd , uk ) and dialysed against eb for at least 2 h at 5 c . sample ( 250 l ) was mixed with 250 l of 5 % ( w / v ) sds and 10 % ( v / v ) -mercaptoethanol in electrophoresis buffer solution ( pharmacia biotech , uk ) in a 1.5 ml eppendorf tube and boiled for 5 min . sds - page ( sodium dodecyl sulphate - polyacrylamide gel electrophoresis ) was performed using phastgel 10 - 15 gradient polyacrylamide gels ( pharmacia ) with a continuous 10 - 15 % gradient and 2 % cross - linking . protein preparations ( 3 l ) were applied and electrophoresis performed at 250v ( 10.0 ma ) for 60 vh . protein markers ( -lactalbumin 14,000 ; trypsin inhibitor , 20,000 ; carbonic anhydrase , 30,000 ; ovalbumin , 43,000 ; bovine serum albumin , 67,000 ; phosphorylase b , 94,000 : amersham biosciences , uk ) were included on each gel for calibration . detection of proteins 
  the proteins were detected by staining with silver stain ( amersham biosciences , uk ) . the gels were washed x 3 with distilled water for two min on a rotary shaker . dry gel solution ( 35 ml ; invitrogen ltd ) was added and shaken for 15 min . the gel was placed between two cellophane sheets ( invitrogen ltd ) and left overnight to dry . the gels were photographed using an alpha imager2200 system ( alpha innotech corporation , uk ) and the r values and the molecular weight of each protein band was automatically calculated using the alpha imager2200 and alpha ease software using the protein markers to calibrate each gel . estimation of protein concentration 
  protein concentration was measured by the bradford method ( 14 ) using bradford reagent and bovine serum albumin as standard ( both from sigma aldrich , uk ) . the strains of s. aureus were cultured for 24 h with and without a sub - inhibitory concentration of agsd and exoproteins isolated from the culture supernatants . total protein remained the same with agsd in the non - responder strains but increased by 6 - 15% with agsd in responder strains . in strain b10 , which does not produce tsst-1 and can not be classified as a responder / non - responder , total exoprotein increased by 5 % in the presence of agsd which proteins were fractionated by sds page . there was no clearly detectable increase in overall protein production in responder strains compared to non - responder strains . there were three patterns of exoproteins produced when cultures were grown with and without agsd . all three strains previously identified as non - responders showed few detectable changes in the proteins ( t1 , b3 , b35 ; figure 1 ) . three strains previously identified as responder strains ( t4 , b54 and b23 ) , showed a visible increase in the relative amounts of some proteins ( arrowed ) . some responder strains showed a reduction with agsd ( rn4282 , b54 ) and two responder strains showed no change ( b6 , b45 ) . strain 8325 - 4 had not previously been classified and b10 did not produce tsst-1 , both showed a reduction with agsd ( figure 1 ) . most strains produced 20 + detectable bands but some bands were unique to particular strains . as the differences may have been due to changes in growth rate between the strains and between different strains the electrophoresis was repeated for strains incubated with and without agsd for 2 to 72 h and the growth curves determined ( figure 2 ) . there was generally little effect of agsd on growth but there were some differences in growth rate between strains ( figure 2 ) . strains b10 , t1 and b45 had similar growth curves and had entered stationary phase by 48h , strain b45 was the only strain to show a decline after 48 h. strains rn4282 , b3 , b23 , b54 and 8325 - 4 had similar growth curves , grew more gently and were still in exponential phase by 72 h. strains b6 , b35 and t4 showed growth decelerating after 24h and were in stationary phase by 48 h ( figure 2 ) . the kinetics of exoprotein production in three representative strains is shown in figures 3 . in the responder strains e.g. t4 , there was inhibition of exoprotein synthesis by agsd up to 4 h , especially of proteins of 60 - 90 kd ( figure 3 ) . stimulation of proteins of 60 and 94 kd occurred after 6h and there was a general stimulation of protein by agsd after 8 h , especially proteins of 22 , 30 , 60 , 70 and 97 kd . there were few differences with and without agsd at later times ( figure 3a ) . in contrast , in the responder strain b6 , production of exoproteins was reduced in the presence of agsd up to 8 h , particularly in the range of 15 - 45 kd . amounts had recovered by 24h and there were few further differences up to 72 h ( figure 3b ) . in non - responder strain t1 , there were few differences during the growth cycle except for reduced amounts of a band at 22 kd at 6h and bands at 40 , 50 and 70 kd after 8 h ( figure 3c ) . it was possible to tentatively identify some of the proteins on the basis of their molecular weight by comparison with published data ( 12 , 13 ) . sds page of extracellular protease from staphylococcus aureus strains culture incubated with and without silver sulphadiazine for 24h . lanes1 and 8 molecular weight markers ( kd ) ; 2 , t4 control ; 3 , t4+agsd ; 4 , t1 control ; 5 , t1+agsd ; 6 , 8325 - 4 control ; 7 , 8325 - 4+agsd ; 9 , b35 control ; 10 , b35+agsd ; 11 , b3 control ; 12 , b3+agsd ; 13 , b54 control ; 14 , b54+agsd lanes 1 and 8 molecular weight markers ( kd ) ; 2 , rn4282 control ; 3 , rn4282+agsd ; 4 , b10 control ; 5 , b10+agsd ; 6 , b6 control ; 7 , b6+agsd ; 9 , b45 control ; 10 , b45 + agsd ; 11 b23 control ; 12 , b23+agsd . growth curves of staphylococcus aureus strains incubated with and without agsd for 2 to 72 hours . t4 control  , t4+agsd  , t1 control  , t1+agsd. rn4282 control  , rn4282+agsd  , b23 control  , b23+agsd . 
