staphylococcus aureus are extremely versatile pathogenic bacteria that cause a wide range of syndromes , ranging from minor skin and soft tissue infections to life - threatening pneumonia and toxinoses . the ability of s. aureus to cause diseases is multifactorial , a combination of many virulent factors including toxins , secreted exoproteins , and cell surface - associated adhesins . the different virulent factors expressed are controlled by a definite locus named the accessory gene regulator ( agr ) and the staphylococcal accessory regulator ( sar ) . the exoproteins or exotoxins include haemolysins , different enzymes , and a family of related pyrogenic toxins . these pyrogenic toxins include staphylococcal enterotoxin ( se ) a-e and g-u subtypes , toxic shock syndrome toxin-1 ( tsst-1 ) , and exfoliative toxins ( et ) a and b . on the other hand , dna sequence analysis of the usa 300 clone identified a genetic region and designated the arginine catabolic mobile element ( acme ) . it was acquired horizontally from s. epidermidis or other coagulase - negative staphylococci   and inhibited polymorphonuclear cell production . acme - encoded arca gene encodes for an arginine deiminase pathway and an oligopeptide permease system enhancing colonization , virulence fitness of the strain , and is associated with invasive diseases ( necrotizing pneumonia ) . s. aureus resistance against a wide variety of antimicrobials is another important factor and is increasing worldwide , including even resistance to vancomycin , the drug of choice for staphylococcal infections . strains resistant to penicillin , however , secrete -lactamase ( through blaz gene ) that hydrolyses the -lactam ring of the antibiotic . resistance to methicillin is encoded by the staphylococcal cassette chromosome mec ( sccmec ) element , composed of the mec gene complex , and the ccr ( cassette chromosome recombinase ) gene complex , encoding for the recombinase gene . sccmec elements have been classified into eight major types ( i  viii ) , some of which are differentiated further into subtypes . sccmec types i , ii , and iii , and types iv and v have been associated with hospital - acquired ( ha ) and community - acquired methicillin resistant s. aureus ( ca - mrsa ) , respectively . typing is crucial in understanding the epidemiology of pathogens and , hence , the development of public health interventions . ribotyping is a dna - based typing method based on the sequencing of the gene coding for rrnas in bacteria . genes encoding for rrna are arranged in an operon in the following order 5-16s-23s-5s-3 and is separated by two spacer regions known as the intergenic transcribed spacer ( its ) . the most commonly used components in ribotyping are 16s rrna gene and the 16s23s its . the its has proved to be more variable than the adjacent 16s and 23s ribosomal genes and may allow efficient identification at the species level due to its variability within a genus . this variability is due partly to differences in the number and type of trna sequences found within the spacer . the use of sequence polymorphisms and length variations found in the its region as a tool for differentiation between closely related organisms is increasing because it can overcome limitations of the resolution of 16s rrna - based phylogenies . this study aims at characterizing 130 s. aureus clinical isolates involved in human diseases based on the determination of antimicrobial resistance profiles , studying the prevalence of exfoliative toxins a ( eta ) and b ( etb ) , enterotoxins ( se ) a-e and g-u , acme , and tsst-1 and through typing the isolates based on the size of the 16s23s dna spacer region and on the patterns obtained by restriction digestion of the amplified spacer region . 