macrolide , lincosamide and streptogramin b ( mlsb ) antibiotics have different structure , but similar mode of action .
these antibiotics inhibit bacterial protein synthesis by binding to 23s rrna in 50s ribosomal subunits .
erythromycin ( a macrolide ) and clindamycin ( a lincosamide ) are widely used in treatment of staphylococcus aureus infections .
firstly , good oral absorption of clindamycin makes it suitable for outpatient therapy or as follow - up after intravenous therapy .
secondly , it has high tissue penetration ( except for the central nervous system ) and accumulation in abscesses , and no need for renal dosing adjustments .
thirdly , clindamycin can be used as an alternative antibiotic in patients allergic to penicillin .
fourthly , community - acquired methicillin - resistant s. aureus , which has rapidly emerged in recent years as a cause of skin and soft - tissue infections , has shown susceptibility to clindamycin .
finally , it has been shown that clindamycin inhibits the production of toxins and virulence factors in gram - positive organisms through inhibition of protein synthesis . however , resistance to erythromycin and clindamycin is increasing among clinical isolates of s. aureus worldwide .
three mechanisms have been reported for resistance to mlsb antibiotics : target site modification , efflux of antibiotics , and drug modification .
methylation of the a2058 residue , located in the conserved domain v of 23s rrna , takes place in target - site modification and prevents the binding of mlsb antibiotics to their ribosomal target .
this phenomenon leads to cross - resistance to these antibiotics , and produces the mlsb phenotype that was encoded by erythromycin ribosome methylases ( erm ) genes . among the 4 major classes of erm genes ( erma , ermb , ermc and ermf ) in different bacteria , erma and ermc are the primary genes responsible for mlsb resistance in s. aureus . on the other hand
, mlsb phenotype can be constitutive ( rrna methylase is always produced ) or inducible ( methylase is produced only in the presence of an inducing agent ) . while strains with constitutive mlsb resistance ( cmlsb ) phenotypes can be detected by routine disk diffusion testing , strains with inducible mlsb resistance ( imlsb ) phenotypes show resistance to erythromycin and sensitivity to clindamycin , similar to strains containing the ms phenotype , which had resistance to only macrolide and streptogramin b , not to clindamycin .
therefore , a special disk diffusion method , the d - test , was developed for the detection of imlsb . in this test
, an erythromycin disk was placed in close proximity to a clindamycin disk . in imlsb - resistant strains ,
resistance to clindamycin is induced by diffusion of erythromycin through the agar , and leads to flattening of the clindamycin zone of inhibition adjacent to the erythromycin disk ( a d - shaped zone ) , while ms phenotype - containing strains form a circular zone around the clindamycin disk .
there are reports of clinical failures of clindamycin in treating patients with imlsb resistance phenotype [ 812 ] , attributed to selection for a mutation in the macrolide - responsive promoter region upstream of the erm gene and emergence of cmlsb - resistant isolates , leading some investigators to recommend that clindamycin therapy be avoided for s. aureus isolates that display the imlsb resistance phenotype . on the other hand , labeling all erythromycin - resistant s. aureus as clindamycin - resistant may prevent the use of clindamycin in cases where it would be effective therapy .
the present investigation was undertaken to determine the prevalence of cmlsb , imlsb and ms resistance phenotypes and primary erm genes ( erma and ermc ) in 126 erythromycin - resistant s. aureus isolates from patients in tehran , iran .
all staphylococcus aureus isolates from various clinical samples ( wounds , abscesses , urine , blood , sterile body fluids , and respiratory tract samples ) , identified from january to june 2008 in 4 university hospitals ( 3 general hospitals and 1 burn hospital ) in tehran , iran , were included in this study .
multiple isolates from the same patient were excluded , even when the site of infection was different .
identification of the isolates as s. aureus was based on colony and microscopic morphology , growth on mannitol salt agar and fermentation of mannitol , and production of catalase , coagulase , and deoxyribonuclease .
moreover , amplification of the species - specific nuc gene was used to confirm phenotypic identification of s. aureus isolates , as described below .
