staphylococcus aureus is one of the most common nosocomial pathogens which can cause a broad spectrum of infections , ranging from mild skin infections to severe abscesses , sepsis , endocarditis , osteomyelitis , urinary tract infections ( uti ) , and fatal necrotizing pneumonia ( 1 ) . staphylococcus aureus strains have the ability to become resistant to different classes of antimicrobial agents such as methicillin ( 2 ) . resistance to methicillin in s. aureus was first reported in 1961 , just one year after its introduction , and methicillin - resistant staphylococcus aureus ( mrsa ) has spread extensively worldwide during the last few decades ( 3 ) . resistance to methicillin is due to the presence of the staphylococcal cassette chromosome mec ( sccmec ) element , which is composed of regulatory genes such as the meca , c , i and r gene complex , and the ccr ( cassette chromosome recombinase ) gene complex , encoding the recombinase gene ( 4 ) . based on the presence of different regulatory and structural genes , the most important feature of mrsa isolates is their resistance to a broad spectrum of antimicrobial agents , which makes infections by these bacteria difficult to treat ( 5 , 6 ) . aminoglycosides are one of the classes of antibiotics that play an important role in the treatment of staphylococcal infections ( 7 ) . the main mechanism of resistance to aminoglycosides is the inactivation of antibiotics by aminoglycoside - modifying enzymes ( ames ) that are encoded by genetic elements ( 7 , 8) . the aac ( 6)-ie + aph ( 2 ) , ant ( 4)-ia , aph ( 3)-iiia , and ant ( 6)-ia genes that encode aminoglycoside-6'-n - acetyltransferase/2"-o - phosphoryltransferase , aminoglycoside-4'-o - nucleotidyltransferase i , aminoglycoside-3'-o- phosphoryltransferase iii , and streptomycin modifying enzyme , respectively , are hence the most important genes in this regard . resistance to gentamicin , kanamycin , and tobramycin in staphylococci is mediated by a bi - functional enzyme displaying aac ( 6 ' ) and aph ( 2 " ) activity . the ant ( 4')-ia enzyme inactivates neomycin , kanamycin , tobramycin , amikacin , and kanamycin , while the aph ( 3')-iii , enzyme inactivates neomycin ( 8 , 9 ) . different genotyping and phenotyping methods are available for the typing of bacteria , and they are useful in studies of the stability and diversity of bacterial populations in investigations . the genotyping methods are highly discriminative , useful , and flexible , although they are very expensive and laborious , especially when comparing data obtained from many isolates ( 10 ) . phenotyping involves methods used in studying the biochemical characterization of bacteria . the phene - plate ( php ) system is an automated phenotyping method based on the quantitative measurement of the kinetics of biochemical reactions formed by bacterial metabolism in the presence of different substrates . compared to genotyping methods such as pfge and mlst , php typing is a very simple , rapid , and cheap method that provides the necessary information required for the subtyping of bacteria . however , for some cases in which php typing could not be performed , genotyping is required in order to obtain the necessary information ( 10 , 11 ) . this study aimed to describe the frequency and the antibiotic susceptibility patterns of clonal groups of gentamicin - resistant strains of mrsa isolated from a tertiary care hospital in tehran , iran . during january to november 2012 , a total of 301 s. aureus strains were collected from a tertiary care hospital in tehran , iran . this hospital is ranked as one of the top hospitals in the country and it is located in the center of tehran . all specimens were collected from hospitalized patients who showed infections 72 hours after admission to the hospital . of the 301 strains , 139 , 99 , 23 , 21 , ten , and nine were isolated from wounds , urine , blood , sputum , cerebrospinal fluid ( csf ) , and eyes , respectively . all of the isolates were cultured on hicrome aureus agar ( himedia , india ) and then identified at the species level using species - specific nuca gene primers as described previously ( 12 ) . the dna of all the