BACKGROUND
immediate breast reconstruction, using tissue expanders and implants, has become a standard of care for almost all women requiring mastectomy for breast cancer  <cit> .
however, after implantation, patients may experience local complications during the ensuing years, with peri-implant infection being one of the most common problems, with an infection rate ranging from  <dig> to  <dig> percent  <cit> .
the most traditional approach to severe or refractory breast periprosthetic infection remains removal of the device.
however, removal makes subsequent reinsertion and re-expansion more difficult, since the soft tissue retracts rapidly to close the expanded pocket  <cit> .
the majority of cases reported identify staphylococcus epidermidis and s. aureus as the most common bacteria responsible for sub-clinical and overt breast implant infections.
bacteria may reach the implants during or after placement and their ability to form biofilms on the implant has been reported as the essential factor in the development and persistence of infection.
biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated.
specific staphylococcal surface proteins impacting adhesion to abiotic surfaces  as well as a vast array of proteins called mscramms  promoting colonization of medical implants have been identified  <cit> .
in s. aureus and s. epidermidis, production of an extracellular polysaccharide promoting intercellular adhesion , also called polymeric n-acetyl-glucosamine  is currently the best-characterised biofilm mechanism  <cit> .
pia/pnag is synthesised by enzymes encoded by the ica operon.
once this operon is activated, four proteins are transcribed icaa, ica d, icab and icac.
the expression of icaa alone induces low enzymatic activity, however, the simultaneous expression of icaa and icad promotes a significant increase in the amount of polysaccharide  <cit> .
however, recent studies have begun to highlight the existence of pia/pnag-independent biofilm mechanisms in both species  <cit> .
accumulation-associated protein  independently or toghether with ica operon, has also been suggested to be important in coagulase-negative staphylococci  <cit> .
in s. aureus and s. epidermidis more additional surface proteins such as sasg, extracellular matrix binding protein , biofilm-associated protein  and the fibronectin-binding proteins fnbpa and b, were implicated in matrix formation.
these findings suggest that other surface proteins may also be involved in biofilm development.
in some strains, biofilm formation may be mediated additionally or exclusively by specific surface proteins   <cit> .
while several studies have extensively described the distribution of genes involved in biofilm formation and virulence in staphylococcal strains causing orthopaedic peri-implant infections, to our knowledge, this is the first study characterizing isolates causing breast peri-implant infections.
RESULTS
antimicrobial susceptibility and meca presence
all s. aureus isolates were susceptible to, oxacillin, co-trimoxazole, daptomycin, gentamicin, linezolid, rifampin, tetracycline and vancomycin.
ciprofloxacin- and erythromycin resistance was detected in one  and three  strains, respectively .
two out of the three erythromycin-resistant s. aureus showed inducible clindamycin resistance.table  <dig>
susceptibility of  <dig> staphylococci isolated from breast implant infections
range
mic50
mic90
%s
*two strains showed inducible clindamycin resistance.
all s. epidermidis were susceptible to, ciprofloxacin, clindamycin, daptomycin, linezolid, rifampin, tetracycline and vancomycin.
oxacillin, erythromycin, gentamicin and co-trimoxazole resistance was detected in 2/ <dig>  5/ <dig>  2/ <dig> and 1/ <dig> strains, respectively.
the oxacillin susceptibility results were confirmed by cefoxitin disk test and meca pcr.
biofilm assays
all s. aureus isolates, and only one out of seven s. epidermidis isolates were classified as slime producers by the congo red agar  plate method, developing brown-black  or black  colonies.
all isolates were classified as biofilm-producing strains by the polystyrene microtiter plates  method.
when grown in brain heart infusion  supplemented with 1% glucose, biofilm development was significantly induced in twelve s. aureus and six s. epidermidis strains while 4% nacl activated biofilm development in five s. aureus and one s. epidermidis strain .
for these six strains nacl was a stronger biofilm inducer than glucose.figure  <dig>
biofilm formation in 13
s. aureus
and 7
s. epidermidis
strains studied.
the strains were grown in bhi medium or in bhi supplemented with 1% glucose or 4% nacl at 37°c for 24 h. biofilm formation in tissue culture-treated 96-well plates was measured three times, and standard deviations, which were less than 20%, are indicated.