130 s. aureus clinical isolates from the american university of beirut medical center ( aub - mc ) were used in this study . the isolates were recovered from different sites of infection , age groups , and years . these isolates were identified by different methods including the api and biolog identification systems and the coagulase test . the following s. aureus reference strains were used as positive controls : tc-142 ( eta positive ) , tc-7 ( etb positive ) , and atcc ( baa-1556d-5 ) ( acme positive ) ; these strains were thankfully donated by dr . reference strains also included the atcc23235 ( sed positive ) , atcc27664 ( see positive ) , nctc 6571 ( seg and sei positive ) , nctc11963 ( tsst positive ) , atcc13565 ( sea positive ) , atcc14458 ( seb positive ) , atcc19095 ( sec positive ) , and nctc10652 ( sej positive ) . all clinical isolates were stored in cryobank tubes ( copan , usa ) at both 20c and 80c . the antimicrobial resistance of the clinical strains to different antibacterial agents was determined by the standard e - test method that comprises a predefined antibiotic gradient strips . strips were used according to the manufacturer instructions ( ab biodisk , sweden ) to determine the minimum inhibitory concentration ( mic ) in g / ml . bacterial suspensions were prepared by selecting colonies from overnight cultures on mannitol salt agar plates . the colonies were suspended to sterile tubes containing 5ml of saline solution ( 0.85% nacl ) to get a suspension with a turbidity similar to the 0.5 mcfarland standard . mueller - hinton agar plates ( oxoid ) were inoculated with the s. aureus clinical isolates , and 18 different antibiotics were used including azithromycin ( az ) , ciprofloxacin ( ci ) , clarithromycin ( ch ) , clindamycin ( cm ) , chloramphenicol ( cl ) , erythromycin ( em ) , gentamicin ( gm ) , levofloxacin ( le ) , nitrofurantoin ( ni ) , norfloxacin ( nx ) , ofloxacin ( of ) , oxacillin ( ox ) , rifampicin ( ri ) , teicoplanin ( tp ) , tetracycline ( tc ) , trimethoprim ( tr ) , trimethoprim / sulfamethoxazol ( ts ) , and vancomycin ( va ) . plates were then incubated for 24  h at 37c . following incubation , mic values that correspond to the edge of the inhibition genomic dna was extracted using qiaamp dna mini kit ( qiagen inc , germany ) following the manufacturer 's instructions . all pcr assays were performed on perkinelmer geneamp 9700 ( perkinelmer , wellesly , massachusetts ) . amplification was performed using the following protocol : an aliquot of 5  l of dna ( 10  ng/l ) was added to 45  l of reaction mixture containing final concentrations of 1x amplitaq buffer , 1.5  mm mgcl2 , 2.5  u of amplitaq gold polymerase ( applied biosystems , roche ) , 200  m each deoxynucleoside triphosphate of either 1  m of each of etaf ( 5-ctagtgcatttgttattcaa-3 ) and etar primers ( 5-tgcattgacaccatagtact-3 ) or 1  m etbf ( 5-acggctatatacattcaatt-3 ) and etbr primers ( 5-tccatcgataatatacctaa-3 ) [ 14 , 15 ] . the amplification was performed with an initial denaturation step for 10  min at 95c followed by 33 cycles of 94c for 2  min , 55c for 1  min , 72c for 1  min , and a final extension at 72c for 7  min . pcr products were separated by electrophoresis of 10  l of reaction product in a 2% agarose gel stained by 0.5  g / ml ethidium bromide , and visualized using a uv bioimaging system ( genesnap system from syngene ) . all pcr assay runs incorporated a negative ( one reagent control without template dna ) and a positive control ( reference strain used for the gene amplified ) . product size was determined by comparison with a 100  bp molecular weight marker ( fermentas , vilnius , lithuania ) . amplification was performed using the following protocol : an aliquot of 2  l of dna was added to 20  l of reaction mixture containing final concentrations of 1x amplitaq buffer , 2.5  mm mgcl2 , 0.1  u of amplitaq gold polymerase ( all from applied biosystems , roche ) , 0.2  mm each deoxynucleoside triphosphate , the junction between acme and orfx was amplified using 0.4  m of the orfx - specific pcr primer xsau325 ( 5-ggatcaaacggcctgcaca-3 ) , and 0.4  m acmer primer ( 5-cctccttcacttagcactg-3 ) . the amplification was performed with an initial denaturation step for 12  min at 95c followed by 30 cycles of 94c for 30  sec , 61c for 30  sec , 72c for 1  min , and a final extension at 72c for 10  min . pcr products were separated by electrophoresis of 10  l of reaction product in a 1.5% agarose gel stained by 0.5  g / ml ethidium bromide , and visualized using a uv bioimaging system ( genesnap system from syngene ) . multiplex pcr assays were used to detect nine staphylococcal enterotoxin ( se ) genes sea , seb , sec , sed , see , seg , seh , sei , sej , and the tst gene . two reaction mixtures were used , primers for sed , see , seg , sei , and tsst   were combined in reaction mixture 1 , and primers for sea , seb - sec , sec , she , and sej were combined in reaction mixture 2 . amplification was performed using 5  l of dna ( 10  ng/l ) added to 45  l of reaction