s. aureus strains was extracted using the boiling method as described previously ( 13 ) . moreover , a high pure pcr template preparation kit ( roche , germany ) was employed for dna extraction from the mrsa strains . all of the 301 s. aureus strains were tested for susceptibility to oxacillin ( 1 g ) and cefoxitin ( 30 g ) ( mast diagnostics , merseyside , united kingdom ) on muller - hinton agar ( merck , germany ) according to the guidelines of the clinical and laboratory standards institute ( clsi ) ( 14 ) . the antibiotics used were : kanamycin ( 30 g ) , amikacin ( 30 g ) , penicillin ( 5 g ) , minocycline ( 30 g ) , erythromycin ( 15 g ) , clindamycin ( 2 g ) , tobramycin ( 10 g ) , rifampin ( 5 g ) , sulfamethoxazole - trimethoprim ( 1.25 - 23.75 g ) , linezolid ( 10 g ) , quinupristin - dalfopristin ( 15 g ) , ciprofloxacin ( 30 g ) , neomycin ( 30 g ) , gentamicin ( 10 g ) , and tetracycline ( 30 g ) . the minimum inhibitory concentrationsof oxacillin , gentamicin , and vancomycin were evaluated using a broth microdilution assay according to the guidelines of the clsi ( 15 ) . the presence of aac ( 6)-ie / aph ( 2  ) , ant ( 4)-ia , aph ( 3)-iiia , and ant ( 6)-ia genes among the mrsa strains was examined using specific primers in a pcr assay as described previously ( 7 , 16 ) . all of the mrsa strains were typed using high resolution php - cs plates ( phplate ab , stockholm , sweden ) . the biochemical fingerprinting method was performed as reported by rahimi et al . during january to november 2012 , a total of 301 s. aureus strains were collected from a tertiary care hospital in tehran , iran . this hospital is ranked as one of the top hospitals in the country and it is located in the center of tehran . all specimens were collected from hospitalized patients who showed infections 72 hours after admission to the hospital . of the 301 strains , 139 , 99 , 23 , 21 , ten , and nine were isolated from wounds , urine , blood , sputum , cerebrospinal fluid ( csf ) , and eyes , respectively . all of the isolates were cultured on hicrome aureus agar ( himedia , india ) and then identified at the species level using species - specific nuca gene primers as described previously ( 12 ) . the dna of all the s. aureus strains was extracted using the boiling method as described previously ( 13 ) . moreover , a high pure pcr template preparation kit ( roche , germany ) was employed for dna extraction from the mrsa strains . all of the 301 s. aureus strains were tested for susceptibility to oxacillin ( 1 g ) and cefoxitin ( 30 g ) ( mast diagnostics , merseyside , united kingdom ) on muller - hinton agar ( merck , germany ) according to the guidelines of the clinical and laboratory standards institute ( clsi ) ( 14 ) . the antibiotics used were : kanamycin ( 30 g ) , amikacin ( 30 g ) , penicillin ( 5 g ) , minocycline ( 30 g ) , erythromycin ( 15 g ) , clindamycin ( 2 g ) , tobramycin ( 10 g ) , rifampin ( 5 g ) , sulfamethoxazole - trimethoprim ( 1.25 - 23.75 g ) , linezolid ( 10 g ) , quinupristin - dalfopristin ( 15 g ) , ciprofloxacin ( 30 g ) , neomycin ( 30 g ) , gentamicin ( 10 g ) , and tetracycline ( 30 g ) . the minimum inhibitory concentrationsof oxacillin , gentamicin , and vancomycin were evaluated using a broth microdilution assay according to the guidelines of the clsi ( 15 ) . the presence of aac ( 6)-ie / aph ( 2  ) , ant ( 4)-ia , aph ( 3)-iiia , and ant ( 6)-ia genes among the mrsa strains was examined using specific primers in a pcr assay as described previously ( 7 , 16 ) . all of the mrsa strains were typed using high resolution php - cs plates ( phplate ab , stockholm , sweden ) . of the 301 s. aureus strains isolated from the clinical samples , 90 strains ( 29.9% ) were confirmed as mrsa using phenotypic and genotypic methods . these isolates originated from wounds ( 46% ) , urine ( 30% ) , sputum ( 13% ) , and blood ( 11% ) , respectively . the isolates were recovered from hospitalized patients and so were classified as hospital - acquired mrsa ( ha - mrsa ) strains . the mrsa strains were resistant to penicillin ( 100% ) , ciprofloxacin ( 100% ) , tobramycin ( 99% ) , kanamycin ( 99% ) , erythromycin ( 97% ) , tetracycline ( 90% ) , clindamycin ( 89% ) , and amikacin ( 82% ) ( figure 1 ) . all of the strains were susceptible to vancomycin , linezolid , and quinupristin - dalfopristin . an , amikacin ; cip , ciprofloxacin ; cd , clindamycin ; e , erythromycin ; gm , gentamicin ; k , kanamycin ; lzd , linezolid ; mn , minocycline ; n , neomycin ; p , penicillin ; tn , tobramycin ; t , tetracycline ; rp , rifampin ; ts , sulfamethoxazole - trimethoprim ; va , vancomycin ; syn , quinupristin - dalfopristin . overall , 22 antibiotic resistance patterns were identified among the mrsa strains , of which 1.1% were resistant to at least three different antibiotics ( table 1 ) . of these moreover , six strains ( 6.7% ) were resistant to all of the antibiotics tested except for vancomycin , linezolid , and quinupristin - dalfopristin . abbreviations : an , amikacin ; cd , clindamycin ; cip , ciprofloxacin ; e , erythromycin ; gm , gentamicin ; k , kanamycin ; mn , minocycline ; n , neomycin ; p , penicillin ; rp , rifampin ; t , tetracycline ; tn , tobramycin ; ts , sulfamethoxazole - trimethoprim . the mic range varied from 16 to 512 g / ml , with 33% and 26% of strains having an mic of 128 g / ml and 256 g / ml for oxacillin , respectively . moreover , one strain had an mic of 16 g / ml . also , for vancomycin , the mics ranged from 0.06 to 0.5 g / ml . further , most of the strains ( 22.2% ) had an mic of 128 g / ml for gentamicin , whilst 11 strains showed an mic  16 g / ml . seven ( 7.8% ) , ten ( 11.1% ) , and 12 ( 13.3% ) strains had an mic of 1024 , 512 , and 256 g / ml , respectively . all of the aminoglycoside - resistant strains were tested for the presence of different genes , with aac ( 6)-ie + aph ( 2 ) being found among 59 ( 65.6% ) strains ( table 2 ) and hence being the most prevalent resistance . abbreviations : an , amikacin ; gm , gentamicin ; k , kanamycin ; n , neomycin ; tn , tobramycin . forty - three ( 47.8% ) isolates harbored the ant ( 6)-ia resistance gene , while ant ( 4)-ia was detected in 38 ( 42.2% ) strains . moreover , the aph ( 3)-iiia gene was present in 18 ( 20% ) strains . also , 14 isolates ( 15.6% ) carried four detected resistance genes together , and none of the genes were positive in 25 ( 27.8% ) strains . on the other hand , aac ( 6)-ie + aph ( 2 ) , aph ( 3)-iiia and ant ( 6)-ia were detected alone in 19 ( 21.1% ) , two ( 2.2% ) and one ( 1.1% ) isolates , respectively . the php typing of the 90 mrsa strains revealed the presence of diverse ( diversity index , di = 0.818 ) php types among the isolates , consisting of seven common types ( ct ) and four single types ( st ) ( data not shown ) . amongst the php types , ct 3 was the dominant type , with 31% of the isolates classified in this pattern . cts 4 and 2 were also found among 24% and 11% of the isolates , respectively . on the other hand , the four sts were common among strains isolated from wounds and urine . the least number of strains belonged to cts 5 - 7 , which contained six isolates ( 7% ) . of the 301 s. aureus strains isolated from the clinical samples , 90 strains ( 29.9% ) were confirmed as mrsa using phenotypic and genotypic methods . these isolates originated from wounds ( 46% ) , urine ( 30% ) , sputum ( 13% ) , and blood ( 11% ) , respectively . the isolates were recovered from hospitalized patients and so were classified as hospital - acquired mrsa ( ha - mrsa ) strains . the mrsa strains were resistant to penicillin ( 100% ) , ciprofloxacin ( 100% ) , tobramycin ( 99% ) , kanamycin ( 99% ) , erythromycin ( 97% ) , tetracycline ( 90% ) , clindamycin ( 89% ) , and amikacin ( 82% ) ( figure 1 ) . all of the strains were susceptible to vancomycin , linezolid , and quinupristin - dalfopristin . an , amikacin ; cip , ciprofloxacin ; cd , clindamycin ; e , erythromycin ; gm , gentamicin ; k , kanamycin ; lzd , linezolid ; mn , minocycline ; n , neomycin ; p , penicillin ; tn , tobramycin ; t , tetracycline ; rp , rifampin ; ts , sulfamethoxazole - trimethoprim ; va , vancomycin ; syn , quinupristin - dalfopristin . overall , 22 antibiotic resistance patterns were identified among the mrsa strains , of which 1.1% were resistant to at least three different