staphylococcus aureus   ( s. aureus ) is one of the most important pathogens in both hospitals and the community and can cause numerous syndromes in humans , such as endocarditis , osteomyelitis , and septicemia [ 1 , 2 ] . greater than 60% of s. aureus   isolates are now resistant to methicillin ( oxacillin ) , and some strains have developed resistance to more than 20 different antimicrobial agents ; new agents are therefore needed for the treatment of s. aureus . for over 100 years , chemical compounds isolated from medicinal plants have served as the models for many clinically proven drugs and are now being reassessed as antimicrobial agents . cryptotanshinone ( ct ) is a major tanshinone of dan shen , the root of salvia miltiorrhiza   bunge ( labiatae ) . ct exhibits antimicrobial activity against a broad range of gram - positive , including s. aureus , and gram - negative bacteria as well as other microorganisms [ 6 , 7 ] . although ct exhibited fairly high levels of activity against s. aureus , there have been no reports related to the inhibitory mechanisms of ct against s. aureus . transcriptional profilings generated with affymetrix genechips   have been used to identify genes of s. aureus   that were induced in response to the antibiotics vancomycin , oxacillin , d - cycloserine , or bacitracin [ 8 , 9 ] . transcriptional profiles of microorganisms treated with an inhibitor could provide valuable information for both pathway characterization , and for determination of the mechanism of inhibition [ 10 , 11 ] . in this report , to investigate the antimicrobial activity and the possible action mechanisms of ct against s. aureus , we tested the minimum inhibitory concentrations ( mics ) of ct against 21 s. aureus   strains , as well as utilizing affymetrix genechip   analysis to identify differentially expressed genes for s. aureus   treated with subinhibitory concentrations of ct . the s. aureus   strains used in this study were comprised of 20 clinical isolates from the first hospital of jilin university , which have different antibiograms against 12 antimicrobial agents ( table 1 ) , and the standard strain atcc 25923 obtained from china medical culture collection center ( cmcc ) . mueller - hinton broth ii ( mhb ii ) and mueller - hinton agar ( mha ) were purchased from bd ( biosciences , inc . , sparks , md ) . ct was purchased from the china medical culture collection centre ( cmcc ) , and stock solutions of varying concentrations were dissolved in dimethyl sulfoxide ( dmso , sigma - aldrich ) . the minimum inhibition concentrations ( mics ) of ct against the 21 s. aureus   strains were determined in triplicate by broth microdilution or broth macrodilution using twofold serial dilutions in mhb ii , according to clsi / nccls m100-s15 . the mics were defined as the lowest concentration at which no visible growth was observed . the minimum concentration of ct that inhibited 90% of the isolates tested was defined as the mic90 . a standard checkerboard assay was performed to assess the interaction of combination ( ct and tmp / sxt ) against s. aureus   strain atcc 25923 by a well - established method . the fractional inhibitory concentration ( fic ) index was calculated by a previously described method . an fic index ( fici ) between two compounds less than or equal to 0.5 is considered synergism , an fic index between 0.5 and 2 is considered indifference , and a fici equal to or more than 2 is considered antagonism . 
