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as biomaterials are used commonly in medical practice , the incidence of infections related to biomaterials is increasing . to prevent biomaterial - related infections , it is essential to understand the pathogenesis of the infection .
biofilm is known to play a key role in the adherence of many pathogenic bacteria to biomaterials . in coagulase negative staphylococci ( cns ) , the most common pathogen of biomaterial - related infections , the later phase of biofilm formation
is mediated by polysaccharide intercellular adhesin ( 1 - 5 ) , a product of the intercellular adhesion ( ica ) operon ( 3 , 6 ) .
it is also known that ica gene of cns is subject to phase variation , which is a process of onoff in the production of biofilm ( 7 , 8) .
the phase variation of cns in biofilm formation is believed to be the mechanism of its long persistence and relapse .
although the whole mechanism of this phase variation have not been understood , some of the phase variation in cns were reported to occur due to the inactivation of ica operon by insertion of the insertion sequence element is256 ( 8) .
staphylococcus aureus is another common pathogen of biomaterial - related infections , and biofilm formation is also known to be associated with its virulence .
deletion of ica genes in s. aureus resulted in the loss of ability to form biofilm .
the presence of phase variation in biofilm formation of s. aureus has been reported before ( 10 ) .
however , the mechanisms of the phenomenon , which might be associated with some changes in ica gene , have not been investigated yet .
furthermore , although the influence of ica gene of s. aureus on the biofilm formation has been evaluated , studies about the impact of changes in ica gene and subsequent biofilm formation on the adhering capacity to biomaterials have not been performed yet .
therefore , this study was carried out to investigate the presence and mechanism of phase variation in ica gene of s. aureus , and to evaluate the relationship between ica gene and the adhering capacity to biomaterials in s. aureus using phase - variant strains .
we screened 38 clinical strains and 44 nasal carriage strains of s. aureus for ica operon and biofilm formation .
biofilm formation was evaluated by biofilm assay using microtiter - plate ( 9 ) and congo red agar method ( 11 ) .
six biofilm - positive strains , which had ica operon and showed consistent results in both of the biofilm formation tests , were tested for selection of biofilm - negative phase - variant strains .
biofilm - positive strains grow on this medium as dark - red colonies , whereas biofilm - negative strains grow on as light - red colonies .
agar plates were incubated overnight at 37 and an additional 24 hr at room temperature .
the variation rate was determined by counting the different colony forms in relation to the total number of colony - forming unit ( cfu ) .
a single colony of a biofilm - negative variant was isolated and incubated at 37 in tryptic soy broth in a tissue culture flask . after 24 hr
this procedure was repeated until a biofilm of adhering bacteria became visible on the bottom of the tissue culture flask ( maximum of 15 days ) . after washing with phosphate - buffered saline ( pbs ) ,
the adhering bacteria cells were scratched from the bottom and streaked on congo red agar .
after incubation at 37 overnight and an additional 24 hr at room temperature , single , dark - red colony was isolated .
dna of biofilm - negative phase - variants , biofilm - positive revertants and their parent strains were compared by pulsed - field gel electrophoresis ( pfge ) .
genes of icaa , icab , and icac were amplified by pcr , and compared with those of the parent and revertant strains .
primers and the amplification conditions of the ica - specific pcr are summarized in table 1 .
changes in ica genes found by pcr were confirmed with southern blot hybridization and dna sequencing .
chromosomal dna was isolated from s. aureus wild - type , biofilm - negative variants , and biofilm - positive revertant and digested with ecori .
the digested dna was separated on 0.8% agarose gel electrophoresis , and then transferred to a hybond - n+ nylon transfer membrane ( amersham life science , buckinghamshire , u.k . ) .
after the membrane was backed at 80 for 2 hr , prehybridization and hybridization were conducted at 42 for 1 hr and overnight , respectively .
southern blot dna - dna hybridization was carried out under high - stringency condition by using icac - specific probe .
the probe dna was labeled by using the ecl direct nucleic acid labelling system ( amersham ) .
washing the membrane with 1ssc containing 0.4% sodium dodecyl sulphate was reacted with detection reagent ( amersham ) and then exposed to radiography film .
a template was mixed terminator ready reaction mix ( perkin elmer cetus , norwalk , u.s.a . ) , which already contained primers , and incubated in thermal at 96 ( 30 sec ) , 50 ( 15 sec ) , and 60 ( 4 min ) .
typically , 25 cycles are required for extensive amplification ( i.e. , 106 fold ) of a specific target . after being denatured by heating for 2 min at 90
, the samples were set abi377 automated dna sequencer ( pe applied biosystems , foster city , ca , u.s.a . ) .