antibiotics ( table 1 ) . of these moreover , six strains ( 6.7% ) were resistant to all of the antibiotics tested except for vancomycin , linezolid , and quinupristin - dalfopristin . abbreviations : an , amikacin ; cd , clindamycin ; cip , ciprofloxacin ; e , erythromycin ; gm , gentamicin ; k , kanamycin ; mn , minocycline ; n , neomycin ; p , penicillin ; rp , rifampin ; t , tetracycline ; tn , tobramycin ; ts , sulfamethoxazole - trimethoprim . the mic range varied from 16 to 512 g / ml , with 33% and 26% of strains having an mic of 128 g / ml and 256 g / ml for oxacillin , respectively . moreover , one strain had an mic of 16 g / ml . also , for vancomycin , the mics ranged from 0.06 to 0.5 g / ml . further , most of the strains ( 22.2% ) had an mic of 128 g / ml for gentamicin , whilst 11 strains showed an mic  16 g / ml . seven ( 7.8% ) , ten ( 11.1% ) , and 12 ( 13.3% ) strains had an mic of 1024 , 512 , and 256 g / ml , respectively . all of the aminoglycoside - resistant strains were tested for the presence of different genes , with aac ( 6)-ie + aph ( 2 ) being found among 59 ( 65.6% ) strains ( table 2 ) and hence being the most prevalent resistance . abbreviations : an , amikacin ; gm , gentamicin ; k , kanamycin ; n , neomycin ; tn , tobramycin . forty - three ( 47.8% ) isolates harbored the ant ( 6)-ia resistance gene , while ant ( 4)-ia was detected in 38 ( 42.2% ) strains . moreover , the aph ( 3)-iiia gene was present in 18 ( 20% ) strains . also , 14 isolates ( 15.6% ) carried four detected resistance genes together , and none of the genes were positive in 25 ( 27.8% ) strains . on the other hand , aac ( 6)-ie + aph ( 2 ) , aph ( 3)-iiia and ant ( 6)-ia were detected alone in 19 ( 21.1% ) , two ( 2.2% ) and one ( 1.1% ) isolates , respectively . the php typing of the 90 mrsa strains revealed the presence of diverse ( diversity index , di = 0.818 ) php types among the isolates , consisting of seven common types ( ct ) and four single types ( st ) ( data not shown ) . amongst the php types , ct 3 was the dominant type , with 31% of the isolates classified in this pattern . cts 4 and 2 were also found among 24% and 11% of the isolates , respectively . moreover , cts 3 and 4 were common among all strains with different origins . on the other hand , the four sts were common among strains isolated from wounds and urine . the least number of strains belonged to cts 5 - 7 , which contained six isolates ( 7% ) . previous studies have revealed that the rate of mrsa in iran varies from 19% to 90% in different cities ( 3 , 13 , 17 - 23 ) . the variation seen in the different reports concerning iran could be in part due to different populations , different geographical locations , and the quality of hospital sampling carried out . of the 15 antibiotics tested in this study , all of the isolates showed susceptibility to vancomycin , linezolid , and quinupristin - dalfopristin , which is consistent with other studies in iran ( 3 , 13 , 17 - 23 ) . although vancomycin is frequently used in a hospital setting , no vancomycin - intermediate s. aureus ( visa ) or vancomycin - resistant s. aureus ( vrsa ) isolates were found in this study , which suggests that the increased use of certain antibiotics is not sufficient to ensure the appearance of resistant strains . yet , most of the isolates were resistant to penicillin ( 100% ) , ciprofloxacin ( 100% ) , kanamycin ( 99% ) , tobramycin ( 99% ) , erythromycin ( 97% ) , tetracycline ( 90% ) , and clindamycin ( 89% ) , which indicates that these antibiotics are no longer effective antibiotics against mrsa infections in tehran . in previous studies ( 3 , 17 , 19 - 23 ) , a high rate of resistance to these antibiotics was reported . these antibiotics are used extensively in hospitals for the treatment of different infections and so the high rate of resistance is not surprising . the rates of resistance tosulfamethoxazole - trimethoprim , rifampin , minocycline , and gentamycin in this study were also higher than those reported in other reports . this could be explained by the high level of these antibiotics being prescribed for the treatment of infections . moreover , gentamicin is one of the most