addition of dnase i did not affect bacterial biofilm stability.
although not statistically significant the lowest percentages of reduction of primary attachment  were observed for those strains producing nacl inducible biofilms .figure  <dig>
susceptibility of s.
aureus
and
s. epidermidis
biofilms to treatment with dnase i. strains sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig> and se  <dig> were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment.
the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to treatment.
effect of dnase i on
s. aureus
and
s. epidermidis
primary attachment.
strains sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig> and se  <dig> were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment.
the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to dilution and treatment.
all nacl-induced biofilms were susceptibile to sodium metaperiodate ) and resistant to proteinase k .
all the remaining biofilms  were resistant to sodium metaperiodate and substantially dispersed by treatment with proteinase k , with the exception of those produced by two s. aureus strains .figure  <dig>
susceptibility of
s. aureus
and
s. epidermidis
biofilms to treatment with sodium metaperiodate and proteinase k. strains sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig>  sa  <dig> and se  <dig> were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment.
the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to treatment.
the different behaviours observed suggested different chemical compositions in the biofilm extracellular matrices.
detection of mscramm and biofilm genes
all s. aureus harboured the icaa/d, atla, clfa, fna, eno and cna genes and the great majority carried sasg , ebps  and fib , while only two strains carried fnb .
no strain harboured the bap and bbp genes.table  <dig>
distribution of mscramm and biofilm genes among
s. aureus
and
s. epidermidis
ebps
eno
atla/e
aae
aap
bap/bhp
sasg
cna
bbp
fnba
fnbb
clfa
clfb
fbe
sdrf
embp
fib
icaa/d
* <dig> strain was icaa negative and icad positive.
two s. epidermidis carried ica genes.
one s. epidermidis  carried both the icaa and icad genes and another strain  was icad positive.
all s. epidermidis harboured the atle, aae, embp genes, the aap and the fbe genes were carried by six strains, while four strains carried the sdrf gene .
no strains harboured the bhp gene .
expression of mscramm and biofilm genes
under the experimental conditions used all strains expressed icaa/d genes with the exception of one s. epidermidis strain which did not express the icad gene .
for all s. aureus strains the rt-pcr amplicons showed positive expression signals for all the remaining genes tested with the exception of fib.
all s. epidermidis strains expressed the atle, aae, embp genes, and six strains out of seven expressed the fbe, aap genes.
detection of s. aureus capsular and leukotoxin genes
six strains were pcr-positive for capsular type  <dig> and seven for capsular type  <dig> genes .
seven out of thirteen s. aureus strains carried the luke/lukd leukotoxin genes, while the pvl and lukm leukotoxins genes were not detected.
randomly amplified polymorphic dna  polymerase chain reaction  profiles
to rule out the possibility of infections due to one epidemic s. aureus or s. epidermidis strain, all isolates  underwent rapd pcr with three and two different primers respectively.
all s. aureus and all s. epidermidis had different rapd profiles .
s. aureus mlst and eburst analysis
as shown in table  <dig>  a total of  <dig> sequence types  were revealed, which were further grouped into  <dig> clonal complexes .table  <dig>
clonal relatedness of 13
s. aureus
isolates and their agr groups
strains
agr group
sequence type
clonal complex*
sa1
sa4
sa7
sa 11
sa18
sa21
sa24
sa 25
sa35
sa 36
sa 38
sa 418
sa 420
*ccs as defined by eburst analysis with a stringent group definition with six of seven loci.
s. aureus agr typing
the distribution of agr alleles among the 13 s. aureus strains is provided in table  <dig>
the agr type i was predominant , followed by agr type ii .
only one s. aureus belonged to agr type iii.
discussion
staphylococcal infection represents a major concern when associated with breast reconstruction, as it may necessitate additional hospitalization, antibiotic treatment, and in more serious cases removal of the device making subsequent reinsertion and re-expansion more difficult  <cit> .
theoretically, both endogenous and hospital-acquired staphylococci may gain access to breast implants during or following placement.
our data  supports a community origin of the infecting microorganisms, in most cases.
after accession to surgical implants, the essential factor in the evolution and persistence of infections is the formation of bacterial biofilm around implanted devices  <cit> .
