BACKGROUND
staphylococcus epidermidis and other coagulase-negative staphylococci  constitute the most frequent causes of hospital-acquired infections and are often associated with the use of medical devices  <cit> .
virulence is mainly attributed to surface colonization and biofilm formation  <cit> .
a biofilm represents an adherent, structured, high density community of bacterial cells  <cit>  embedded in an extracellular matrix, previously called slime.
polysaccharide intercellular adhesin , a homoglycan composed of β- <dig> -linked 2-deoxy-2-amino-d-glucopyranosyl residues, is considered to be the major functional component mediating intercellular adhesion in s. epidermidis biofilms  <cit> .
biofilm formation mediated by pia is a major virulence factor in experimental biomaterial-associated infection  <cit>  and provides also protection against opsonophagocytosis and activity of anti-microbial peptides  <cit> .
the genes encoding pia production are organized in the icaadbc operon  <cit> .
moreover, a polysaccharide molecule with 20-kda average molecular mass, defined as 20-kdaps, was isolated from s. epidermidis atcc <dig> , atcc <dig>  and clinical biofilm-producing strains by ion-exchange chromatography and gel filtration  <cit> .
its purity, charge density and molecular integrity have been confirmed by reverse polarity capillary electrophoresis  <cit> .
20-kdaps consists mainly of glucose and n-acetylglucosamine, and is partially sulfated.
proposed structure of 20-kdaps is 30– <dig> molecules of glucose, 1– <dig> molecules of xylose and fucose, 61– <dig> molecules of glucosamine   and 3– <dig> molecules of glucuronic acid  <cit> .
this polysaccharide represents 60-65% of total slime carbohydrate and seems to be one of the main antigenic components of slime  <cit> .
immunization of rabbits with purified 20-kdaps elicits production of antibodies reacting specifically with 20-kdaps and biofilm-producing reference strain atcc <dig>  and other biofilm-producing clinical s. epidermidis strains, but not with other cons or s. aureus clinical isolates  <cit> .
protective value of 20-kdaps antibodies has been proven in experimental keratitis protocols, where passive and active immunization of rabbits with 20-kdaps antigen and anti-20-kdaps exhibit beneficial properties  <cit> .
administration of intravenous immunoglobulin preparations with high anti-20-kdaps titers in preterm neonates reduces risk of bacteraemia caused by biofilm-producing s. epidermidis <cit> .
finally, experimental data suggest that 20-kdaps is associated with attachment of s. epidermidis to endothelial cells  <cit> .
several other polysaccharide molecules have been associated with biofilm accumulation or initial adherence on surfaces, such as ps/a  or pnsg , finally defined as pnag  <cit> , and saa   <cit> .
as other polysaccharide molecules associated with s. epidermidis’ pathogenesis turned out to be identical or related to pia  <cit> , the aim of this study was to define the relation of 20-kdaps and pia using isogenic mutants with tn917-insertions in various locations in icaadbc, specific antisera and specific glycosidase and chemical treatments.
in addition, in vitro experiments were conducted exploring 20-kdaps biological interference in phagocytosis by human macrophages.
RESULTS
detection of 20-kdaps, pia expression and icaadbc-genotype in clinical cons isolates
among fifty  clinical s. epidermidis strains, eighteen  were found ica+ biofilm+ 20-kdaps+, ten  ica- biofilm- 20-kdaps-, six  ica+ biofilm- 20-kdaps+, six  ica- biofilm- 20-kdaps+, five  ica+ biofilm- 20-kdaps- and five  strains ica+ biofilm+ 20-kdaps-.
all other cons  were ica- biofilm- 20-kdaps-.
all ica+ biofilm+s.
epidermidis strains were pia-positive by specific immunofluorescence test, whereas, ica- biofilm- or ica+ biofilm- strains were pia-negative.
in our s. epidermidis strain collection, 46%  were pia positive and 60%  were 20-kdaps positive.
icaadbc prevalence in our collection was 68%, whereas 46% of s. epidermidis strains were biofilm-producing.
20-kdaps expression among ica+s.
epidermidis strains was 70% , whereas, 20-kdaps expression among ica- strains was 37% .
