staphylococcus aureus is an important bacterial pathogen that may cause a variety of diseases , ranging from common skin and soft tissue infections to very serious and lethal diseases such as septic shock , toxic shock syndrome or pneumonia ( 1 , 2 ) . although antibiotic therapy is currently used to treat s. aureus infections , the emergence of antibiotic - resistant strains is rapidly exhausting available treatment options ( 1 , 2 ) . to date , resistance of s. aureus strains to methicillin is categorized as one the main causes of hospital infections . furthermore , treatment of methicillin - resistant s. aureus ( mrsa ) infections often requires longer hospital stays and imposes a tremendous financial burden ( 3 ) . severe mrsa infections therapy is complicated by the fact that these strains are susceptible to only a few antimicrobials such as vancomycin . unfortunately , in the past decade , vancomycin resistant s. aureus is isolated , highlighting the ability of the bacteria to adapt to the new antibiotics ( 4 , 5 ) . the main resistance mechanism of s. aureus to methicillin is the acquisition of the meca gene and encoding a new transpeptidase enzyme ( pbp ) that has a reduced tendency to beta lactam antibiotics . since meca gene has high homology among mrsa strains and coagulase negative staphylococcus resistant to methicillin , it is considered as a proper molecular marker to determine methicillin resistance in all staphylococcal species ( 6 , 7 ) . these differences appear to be due to various distribution of this gene at different geographical regions or their identification method . increased spread of meca is the common issue among all these studies , indicating a potential threat that make staphylococcal infections resistant to methicillin and other globally used antibiotics ( 9 ) . nowadays , phenotypic methods such as disk diffusion and minimum inhibitory concentration ( mic ) , as well as genotypic methods such as pcr are used to diagnose mrsa strains . these methods have some limitations and there is no perfect method to diagnose these strains ( 10 ) . as mentioned above , since mrsa strains are among the important human pathogens , it is necessary to find a reliable and simple method as an alternative or complementary method to diagnose mrsa strains especially during the prevalence of the strains or performance of a health plan . for instance , sucrose is used by almost 98% of s. aureus strains , producing acid during its fermentation process . the current study assayed the turanose metabolism at different dilutions as a new rapid phenotypic method to identify mrsa isolates . the obtained results indicated a considerable difference between the fermentation of turanose in mrsa and methicillin - sensitive s. aureus ( mssa ) isolates at 0.7% dilution . therefore , a simple phenotypic test based on the turanose fermentation is suggested as a useful method to diagnose mrsa strains . due to the importance of methicillin - resistant s. aureus ( mrsa ) infections , the current study aimed to assay the turanose metabolism at different dilutions as a rapid phenotypic method to identify mrsa isolates . samples were collected from tehran medical centers , within three months ( october - december 2011 ) . isolation of s. aureus was performed according to the standard microbial and biochemical tests , such as fermentation of mannitol , coagulase test and dnase ( merck , kgaa , darmstadt , germany ) . the disk diffusion method was performed using a disk containing 1 g oxacillin ( bbl , sensi disk , usa ) to diagnose mrsa isolates ( 12 ) . this method was performed according to the clinical and laboratory standards institute ( clsi ) guidelines ( 13 ) . for this purpose , bacterial suspensions equal to 0.5 mcfarland standard were scattered on agar mueller - hinton medium ( merck , kgaa , darmstadt , germany ) . then , the oxacillin disk was put on the incubated plates and after 24 hours the inhibition zone diameter around the disk was measured . dna extraction of s. aureus isolates was conducted by dna extraction kit ( qiagen gmbh , hilden , germany ) to identify the presence or absence of meca gene in the s. aureus isolates , the following pairs of primers were used for the amplification ( 14 ) : meca f : 5'-tagaaatgactgaacgtccgataa-3 ' meca r : 5'-ccaattccatgtttcggtctaa-3 ' pcr reactions were carried out in a final volume of 50 l containing 2 l of dna template , 5 l of 10x reaction buffer , 2 l of dntps ( 10 mm ) , 2 l of mgcl2 ( 50 mm ) , 2 l of each primer ( 10 pmol ) , and 1u of taq dna polymerase ( fermentas ; vilnius , lithuania ) . the pcr conditions were as follow : an initial denaturation for 5 minutes at 94c , followed by 30 cycles , each consisting of 60 seconds at 94c , 60 seconds at 55c , and 60 seconds at 72c with a final extension step at 72c for 5 minutes . after amplification of meca gene , 10 l of the samples were subjected to electrophoresis on a 1% agarose gel to confirm the presence of the amplified products . five grams of turanose was dissolved in 100 ml of distilled water and sugar with 5% dilution was prepared . to complete tantalization process of the turanose , the mixture was placed in a boiling water bath ( 80c ) for sterilization , for an hour per day on three consecutive