 s. aureus   strain atcc 25923 was grown to an optical density of 0.3 at 600 nm in mhb ii , and 100 ml aliquots were then distributed into five 500 ml erlenmeyer flasks . ct ( dissolved in dmso ) was added to four of the cultures to obtain final concentrations of 1/4       mic ( 1 gml ) , 1/2       mic ( 2 gml ) , mic ( 4 gml ) , and 2       mic ( 8 gml ) , respectively . the final dmso concentration used was 1% ( vol / vol ) , and the control culture was supplemented with 1% dmso . the cultures were incubated , and cell growth was spectrophotometrically monitored as the optical density at 600 nm , which was recorded at certain time intervals . s. aureus strain atcc 25923 was grown overnight at 200 rpm in a rotary shaker at 37c in 10 ml of mhb ii . six 250-ml erlenmeyer flasks , each containing 100 ml of mhb ii , were inoculated with an overnight culture to an initial od600   of 0.05 . the bacteria were then grown at 37c at 200 rpm to an od600   of 0.3 . subsequently , 500 l of a 12 800 gml ct stock solution , prepared in dimethyl sulfoxide ( dmso ) , was added to three of the cultures ( experimental cultures ) , yielding a final concentration of 1/2       mic ( 2 gml ) . hence , the final concentration of solvent in each ct treatment was 1% ( vol / vol ) dmso , which did not alter the ph of the medium . the other three cultures lacking ct and supplemented with 1% ( vol / vol ) dmso were used as the control . all bacterial suspensions ( both ct treatment or control condition ) the genechip s. aureus   genome array ( antisense ) was provided by capitalbio corporation ( http://www.capitalbio.com/index.asp , beijing , china ) , a service provider authorized by affymetrix inc . the array contains probe sets to over 3 300 s. aureus   orfs and over 4 800 intergenic regions . the raw data from the array scans were normalized by median - centering genes for each array , followed by log transformation . expressed genes were identified using affymetrix genechip operating software ( gcos , ver.1.0 ) , which utilizes statistical criteria to generate a present  or  absent  call for genes represented by each probe set on the array . absent  scores were filtered out of the dataset , and the remaining genes were analyzed . to identify genes that are differentially expressed in ct - treated samples compared to controls , the significance analysis of microarrays ( sam ) software ( http://www-stat.stanford.edu/~tibs/sam/index.html ) was used . to select the differentially expressed genes , we used threshold values of   1.5- and   1.5-fold change between three rh treatment samples and three control samples ; the fdr significance level was   < 5% . quantitative real - time reverse transcription ( rt)-pcr was used to verify the microarray results . aliquots of the rna preparations from ct - treated and control samples used in the microarray experiments were also used for quantitative real - time rt - pcr follow - up studies . the cdna was subjected to real - time pcr using the primer pairs listed in table 2 . quantitative real - time pcr was performed in triplicate using the 7000 sequence detection system ( applied biosystems , foster city , ca , usa ) , as previously described . in this experiment , the mics of ct against 21 s. aureus   strains ranged from 4 to 64 gml ( table 1 ) , and the mic90   was 16 gml . the mic value of s. aureus   strain atcc 25923 versus ct was 4 gml . we performed growth curve of s. aureus   atcc 25923 with ct ( 1 , 2 , 4 , 8 , and 16 gml ) added at the time point of od600   = 0.3 . bacterial cells samples were collected at certain time intervals in this experiment with a time course of 1440 minutes , and the growth curves of ct - treated and untreated strain were measured spectrophotometrically . with 1 and 2 gml of ct and controls , we found the optical density increased steadily , while with 4 gml , 8 gml , and 16 gml of ct , we did not observe increase in the optical density ( figure 1 ) . this result showed that ct displays bacteriostatic action against s. aureus , which is consistent with previous reports [ 15 , 16 ] . genechip analysis revealed that a substantial number of genes ( 185 ) were differentially regulated in response to ct . of these , 100 genes exhibited a significant increase in transcript abundance , and 85 exhibited a significant decrease in transcript abundance . the microarray - related data were submitted to gene expression omnibus ( geo ) under the accession number : gse13203 . the distribution of ct - responsive genes and their biological roles are shown in figure 2 . a complete list of all genes differentially expressed by ct can be found in the supplemental material ( see supplementary table s1 in supplementary material available online at doi : 10.1155/2009/617509 ) . herein , our interest mainly focused on specific genes that may allow the organism to survive in the presence of ct . in the present study , the sortase - encoding gene srtb   ( sa0982 ) was repressed 3-fold , and some surface protein - encoding genes of isdabdefg   were also significantly inhibited in response to ct . the isd cluster of s. aureus   is composed of 10 genes ( isdabcdefghi   and srtb ) . pathogens , such as s. aureus , require iron for survival and have evolved specialized proteins , such as isd , to scavenge heme from their host . therefore , the staphylococcal isd system can be viewed as a pathogenic strategy for scavenging heme iron during infection by tapping into the iron rich source of hemoproteins . our results suggest that ct significantly affected the staphylococcal isd system . in our study , the transcriptional level of srtchi   was not differentially modulated by a factor of   2.0 or   2.0 upon exposure to ct . the expression of some genes involved in anaerobic respiration and fermentation was induced by ct during this study , including   fdab , pflb , pfla , nirb , nird , narg , narh , and nari . the transcription of the fdab   gene , which is involved in glycolysis , was upregulated , indicating enhanced glycolytic activity by ct . the   pflb   and pfla   genes encode enzymes of the fermentation pathways , which are involved in acetate and ethanol formation   . prior studies demonstrated that the pfla / pflb   pair is involved in energy metabolism when e. coli   grows under oxygen - limiting conditions . narg ( respiratory nitrate reductase , alpha subunit ) , narh ( respiratory nitrate reductase , beta subunit ) , and nari ( respiratory nitrate reductase , gamma subunit ) have been shown to be involved in nitrate respiration in e. coli   . nirb ( nitrite reductase , large subunit ) and nird ( nitrite reductase , small subunit ) are essential and sufficient for nadh - dependent nitrite reduction . furthermore , s. aureus   is able to use nitrate or nitrite as alternative electron acceptors [ 19 , 23 ] . transcripts of the nar   and   nir   operon were found to be present at elevated levels under anaerobic conditions . additionally , ct also induced the arcabcd   genes , which are associated with energy metabolism . the s. aureus   arginine deiminase ( adi ) metabolic pathway genes are organized in an operon arcabdc   . the proteins encoded by arcabcd   are responsible for the adi pathway , which enables arginine - dependent anaerobic growth . oxygen deficiency promotes the autophosphorylation of arcb that activates phosphorylation of arca and results in regulation of numerous operons that provide control of carbon catabolism and cellular redox status . consequently , our results described herein imply that ct may induce anaerobic respiration and fermentation in s. aureus . expression of some genes involved in oxidative stress resistance was affected by ct , including ahpc , ahpf , kata , and sodm . the sodm   gene encodes superoxide dismutase , and   kata   encodes the catalase enzyme . kata   is the only major catalase in s. aureus , accumulates extracellularly , detoxifies h2o2 , and was proposed to be a major virulence determinant in s. aureus   [ 27 , 28 ] . our results showed upregulation of the kata   gene and downregulation of the sodm   gene , thus connecting ct to oxidative - stress response . surprisingly , a similar transcriptional response also occurs when cells are subjected to 1 mm peracetic acid . the ahpc gene encodes alkyl hydroperoxide reductase subunit c , and ahpf encodes alkyl hydroperoxide reductase subunit f. our results also demonstrated an upregulation of the ahpc   and ahpf   genes . alkyl hydroperoxide reductase subunit c ( ahpc ) protects cells against oono2 , which is generated within neutrophils and macrophages . recent report suggested that superoxide radical formation might be the cause of antibacterial activity of ct . as mentioned above , our results showed clearly that genes fdab , pflb , pfla , nirb , nird , narg , narh , and nari   involved in anaerobic respiration and fermentation were upregulated , and genes ahpc , ahpf , and   kata   involved in oxidative stress resistance were upregulated by ct . chang et al . indicated that hydrogen peroxide , a reactive oxygen species , led to genes pflba   and arcbc   increases in transcription levels ; they suggested that s. aureus   might undergo an oxygen - limiting state in response to hydrogen peroxide - driven oxidative stress . further , previous result showed that in e. coli   pfl   is significantly induced by shifting the culture condition from an aerobic to a microaerobic state . moreover , transcriptome and proteome analysis of bacillus subtilis   gene expression in response to superoxide and peroxide stress showed that genes kata   and ahpcf   were significantly induced . thus , our microarray result is consistent with previous observations which revealed that ct might act as superoxide radicals generator ; lee et al . proposed that this phenomenon benefited s. aureus   by preventing further cytotoxicity arising from reactive oxygen species produced during oxygen respiration . lactoquinomycin a ( lqma ) , an antibiotic having a quinone moiety like ct , also generated superoxide radicals during reduction of the quinone moiety by quinone reductase and resulted in other active oxygens . genechip analysis showed that several antibiotic resistance genes were differentially regulated by ct exposure , including dfra , drp35 , cdsa , and pgsa . among these , the transcription of dfra   was upregulated , whereas the transcription of drp35 ,   pgsa   ( 2.0-fold ) , and cdsa   was significantly downregulated by ct exposure . the dfra   gene encodes dihydrofolate reductase , which is responsible for trimethoprim