all isolates studied were able to form biofilm on polystyrene surfaces but, in contrast to s. aureus, the great majority of s. epidermidis isolates did not carry the icaa or icad genes.
our results are in agreement with previous studies showing that the ica-operon is present and expressed in almost all s. aureus isolates, although the absence of ica-operon activity does not necessarily impact on the ability of s. epidermidis to cause clinical biofilm disease.
in this study, only one out of seven s. epidermidis strains carried both icaa and icad.
in a recent letter, persichetti and colleagues  <cit> , described 10 s. epidermidis causing periprosthetic breast infections and capsular contracture, yet they reported only three icaa and icad positive strains.
although the number of cases analysed in both studies is limited, it seems that ica-independent biofilm formation plays a major role compared to ica-dependent mechanisms in s. epidermidis breast peri-implant infections.
in contrast, all s. epidermidis studied carried and expressed the gene for aap protein, a protein that has been shown to mediate biofilm formation in strains lacking the ica genes.
biofilm induction by glucose or nacl  had different effects on our strains and revealed that our strains had different mechanisms of biofilm development, irrespective of ica operon carriage.
proteinase k and sodium metaperiodate treatment confirmed that strains causing breast peri-implant infection can produce different types of biofilm extracellular matrix .
although not statistically significant dnase i appeared less effective in reducing bacterial attachment in those strains producing nacl-induced biofilm, suggesting a minor role of extracellular dna in these isolates.
our data adds further evidence that, in the context of peri-implant infections, biofilm formation is a complex, phenomenon involving distinct molecules, whose relative importance may vary depending on the different clinical settings.
addition of dnase i did not affect bacterial biofilm stability as already reported  <cit> .
genes coding for biofilm-associated molecules have been thoroughly studied  <cit>  in s. aureus and s. epidermidis isolated from prosthetic joint infections .
the most striking difference observed between s. aureus strains isolated from pji and breast implant infections was the rate of surface adhesin  and collagen adhesin  genes found.
the isolation of a cna-positive s. aureus strain was more probable in isolates from breast implant infections than from pji  indicating that cna is of major importance in the specific context of breast implant infections.
other authors  <cit>  have shown the prevalence of the cna gene to be much higher in methicillin-resistant s. aureus   than methicillin-sensible s. aureus  clones  and that cna positive strains appeared to be associated with capsular type  <dig>  however, our strains were all methicillin sensible  and  <dig> out of  <dig> strains carried the cps <dig> gene for capsular type  <dig> and the remaining cps <dig>  suggesting that the site of infection may have been the driving force in selecting a subpopulation of cna positive s. aureus strains irrespective of other characteristics such as methicillin resistance and capsular type.
an elevated breast collagen concentration has been shown to be one of the greatest risk factors for developing breast carcinoma  <cit> .
breast implants, especially in oncologic patients, due to the increased collagen deposition, could be easily covered by a more dense collagen matrix and thus become more prone to adhesion of s. aureus strains able to bind collagen.
in contrast to s. aureus causing pji, the great majority of our strains carried and expressed the sasg gene .
this difference in distribution according to the site of infection, is remarkable as its s. epidermidis homologue aap  appears to be almost ubiquitously present.
further studies are needed to better characterize this protein, its function/s and to understand its role in different types of infections.
the gene coding for bap, a cell wall protein found in bovine mastitis s. aureus isolates, involved in intercellular adhesion and biofilm formation was absent in all s. aureus isolates studied.
our results support the suggestion of other authors, that the role of bap in human infections is doubtful  <cit> .
besides the ability to form biofilms, other authors have investigated the incidence of genes coding for leukocidal toxins in s. aureus isolated from implant orthopaedic infections.
panton-valentine, luke/lukd and lukm leukocidins are known to be associated with necrotic skin and soft tissue infections and could promote damage to peri-implant tissues.
luke/lukd was the only gene found in strains from breast implant infections .
leukotoxin genes displayed a similar prevalence in strains from breast implants and pjis, suggesting that luke/lukd could be the only relevant leukocidin in both types of implant-related infections.
the great majority of s. epidermidis analysed carried atle, aap, aae, embp and fbe genes, a condition already described for s. epidermidis isolated from catheter-related bloodstream infections and pjis as well as for commensal s. epidermidis, indicating that these proteins are valuable during both infection and colonization.