20-kdaps expression in relation to biofilm formation reveals that 78% of biofilm-producing s. epidermidis strains expressed 20-kdaps , whereas, 44% of biofilm-negative strains were 20-kdaps positive .
these results show that the majority of clinical s. epidermidis isolates express 20-kdaps and that there is no strict correlation of icaadbc-genotype or biofilm phenotype and expression of 20-kdaps.
expression of 20-kdaps and pia by s. epidermidis strains with known genetic backgrounds
using an indirect immunofluorescence test with specific anti-pia antiserum s. epidermidis strains  <dig>   <dig>  and  <dig> were shown to express pia, while the isogenic icaa-insertion mutants 1457-m <dig>  m <dig> and 8400-m <dig> and isogenic icac-insertion mutants m <dig> and m <dig> did not express pia.
similarly, s. epidermidis  <dig>  5179r <dig> and  <dig> did not synthesize pia as in the former two strains icaadbc is inactivated through insertion of is <dig> <cit> , while  <dig> is icaadbc-negative.
using specific anti-20-kdaps antiserum s. epidermidis  <dig>  1457-m <dig>  m <dig>  m <dig>  m <dig>   <dig>  8400-m <dig>   <dig>   <dig>  5179r <dig> were 20-kdaps positive, whereas, s. epidermidis strain  <dig> was 20-kdaps negative.
a representative immunofluorescence test with anti-pia and anti-20-kdaps antisera, comparing s. epidermidis  <dig> and 1457-m <dig>  is displayed in figure  <dig>  an identical expression pattern of 20-kdaps was independently demonstrated for these strains using specific elisa, excluding that there are significant quantitative differences in 20-kdaps antigen expression between the isogenic mutant strain pairs .
20-kdaps detection in transposon mutants of s. epidermidis 1457-m <dig>  m <dig>  m <dig>  m <dig> is shown in figure  <dig>  inactivation of icaa in mutant 1457-m <dig> and of icac in mutants m <dig> and m <dig> lead to biofilm negative and pia negative phenotype, but did not alter 20-kdaps antigen detection.
the fact that mutant m <dig>  where the transposon is oriented in the opposite transcriptional direction than the icaadbc operon and no ica specific transcript can be identified, still expressed 20-kdaps provide clear proof that 20-kdaps synthesis is independent of the icaadbc operon.
influence of chemical and enzymatic treatments on antigen detection by immunofluorescence and on biofilm integrity
periodate oxidation led to abolishment of antigenic reactivity of pia, whereas 20-kdaps preserved its antigenic properties .
treatment with dispersin b  completely destroyed antigenic reactivity of pia within one hour of incubation.
dspb is a hexosaminidase  produced by the oral pathogen aggregatibacter actinomycetemcomitans, which specifically cleaves β- <dig> -linked n-acetylglucosamine polymer disrupting pia chain  <cit> .
in contrast, dspb does not alter 20-kdaps antigenic properties .
parallel to pia destruction, biofilm structure is disrupted after periodate oxidation and dspb treatments and large clumps are substituted by small clumps or single and double cells, still detectable by anti-20-kdaps antiserum .
finally, the fact that pia and 20-kdaps retain their antigenic properties after proteinase k digestion is consistent with their polysaccharide nature .
integrity of biofilm, formed on 96-well cell culture plates, to treatment with proteinase k, sodium meta-periodate and dspb was also studied.
all biofilms were susceptible to sodium meta-periodate and dspb, whereas, addition of proteinase k did not affect biofilm stability.
thus, biofilm production in our strain collection is mediated mainly through pia, as was shown in other studies  <cit> .
in addition, 20-kdaps presence does not relate to biofilm formation as agents, such as sodium meta-periodate and dspb that destroy biofilm integrity, do not affect antigenic properties of 20-kdaps.
lack of co-purification of 20-kdaps with pia polysaccharide i in q-sepharose anion-exchange chromatography
clarified crude bacterial extracts obtained after bacterial sonication were tested for presence of pia and 20-kdaps reactivity by elisa using anti-pia and anti-20-kdaps rabbit antisera, respectively .
under the conditions employed, in the crude extract consistently higher absorbance values were obtained with the 20-kdaps specific antiserum as compared to the anti-pia specific antiserum.
the crude extract was applied to a q-sepharose column as described in materials and methods.
under these conditions the majority of pia  did not bind to the columns, but was immediately eluted.
this pia antigen fraction is referred to as polysaccharide i of pia  <cit> .
however, in the fractions representing the pia antigenic peak reactivity with the specific anti-20-kdaps antiserum was negligible indicating that 20-kdaps does not co-purify with polysaccharide i of pia.
additionally, this excludes significant cross reactivity of the 20-kdaps antiserum with epitopes present on pia.
pia and 20-kdaps antisera do not cross-react with each-other
in order to identify any cross reactivity among 20-kdaps antiserum and pia antigen and vice versa, absorption studies were performed.
pia-specific antiserum was absorbed by s. epidermidis  <dig>  strain, as described in methods.
absorbed antiserum was incubated with  <dig> on immunofluorescence slides and achievement of complete absorption was confirmed.
furthermore, absorbed antiserum did not detect pia on rp <dig> ,  <dig>  and  <dig>  s. epidermidis strains.
pia-specific antiserum was also absorbed by s. epidermidis  <dig>  and immunofluorescence tests performed with s. epidermidis rp <dig>   <dig> and  <dig>  no remaining anti-pia reactivity was observed with any strain using the absorbed antiserum.