resistance ( tp ) . in present study , we found that ct has high mic values in trimethoprimsulfamethoxazole ( tmp / sxt ) susceptible and resistant strains . in order to test the interaction between ct and tmp / sxt , we conducted additional experiment to assay the vitro antimicrobial activity of ct against s. aureus   strain atcc 25923 in combination with tmp / sxt using checkerboard microdilution method . the result showed that there is an antagonism in combination of ct and tmp / sxt against s. aureus   25923 , with fici of 4 . it is indicated that the increased expression of gene dfra   induced by ct might increase the resistance of the s. aureus   to tmp / sxt . this result suggested that it is necessary to avoid simultanous use of ct and tmp / sxt . the drp35   gene encodes drp35 , which possesses calcium - dependent lactonase activity and is a cytoplasmic protein induced by cell wall - affecting antibiotics or detergents . antibiotic susceptibility experiments using a drp35-defective strain and an overexpressing strain of s. aureus   revealed that drp35 is correlated with bacitracin resistance . two essential genes , cdsa   and pgsa , encoding phosphatidate cytidylyltransferase and phosphatidylglycerophosphate synthase , respectively , are required for synthesis of phosphatidylglycerol phosphate , which might be converted into phosphoglycerol by a nonspecific phosphatase . conditional lethal alleles of genes involved in phospholipid biosynthesis in s. aureus , including pgsa   and cdsa , have also been found to confer hypersusceptibility to macrolides . we found that 2 carotenoid pigment - associated genes , crtm   and crtn , of s. aureus   were upregulated in response to ct . the species description of s. aureus   reflects the colony color ( l. aureus : golden or orange ) and distinguishes this species from staphylococcus epidermidis   ( formerly staphylococcus albus ) . additionally , ct exposure also induced the ald   gene that encodes aldehyde dehydrogenase , which is associated with biosynthesis of c30 carotenoids in s. aureus . the ald   gene was located between crtn   and crtnb   in the same gene cluster . moreover , some putative transporter genes were differentially regulated by ct exposure . in our study , a large number of putative transporter genes ( 39 ) were differentially regulated upon exposure to ct ; 12 of these genes were upregulated , while 27 were downregulated . these genes included 26 atp - dependent transporter genes ( all involved in the atp - binding cassette ( abc ) superfamily ) and 12 secondary transporter genes ( 3 genes are associated with the major facilitator superfamily ( mfs ) ) . in addition , a phosphotransferase system ( pts ) gene , mtla   ( sa1962 ) , was expressed at an increased level . among the putative transporter genes regulated by ct , the semb   gene is a multidrug resistance ( mdr ) transporter , which was expressed at an increased level of 2.8-fold . real - time quantitative rt - pcr was conducted to validate microarray data using the same rna from the original microarray experiment , and 13 genes were selected for this analysis ( i.e. , riba ,   isda ,   moda , seo , sa1831 , srtb , glpt , oppf , sa2135 , msmx , opp-1f , dps , and sa0302 ) . in general , there were positive correlations between microarray data and real - time rt - pcr data for all 13 genes ( table 3 ) . however , expression of the genes sa1831 and sa0302 was observed at greater n - fold changes by real - time rt - pcr than with microarray analysis , indicating that real - time rt - pcr analysis may be more sensitive to changes than microarray analysis in transcript levels . for the remaining 11 genes ( i.e. , riba , isda , moda , seo , srtb , glpt , oppf , sa2135 , msmx , opp-1f , and   dps ) , the levels of gene induction did not differ markedly between microarray data and real - time rt - pcr data . in summary , the presented antibacterial activity and growth curve experiments demonstrated that ct is a bacteriostatic agent against s. aureus . transcriptional profiling revealed that the action mechanism of ct on s. aureus   is correlated to its action as active oxygen radical generator ; s. aureus   might undergo an oxygen - limiting state upon exposure to ct . to our knowledge , this genome - wide transcriptomic approach revealed the first insights into the response of s. aureus   to ct challenge . recognition of ct lays the groundwork for developing new agents owning good activity based on chemical structure modification of the lead compound .
staphylococcus aureus ( s. aureus ) strains with multiple antibiotic resistances are increasingly widespread , and new agents are required for the treatment of s. aureus . 
 cryptotanshinone ( ct ) , a major tanshinone of medicinal plant salvia miltiorrhiza bunge , demonstrated effective in vitro antibacterial activity against all 21 s. aureus strains tested in this experiment . 
 affymetrix genechips were utilized to determine the global transcriptional response of s. aureus atcc 25923 to treatment with subinhibitory concentrations of ct . 
 transcriptome profiling indicated that the antibacterial action of ct may be associated with its action as active oxygen radical generator ; s. aureus undergoes an oxygen - limiting state upon exposure to ct .