a total of 107 s. epidermidis strains isolated from blood cultures of 
 patients hospitalised at a teaching hospital at botucatu medical school ( fmb ) , so paulo 
 state university ( unesp ) , state of so paulo , brazil , between 2005 - 2010 were studied . the university hospital of fmb provides tertiary care and possesses 385 beds , including 
 52 in intensive care units ( icus ) ( 30 adult , 15 neonatal and 7 paediatric icu beds ) . the 
 isolates are stored in the culture collection of the department of microbiology and 
 immunology , botucatu institute of biosciences , unesp , and were obtained from adult and 
 paediatric patients of both genders hospitalised in different units of the hospital . table i describes the characteristics of the 
 patients and the year of s. epidermidis isolation . the criteria 
 proposed by the centers for disease control and prevention / national healthcare safety 
 network ( cdc / nhsn 2014 ) were used to determine 
 the inclusion of blood cultures . table igeneral data of the patients included in the study and year of isolation of 
 staphylococcus epidermidis from blood culturesunit 
 patients ( n)er 33neo - icu 23nursery 15er - icu 9paed 5dialysis 4central - icu 3im 4paed - icu 2cardio 2cor - icu 1ipd 1no data 2 

 mean 
 age53 years6 days12.3 days64.7 years120 daysnana37 yearsna59nananagender ( n)male191906320121010female133149223301300na1110000000002 

 year ( n)2005 ( 17)17011011110102006 ( 18)34143011100002007 ( 18)010100411000012008 ( 19)92430000000002009 ( 20)60911001011002010 ( 15)14000000000001cardio : cardiology ward ; cor - icu : coronary intensive care unit ; er : 
 emergency room ; i m : internal medicine ; ipd : infectious and parasitic 
 diseases ; na : not available ; neo : neonatal ; paed : paediatric . cardio : cardiology ward ; cor - icu : coronary intensive care unit ; er : 
 emergency room ; i m : internal medicine ; ipd : infectious and parasitic 
 diseases ; na : not available ; neo : neonatal ; paed : paediatric . species of the genus staphylococcus were isolated and identified as 
 described by baker ( 1984 ) and koneman et al . dna was extracted from isolates identified as s. 
 epidermidis using the illustra kit ( ge healthcare ) . amplification of the internal transcribed spacer region - polymerase chain reaction ( pcr ) , 
 as described by barry et al . ( 2001 ) , was used to confirm that the 
 isolates belonged to the species s. epidermidis . the disk diffusion test employing oxacillin ( 1 g ) and cefoxitin ( 30 g ) disks was used 
 for the phenotypic detection of methicillin resistance according to the recommendations 
 of the clinical and laboratory standards institute ( clsi 
 2007 , 2012 ) . for genotypic analysis , international reference strains were included in all 
 reactions as positive ( s. aureus atcc 33591 ) and negative ( s. 
 aureus atcc 25923 ) controls , according to the clsi ( 2012 ) . the strains that were positive for the meca 
 gene were subjected to sccmec typing by multiplex pcr , 
 according to machado et al . the presence of the icaa , icad , icab 
 and icac genes was determined by pcr ( arciola et al . 2001 , 2005 ) and the 
 results were validated using international reference strains as positive ( s. 