mixture containing final concentrations of 1x amplitaq buffer , 4  mm mgcl2 , 2  u of amplitaq gold polymerase ( applied biosystems , roche ) , 400  m each deoxynucleoside triphosphate , 300  nm of each exotoxin primer set . the amplification was performed with an initial denaturation step for 10  min at 95c followed by 15 cycles of 95c for 1  min , 68c for 45  s , 72c for 1  min , 20 cycles of 95c for 1  min , 64c for 45  s , 72c for 1  min , and a final extension at 72c for 10  min . pcr products were separated by electrophoresis of 10  l of reaction product in a 2.5% agarose gel stained by 0.5  g / ml ethidium bromide , at 100  v for 100  min , and visualized using a uv bioimaging system ( digidoc - it system , version 2.2.0 , 2003 ) . the 16s23s its region was amplified using the following protocol : an aliquot of 2  l of dna was added to 20  l of reaction mixture containing final concentrations of 1x amplitaq buffer , 2.5  mm  mgcl2 , 0.1  u of amplitaq gold polymerase ( applied biosystems , roche ) , 0.2  mm each deoxynucleoside triphosphate , 0.4  m staph its - f primer ( 5-agagtttgatcctggctcag-3 ) , 0.4  m staph its - r primer ( 5-caaggcatccaccgt-3 ) . the amplification was performed with an initial denaturation step for 12  min at 95c followed by 30 cycles of 94c for 30  sec , 42c for 30  sec , 72c for 1  min , and a final extension at 72c for 10  min . pcr products were separated by electrophoresis of 10  l of reaction product in a 1.5% agarose gel stained with 0.5  g / ml ethidium bromide , and visualized using a uv bioimaging system ( genesnap system from syngene ) . the amplified 16s23s its rrna gene fragments were digested with 10  u taqi ( mbi fermentas ) restriction enzyme . 1  l of the enzyme was added to 12  l of the its pcr products , 16  l water , along with 2  l 10x taqi buffer for 3  h at 65c ( fermentas ) . products were resolved on a 2% agarose gels in 1x tris - borate - edta buffer and were subsequently visualized by uv illumination after ethidium bromide staining . gel extraction of the 16s23s dna spacer region primary product ( 1800  bp ) was performed using qiaquick gel extraction kit ( qiagen inc , germany ) following the manufacturer 's instructions . a 500  bp of the purified dna was subjected to direct sequencing using the big dye terminator kit ( abi 3130xi genetic analyzer , applied biosystems ) . sequencing was performed in separate tubes , and each reaction ( 10  l ) consisted of 5  l of purified dna and 4  l of terminator ready reaction mix and was amplified using 2  pmol of the staph its - f ( 5-aga gtt tga tcc tgg ctc ag-3 ) and staph its - r ( 5-caaggcatccaccgt-3 ) primers in separate reactions . sequence homology and identity comparison to other sequences was performed with the ncbi - blast sequence search . phylogenetic tree was constructed by the neighbor  joining method using the clc main workbench version . the prevalence of eta within both groups was 11% while it was 3% for etb in mrsa and 9% in mssa . the eta gene ( n = 14 ; 10.8% ) was more prevalent than etb ( n = 6 ; 4.61% ) , and only three ( 2% ) of the tested isolates harbored both genes . the sizes of amplicons obtained for the exotoxin genes were 119 and 200  bp for eta and etb genes , respectively , and 333  bp for the acme , being same as those obtained for the reference strains tc-7 ( for eta ) , tc-142 ( for etb ) , and baa-1556d-5 ( for acme ) . majority of the isolates , which were previously typed by tokajian et al .   carrying the eta gene , belonged to spa cc044 ( 21% ) and spa cc008 ( 21% ) while the etb - positive isolates were detected mainly in cce spa - clonal complex ( 33% ) and the acme - positive isolate was of spa ccd type ( table 1 ) . isolates were also tested for the presence of nine enterotoxin genes in addition to the tsst-1 gene . the nine enterotoxin genes were randomly distributed among the mssa and the mrsa with the number of toxins detected / isolate ranging between 0 and 4 ( table 3 ) . most of the isolates having more than one enterotoxin ( 4 genes ) were resistant on average to only one antibiotic . the percentage of resistant isolates to each of the tested antibiotics is shown in figure 1 . all isolates tested showed no resistance to vancomycin , 2% to chloramphenicol and only one showed resistance to nitrofurantoin and teicoplanin . the highest percentage of resistance was to oxacillin ( 32% ; n = 41 ) and