disk diffusion testing was used to determine antibiotic resistance according to the guidelines of the clinical and laboratory standards institute ( clsi ) , with s. aureus atcc 25923 as control . for detecting erythromycin and clindamycin resistance ,
15 g erythromycin disks and 2 g clindamycin disks ( purchased from mast co. , merseyside , uk ) were used .
interpretation of the diameters of zones of inhibition was as follows : for erythromycin = 23 mm ; s , 1422 mm ; i , = 13 mm ; r , and for clindamycin = 21 mm ; s , 1520 mm ; i , = 14 mm ; r .
d - testing was performed for erythromycin - resistant s. aureus strains according to the guidelines of the clsi .
suspension equivalent to 0.5 mcfarland of each freshly cultured isolate in normal saline was prepared and used for inoculation of mueller - hinton agar ( merck - hampshire , england ) plates .
erythromycin and clindamycin disks were placed on inoculated plates 15 mm apart ( edge - to - edge ) .
plates were read after 18 h of incubation at 35c and the shape of the clindamycin zone was verified .
strains with flattening of the susceptible zone of inhibition to clindamycin adjacent to the erythromycin disk ( d - shape ) were considered to contain imlsb - resistance , while strains with circular zones were considered to contain ms resistance .
dna was extracted from all erythromycin - resistant s. aureus strains by rapid dna extraction method .
five colonies of each isolate s overnight growth on brain - heart infusion agar were suspended in 300 l of sterile distilled water and heated for 15 min at 100c .
after centrifugation at 14 000 rpm for 5 min , supernatant was collected and used as the dna template in each pcr run .
pcr was performed with primers specific for erma , ermc , meca and nuc genes according to previous studies .
primers were purchased from cinnagen , iran , and their sequences , thermal cycling profile and pcr fragment size are shown in table 1 .
pcr reaction was performed in a 20 l volume , and 2 l of dna template was added to 18 l of pcr mixture consisting of 2 l of pcr buffer ( 10 ) , 1 l of mgcl2 ( 50 mm ) , 4 l of dntps ( 1 mm ) , 1 l of each primers ( 10 pmol ) , 0.25 l of taq dna polymerase ( 5 u/l ) and 8.75 l of double - distilled water .
dna amplification was carried out in a thermocycler ( touchgene gradient , techne , uk ) . in each thermal cycling profile
, there was an initial denaturation step at 94c for 5 min , and a final extension step at 72c for 5 min .
after pcr amplification , 5 l of pcr product was subjected to agarose gel electrophoresis ( 2% agarose , 1 tris - acetate - edta , 100 v , 100 min ) . the gel was stained with ethidium bromide , and a pcr fragment was visualized using a gel documentation system by comparison with a molecular size marker ( 100 bp ladder , eurobio , uk ) .
positive control strains for erma and ermc donated by mohammad emaneini ( faculty of medicine , tehran university of medical sciences , tehran , iran ) and molecular grade water instead of template dna as the negative control were included in each run .
s. aureus atcc29213 ( without erma , ermc , and meca genes ) was also used as a negative control .
all staphylococcus aureus isolates from various clinical samples ( wounds , abscesses , urine , blood , sterile body fluids , and respiratory tract samples ) , identified from january to june 2008 in 4 university hospitals ( 3 general hospitals and 1 burn hospital ) in tehran , iran , were included in this study .
multiple isolates from the same patient were excluded , even when the site of infection was different .
identification of the isolates as s. aureus was based on colony and microscopic morphology , growth on mannitol salt agar and fermentation of mannitol , and production of catalase , coagulase , and deoxyribonuclease .
moreover , amplification of the species - specific nuc gene was used to confirm phenotypic identification of s. aureus isolates , as described below .
disk diffusion testing was used to determine antibiotic resistance according to the guidelines of the clinical and laboratory standards institute ( clsi ) , with s. aureus atcc 25923 as control . for detecting erythromycin and clindamycin resistance ,
15 g erythromycin disks and 2 g clindamycin disks ( purchased from mast co. , merseyside , uk ) were used .