the assay for the quantitative determination of biofilm formation was performed with polyurethane ( pu ) films .
bacterial cultures in tryptic soy broth , containing 10cfu / ml of sa 204 wild - type strain and phase variant , respectively , incubated with 100 rpm agitation at 37 with 4 sheets of pu films .
after 5 days , pu films were washed three times with 4 ml of pbs .
remaining bacteria on pu films were resuspended by vigorous vortexing and 0.1 ml of a diluted culture was seeded per blood agar plate .
from a strain among the 6 strains tested , potential biofilm - negative subcolonies were grown at a frequency of 110 - 510 .
two strains ( sa204/v-5 , sa204/v-6 ) from the subcolonies showed biofilm negative on biofilm assay . from one strain among them , a biofilm - positive revertant was obtained after 3 - 4 passages .
congo red agar test and biofilm assay of the strains are compared with those of their parent strain in fig . 1 and fig
. 2 , biofilm forming strains , sa204 and sa204/r-6 , grow on congo red agar medium as dark - red color , and show red color on microtiter plate .
whereas biofilm - negative phase - variant strains , sa204/v-5 and sa204/v-6 , grow on congo red agar medium as light - red color . sa204/v-5 and sa204/v-6 showed the same pfge patterns with that of the parent strain and the revertant strain ( fig .
3 ) . while pcr products of icaa and icab genes of the variant strains were the same in size with those of the parent strain and the revertant strain , icac - specific pcr products showed different sized bands ( fig .
southern hybridization analysis with the icac - specific dna probe showed a larger restriction fragment also ( fig .
the increased size of the shifted icac gene was about 1.3 kb , which was the same in size with that of the is256 .
nucleotide sequence of the enlarged pcr fragments perfectly matched the published sequence of the insertion sequence element is256 .
the adhesion capacity to pu film of sa204/v-5 and its parent strain showed a significant difference statistically ( 10 vs. 10 , p<0.02 ) .
in s. epidermidis , phase variation of biofilm formation is caused by alternating insertion and excision of the insertion sequence element is256 into different sites of the ica gene cluster ( 7 , 8) .
specifically , 11 out of 13 insertion mutants carry the element in the icac gene , one mutant was an icaa and another one an icab insertional mutant ( 8) . in this study
, we obtained two phase - variant strains that are insertional mutants the inactivation of icac gene by is256 .
these investigations appear to suggest that the icac nucleotide sequence seems to represent a preferred insertion site of is256 . as far as we know ,
this is the first report about the role of is256 in the mechanism of phase variation in biofilm formation of s. aureus .
we did not evaluate the prevalence of phase variant strains , nor the proportion of is256 contributing to the phase variation of ica gene in s. aureus . to figure out the frequency of phase variation in biofilm and insertion of is256 , studies with more strains
due to the fact that a biofilm - variant strain had a low capacity of adhering to pu films , we could conclude that ica gene and biofilm play a role in the adherence of s. aureus to biofilm materials .
we used phase - variant strains to compare the adherence ability to pu films between biofilm - forming and biofilm - nonforming s. aureus strains , on the premise that the phase - variant s. aureus strains had changed only in ica genes .
it was not feasible to evaluate all other virulence factors in phase - variant strains . to exclude the potential influence of other virulence factors ,
studies using mutant strains with inactivated ica genes specifically are warranted . moreover , considering the influence of host components in adherence of s. aureus to biomaterials , in vivo studies using animal models are also warranted .
while ica gene of staphylococcus epidermidis is known to undergo phase variation by insertion of is256 , the phenomenon in staphylococcus aureus has not been evaluated . 
 six biofilm - positive strains were tested for the presence of biofilm - negative phase - variant strains by congo red agar test . for potential phase - variant strains , 
 pulsed - field gel electrophoresis was done to exclude the possibility of contamination . to investigate the mechanism of the biofilm - negative phase variation , pcr for each ica genes 
 were done . 
 changes of ica genes detected by pcr were confirmed by southern hybridization , and their nucleotides were analyzed by dna sequencing . 
 influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane surface among a biofilm - negative phase - variant strain and its parent strain . 
 a biofilm - negative phase - variant s. aureus strain was detected from 6 strains tested . 
 icac gene of the phase - variant strain was found to be inactivated by insertion of additional gene segment , is256 . 
 the biofilm - negative phase - variant strain showed lower adhering capacity to polyurethane than its parent strain . 
 this study shows that phase variation of ica gene occurs in s. aureus by insertion of is256 also , and this biofilm - negative phase variation reduces adhering capacity of the bacteria .