days . the prepared 5% dilution of turanose was used to provide 1% , 0.7% and 0.5% dilutions . preparation of microbial suspensions of each s. aureus strain was performed in sterile glass vials and their turbidity was adjusted to 0.5 mcfarland . in triplicate , 150 l of nutrient broth ( sugar free ) medium containing different turanose dilutions ( 0.5% , 0.7% and 1% ) were inoculated to each well in microplates ( greiner , germany ) and 150 l of each microbial suspension was added to the microplates . furthermore , medium as well as turanose without microbial suspension were used as a negative control and blank , respectively . the growth of susceptible and resistant isolates to methicillin was assayed in the presence of different dilutions of turanose by measurement of their light absorption with enzyme - linked immunosorbent assay ( elisa ) reader ( awareness technology inc . ) at 610 nm . to calculate the average , one - way anova , students t - test and tukey hsd test were used to compare the differences between the mean values of the groups using spss software . samples were collected from tehran medical centers , within three months ( october - december 2011 ) . isolation of s. aureus was performed according to the standard microbial and biochemical tests , such as fermentation of mannitol , coagulase test and dnase ( merck , kgaa , darmstadt , germany ) . the disk diffusion method was performed using a disk containing 1 g oxacillin ( bbl , sensi disk , usa ) to diagnose mrsa isolates ( 12 ) . this method was performed according to the clinical and laboratory standards institute ( clsi ) guidelines ( 13 ) . for this purpose , bacterial suspensions equal to 0.5 mcfarland standard were scattered on agar mueller - hinton medium ( merck , kgaa , darmstadt , germany ) . then , the oxacillin disk was put on the incubated plates and after 24 hours the inhibition zone diameter around the disk was measured . dna extraction of s. aureus isolates was conducted by dna extraction kit ( qiagen gmbh , hilden , germany ) to identify the presence or absence of meca gene in the s. aureus isolates , the following pairs of primers were used for the amplification ( 14 ) : meca f : 5'-tagaaatgactgaacgtccgataa-3 ' meca r : 5'-ccaattccatgtttcggtctaa-3 ' pcr reactions were carried out in a final volume of 50 l containing 2 l of dna template , 5 l of 10x reaction buffer , 2 l of dntps ( 10 mm ) , 2 l of mgcl2 ( 50 mm ) , 2 l of each primer ( 10 pmol ) , and 1u of taq dna polymerase ( fermentas ; vilnius , lithuania ) . the pcr conditions were as follow : an initial denaturation for 5 minutes at 94c , followed by 30 cycles , each consisting of 60 seconds at 94c , 60 seconds at 55c , and 60 seconds at 72c with a final extension step at 72c for 5 minutes . after amplification of meca gene , 10 l of the samples were subjected to electrophoresis on a 1% agarose gel to confirm the presence of the amplified products . five grams of turanose was dissolved in 100 ml of distilled water and sugar with 5% dilution was prepared . to complete tantalization process of the turanose , the mixture was placed in a boiling water bath ( 80c ) for sterilization , for an hour per day on three consecutive days . the prepared 5% dilution of turanose was used to provide 1% , 0.7% and 0.5% dilutions . preparation of microbial suspensions of each s. aureus strain was performed in sterile glass vials and their turbidity was adjusted to 0.5 mcfarland . in triplicate , 150 l of nutrient broth ( sugar free ) medium containing different turanose dilutions ( 0.5% , 0.7% and 1% ) were inoculated to each well in microplates ( greiner , germany ) and 150 l of each microbial suspension was added to the microplates . furthermore , medium as well as turanose without microbial suspension were used as a negative control and blank , respectively . the growth of susceptible and resistant isolates to methicillin was assayed in the presence of different dilutions of turanose by measurement of their light absorption with enzyme - linked immunosorbent assay ( elisa ) reader ( awareness technology inc . ) at 610 nm . to calculate the average , one - way anova , students t - test and tukey hsd test were used to compare the differences between the mean values of the groups using spss software . out of the 150 staphylococcus isolates , 80 were identified as s. aureus . according to the disk diffusion results , 40 s. aureus isolates electrophoresis of pcr products showed that the length of pcr fragment of the meca gene was approximately 310 bp ( figure 1 ) . the growth of mrsa and mssa strains was evaluated in the presence of turanose with 0.5% , 0.7% and 1% dilutions to find significant differences in metabolism of turanose among the strains . the result of turanose metabolism at the dilutions showed significant difference among mrsa and mssa isolates in 0.7% dilution of turanose ( p < 0.003 ) ( figure 2 ) . however , the differences between mrsa and mssa isolates in 1% and 0.5% dilutions were not statistically significant ( p > 0.05 ) ( figure 2 ) . the metabolism of turanose was evaluated at ( a ) 0.7% dilution for mrsa and ( b ) mssa , ( c ) 1% dilution for mrsa and ( d ) mssa , and ( e ) 0.5% dilution for mrsa and ( f ) mssa strains . numbers 1 - 40 : the mrsa or mssa