 epidermidis atcc 35983 ) and negative ( s. epidermidis atcc 
 12228 ) controls . biofilm production by the isolates that were positive for icaadbc was 
 detected by the polystyrene plate method described by christensen et al . all 
 isolates were screened for reduced susceptibility to vancomycin by growth on agar 
 containing 4 and 6 g ml of the antibiotic , as described by hiramatsu ( 2001 
 ) and clsi ( 2012 ) , but 
 using an inoculum size of 2.0 mcfarland standards . international reference strains 
 ( enterococcus faecalis atcc 29212 and atcc 51299 ) were used as 
 negative and positive controls , respectively . the presence of the vana 
 and vanb genes was determined by pcr ( clark et al . 1993 ) and the results were validated using 
 international reference strains as positive ( e. faecalis atcc 51299 ) 
 and negative ( e. faecalis atcc 29212 ) controls . vancomycin minimum inhibitory concentrations ( mics ) were determined by a standardised 
 broth microdilution method according to clsi recommendations ( clsi 2012 ) and using 
 panels prepared in - house . the following vancomycin concentrations were prepared with 
 cation - adjusted mueller - hinton broth : 16 , 8 , 4 , 2 , 1 , 0.5 , 0.25 and 0.125 g 
 ml . after inoculation , each well contained approximately 5 x 105 
 colony - forming unit ml . the mic was defined as the concentration that 
 completely inhibited bacterial growth after 24 h of incubation at 35c . however , for 
 comparison , the plates were also analysed after 48 h. the following international 
 reference strains were used to validate the results : e. faecalis atcc 
 51299 , e. faecalis atcc 29212 and s. aureus atcc 
 29213 . the results were compared by the chi - squared test , adopting a level of significance of 
 < 0.05 ( curi 1997 ) . seventy - nine ( 73.8% ) of the 107 s. epidermidis isolates studied were 
 resistant to methicillin based on the disk diffusion method using cefoxitin or oxacillin 
 disks ; 87 ( 81.3% ) of the isolates were positive for the meca gene . the 
 sensitivity and specificity of the two phenotypic methods were 87.3% and 85% , 
 respectively . twenty - one ( 24.1% ) of the 87 isolates were classified 
 as sccmec type i , one ( 1.1% ) as type ii , 42 ( 48.2% ) as type iii and 18 
 ( 20.7% ) as type iv ; five ( 5.7% ) could not be typed by this technique . a decrease in 
 strains carrying sccmec type iii was observed over the period from 
 2005 - 2007 ( 56.8% ) and from 2008 - 2010 ( 39.5% ) , whereas the prevalence of type iv 
 increased almost threefold ( from 11.3 to 30.3% ) . vancomycin susceptibility testing showed that 51 ( 47.7% ) isolates were able to grow on 
 agar plates containing 4 g ml of the antibiotic , whereas three ( 2.8% ) 
 isolates were able to grow on agar containing 6 g ml . the colonies were 
 confirmed to be cons by gram staining as well as catalase and coagulase tests to rule 
 out the possibility of contamination . dna was extracted from the colonies to determine 
 the presence of the vana and vanb genes and all of 
 them were negative for these genes . the vancomycin mics obtained for subpopulations that grew on vancomycin screen agar and 
 for the original populations are shown in table 
 ii . the mic was  4 g ml in 64.7% of the isolates that grew on 
 agar plates containing 4 g ml vancomycin , with an mic of 8 g 
 ml in 15.7% of these isolates and an mic of 16 g ml in one 
 isolate ( 2% ) . mics of 8 and 4 g ml were observed in two ( 66.6% ) and one of 
 the three strains that grew on agar plates with 6 g ml vancomycin , 
 respectively . a comparison of mics between the original isolates and the subpopulations 
 that grew on vancomycin screen agar showed an increase in mic in 75.9% of the isolates 
 after 24 h of incubation . a two , four or eight - fold increase was observed in 96.3% of 
 the strains after 48 h. 
 table iivancomycin minimum inhibitory concentrations ( mics ) obtained after 24 h and 
 48 h of incubation of staphylococcus epidermidis strains and 
 subpopulations that grew on vancomycin screen agar  strains n ( % ) 
 subpopulation on 
 vancomycin agar n ( % ) 

 4 g ml 
 6 g ml 


 mic ( g 
 ml)24 h48 h24 h48 h24 h48 h117 ( 32.7)14 ( 26.9)0 ( 0)0 ( 0)0 ( 0)0 ( 0)234 ( 65.4)30 ( 57.7)19 ( 37.3)4 ( 7.8)0 ( 0)0 ( 0)41 ( 1.9)8 ( 15.4)24 ( 47)31 ( 60.8)1 ( 33.3)0 ( 0)80 ( 0)0 ( 0)8 ( 15.7)16 ( 31.4)2 ( 66.6)3 ( 100)160 ( 0)0 ( 0)1 ( 2)1 ( 2)0 ( 0)0 ( 0 ) 

 total52 ( 100)52 ( 100)51 ( 100)51 ( 100)3 ( 100)3 ( 100)a : parental strains . 
  regarding the presence of ica genes , at least one ica 
 gene was detected in 96 ( 89.7% ) of the isolates . forty - one ( 38.3% ) s. 