finally, pia-specific antiserum absorbed with s. epidermidis  <dig>  retains all reactivity to s. epidermidis  <dig>  rp <dig> and  <dig> strains.
in case that pia antiserum reacted - even weakly - with 20-kdaps antigen, incubation of pia antiserum with strain  <dig> bearing 20-kdaps antigen, would lead to absorption of anti-pia antibodies and no anti-pia reactivity would remain.
a selection of analogous experiments was performed regarding anti-20kdaps serum, as shown in table  <dig>
a strains used for absorption of specific antisera, b strains applied on immunofluorescence slide, c no fluorescence indicated no residual reactivity for specific antigen, d fluorescence indicated reactivity for specific antigen.
synthesis of 20-kdaps and pia in different culture media
in order to explore possible polysaccharide synthesis dependence on certain constituents of culture media, 20-kdaps and pia presence upon prolonged culture in different culture media was studied.
20-kdaps expression was not abolished after long time incubation of bacteria in any of the selected media .
20-kdaps antiserum revealed strong reactivity to bacterial cells growing in all media with the exception of tsb w/o dextrose where only a percentage of bacterial cells express 20-kdaps.
regarding pia synthesis, tsb seems superior to rpmi  <dig>  rpmi 1640 + glutamine and imdm upon prolonged consecutive subcultures, whereas pia expression was almost abolished in tsb lacking dextrose, in accordance to previous reports  <cit> .
in addition, pia presence was strongly associated to biofilm formation.
biofilms formed in rpmi <dig>  rpmi1640 + glutamine and imdm were more susceptible to mechanic disruption following agitation by vortex and disintegration into small clumps .
* small clumps, ° few cells, ++ strong fluorescence, - no fluorescence.
impact of 20-kdaps on bacterial endocytosis
differences in phagocytosis between s. epidermidis reference strain atcc <dig> and the clinical 20-kdaps negative strain  <dig> were observed .
phagocytosis experiments were performed without addition of exogenous complement.
preincubation of non-20kdaps-producing strain with different concentrations of 20-kdaps inhibits endocytosis .
specifically, preincubation of non-20kdaps-producing strain with 20-kdaps  reduces the number of endocytosed bacteria from  <dig>  ±  <dig>  to  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> bacteria/well, respectively.
differences are statistically significant in all above 20-kdaps concentrations.inhibition of endocytosis takes place at a dose dependent manner between  <dig> and 60 μg/ml .
on the contrary, 20-kdaps antiserum increases endocytosis of 20-kdaps-producing atcc <dig> strain ca  <dig> fold, as compared to bacteria preincubated with preimmune serum .
preincubation with preimmune antiserum did not alter endocytosis, as compared to bacteria preincubated with pbs .
in terms of s. epidermidis clinical isolate  <dig>  preincubation with preimmune antiserum seems to enhance endocytosis, as compared to bacteria preincubated with pbs , but preincubation with 20-kdaps antiserum does not further increase endocytosis, as compared to bacteria preincubated with preimmune serum .
this phenomenon may be associated with the presence of other anti-staphylococcal antibodies in rabbit serum.
prior to immunization, rabbit serum was collected and tested by elisa for reactivity to 20-kdaps in order to exclude pre-existence of 20-kdaps specific antibodies.
low titers of antibodies to various staphylococcal strains, s. epidermidis and s. aureus, are present in preimmune serum  and may be responsible for the observed effect.
a representative experiment of five similar ones is presented in figure  <dig>
discussion
staphylococcus epidermidis is an important pathogen  <cit>  and extracellular polysaccharides as well as a number of surface proteins contributing to bacterial attachment and biofilm formation have been extensively studied.
analysis of s. epidermidis’ polysaccharides has been associated with difficulties, however, it is now clear that, despite some possible variation, pia, and other analogue polysaccharides such as ps/a, pnsg, pnag, and saa are chemically closely related if not identical and represent the same chemical entity, namely pia.
this is the first time shown that 20-kdaps is discrete from pia and this statement is based on concrete basis.
transposon insertion in icaadbc, the locus encoding synthetic enzymes for pia synthesis, does not abrogate production of 20-kdaps.
in mutant 1457-m <dig> in which tn <dig> was inserted in icaa in the same transcriptional orientation, outward directed transcription resulted in transcripts comprising the complete sequences of icadicab and icac <cit> .
expression of 20-kdaps in mutant 1457-m <dig> where icaa synthesis is inhibited and in mutant m <dig> and m <dig> where icac expression was inhibited shows that 20-kdaps synthesis does not require an intact icaa or icac gene.
the fact that 20-kdaps was detected in m <dig>  where tn <dig> was inserted in the opposite transcriptional direction to the ica operon and no-ica specific transcripts were identified  <cit> , provides evidence that 20-kdaps synthesis is independent of ica operon.