tetracycline ( 42% ; n = 54 ) . resistance to other antibiotics was intermediate , ranging between 7% to rifampicin and 25% ( n = 33 ) to azithromycin . multiple drug resistance was detected in both mssa and mrsa with the maximum number being to 14 drugs ( table 3 ) . resistance to 1014 drugs was mainly detected in mrsa mec subtypes ii and iii except for one isolate being resistant to 13 and having mec subtype iv . the 16s - its rrna gene was amplified in 15 s. aureus isolates representing the major spa types . almost 87% of the isolates belonged to ribotype i with two amplification products size of which were 1500 and 1800  bp . ribotype ii represented 13% of the isolates and gave one band with a size of 1800  bp ( figure 2 ) . all the isolates gave an intense band at 1800  bp , which was designated as the primary product , and most had an additional band , which appeared weaker and was designated as secondary product . 16s23s its - amplified products were digested using taqi restriction enzyme ( table 4 and figure 3 ) . electrophoresis and fragment size analysis of restriction products revealed the presence of five banding patterns [ four patterns in ribotype i ( lanes 3 , 5 , 7 , and 13 ) and only one in ribotype ii ( lanes 14 and 16 ) ] , but the difference only between ribotype i and ii was significant enough totaling to three bands ( figure 3 ) . to evaluate the phylogenetic relationships between the 15 s. aureus representative isolates , we extracted and partially sequenced the 16s23s its rdna primary product ( 1800  bp ) that was detected in all . two main clusters were detected and designated as a and b. the first contained s78 ( mssa spa t1149 ) and s89 ( mssa spa t937 ) isolates , and the second contained s1 ( mrsa - iv spa t044 ) , s3 ( mrsa - ii spa t002 ) , s12 ( mssa spa t008 ) , s19 ( mrsa - iv spa t068 ) , s34 ( mrsa - v spa t267 ) , s38 ( mrsa - iii spa t044 ) , s40 ( mrsa - iv spa t537 ) , s75 ( mssa spa t021 ) , s88 ( mssa t055 ) , s111 ( mrsa - iv spa t4098 ) , s113 ( mrsa - iv spa t279 ) , s121 ( mrsa - iv spa t660 ) , and s130 ( mrsa - iv spa t044 ) isolates . in cluster sequencing of the primary product revealed that ribotype i representatives belonged to cluster a , while all other isolates representing ribotype ii were in cluster b. additionally strains showing similar restriction patterns were clustered together ( s1 , s34 , s38 , and s40 all had the same restriction pattern the same applies for s78 and s89 ) . s. aureus is one of the most common causes of ca- and ha - localized and -systemic infections . nearly all isolates of s. aureus produce enzymes and a range of more than 30 different extracellular proteins , most of which play a direct role in pathogenesis . this study aimed at defining the prevalence of virulent determinants ( exfoliative toxins , enterotoxins , tsst-1 , and acme ) , drug resistance and typing of a diverse set of 130 s. aureus clinical isolates . the eta gene ( 11% ; n = 14 ) was more frequently detected than the etb gene ( 5% ; n = 6 ) , and only 2.3% ( n = 3 ) of all isolates tested harbored both genes . .   in the united kingdom and ireland , showed that 32% of s. aureus isolates produced eta and only 12% produced etb . the higher prevalence of the eta gene in staphylococci could be explained by its greater immunogenicity . the majority of previous reports confirmed that eta was the predominant et isoform in europe , north america , and africa which was similar to our findings , whereas etb - producing isolates were shown to be more frequent in japan [ 2023 ] . acme contributed to the success in disseminating the usa300 isolate through enhancing virulence and colonization of humans . in this study , acme was detected in only one mssa isolate that was recovered from a skin infection . however , also revealed that only 2 out of 22 usa300 mssa isolates from california and nevada were acme positive . to date , acme has been reported predominantly among usa300 isolates harboring specific sccmec types ( iva and ii ) and mlst clonal complexes ( ccs ) ( cc8 and cc5 ) genetic background , and it has been detected also in england among s. aureus isolates with sccmec type iva [ 16 , 24 ] . the three virulent determinants ( eta , etb , and acme ) in this study were variably distributed among 16 different spa types belonging to 12 different spa ccs , possibly because they are located on prophage , plasmid , and a pathogenicity island , which are mobile vectors of horizontal transfer . moreover , the most prevalent enterotoxin gene among the tested isolates in this study was sei ( 59% ) , which agreed with the findings of becker et al .   