 figure 3 . sds - page of extracellular proteins produced by staphylococcus aureus strains at different times 2 , 4 , 6 , 8 , 24 , 48 , 72 hours of incubation in the presence and absence of silver sulphadiazine ( 25 g ml-1agsd ) . sds - page of extracellular proteins produced by strains t4 , at different times in the presence and absence of silver sulphadiazine sds - page of extracellular proteins produced by strain b6 , at different times in the presence and absence of silver sulphadiazine sds - page of extracellular proteins produced by strain t1 , at different times in the presence and absence of silver sulphadiazine the effects of sub - inhibitory concentrations of silver sulphadiazine on exoprotein synthesis were complex . there were three classes of response , responder strains that showed enhancement of synthesis of certain proteins , non - responder strains that showed no change and responder strains that showed a general decrease in exoprotein production in the early stages of growth . some of the exoproteins were produced throughout growth , some were only produced in exponential phase , some were produced only in late exponential phase and some were only produced in stationary phase in both responder and non - responder strains . expression of hla , tst and spa was strongly repressed in the presence of sodium chloride ( 1 m ) or sucrose ( 20 mm ) , expression of tst requires o2 and co2 and is affected by concentration of mg ( 15 - 19 ) . the synthesis of many exoproteins is regulated by agr and sar , so that is highest in stationary phase which was reached by most stains at 48 h in the present study . the cell density was approximately 10 to 20 times lower than in shaker ( aerated ) cultures ( unpublished observations ) but cell densities are still higher than the1.2 x 10 cfu ml needed for induction of agr by 24 - 48h depending on the strain . the control of exoprotein gene expression is a complex interplay between different regulatory systems ( 20 - 28 ) . sar regulates expression of agr mainly during the transition between exponential and stationary phases of growth and also has direct effects on expression of some exoproteins independent of agr ( 29 - 32 ) . it has also been reported that strains that produce tsst-1 produce lower amounts of extracellular proteins ( 32 - 34 ) . the tsst-1 negative strain b10 did not produce significantly more exoprotein than the tsst-1 producing strains . the reduction in exoprotein production in tsst-1 positive strains may not be a general phenomenon or may not have been expressed under the growth conditions used here . the increases seen in this study were modest compared to the 8 - 32 fold increases in tsst-1 and changes in proteases seen in our previous study ( 12 , 13 ) . one difference is that static cultures were used in present study , whereas shaken cultures ( aeration ) were used in previous studies . the production of tsst-1 has been shown to be affected by aeration and co2 levels ( 18 , 19)and specific growth rate ( 33 ) and there is clearly an interaction with the stimulation of tsst-1 synthesis by agsd and aeration which is not simply due to the higher cell densities formed by aerated cultures . overall the effects of sub - inhibitory concentrations of agsd were complex with three classes of responses recorded in the strains tested . this may reflect differences in susceptibility to agsd and , although mic s were similar , this could be tested by investigating the effects of different concentrations of agsd . ( 34 ) who showed that 70 g / ml of ag decreased synthesis of haemolysin , deoxyribonuclease and protein a but increased coagulase production in that the effect of ag are dependent on exoprotein . some of the proteins were tentatively identified on the basis of their apparent molecular weight when compared to published data but there were a number of proteins that could not be identified . the pattern of exoproteins will depend on whether a particular strain carries the gene for a particular protein and may be complicated by activation of exoenzymes by cleavage by proteases e.g. lipase and serine protease ( 35 ) and by modification of surface proteins such as fnba and protein a by proteoytic cleavage ( 27 ) the cleavage products of which will be present in the medium . the proteins that could not be identified from results published in previous studies may reflect the use of different strains and culture followed by maldi / tof ( matrix assisted laser desorption ionization/ time of flight ) mass spectroscopy should allow identification of the proteins .
previous studies had shown that sub - inhibitory concentrations of silver sulphadiazine ( agsd ) stimulated the production of toxic shock syndrome toxin-1 in certain strains ( responder strains ) of staphylococcus aureus and that protease production was also affected . 
 no changes were detected in other strains ( non - responders ) . 
 extracellular proteins from eleven responder and non - responder strains grown with and without agsd were separated by sds page . 
 there were three classes of response , responder strains that showed enhancement of synthesis of certain proteins , non - responder strains that showed no change and responder strains that showed a general decrease in exoprotein production in the early stages of growth . 
 the results showed that the effects of agsd were complex and that s. aureus strains were heterogenous with respect to their response to sub - inhibitory concentrations of agsd .