in contrast, pia synthesis is completely inhibited not only by the disruption of the entire icaadbc operon but also by the isolated inhibition of icaa  and icac  gene expression.
proteinase k does not disrupt antigenic properties of 20-kdaps reconfirming its polysaccharide nature.
furthermore, dspb, which specifically cleaves β- <dig> -linked n-acetylglucosamine polymer disrupting pia chain  <cit> , did not affect 20-kdaps.
although sodium meta-periodate is an agent commonly used to disrupt polysaccharide molecules, it did not affect integrity of 20-kdaps antigen.
taking into account that periodate preferably degrades cis-diols, it is suggested that monomeric units of the polysaccharide core form glycosidic bonds between the anomeric c- <dig> and the c- <dig> or c- <dig>  this is not the case for pia, where a β- <dig> -glycosidic bond is present leaving free vicinal hydroxyl groups of glucosamine at c- <dig> and c- <dig>  the above structural data suggest that 20-kda ps and pia are two discrete and different polysaccharides.
preliminary data in our laboratories showed that 20-kdaps is not affected upon treatment with glycosaminoglycan- degrading enzymes , suggesting a non glycosaminoglycan-related structure.
absence of 20-kdaps in q-sepharose fractions containing maximum pia reactivity is due to different physicochemical properties among the two molecules.
q-sepharose is a strong anion-exchanger which retains negatively charged molecules.
whereas pia is eluting, 20-kdaps may be strongly retained by the column due to its negative charges.
aforementioned differentiation was expected as different isolation procedures are used for the two polysaccharides.
as previously described  <cit> , 20-kdaps is obtained from bacterial extracellular matrix using a linear nacl gradient on deae-sephacel and elutes at  <dig> - <dig>  m nacl.
presented data suggest that 20-kdaps inhibits endocytosis of s. epidermidis bacterial cells at a dose-dependent manner.
similarly, pia provides protection against opsonophagocytosis and activity of anti-microbial peptides  <cit> .
in the absence of specific opsonizing antibodies, macrophages are able to clear pathogens by innate immune receptors, such as the group of molecular pattern recognition receptors , collectively known as scavenger receptors  <cit> .
20-kdaps may interfere with or mask staphylococcal antigen promoting phagocytosis  <cit> ; on the other hand, it may interact with a receptor that does not facilitate phagocytosis.
adhesion receptors and phagocytosis receptors can both activate and inhibit each other functions  <cit> .
it has been previously shown that 20-kdaps promotes adhesion to human endothelial cells and this interaction is blocked upon addition of anti-20kdaps antibodies.
comparable data were acquired by using human macrophages , indicating the presence of a specific ligand for 20-kdaps on human cells.
adherence of unopsonized bacteria to macrophages does not preclude internalization  <cit> .
nonopsonic binding of pathogens to host phagocytic cells may not always result in phagocytosis, however, it may serve an important role in the immune response  <cit>
nevertheless, phagocytic activity of macrophages is greatly enhanced if specific antibodies are attached to the pathogen  <cit> .
20-kdaps antiserum do not exhibit any cross reactivity with pia.
antibodies against pnsg and pia have been found completely cross-reactive  <cit> .
as 20-kdaps antiserum reacts specifically and strictly with 20-kdaps, observed biologic properties concern exclusively this entity.
our data show that 20-kdaps antiserum exhibits opsonic properties as it increases endocytosis of s. epidermidis atcc <dig> by human macrophages.
several surface molecules have been studied as potential antibody targets in order to enhance phagocytic potential of monocytes/macrophages.
opsonic activity of antibodies to s. epidermidis fbe and atle has been demonstrated in a study where fresh alveolar macrophages from rat ingested and killed s. epidermidis opsonized with anti-fbe antibodies  to a much higher extent than they ingested and killed nonopsonized bacteria or bacteria opsonized with antibodies directed against atle or embp  <cit> .
also, a chimerized  monoclonal antibody against lipoteichoic acid that was proven protective for cons and s. aureus bacteremia in animal models has been also tested to humans  <cit> .
in contrast, antibodies to accumulation-associated protein and lipoteichoic acid had no opsonic activity in vitro and did not protect mice against experimental biomaterial-associated infections  <cit> .
although, conjugate vaccines based on pia/pnag have been shown to be beneficial in animal models  <cit> , several doubts for their use in human trials have been documented  <cit> .
thus, more and extensive investigations are needed to evaluate the potential use of 20-kdaps in conjugate vaccines.
CONCLUSIONS
this is the first study providing concrete data that 20-kdaps is a unique polysaccharide molecule discrete from pia.
20-kdaps exhibits antiphagocytic properties that may be shown to play a role in pathogenicity.
further work is in progress to establish a role in conjugate vaccine development.