in contrast to s. aureus only three s. epidermidis strains carried a gene  coding for a surface protein binding collagen.
this is not surprising, since adhesins that recognize host proteins coating the device seem to be the primary mechanism of adhesion in s. aureus, while s. epidermidis initial adherence is probably multifactorial  <cit> .
breast reconstruction has become a standard of care for almost all women requiring mastectomy for breast cancer.
reduction of mutilation greatly improves the psychological status of women without interfering with oncological treatment, however, the reconstructive approach may be compromised by early or late implant infection in 8% of patients in prospective series  <cit> .
further studies are needed to confirm our observation and to verify if patients colonized by cna positive strains are at higher risk of breast peri-implant infections than those colonized by cna negative s. aureus.
if this holds true, screening of patients before and after reconstructive surgery at regular intervals could be extremely useful to identify those colonized and thus concentrate management, and resources.
should colonization with cna-positive strains confirm to be predictive for higher risk of implant infection, effective prophylactic strategies/treatments could be devised to reduce the risk of infection of reconstruction breast implants.
CONCLUSIONS
s. aureus and s. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms.
since the collagen adhesin  gene is not ubiquitously distributed among s. aureus, this protein could have an important role in the cause of breast peri-implant infections.
further studies are needed to confirm our observation and eventually recommend screening of patients before and after reconstructive surgery to identify those colonized by cna-positive s. aureus and at higher risk of peri-implant infections.

 immediate breast reconstruction, using tissue expanders and implants, has become a standard of care for almost all women requiring mastectomy for breast cancer. however, after implantation, patients may experience local complications during the ensuing years, with peri-implant infection being one of the most common problems, with an infection rate ranging from to percent. the most traditional approach to severe or refractory breast periprosthetic infection remains removal of the device. however, removal makes subsequent reinsertion and re-expansion more difficult, since the soft tissue retracts rapidly to close the expanded pocket. the majority of cases reported identify staphylococcus epidermidis and s. aureus as the most common bacteria responsible for sub-clinical and overt breast implant infections. bacteria may reach the implants during or after placement and their ability to form biofilms on the implant has been reported as the essential factor in the development and persistence of infection. biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. specific staphylococcal surface proteins impacting adhesion to abiotic surfaces as well as a vast array of proteins called mscramms promoting colonization of medical implants have been identified. in s. aureus and s. epidermidis, production of an extracellular polysaccharide promoting intercellular adhesion, also called polymeric n-acetyl-glucosamine is currently the best-characterised biofilm mechanism. pia/pnag is synthesised by enzymes encoded by the ica operon. once this operon is activated, four proteins are transcribed icaa, ica d, icab and icac. the expression of icaa alone induces low enzymatic activity, however, the simultaneous expression of icaa and icad promotes a significant increase in the amount of polysaccharide. however, recent studies have begun to highlight the existence of pia/pnag-independent biofilm mechanisms in both species. accumulation-associated protein independently or toghether with ica operon, has also been suggested to be important in coagulase-negative staphylococci. in s. aureus and s. epidermidis more additional surface proteins such as sasg, extracellular matrix binding protein, biofilm-associated protein and the fibronectin-binding proteins fnbpa and b, were implicated in matrix formation. these findings suggest that other surface proteins may also be involved in biofilm development. in some strains, biofilm formation may be mediated additionally or exclusively by specific surface proteins. while several studies have extensively described the distribution of genes involved in biofilm formation and virulence in staphylococcal strains causing orthopaedic peri-implant infections, to our knowledge, this is the first study characterizing isolates causing breast peri-implant infections.  