strains and number 41 : the negative control . out of the 150 staphylococcus isolates , 80 were identified as s. aureus . according to the disk diffusion results , 40 s. aureus isolates electrophoresis of pcr products showed that the length of pcr fragment of the meca gene was approximately 310 bp ( figure 1 ) . the growth of mrsa and mssa strains was evaluated in the presence of turanose with 0.5% , 0.7% and 1% dilutions to find significant differences in metabolism of turanose among the strains . the result of turanose metabolism at the dilutions showed significant difference among mrsa and mssa isolates in 0.7% dilution of turanose ( p < 0.003 ) ( figure 2 ) . however , the differences between mrsa and mssa isolates in 1% and 0.5% dilutions were not statistically significant ( p > 0.05 ) ( figure 2 ) . the metabolism of turanose was evaluated at ( a ) 0.7% dilution for mrsa and ( b ) mssa , ( c ) 1% dilution for mrsa and ( d ) mssa , and ( e ) 0.5% dilution for mrsa and ( f ) mssa strains . numbers 1 - 40 : the mrsa or mssa strains and number 41 : the negative control . staphylococcus aureus is one of the major causes of nosocomial and community - acquired infections that results in substantial morbidity and mortality ( 1 , 2 ) . furthermore , due to multi - drug resistance and the ability to acquire resistance to new antibiotics quickly , it is more and more difficult to treat s. aureus infection , especially with the emergence of vancomycin resistant s. aureus ( 15 , 16 ) . after the growth of mrsa at hospitals , these infections gradually go out of hospitals , which worsen their treatment complexity ( 4 ) . for example , the prevalence of mrsa was reported 2% in switzerland and the netherlands or 80% in china ( 17 ) . similarly , high prevalence of mrsa was reported in different parts of iran ( 18 , 19 ) . the differences in prevalence of the mrsa strains may be due to long - term antibiotic treatment of patients suffering from severe infections , with long hospitalization and increased selective pressure ( 20 ) . , meca gene is detected by pcr in most studies to confirm phenotypic tests in mrsa diagnosis ( 20 ) . in the current study , rodriguez reported that all mrsa strains contain the meca gene , similar to the current study . however , there are reports on the absence of this gene in mrsa strains . in a study , it was shown that 2.25% of mrsa strains lack meca gene ( 12 ) . this discrepancy may be due to bad quality of the pcr test or genetic mutation . although , genotypic methods such as pcr are in the category of rapid methods to diagnose resistant strains , they are not perfect enough to diagnose resistance . for example , whereas detection of the meca gene is the gold standard of identification of mrsa strains , the required facilities and high costs do not let many laboratories use the method to identify mrsa ( 21 ) . furthermore , it is documented that some resistant strains have different genetic sequences and the designed primers fail to identify the mrsa strains in pcr reaction . this difference in genetic sequence indicates that the necessary precaution should be noted in the application of genotypic methods to detect mrsa . in addition to being time - consuming and expensive , phenotypic methods such as disk diffusion have difficulties to diagnose resistant strains ( 22 ) . despite extensive efforts , a diagnostic method identifying all mrsa is currently missing and there is a need to make future diagnostic test evaluations to develop a sensitive , specific and practical method to detect the strains ( 22 ) . phenotypic diagnostic methods of bacteria are on the basis of quantitative assessment of products obtained from biochemical reactions or metabolism of initial substrates . since biochemical and phenotypic methods completely depend on responsible genes on the bacterial dna , these methods are a kind of indirect assessment of bacterial genetics . although , phenotypic methods are usually simpler than genotypic methods , genotypic methods appear to be necessary in some cases in order to confirm phenotypic findings ( 9 , 10 ) . the current study was designed based on the kinetic assessment of biochemical reactions . some studies showed that genes are responsible for resistance to methicillin make some changes in the walls of gram - positive bacteria and there is a relationship between the wall changes made by resistance genes and metabolism of turanose in mrsa strains ( 23 ) . thus , the relationship between metabolism of turanose and resistance to methicillin in s. aureus strains was evaluated in the current study . the results showed a significant relationship between the metabolism of turanose at 0.7% dilution and resistance to methicillin in s. aureus strains . by turanose metabolism as a phenotypic test , it was found that a simple diagnosis method can be achieved to identify these strains . therefore , such studies that can lead to find a rapid way to diagnose antibiotic resistance is highly valuable . it is likely to introduce rapid diagnostic kits of these bacteria by analyzing bigger populations of the strains . in conclusion , the current study suggests a new method for rapid and simple diagnosis of mrsa strains at laboratories by measuring the metabolism of turanose .