interpretation of the diameters of zones of inhibition was as follows : for erythromycin = 23 mm ; s , 1422 mm ; i , = 13 mm ; r , and for clindamycin = 21 mm ; s , 1520 mm ; i , = 14 mm ; r .
d - testing was performed for erythromycin - resistant s. aureus strains according to the guidelines of the clsi .
suspension equivalent to 0.5 mcfarland of each freshly cultured isolate in normal saline was prepared and used for inoculation of mueller - hinton agar ( merck - hampshire , england ) plates .
erythromycin and clindamycin disks were placed on inoculated plates 15 mm apart ( edge - to - edge ) .
plates were read after 18 h of incubation at 35c and the shape of the clindamycin zone was verified .
strains with flattening of the susceptible zone of inhibition to clindamycin adjacent to the erythromycin disk ( d - shape ) were considered to contain imlsb - resistance , while strains with circular zones were considered to contain ms resistance .
dna was extracted from all erythromycin - resistant s. aureus strains by rapid dna extraction method .
five colonies of each isolate s overnight growth on brain - heart infusion agar were suspended in 300 l of sterile distilled water and heated for 15 min at 100c .
after centrifugation at 14 000 rpm for 5 min , supernatant was collected and used as the dna template in each pcr run .
pcr was performed with primers specific for erma , ermc , meca and nuc genes according to previous studies .
primers were purchased from cinnagen , iran , and their sequences , thermal cycling profile and pcr fragment size are shown in table 1 .
pcr reaction was performed in a 20 l volume , and 2 l of dna template was added to 18 l of pcr mixture consisting of 2 l of pcr buffer ( 10 ) , 1 l of mgcl2 ( 50 mm ) , 4 l of dntps ( 1 mm ) , 1 l of each primers ( 10 pmol ) , 0.25 l of taq dna polymerase ( 5 u/l ) and 8.75 l of double - distilled water .
dna amplification was carried out in a thermocycler ( touchgene gradient , techne , uk ) . in each thermal cycling profile
, there was an initial denaturation step at 94c for 5 min , and a final extension step at 72c for 5 min .
after pcr amplification , 5 l of pcr product was subjected to agarose gel electrophoresis ( 2% agarose , 1 tris - acetate - edta , 100 v , 100 min ) .
the gel was stained with ethidium bromide , and a pcr fragment was visualized using a gel documentation system by comparison with a molecular size marker ( 100 bp ladder , eurobio , uk ) .
positive control strains for erma and ermc donated by mohammad emaneini ( faculty of medicine , tehran university of medical sciences , tehran , iran ) and molecular grade water instead of template dna as the negative control were included in each run .
s. aureus atcc29213 ( without erma , ermc , and meca genes ) was also used as a negative control .
during the 6-month study period , 186 staphylococcus aureus strains were isolated from patients admitted to 4 university hospitals in tehran , iran .
as expected , all phenotypically detected s. aureus strains showed species - specific nuc gene amplification in pcr , as reported by zhang k. , et al . .
in disk diffusion testing , 126 strains ( 67.7% ) showed resistance to erythromycin and were selected for further study . from these , 117 strains ( 92.9% ) showed resistance to clindamycin in disk diffusion testing and 9 strains were clindamycin - susceptible .
in d - testing , 117 strains ( 92.9% ) exhibited cmlsb resistance , 8 strains ( 6.3% ) imlsb , and 1 strain ( 0.8% ) had ms resistance phenotype ( figure 1 ) .
therefore , from 9 clindamycin susceptible strains in disk diffusion testing , 8 strains had imlsb and 1 strain had ms resistance phenotypes . in pcr testing ,
76 and 69 strains ( 60.3% and 54.8% ) showed erma and ermc genes amplification , respectively ; 84 strains ( 66.7% ) contained 1 or 2 of the studied erm genes and 42 strains ( 33.3% ) did not contain any studied erm genes . both genes ( erma and ermc ) were co - present in 61 strains ( 48.4% ) .
table 2 shows the difference in mlsb resistance phenotypes in relation to presence of erma and ermc genes . in strains with cmlsb and imlsb resistance phenotypes ,
the most prevalent genotype was erma + ermc , while strains with the ms resistance phenotype had only the erma gene .