 staphylococcus epidermidis and other coagulase-negative staphylococci constitute the most frequent causes of hospital-acquired infections and are often associated with the use of medical devices. virulence is mainly attributed to surface colonization and biofilm formation. a biofilm represents an adherent, structured, high density community of bacterial cells embedded in an extracellular matrix, previously called slime. polysaccharide intercellular adhesin, a homoglycan composed of β- -linked 2-deoxy-2-amino-d-glucopyranosyl residues, is considered to be the major functional component mediating intercellular adhesion in s. epidermidis biofilms. biofilm formation mediated by pia is a major virulence factor in experimental biomaterial-associated infection and provides also protection against opsonophagocytosis and activity of anti-microbial peptides. the genes encoding pia production are organized in the icaadbc operon. moreover, a polysaccharide molecule with 20-kda average molecular mass, defined as 20-kdaps, was isolated from s. epidermidis atcc, atcc and clinical biofilm-producing strains by ion-exchange chromatography and gel filtration. its purity, charge density and molecular integrity have been confirmed by reverse polarity capillary electrophoresis. 20-kdaps consists mainly of glucose and n-acetylglucosamine, and is partially sulfated. proposed structure of 20-kdaps is 30– molecules of glucose, 1– molecules of xylose and fucose, 61– molecules of glucosamine and 3– molecules of glucuronic acid. this polysaccharide represents 60-65% of total slime carbohydrate and seems to be one of the main antigenic components of slime. immunization of rabbits with purified 20-kdaps elicits production of antibodies reacting specifically with 20-kdaps and biofilm-producing reference strain atcc and other biofilm-producing clinical s. epidermidis strains, but not with other cons or s. aureus clinical isolates. protective value of 20-kdaps antibodies has been proven in experimental keratitis protocols, where passive and active immunization of rabbits with 20-kdaps antigen and anti-20-kdaps exhibit beneficial properties. administration of intravenous immunoglobulin preparations with high anti-20-kdaps titers in preterm neonates reduces risk of bacteraemia caused by biofilm-producing s. epidermidis. finally, experimental data suggest that 20-kdaps is associated with attachment of s. epidermidis to endothelial cells. several other polysaccharide molecules have been associated with biofilm accumulation or initial adherence on surfaces, such as ps/a or pnsg, finally defined as pnag, and saa. as other polysaccharide molecules associated with s. epidermidis’ pathogenesis turned out to be identical or related to pia, the aim of this study was to define the relation of 20-kdaps and pia using isogenic mutants with tn917-insertions in various locations in icaadbc, specific antisera and specific glycosidase and chemical treatments. in addition, in vitro experiments were conducted exploring 20-kdaps biological interference in phagocytosis by human macrophages.  detection of 20-kdaps, pia expression and icaadbc-genotype in clinical cons isolates among fifty clinical s. epidermidis strains, eighteen were found ica+ biofilm+ 20-kdaps+, ten ica- biofilm- 20-kdaps-, six ica+ biofilm- 20-kdaps+, six ica- biofilm- 20-kdaps+, five ica+ biofilm- 20-kdaps- and five strains ica+ biofilm+ 20-kdaps-. all other cons were ica- biofilm- 20-kdaps-. all ica+ biofilm+s. epidermidis strains were pia-positive by specific immunofluorescence test, whereas, ica- biofilm- or ica+ biofilm- strains were pia-negative. in our s. epidermidis strain collection, 46% were pia positive and 60% were 20-kdaps positive. icaadbc prevalence in our collection was 68%, whereas 46% of s. epidermidis strains were biofilm-producing. 20-kdaps expression among ica+s. epidermidis strains was 70%, whereas, 20-kdaps expression among ica- strains was 37%. 20-kdaps expression in relation to biofilm formation reveals that 78% of biofilm-producing s. epidermidis strains expressed 20-kdaps, whereas, 44% of biofilm-negative strains were 20-kdaps positive. these results show that the majority of clinical s. epidermidis isolates express 20-kdaps and that there is no strict correlation of icaadbc-genotype or biofilm phenotype and expression of 20-kdaps. expression of 20-kdaps and pia by s. epidermidis strains with known genetic backgrounds using an indirect immunofluorescence test with specific anti-pia antiserum s. epidermidis strains and were shown to express pia, while the isogenic icaa-insertion mutants 1457-m m and 8400-m and isogenic icac-insertion mutants m and m did not express pia. similarly, s. epidermidis 5179r and did not synthesize pia as in the former two strains icaadbc is inactivated through insertion of is, while is icaadbc-negative. using specific anti-20-kdaps antiserum s. epidermidis 1457-m m m m 8400-m 5179r were 20-kdaps positive, whereas, s. epidermidis strain was 20-kdaps negative. a representative immunofluorescence test with anti-pia and anti-20-kdaps antisera, comparing s. epidermidis and 1457-m is displayed in figure an identical expression pattern of 20-kdaps was independently demonstrated for these strains using specific elisa, excluding that there are significant quantitative differences in 20-kdaps antigen expression between the isogenic mutant strain pairs. 