 epidermidis isolates carried the complete ica operon , 43 
 ( 40% ) carried the icaa+icad+icac genes , 47 ( 43.9% ) concomitantly 
 carried the icaa and icad genes and 11 ( 10.9% ) were 
 negative for all genes of the operon . all icaa gene - positive isolates 
 were positive for icad ( table 
 iii ) . twenty - four ( 58.5% ) of the isolates carrying the complete 
 ica operon produced a biofilm ; of these , 15 ( 36.6% ) were classified 
 as weakly adherent and nine ( 22% ) as strongly adherent . table iiifrequency of the meca and ica genes , 
 biofilm formation , reduced susceptibility to vancomycin , increased vancomycin 
 minimum inhibitory concentrations ( mics ) on screen agar and associations 
 found  isolates ( n ) 
 ica 
  ( 96 ) 
 icaa+icad ( 47 ) 
 icaa+icad+icac ( 43 ) 
 icaadbc ( 41 ) 
 icaadbc/ biofilm production ( 24)reduced 
 susceptibility to vancomycin ( 52)subpopulation with 
 increased vancomycin mic ( 39)mrsetotal ( 87)7945 
 41 
 39 
 24 
 4433i ( 21)17666373ii ( 1)1111111iii ( 42)3924 
 2019142824 
 iv ( 18)17111110685nt ( 5)5333000 

 msse ( 20)17222086mrse : methicillin - resistant staphylococcus epidermidis 
 based on the presence of the meca gene ; msse : 
 methicillin - sensitive s. epidermidis ; nt : no typed ; 
 a : at least one ica gene ; 
 b : significantly associated with meca 
 gene positivity ( p < 0.05 ) ; c : significantly associated 
 with the presence of staphylococcal cassette chromosome mec 
 type iii ( p < 0.05 ) . mrse : methicillin - resistant staphylococcus epidermidis 
 based on the presence of the meca gene ; msse : 
 methicillin - sensitive s. epidermidis ; nt : no typed ; 
 a : at least one ica gene ; 
 b : significantly associated with meca 
 gene positivity ( p < 0.05 ) ; c : significantly associated 
 with the presence of staphylococcal cassette chromosome mec 
 type iii ( p < 0.05 ) . eight ( 33.3% ) of the isolates that produced a biofilm and carried the 
 icaadbc operon exhibited vancomycin mics  8 g ml , 
 whereas this level of resistance was observed in only nine ( 8.4% ) of all isolates 
 studied . the only isolate that exhibited a vancomycin mic equal to 16 g ml 
 produced a strongly adherent biofilm . comparisons of the isolates that exhibited an increase in the vancomycin mic after 24 h 
 of incubation ( n = 39 ; 36.4% ) with those that did not grow ( n = 55 ; 51.4% ) and those 
 that did grow , but maintained the same mic ( n = 13 ; 12.1% ) , revealed that the increase 
 in vancomycin mic was positively associated with methicillin resistance using oxacillin 
 or cefoxitin disks ( p = 0.02 ) and the presence of sccmec type iii ( p = 
 0.0003 ) . an association was observed between the presence of the meca gene and 
 the presence of the icaa+icad genes ( p = 0.001 ) : most isolates carrying 
 the meca gene were also positive for the 
 icaa+icad+icac genes ( p = 0.002 ) and also for the complete 
 icaadbc operon ( p = 0.004 ) . in addition , the presence of 
 icaa and icad was associated with sccmec 
 type iii ( p = 0.030 ) ( table iii ) . furthermore , the presence of the complete ica operon and biofilm 
 production were significantly associated with the presence of the meca 
 gene ( 100% ; p = 0.006 ) and with growth on vancomycin screen agar ( 66.7% ; p = 0.05 ) when 
 compared to isolates that did not carry the complete operon or that did carry the 
 operon , but did not produce a biofilm . formerly considered to be an innocuous commensal bacterium of human skin , s. 
 epidermidis is now recognised as an important opportunistic pathogen . this 
 microorganism is one of the most common causative agents of medical device - related 
 infections ( sievert et al . we isolated 107 
 s. epidermidis strains from blood cultures of patients seen at a 
 brazilian teaching hospital and ana- lysed antimicrobial resistance patterns , the 
 presence of ica operon genes and biofilm formation . the results showed 
 a high prevalence of methicillin resistance as detected by both phenotypic and genotypic 
 methods ( 73.8% and 81.3% , respectively ) . this finding was expected because the 
 prevalence of methicillin resistance is high ( 70 - 80% ) among cons in latin america ( diekema et al . 2001 ) . the prevalence of sccmec types iii ( 48.2% ) and iv ( 20.7% ) was high , 
 which is a finding that was also reported by wisplinghoff et al . sccmec type iv is smaller and 
 the metabolic cost of its transfer is thus lower ; as a consequence , its prevalence is 
 expected to increase over time . indeed , in the present study , the proportion of 
 sccmec type iv increased three - fold over a period of six years . conversely , the prevalence of type iii decreased during the same period , although its 
 prevalence continues to be high . this high proportion of sccmec type 
 iii might be related to the selection of this type in the hospital environment due to 
 the multidrug resistance of these strains . ( 2007 ) , suggesting 
 that mr s. epidermidis ( mrse ) carrying this sccmec 
 type is not prevalent in brazil . sccmec type i , a hospital - associated 
 type , was frequent ( 24.1% ) ; this finding stands in contrast to previous studies in which 
 type i was found to be rare ( ibrahem et al . 2011 ) , but is similar to the findings reported in other 
 brazilian studies ( machado et al . these findings might be related to the local 
 epidemiology of mrse , indicating a higher frequency of sccmec type i in 
 brazilian hospitals . these data indicate the existence of a vast reservoir of 
 sccmec among s. epidermidis strains ( wisplinghoff et al . although these 
 similarities prevail , the distribution of sccmec types depends on 
 several factors , including geographic location and the use of antimicrobial agents . the incidence of the ica genes was high in the present study , with 
 90.6% of the isolates being positive for at least one ica gene and 
 38.3% being positive for all genes . biofilm production was detected by the phenotypic 
 method in 58.5% of the isolates carrying the complete ica operon . similarly , oliveira and cunha ( 2010 ) observed that 56.6% of strongly adherent cons 
 carried the icaa+icac+icad genes . the regulation of the 
 ica operon is complex and the expression of the ica 
 genes is variable : it can be activated or deactivated according to in vivo conditions . 