the meca gene was detected in 86 strains by pcr , thus 68.3% of strains were considered methicillin - resistant staphylococcus aureus ( mrsa ) .
most strains with the cmlsb resistance phenotype were mrsa ( 69.2% ) . also , among 8 strains with imlsb resistance phenotypes , 5 strains were mrsa , while 1 strain with the ms resistance phenotype was mssa .
of 86 mrsa strains , 70 strains contained the erma gene , while in 40 mssa strains only 6 strains contained this gene .
prevalence of the erma gene in mrsa and mssa strains was 81.4% and 15% , respectively , and for the ermc gene prevalence was 74.4% and 12.5% , respectively .
erma and ermc genes were co - present in 58 ( 67.4% ) of mrsa strains and in 3 ( 7.5% ) of the mssa strains .
therefore , erma and ermc genes were more common in mrsa erythromycin - resistant strains than in mssa strains ( table 3 ) .
the increasing frequency of s. aureus infections and their antimicrobial resistance have led to renewed interest in the use of mlsb antibiotics , especially clindamycin , for treatment of these infections in many countries . for appropriate therapy decision - making ,
however , only a few published articles are available on the prevalence of erythromycin and clindamycin resistance in iranian isolates of s. aureus .
moreover , false susceptibility results for clindamycin may be obtained if isolates are not tested for imlsb resistance by d - testing , because it can not be determined using standard susceptibility tests .
recognition of this type of resistance is important because treatment of patients harboring imlsb - resistant s. aureus with clindamycin leads to the development of constitutive resistance , subsequently leading to therapeutic failure [ 812 ] . in this study ,
resistance to erythromycin ( 67.7% ) was higher than in studies from other countries , such as the study of schmitz et al . on s. aureus isolated from patients in 20 european university hospitals with rates of 39% .
in another study in tehran , resistance to erythromycin in clinical isolates of s. aureus was also high ( 56.2% ) .
most erythromycin - resistant strains ( 92.9% ) in this study also showed clindamycin resistance and were mrsa ( 68.3% ) . in the present study , prevalence of cmlsb , imlsb and ms resistance phenotypes among erythromycin - resistant s. aureus was 92.9% , 6.3% and 0.8% , respectively .
there is only 1 previous study of mlsb resistance among s. aureus isolated in iran for comparison , reporting 9.7% of s. aureus strains isolated from patients in milad hospital , tehran , had the imlsb resistance phenotype . in the present study ,
resistant s. aureus isolates , in agreement with the results of the 3-year study by spiliopoulou et al . on 173 erythromycin - resistant s. aureus strains isolated from patients in a university hospital in greece , which reported 61.3% , 30.6% and 7.5% prevalence to cmlsb , imlsb and ms resistance phenotypes , respectively .
higher prevalence of cmlsb than imlsb resistance phenotype among s. aureus isolates were also reported in other studies .
low prevalence of the ms resistance phenotype seen in the present study has also been shown in other studies performed in turkey , the neighboring country of iran ; although in the previously mentioned european study , it was relatively high ( 13% ) .
such differences in the mlsb resistance pattern could be caused by differences in guidelines for drug usage in iran , where mlsb antibiotics are widely used in treating s. aureus infections .
however , since the occurrence of imlsb varies widely by hospital and geographic region , it is necessary to perform the d - zone test for erythromycin - resistant , clindamycin - susceptible s. aureus isolates .
we studied the distribution of 2 erm genes ( erma and ermc ) among erythromycin - resistant s. aureus isolates .
these genes were reported as being the most prevalent genes responsible for resistance to mlsb antibiotics within s. aureus strains in other studies , including the study by lina et al in french hospitals . in the present study , erma and ermc were also prevalent in erythromycin - resistant s. aureus isolates ( 60.3% and 54.8% , respectively ) , although there was no significant difference between their presence .
it should be noted the prevalences of these genes were variable in different studies , and in some studies erma was more prevalent than ermc , while in other studies the reverse was found . in research by martineau et al , the prevalence of erma and ermc in erythromycin - resistant s. aureus strains was 21% and 10.2% , respectively .