20-kdaps detection in transposon mutants of s. epidermidis 1457-m m m m is shown in figure inactivation of icaa in mutant 1457-m and of icac in mutants m and m lead to biofilm negative and pia negative phenotype, but did not alter 20-kdaps antigen detection. the fact that mutant m where the transposon is oriented in the opposite transcriptional direction than the icaadbc operon and no ica specific transcript can be identified, still expressed 20-kdaps provide clear proof that 20-kdaps synthesis is independent of the icaadbc operon. influence of chemical and enzymatic treatments on antigen detection by immunofluorescence and on biofilm integrity periodate oxidation led to abolishment of antigenic reactivity of pia, whereas 20-kdaps preserved its antigenic properties. treatment with dispersin b completely destroyed antigenic reactivity of pia within one hour of incubation. dspb is a hexosaminidase produced by the oral pathogen aggregatibacter actinomycetemcomitans, which specifically cleaves β- -linked n-acetylglucosamine polymer disrupting pia chain. in contrast, dspb does not alter 20-kdaps antigenic properties. parallel to pia destruction, biofilm structure is disrupted after periodate oxidation and dspb treatments and large clumps are substituted by small clumps or single and double cells, still detectable by anti-20-kdaps antiserum. finally, the fact that pia and 20-kdaps retain their antigenic properties after proteinase k digestion is consistent with their polysaccharide nature. integrity of biofilm, formed on 96-well cell culture plates, to treatment with proteinase k, sodium meta-periodate and dspb was also studied. all biofilms were susceptible to sodium meta-periodate and dspb, whereas, addition of proteinase k did not affect biofilm stability. thus, biofilm production in our strain collection is mediated mainly through pia, as was shown in other studies. in addition, 20-kdaps presence does not relate to biofilm formation as agents, such as sodium meta-periodate and dspb that destroy biofilm integrity, do not affect antigenic properties of 20-kdaps. lack of co-purification of 20-kdaps with pia polysaccharide i in q-sepharose anion-exchange chromatography clarified crude bacterial extracts obtained after bacterial sonication were tested for presence of pia and 20-kdaps reactivity by elisa using anti-pia and anti-20-kdaps rabbit antisera, respectively. under the conditions employed, in the crude extract consistently higher absorbance values were obtained with the 20-kdaps specific antiserum as compared to the anti-pia specific antiserum. the crude extract was applied to a q-sepharose column as described in materials and methods. under these conditions the majority of pia did not bind to the columns, but was immediately eluted. this pia antigen fraction is referred to as polysaccharide i of pia. however, in the fractions representing the pia antigenic peak reactivity with the specific anti-20-kdaps antiserum was negligible indicating that 20-kdaps does not co-purify with polysaccharide i of pia. additionally, this excludes significant cross reactivity of the 20-kdaps antiserum with epitopes present on pia. pia and 20-kdaps antisera do not cross-react with each-other in order to identify any cross reactivity among 20-kdaps antiserum and pia antigen and vice versa, absorption studies were performed. pia-specific antiserum was absorbed by s. epidermidis strain, as described in methods. absorbed antiserum was incubated with on immunofluorescence slides and achievement of complete absorption was confirmed. furthermore, absorbed antiserum did not detect pia on rp, and s. epidermidis strains. pia-specific antiserum was also absorbed by s. epidermidis and immunofluorescence tests performed with s. epidermidis rp and no remaining anti-pia reactivity was observed with any strain using the absorbed antiserum. finally, pia-specific antiserum absorbed with s. epidermidis retains all reactivity to s. epidermidis rp and strains. in case that pia antiserum reacted - even weakly - with 20-kdaps antigen, incubation of pia antiserum with strain bearing 20-kdaps antigen, would lead to absorption of anti-pia antibodies and no anti-pia reactivity would remain. a selection of analogous experiments was performed regarding anti-20kdaps serum, as shown in table a strains used for absorption of specific antisera, b strains applied on immunofluorescence slide, c no fluorescence indicated no residual reactivity for specific antigen, d fluorescence indicated reactivity for specific antigen. synthesis of 20-kdaps and pia in different culture media in order to explore possible polysaccharide synthesis dependence on certain constituents of culture media, 20-kdaps and pia presence upon prolonged culture in different culture media was studied. 20-kdaps expression was not abolished after long time incubation of bacteria in any of the selected media. 