important antibiotics used in combination with other antibiotics worldwide for treatment of s. aureus infections ( 20 , 24 - 26 ) . in this study , similar to the findings of other reports ( 20 , 24 - 27 ) , the aac ( 6)-ie + aph ( 2 ) gene was dominant among the gentamicin - resistant strains of mrsa , and the isolates that were positive for this gene showed a high level resistance to gentamicin , which is consistent with other reports ( 16 , 28 - 30 ) . we also found 16 gentamicin - susceptible strains that harbored the aac ( 6)-ie + aph ( 2 ) gene . although these isolates harbored the aac ( 6)-ie + aph ( 2 ) gene considered to be resistant to gentamicin and all aminoglycosides , we also found strains that were susceptible to gentamicin and showed resistance to other aminoglycosides , which is consistent with other reports ( 7 ) . detection of resistance genes in antibiotic susceptible strains is due to amplification of repressed antibiotic resistance gene or ame of these strains display lower enzymatic activity ( 7 ) . moreover , the prevalence rate of the ant ( 4)-ia gene was higher than in other reports ( 7 , 16 , 20 , 26 ) , which could be due to the high resistance to kanamycin , with 89 out of 90 strains being kanamycin - resistant . also , we found a strain that was susceptible to all of the aminoglycoside antibiotics tested and was also not positive for all of the genes . differences between reports from different countries could be due to differences among the isolates and different geographical regions . our results illustrated that all of the aminoglycosides tested in this study are no longer effective agents against mrsa strains . the results of the php typing showed the presence of diverse php types consisting of seven cts and four sts , indicating that the presence of mrsa strains in this hospital in tehran is attributable to the spread of a limited number of clonal types . cts 3 and 4 were common between mrsa strains with different origins , which further supports the spread of these clonal types among strains collected from this hospital in tehran . in another study in tehran , more diverse php types ( consisting of 18 cts and 15 sts ) were reported among the mrsa strains isolated from a tertiary care hospital ( 3 ) . on the other hand , 16 cts and 13 sts were identified in mrsa strains isolated from sewage treatment plants in tehran ( 31 ) . javidnia et al . reported six cts and 13 sts among mrsa strains in tehran ( 19 ) . seven cts identified in this study were also common among the mrsa isolates reported in other studies , in which ct 3 was the predominant type and its dissemination is consistent with previous studies in tehran ( 3 , 9 , 31 , 32 ) . in this study , different php types were common among different samples , which indicates the prevalence of mrsa strains in different wards of the hospital and also highlights the presence of common cts in this hospital . the mrsa strains isolated from wounds and urine were grouped in different cts and showed the highest frequency . a certain php mrsa clonal type , i.e. ct 3 ( comprising 31% of the isolates ) , was identified in various samples tested , indicating an epidemiological link between the mrsa strains isolated from different sources . the results indicate that mrsa strains are endemic in this hospital in tehran ; therefore , contamination of patients during the hospitalization process may be responsible for these infections . as previously reported , the aminoglycoside - resistant ha - mrsa strains have spread widely through the community and identical ha - mrsa strains from different sources in iran have been isolated , indicating the hospital origin of these strains . in conclusion , our results showed that the php typing method provided useful information for clonal dissemination and also for the epidemiological links of clonal groups of aminoglycoside - resistant strains of mrsa in hospitals in tehran . moreover , the presence of predominant clonal types among all strains isolated from different sources with different antimicrobial resistance patterns suggested that cross - infection was a factor in the maintenance of these strains in hospitals in tehran .