schmitz et al .   found erma was more prevalent than ermc ( 67% and 23% , respectively ) . on the other hand , 16% and 84% of s. aureus strains isolated in denmark were carrying erma and ermc , respectively . in the study by spiliopoulou et al .
a notable finding of the present study was the co - existence of erma and ermc in a significant number of erythromycin - resistant s. aureus strains ( 48.4% ) , similar to results from 2 studies in turkey ( 37.5% and 18.6% ) , and in contrast to 2 studies in european countries ( 0.6% and 3% ) .
we also found that a significant number of erythromycin - resistant s. aureus isolates ( 33.3% ) did not carry erma and ermc , therefore other genes also have a significant role in resistance to erythromycin .
we found prevalence of 81.4% for mrsa strains and 15% for mssa isolates for erma .
prevalence of ermc in mrsa and mssa isolates were 74.4% and 12.5% , respectively ; therefore , both erma and ermc were more prevalent among mrsa than mssa isolates in tehran , iran .
these results regarding predominance of the erma among mrsa isolates are consistent with previous reports , but predominance of the ermc among mrsa isolates has not been reported , except from greece .
however , prevalence of these genes among mrsa and mssa strains were variable in different studies . a multicenter study in 24 european university hospitals   revealed that in s. aureus , the erma gene was more common among mrsa isolates ( 88% ) than in mssa isolates ( 38% ) . in contrast , ermc was more common in mssa ( 47% ) than in mrsa ( 5% ) .
otsuka et al .   found the erma gene was also predominant among erythromycin - resistant isolates of mrsa compared to mssa strains ( 95.0% and 53.3% , respectively ) , while the ermc gene were more prevalent among mssa than mrsa strains ( 42.0% and 11.5% , respectively ) .
high prevalence of erm genes in mrsa strains emphasizes the need for performing antimicrobial susceptibility testing when clindamycin is considered for use in treatment of infections caused by mrsa .
although there are some reports of mlsb resistance phenotypes in iranian isolates of s. aureus , to our knowledge this is the first report of the involved genes in iran .
we found erma + ermc related resistance was the most prevalent in erythromycin - resistant s. aureus isolates , and constitutive resistance was particularly predominant among mrsa strains .
 summarybackgroundresistance to antimicrobial agents among staphylococcus aureus is an increasing problem . 
 two common genes responsible for resistance to macrolide , lincosamide and streptogramin b ( mlsb ) antibiotics are the erma and ermc genes . 
 three resistance phenotypes have been detected to these antibiotics : strains containing cmlsb ( constitutive mlsb ) and imlsb ( inducible mlsb ) , which are resistant to macrolide , lincosamide and streptogramin b antibiotics , and ms , which is only resistant to macrolide and streptogramin b antibiotics . 
 the aim of this study was to determine the prevalence of mlsb phenotypes and genotypes in erythromycin - resistant strains of s. aureus isolated from patients in 4 university hospitals in tehran , iran.material/methodss . 
 aureus strains were isolated from various clinical specimens and identified by routine phenotypic methods and pcr for nuc gene . 
 erythromycin resistance was determined by disk diffusion testing . 
 prevalence of mlsb phenotypes was determined by use of the d - test . 
 erma and ermc genes were detected by pcr.resultsaltogether , 126 erythromycin - resistant strains of s. aureus were detected . 
 prevalence of cmlsb , imlsb and ms resistance phenotypes were 92.8% , 6.4% , and 0.8% , respectively ; 60.3% of strains had erma gene and 54.8% ermc gene ; 61 strains ( 48.4% ) contained 2 studied erm genes and 42 strains ( 33.3% ) did not have any studied erm genes.conclusionsdue to the high prevalence of clindamycin resistance among s. aureus isolated from patients in iran , we recommend clindamycin therapy only after proper antimicrobial susceptibility testing . 
Background
Material and Methods
Bacterial strains
Phenotypic determination of antibiotic resistance
Polymerase Chain Reaction (PCR)
Results
Discussion
Conclusions