20-kdaps antiserum revealed strong reactivity to bacterial cells growing in all media with the exception of tsb w/o dextrose where only a percentage of bacterial cells express 20-kdaps. regarding pia synthesis, tsb seems superior to rpmi rpmi 1640 + glutamine and imdm upon prolonged consecutive subcultures, whereas pia expression was almost abolished in tsb lacking dextrose, in accordance to previous reports. in addition, pia presence was strongly associated to biofilm formation. biofilms formed in rpmi rpmi1640 + glutamine and imdm were more susceptible to mechanic disruption following agitation by vortex and disintegration into small clumps. * small clumps, ° few cells, ++ strong fluorescence, - no fluorescence. impact of 20-kdaps on bacterial endocytosis differences in phagocytosis between s. epidermidis reference strain atcc and the clinical 20-kdaps negative strain were observed. phagocytosis experiments were performed without addition of exogenous complement. preincubation of non-20kdaps-producing strain with different concentrations of 20-kdaps inhibits endocytosis. specifically, preincubation of non-20kdaps-producing strain with 20-kdaps reduces the number of endocytosed bacteria from  ±  to  ± ,  ± ,  ± ,  ±  bacteria/well, respectively. differences are statistically significant in all above 20-kdaps concentrations.inhibition of endocytosis takes place at a dose dependent manner between and 60 μg/ml. on the contrary, 20-kdaps antiserum increases endocytosis of 20-kdaps-producing atcc strain ca fold, as compared to bacteria preincubated with preimmune serum. preincubation with preimmune antiserum did not alter endocytosis, as compared to bacteria preincubated with pbs. in terms of s. epidermidis clinical isolate preincubation with preimmune antiserum seems to enhance endocytosis, as compared to bacteria preincubated with pbs, but preincubation with 20-kdaps antiserum does not further increase endocytosis, as compared to bacteria preincubated with preimmune serum. this phenomenon may be associated with the presence of other anti-staphylococcal antibodies in rabbit serum. prior to immunization, rabbit serum was collected and tested by elisa for reactivity to 20-kdaps in order to exclude pre-existence of 20-kdaps specific antibodies. low titers of antibodies to various staphylococcal strains, s. epidermidis and s. aureus, are present in preimmune serum and may be responsible for the observed effect. a representative experiment of five similar ones is presented in figure discussion staphylococcus epidermidis is an important pathogen and extracellular polysaccharides as well as a number of surface proteins contributing to bacterial attachment and biofilm formation have been extensively studied. analysis of s. epidermidis’ polysaccharides has been associated with difficulties, however, it is now clear that, despite some possible variation, pia, and other analogue polysaccharides such as ps/a, pnsg, pnag, and saa are chemically closely related if not identical and represent the same chemical entity, namely pia. this is the first time shown that 20-kdaps is discrete from pia and this statement is based on concrete basis. transposon insertion in icaadbc, the locus encoding synthetic enzymes for pia synthesis, does not abrogate production of 20-kdaps. in mutant 1457-m in which tn was inserted in icaa in the same transcriptional orientation, outward directed transcription resulted in transcripts comprising the complete sequences of icadicab and icac. expression of 20-kdaps in mutant 1457-m where icaa synthesis is inhibited and in mutant m and m where icac expression was inhibited shows that 20-kdaps synthesis does not require an intact icaa or icac gene. the fact that 20-kdaps was detected in m where tn was inserted in the opposite transcriptional direction to the ica operon and no-ica specific transcripts were identified, provides evidence that 20-kdaps synthesis is independent of ica operon. in contrast, pia synthesis is completely inhibited not only by the disruption of the entire icaadbc operon but also by the isolated inhibition of icaa and icac gene expression. proteinase k does not disrupt antigenic properties of 20-kdaps reconfirming its polysaccharide nature. furthermore, dspb, which specifically cleaves β- -linked n-acetylglucosamine polymer disrupting pia chain, did not affect 20-kdaps. although sodium meta-periodate is an agent commonly used to disrupt polysaccharide molecules, it did not affect integrity of 20-kdaps antigen. taking into account that periodate preferably degrades cis-diols, it is suggested that monomeric units of the polysaccharide core form glycosidic bonds between the anomeric c- and the c- or c- this is not the case for pia, where a β- -glycosidic bond is present leaving free vicinal hydroxyl groups of glucosamine at c- and c- the above structural data suggest that 20-kda ps and pia are two discrete and different polysaccharides. preliminary data in our laboratories showed that 20-kdaps is not affected upon treatment with glycosaminoglycan- degrading enzymes, suggesting a non glycosaminoglycan-related structure. absence of 20-kdaps in q-sepharose fractions containing maximum pia reactivity is due to different physicochemical properties among the two molecules. q-sepharose is a strong anion-exchanger which retains negatively charged molecules. whereas pia is eluting, 20-kdaps may be strongly retained by the column due to its negative charges. aforementioned differentiation was expected as different isolation procedures are used for the two polysaccharides. as previously described, 20-kdaps is obtained from bacterial extracellular matrix using a linear nacl gradient on deae-sephacel and elutes at -  m nacl. presented data suggest that 20-kdaps inhibits endocytosis of s. epidermidis bacterial cells at a dose-dependent manner. similarly, pia provides protection against opsonophagocytosis and activity of anti-microbial peptides. in the absence of specific opsonizing antibodies, macrophages are able to clear pathogens by innate immune receptors, such as the group of molecular pattern recognition receptors, collectively known as scavenger receptors. 