 some events , such as the addition of the insertion sequence is256 , all isolates that were positive for the icaa gene 
 were also positive for icad , in agreement with arciola et al . ( 2004 ) showed that 
 the icad gene was always expressed in s. epidermidis , 
 but that phenotypic biofilm production only occurred when icaa was 
 expressed simultaneously . this relationship might be explained by the selective pressure 
 exerted by biofilm production because , when expressed , the icad gene 
 alone does not induce transferase activity and icaa induces little 
 activity ; in contrast , the combined expression of icaa and 
 icad produces large amounts of pia ( gerke et al . although the icaa and icad 
 genes overlap and are co - transcribed , the fact that the isolates were more 
 frequently positive for icad than icaa , 
 icab and icac in the present study and in previous studies 
 ( zhou et al . 2013 ) may be related to the loss 
 or mutation of one of these genes . the meca gene was found to be associated with the presence of the 
 icaadbc operon and biofilm production ( p < 0.05 ) . in addition , 
 the concomitant presence of icaa+icad was associated 
 with sccmec type iii . zhou et al . 
 ( 2013 ) also detected icad more frequently than 
 icaa and observed a significant association between 
 icad and the meca gene in s. 
 epidermidis . furthermore , other studies have demonstrated that methicillin 
 resistance is correlated with biofilm production and the presence of 
 ica genes ( alcarz et al . these associations might be a 
 consequence of the intimate contact between biofilm bacteria , facilitating the 
 horizontal transfer of genetic material such as antimicrobial resistance genes ( klingenberg et al . 2005 ) , which would explain the 
 horizontal transfer of meca to ica gene - positive 
 strains ( kozitskaya et al . the present 
 results suggest the presence of the icaadbc operon in conjunction with 
 biofilm production and the presence of the meca gene to be an important 
 marker of nosocomial strains with the potential to cause infection . the indiscriminate use of antibiotics has led to the selection of bacterial populations 
 that contain cells with distinct levels of susceptibility to antimicrobial agents , 
 including resistant cells . intermediate and complete resistance to vancomycin in 
 s. epidermidis has been reported ( palazzo et al . 2005 ) and biofilm formation is known to significantly reduce 
 the effects of this antibiotic on bacteria ( raad et al . 2007 ) . in the present study , 47.7% and 2.8% of the s. 