antimicrobial susceptibility and meca presence all s. aureus isolates were susceptible to, oxacillin, co-trimoxazole, daptomycin, gentamicin, linezolid, rifampin, tetracycline and vancomycin. ciprofloxacin- and erythromycin resistance was detected in one and three strains, respectively. two out of the three erythromycin-resistant s. aureus showed inducible clindamycin resistance.table susceptibility of staphylococci isolated from breast implant infections range mic50 mic90 %s *two strains showed inducible clindamycin resistance. all s. epidermidis were susceptible to, ciprofloxacin, clindamycin, daptomycin, linezolid, rifampin, tetracycline and vancomycin. oxacillin, erythromycin, gentamicin and co-trimoxazole resistance was detected in 2/ 5/ 2/ and 1/ strains, respectively. the oxacillin susceptibility results were confirmed by cefoxitin disk test and meca pcr. biofilm assays all s. aureus isolates, and only one out of seven s. epidermidis isolates were classified as slime producers by the congo red agar plate method, developing brown-black or black colonies. all isolates were classified as biofilm-producing strains by the polystyrene microtiter plates method. when grown in brain heart infusion supplemented with 1% glucose, biofilm development was significantly induced in twelve s. aureus and six s. epidermidis strains while 4% nacl activated biofilm development in five s. aureus and one s. epidermidis strain. for these six strains nacl was a stronger biofilm inducer than glucose.figure biofilm formation in 13  s. aureus and 7  s. epidermidis strains studied. the strains were grown in bhi medium or in bhi supplemented with 1% glucose or 4% nacl at 37°c for 24 h. biofilm formation in tissue culture-treated 96-well plates was measured three times, and standard deviations, which were less than 20%, are indicated. addition of dnase i did not affect bacterial biofilm stability. although not statistically significant the lowest percentages of reduction of primary attachment were observed for those strains producing nacl inducible biofilms.figure susceptibility of s. aureus and s. epidermidis biofilms to treatment with dnase i. strains sa sa sa sa sa and se were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment. the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to treatment. effect of dnase i on s. aureus and s. epidermidis primary attachment. strains sa sa sa sa sa and se were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment. the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to dilution and treatment. all nacl-induced biofilms were susceptibile to sodium metaperiodate ) and resistant to proteinase k. all the remaining biofilms were resistant to sodium metaperiodate and substantially dispersed by treatment with proteinase k, with the exception of those produced by two s. aureus strains.figure susceptibility of s. aureus and s. epidermidis biofilms to treatment with sodium metaperiodate and proteinase k. strains sa sa sa sa sa and se were grown in bhi supplemented with 4% nacl at 37°c for 24 h prior to treatment. the remaining strains were grown in bhi supplemented with 1% glucose at 37°c for 24 h prior to treatment. the different behaviours observed suggested different chemical compositions in the biofilm extracellular matrices. detection of mscramm and biofilm genes all s. aureus harboured the icaa/d, atla, clfa, fna, eno and cna genes and the great majority carried sasg, ebps and fib, while only two strains carried fnb. no strain harboured the bap and bbp genes.table distribution of mscramm and biofilm genes among s. aureus and s. epidermidis ebps eno atla/e aae aap bap/bhp sasg cna bbp fnba fnbb clfa clfb fbe sdrf embp fib icaa/d * strain was icaa negative and icad positive. two s. epidermidis carried ica genes. one s. epidermidis carried both the icaa and icad genes and another strain was icad positive. all s. epidermidis harboured the atle, aae, embp genes, the aap and the fbe genes were carried by six strains, while four strains carried the sdrf gene. no strains harboured the bhp gene. expression of mscramm and biofilm genes under the experimental conditions used all strains expressed icaa/d genes with the exception of one s. epidermidis strain which did not express the icad gene. for all s. aureus strains the rt-pcr amplicons showed positive expression signals for all the remaining genes tested with the exception of fib. all s. epidermidis strains expressed the atle, aae, embp genes, and six strains out of seven expressed the fbe, aap genes. detection of s. aureus capsular and leukotoxin genes six strains were pcr-positive for capsular type and seven for capsular type genes. seven out of thirteen s. aureus strains carried the luke/lukd leukotoxin genes, while the pvl and lukm leukotoxins genes were not detected. randomly amplified polymorphic dna polymerase chain reaction profiles to rule out the possibility of infections due to one epidemic s. aureus or s. epidermidis strain, all isolates underwent rapd pcr with three and two different primers respectively. all s. aureus and all s. epidermidis had different rapd profiles. s. aureus mlst and eburst analysis as shown in table  a total of sequence types were revealed, which were further grouped into clonal complexes.table clonal relatedness of 13  s. aureus isolates and their agr groups strains agr group sequence type clonal complex* sa1 sa4 sa7 sa 11 sa18 sa21 sa24 sa 25 sa35 sa 36 sa 38 sa 418 sa 420 *ccs as defined by eburst analysis with a stringent group definition with six of seven loci. s. aureus agr typing the distribution of agr alleles among the 13 s. aureus strains is provided in table  the agr type i was predominant, followed by agr type ii. only one s. aureus belonged to agr type iii. discussion staphylococcal infection represents a major concern when associated with breast reconstruction, as it may necessitate additional hospitalization, antibiotic