in germany ( 55% ) while it was the sea in jordan ( 65% ) . percentage of isolates positive for seb was 8 , which was again in harmony with the results obtained by becker et al . . in contrast , 25% of the isolates harbored the seg gene compared to only 1.4% in japan . the gene coding for tsst-1 , however , was detected only in 7% of the isolates in this study , which matched with the results of srinivasan et al . in the united states but was lower than the percentage reported by becker et al . it 's interesting to note that none of the isolates undertaken in this study had the see gene , which was the case with clinical isolates studied in jordan . on the other hand , isolates harboring the enterotoxin genes were randomly distributed among males and females , age categories , site and date of infection , and type ( mssa versus mrsa ) . it was notable that tst - positive isolates were all mssa , being recovered from abscesses , pus , sputum , or blood . finally , isolates harboring toxin genes were not multiple drug resistant , and the opposite was also true . multidrug resistance is very common in s. aureus , which is now considered as a leading cause of nosocomial infections . the widespread s. aureus isolates that are resistant to many antimicrobial agents such as the semisynthetic penicillins ( methicillin , oxacillin , and nafcillin ) , macrolides , tetracyclines , and aminoglycosides made the treatment of staphylococcal infections a global challenge . in this study , resistance to tetracycline was the highest ( 48% ) with similar result being previously obtained by hamze et al .   with samples collected from north lebanon ( 44% ) . percentage of resistant strains , however , was higher in australia ( 80% ) , while lower in the united states ( 5% ) . almost 32% of the isolates undertaken in this study were oxacillin resistant which was significantly lower than the percentage detected in belgium ( almost 99% ) by maskell et al . . percentage of resistance to azithromycin ( 25% ) , trimethoprim ( 24% ) , and erythromycin ( 24% ) was almost the same and was in harmony with resistance to azithromycin in the uk ( 23% ) and the united states ( 26% ) [ 31 , 32 ] ; it was lower for azithromycin ( 12% ) in japan   and higher to trimethoprim in the uk ( 69% )   and australia ( 82% ) . this study also revealed an increase in the percentage of strains resistant to erythromycin ( 24% ) compared to results obtained by baddour et al .   where the percentage of resistant strains were much lower being only 7% . resistance reported for erythromycin was higher in the uk   and australia   being 90% and 89% , respectively , while only 12% in japan . it 's noteworthy that almost 15% of the isolates were resistant to trimethoprim / sulfamethoxazol , which agreed with results obtained by baddour et al . . a significant increase was also detected in this study with the percentage of isolates resistant to clindamycin ( 22% ) and gentamicin ( 9% ) compared to what was reported previously   with only 4% of the strains being resistant to clindamycin and 3% to gentamicin . the percentage of strains resistant to teicoplanin , nitrofurantoin , chloramphenicol , and vancomycin detected in this study was either very low or absent . similar results were also previously reported for both teicoplanin and vancomycin [ 3436 ] while it was higher to chloramphenicol [ 34 , 35 ] . multiple drug resistance was detected in both mssa and mrsa with the maximum number being to 14 drugs . resistance to 1014 drugs was mainly detected in mrsa mec subtypes ii and iii except for one isolate being resistant to 13 and having mec subtype iv . these findings confirm along with the mec subtyping that the majority of the multiple drug - resistant isolates probably were hospital acquired . the 16s23s internal transcribed spacer ( its ) pcr amplification can be used for typing s. aureus strains . this region is known to be extremely variable between species , strains , and even among different operons within the same cell and has been widely used to study evolutionary relationships among closely related microorganisms [ 37 , 38 ] . ribotype i gave two amplification products with a size of 1500 and 1800  bp , which was consistent with the findings of sudagidan et al . ribotype i was the most frequent accounting for 87% of the isolates , whereas ribotype ii represented only 13% . most of the isolates gave additional weaker band , and the source of this secondary product might be the presence of more than one rrna operon in those isolates with the upper band ( 1800  bp ) containing one or two trnas and the lower band ( 1500  bp ) none or only one trna - encoding gene [ 39 , 40 ] . couto et al .   