 epidermidis isolates grew colonies on screen agar plates containing 4 and 6 
 g ml vancomycin , respectively . one - third of the isolates that were biofilm 
 producers also exhibited vancomycin mics  8 g ml after growing on 
 vancomycin screen agar , characteristics that can lead to treatment failure and may be a 
 precursor of glycopeptide resistance . the term vancomycin heteroresistance refers to strains that contain a subpopulation of 
 cells with different levels of susceptibility to this antibiotic , including resistant 
 cells and cells with intermediate susceptibility ( raad 
 et al . our analysis of colonies grown on vancomycin screen agar showed 
 an increase in mics in 75.9% of the isolates , with an increase from 1 to  4 g 
 ml in 64.7% of the isolates . mic increases were also observed in 100% of 
 the colonies that grew on agar with 6 g ml vancomycin . using isolates that 
 grew on screen agar with 4 g ml vancomycin , ma et al . ( 2011 ) reported slightly lower rates : 64.7% of strains 
 showed increased mics and 35.3% showed mics  4 g ml . mics  4 g 
 ml were observed in 85.7% of the subpopulations that grew on screen agar 
 with 6 g ml vancomycin . in our study , increased mics were detected in 
 96.3% of the isolates after 48 h of incubation . furthermore , the observation of 
 subpopulations with mics > 4 g ml ( 64.7% ) , as well as mics of 8 g 
 ml ( 15.7% ) and 16 g ml ( 2% ) , classifying the isolates as 
 intermediate resistant , indicates that infections caused by these microorganisms will be 
 difficult to treat . indeed , reports have shown higher mortality among patients infected 
 with heteroresistant bacteria and strains with higher mics compared to those infected 
 with susceptible strains ( wong et al . however , therapeutic alternatives for infections caused by staphylococci with reduced 
 vancomycin resistance have been approved by the us food and drug administration and 
 include linezolide , daptomycin , tigecycline and quinupristin / dalfopristin ( micek 2007 ) . two next - generation cephalosporins , 
 ceftobiprole and ceftaroline fosamil , are currently available for the treatment of 
 patients carrying mr and vancomycin - intermediate staphylococci ( saravolatz et al . reduced susceptibility to vancomycin was significantly associated 
 with methicillin resistance detected by phenotypic methods ( p < 0.05 ) . other studies 
 ( wong et al . 1999 , loomba et al . 2010 ) have shown that the isolation of mr 
 staphylococci is significantly more common among patients with bacteraemia due to 
 vancomycin - heteroresistant staphylococci . although the mechanism underlying the 
 association between methicillin resistance and reduced vancomycin susceptibility remains 
 unclear , it might be related to the treatment of infections caused by mr bacteria with 
 vancomycin , thereby selecting heterogeneous populations and strains with low 
 susceptibility . the proportion of strains with reduced susceptibility to vancomycin was higher among the 
 isolates harbouring sccmec type iii . ( 2007 ) , who found that 
 higher vancomycin mics were significantly associated with sccmec type 
 ii in s. aureus , whereas lower mics were related to 
 sccmec type iv . the association between reduced vancomycin 
 susceptibility and sccmec type iii observed in the present study may be 
 due to the high prevalence of sccmec type iii in the hospital 
 environment and the frequent use of vancomycin . the presence of subpopulations with higher vancomycin mics and intermediate resistance 
 demonstrates the heterogeneity of s. epidermidis strains in terms of 
 susceptibility to this antibiotic . because the main genes responsible for vancomycin 
 resistance ( vana and vanb ) were not detected , this 
 reduced susceptibility may be related to a thickening of the bacterial cell wall that 
 prevents uptake of the antibiotic by the microorganism ( billot - klein et al . 1996 ) . however , further studies are needed to elucidate 
 how these events occur in staphylococci . the present study demonstrated an association between the presence of the 
 icaadbc operon , biofilm formation and antimicrobial resistance in 
 s. epidermidis strains isolated from blood cultures . furthermore , 
 the detection of s. epidermidis subpopulations with intermediate 
 vancomycin resistance associated with methicillin resistance highlights the role of this 
 species as an important multidrug - resistant microorganism and the consequent need to 
 implement measures for the control of antibiotic use .
this study aimed to correlate the presence of ica genes , biofilm 
 formation and antimicrobial resistance in 107 strains of staphylococcus 
 epidermidis isolated from blood cultures . 
 the isolates were analysed to 
 determine their methicillin resistance , staphylococcal cassette chromosome 
 mec ( sccmec ) type , ica genes 
 and biofilm formation and the vancomycin minimum inhibitory concentration ( mic ) was 
 measured for isolates and subpopulations growing on vancomycin screen agar . 
 the 
 meca gene was detected in 81.3% of the s. 
 epidermidis isolated and 48.2% carried sccmec type iii . 
 the complete icaadbc operon was observed in 38.3% of the isolates ; 
 of these , 58.5% produced a biofilm . 
 furthermore , 47.7% of the isolates grew on 
 vancomycin screen agar , with an increase in the mic in 75.9% of the isolates . 
 determination of the mic of subpopulations revealed that 64.7% had an mic  4 g 
 ml-1 , including 15.7% with an mic of 8 g ml-1 and 2% with 
 an mic of 16 g ml-1 . 
 the presence of the icaadbc operon , 
 biofilm production and reduced susceptibility to vancomycin were associated with 
 methicillin resistance . 
 this study reveals a high level of methicillin resistance , 
 biofilm formation and reduced susceptibility to vancomycin in subpopulations of 
 s. epidermidis . 
 these findings may explain the selection of 
 multidrug - resistant isolates in hospital settings and the consequent failure of 
 antimicrobial treatment .