treatment, and in more serious cases removal of the device making subsequent reinsertion and re-expansion more difficult. theoretically, both endogenous and hospital-acquired staphylococci may gain access to breast implants during or following placement. our data supports a community origin of the infecting microorganisms, in most cases. after accession to surgical implants, the essential factor in the evolution and persistence of infections is the formation of bacterial biofilm around implanted devices. all isolates studied were able to form biofilm on polystyrene surfaces but, in contrast to s. aureus, the great majority of s. epidermidis isolates did not carry the icaa or icad genes. our results are in agreement with previous studies showing that the ica-operon is present and expressed in almost all s. aureus isolates, although the absence of ica-operon activity does not necessarily impact on the ability of s. epidermidis to cause clinical biofilm disease. in this study, only one out of seven s. epidermidis strains carried both icaa and icad. in a recent letter, persichetti and colleagues, described 10 s. epidermidis causing periprosthetic breast infections and capsular contracture, yet they reported only three icaa and icad positive strains. although the number of cases analysed in both studies is limited, it seems that ica-independent biofilm formation plays a major role compared to ica-dependent mechanisms in s. epidermidis breast peri-implant infections. in contrast, all s. epidermidis studied carried and expressed the gene for aap protein, a protein that has been shown to mediate biofilm formation in strains lacking the ica genes. biofilm induction by glucose or nacl had different effects on our strains and revealed that our strains had different mechanisms of biofilm development, irrespective of ica operon carriage. proteinase k and sodium metaperiodate treatment confirmed that strains causing breast peri-implant infection can produce different types of biofilm extracellular matrix. although not statistically significant dnase i appeared less effective in reducing bacterial attachment in those strains producing nacl-induced biofilm, suggesting a minor role of extracellular dna in these isolates. our data adds further evidence that, in the context of peri-implant infections, biofilm formation is a complex, phenomenon involving distinct molecules, whose relative importance may vary depending on the different clinical settings. addition of dnase i did not affect bacterial biofilm stability as already reported. genes coding for biofilm-associated molecules have been thoroughly studied in s. aureus and s. epidermidis isolated from prosthetic joint infections. the most striking difference observed between s. aureus strains isolated from pji and breast implant infections was the rate of surface adhesin and collagen adhesin genes found. the isolation of a cna-positive s. aureus strain was more probable in isolates from breast implant infections than from pji indicating that cna is of major importance in the specific context of breast implant infections. other authors have shown the prevalence of the cna gene to be much higher in methicillin-resistant s. aureus than methicillin-sensible s. aureus clones and that cna positive strains appeared to be associated with capsular type however, our strains were all methicillin sensible and out of strains carried the cps gene for capsular type and the remaining cps suggesting that the site of infection may have been the driving force in selecting a subpopulation of cna positive s. aureus strains irrespective of other characteristics such as methicillin resistance and capsular type. an elevated breast collagen concentration has been shown to be one of the greatest risk factors for developing breast carcinoma. breast implants, especially in oncologic patients, due to the increased collagen deposition, could be easily covered by a more dense collagen matrix and thus become more prone to adhesion of s. aureus strains able to bind collagen. in contrast to s. aureus causing pji, the great majority of our strains carried and expressed the sasg gene. this difference in distribution according to the site of infection, is remarkable as its s. epidermidis homologue aap appears to be almost ubiquitously present. further studies are needed to better characterize this protein, its function/s and to understand its role in different types of infections. the gene coding for bap, a cell wall protein found in bovine mastitis s. aureus isolates, involved in intercellular adhesion and biofilm formation was absent in all s. aureus isolates studied. our results support the suggestion of other authors, that the role of bap in human infections is doubtful. besides the ability to form biofilms, other authors have investigated the incidence of genes coding for leukocidal toxins in s. aureus isolated from implant orthopaedic infections. panton-valentine, luke/lukd and lukm leukocidins are known to be associated with necrotic skin and soft tissue infections and could promote damage to peri-implant tissues. luke/lukd was the only gene found in strains from breast implant infections. leukotoxin genes displayed a similar prevalence in strains from breast implants and pjis, suggesting that luke/lukd