characterized the spacer sequences of s. aureus strains and identified 9 rrna operons , and reported that 3 of these spacers contained the trna gene and 2 contained both the trna and the trna genes , while the remaining 4 16s23s spacers have no trna gene . it is noteworthy that the observed its size variability alone was not enough to subtype s. aureus isolates at the strain level , and consequently the amplified 16s23s dna spacer region was subjected to restriction digestion using taqi restriction enzyme . previously , its rflp using taqi has been employed for the identification of bacteria of diverse origin including lactobacilli , thermophilic bacilli , and alkalophilic bacilli   and was also used by sudagidan et al .   for subtyping 16 staphylococcal species in turkey . our results revealed 5 distinct rflp haplotypes , with those belonging to ribotype i having 4 restriction patterns that matched with the results of sudagidan et al . , while isolates belonging to different ribotypes generated different restriction patterns . this study did n't reveal any correlation between its ribotyping and prevalence of the virulent determinants . combining 16s23s its pcr - rflp with partial sequencing of the primary product and the construction of a phylogenetic tree revealed the presence of 2 clusters a and b. isolates belonging to each cluster harbored the same its amplicon sizes and similar restriction patterns . these findings matched with the results of osorio et al . , who reported that the intraspecies evolutionary divergence is due to polymorphisms clustered within the its variable region where a number of insertion and deletion events occur . the its - based identification method can be used to generate molecular fingerprints of s. aureus for characterization of isolates and could have useful applications for evolutionary studies . the major drawback , however , is the absence of sufficient information about rrna copy number and size , the number of trna encoding genes and its sequence and size in s. aureus isolates . to the extent of our knowledge , this is the first study that demonstrates the prevalence of virulent determinants and describes the use of 16s23s its pcr - rflp and its partial sequencing for the subtyping s. aureus isolates in lebanon . the results show that the virulence gene content of s. aureus isolated from lebanon varies widely . the use of efficient and accurate epidemiological typing methods is a prerequisite for monitoring and for limiting the occurrence and spread of epidemic clones within and between hospitals .
molecular characterization of staphylococcus aureus is of both clinical and infection control importance . 
 virulence determinants using pcr and multiple drug resistance profiles were studied in 130 s. aureus isolates . 
 pcr - rflp analysis of the 16s23s dna spacer region was done to investigate the level of 16s23s its ( internal transcribed spacer ) polymorphism . 
 methicillin - resistant s. aureus ( mrsa ) , which represented 72% of the studied isolates , showed multiple drug resistance with 18% being resistant to 1018 of the drugs used compared to a maximum resistance to 9 antibiotics with the methicillin sensitive s. aureus ( mssa ) isolates . 
 exfoliative toxin a ( eta ) was more prevalent than b ( etb ) with virulent determinants being additionally detected in multiple drug - resistant isolates . 
 16s23s its pcr - rflp combined with sequencing of the primary product was successful in generating molecular fingerprints of s. aureus   and could be used for preliminary typing . 
 this is the first study to demonstrate the incidence of virulent genes , acme , and genetic diversity of s. aureus isolates in lebanon . 
 the data presented here epitomize a starting point defining the major genetic populations of both mrsa and mssa in lebanon and provide a basis for clinical epidemiological studies .