could be the only relevant leukocidin in both types of implant-related infections. the great majority of s. epidermidis analysed carried atle, aap, aae, embp and fbe genes, a condition already described for s. epidermidis isolated from catheter-related bloodstream infections and pjis as well as for commensal s. epidermidis, indicating that these proteins are valuable during both infection and colonization. in contrast to s. aureus only three s. epidermidis strains carried a gene coding for a surface protein binding collagen. this is not surprising, since adhesins that recognize host proteins coating the device seem to be the primary mechanism of adhesion in s. aureus, while s. epidermidis initial adherence is probably multifactorial. breast reconstruction has become a standard of care for almost all women requiring mastectomy for breast cancer. reduction of mutilation greatly improves the psychological status of women without interfering with oncological treatment, however, the reconstructive approach may be compromised by early or late implant infection in 8% of patients in prospective series. further studies are needed to confirm our observation and to verify if patients colonized by cna positive strains are at higher risk of breast peri-implant infections than those colonized by cna negative s. aureus. if this holds true, screening of patients before and after reconstructive surgery at regular intervals could be extremely useful to identify those colonized and thus concentrate management, and resources. should colonization with cna-positive strains confirm to be predictive for higher risk of implant infection, effective prophylactic strategies/treatments could be devised to reduce the risk of infection of reconstruction breast implants.  s. aureus and s. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms. since the collagen adhesin gene is not ubiquitously distributed among s. aureus, this protein could have an important role in the cause of breast peri-implant infections. further studies are needed to confirm our observation and eventually recommend screening of patients before and after reconstructive surgery to identify those colonized by cna-positive s. aureus and at higher risk of peri-implant infections. 
since the collagen adhesin gene is not ubiquitously distributed among s. aureus, this protein could have an important role in the cause of breast peri-implant infections. biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. proteinase k and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation. staphylococcus epidermidis and s. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. to our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections. thirteen s. aureus and seven s. epidermidis causing breast peri-implant infections were studied. s. aureus and s. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms. all s. aureus strains harboured and expressed the atla, clfa, fna, eno and cna genes and the majority also carried and expressed the sasg, ebps genes. biofilm development was glucose- and nacl-induced or glucose-induced. all s. epidermidis strains harboured and expressed the atle, aae, embp genes, and the majority also carried and expressed the fbe, aap genes. methicillin resistance was detected in two s. epidermidis. all strains had different rapd profiles and were able to produce biofilms in microtitre plate assays but, while all s. aureus carried and were able to express icaa and icad genes, this was only true for one s. epidermidis. the only leukotoxin genes detected were luke/lukd. genes for s. aureus capsular types and were almost equally distributed.
 staphylococcus epidermidis and s. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. to our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections.  thirteen s. aureus and seven s. epidermidis causing breast peri-implant infections were studied. using the broth microdilution method and the e-test, the majority of the strains were susceptible to all antibiotics tested. methicillin resistance was detected in two s. epidermidis. all strains had different rapd profiles and were able to produce biofilms in microtitre plate assays but, while all s. aureus carried and were able to express icaa and icad genes, this was only true for one s. epidermidis. biofilm development was glucose- and nacl-induced or glucose-induced. proteinase k and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation. all s. aureus strains harboured and expressed the atla, clfa, fna, eno and cna genes and the majority also carried and expressed the sasg, ebps genes. all s. epidermidis strains harboured and expressed the atle, aae, embp genes, and the majority also carried and expressed the fbe, aap genes. genes for s. aureus capsular types and were almost equally distributed. the only leukotoxin genes detected were luke/lukd.  s. aureus and s. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms. since the collagen adhesin gene is not ubiquitously distributed among s. aureus, this protein could have an important role in the cause of breast peri-implant infections. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords implant infectionsbiofilmstaphylococcal infectionsica genebiofilm-related genesmscrammsissue-copyright-statement© the author 2015 