20-kdaps may interfere with or mask staphylococcal antigen promoting phagocytosis; on the other hand, it may interact with a receptor that does not facilitate phagocytosis. adhesion receptors and phagocytosis receptors can both activate and inhibit each other functions. it has been previously shown that 20-kdaps promotes adhesion to human endothelial cells and this interaction is blocked upon addition of anti-20kdaps antibodies. comparable data were acquired by using human macrophages, indicating the presence of a specific ligand for 20-kdaps on human cells. adherence of unopsonized bacteria to macrophages does not preclude internalization. nonopsonic binding of pathogens to host phagocytic cells may not always result in phagocytosis, however, it may serve an important role in the immune response nevertheless, phagocytic activity of macrophages is greatly enhanced if specific antibodies are attached to the pathogen. 20-kdaps antiserum do not exhibit any cross reactivity with pia. antibodies against pnsg and pia have been found completely cross-reactive. as 20-kdaps antiserum reacts specifically and strictly with 20-kdaps, observed biologic properties concern exclusively this entity. our data show that 20-kdaps antiserum exhibits opsonic properties as it increases endocytosis of s. epidermidis atcc by human macrophages. several surface molecules have been studied as potential antibody targets in order to enhance phagocytic potential of monocytes/macrophages. opsonic activity of antibodies to s. epidermidis fbe and atle has been demonstrated in a study where fresh alveolar macrophages from rat ingested and killed s. epidermidis opsonized with anti-fbe antibodies to a much higher extent than they ingested and killed nonopsonized bacteria or bacteria opsonized with antibodies directed against atle or embp. also, a chimerized monoclonal antibody against lipoteichoic acid that was proven protective for cons and s. aureus bacteremia in animal models has been also tested to humans. in contrast, antibodies to accumulation-associated protein and lipoteichoic acid had no opsonic activity in vitro and did not protect mice against experimental biomaterial-associated infections. although, conjugate vaccines based on pia/pnag have been shown to be beneficial in animal models, several doubts for their use in human trials have been documented. thus, more and extensive investigations are needed to evaluate the potential use of 20-kdaps in conjugate vaccines.  this is the first study providing concrete data that 20-kdaps is a unique polysaccharide molecule discrete from pia. 20-kdaps exhibits antiphagocytic properties that may be shown to play a role in pathogenicity. further work is in progress to establish a role in conjugate vaccine development. 
here we provide experimental evidence that 20-kdaps and pia represent distinct molecules and that 20-kdaps is implicated in endocytosis of s. epidermidis bacterial cells by human monocyte-derived macrophages. 20-kdaps antiserum prevents adhesion of s. epidermidis on endothelial cells and development of experimental keratitis in rabbits. preincubation of non-20-kdaps-producing clinical strain with increasing amounts of 20-kdaps inhibits endocytosis by human macrophages, whereas, preincubation of 20-kdaps-producing strain atcc with 20-kdaps antiserum enhances bacterial endocytosis by human macrophages. 20-kdaps exhibits anti-phagocytic properties, whereas, 20-kdaps antiserum may have a beneficial effect on combating infection by 20-kdaps-producing s. epidermidis. while periodate oxidation and dispersin b treatments abolish immuno-reactivity and intercellular adhesive properties of pia, no abrogative activity is exerted towards 20-kdaps immunochemical reactivity following these treatments. pia polysaccharide i-containing fractions eluting from q-sepharose were devoid of detectable 20-kdaps using specific elisa. in conclusion, icaadbc is not involved in 20-kdaps synthesis, while the chemical and chromatographic properties of pia and 20-kdaps are distinct. the polysaccharide intercellular adhesin, a homoglycan composed of β- -linked n-acetylglucosamine residues, synthesized by enzymes encoded in icaadbc is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated s. epidermidis infection. tn917-insertion in various locations in icaadbc in mutants m m m and m of s. epidermidis are abolished for pia synthesis, while 20-kdaps expression appears unaltered as compared to wild-type strains using specific anti-pia and anti-20-kdaps antisera. extracellular mucous layer extracts of s. epidermidis contain another major polysaccharide, referred to as 20-kda polysaccharide, composed mainly out of glucose, n-acetylglucosamine, and being partially sulfated. analysis of clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kdaps is expressed exclusively in s. epidermidis but not in other coagulase-negative staphylococcal species.
 the skin commensal and opportunistic pathogen staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. the polysaccharide intercellular adhesin, a homoglycan composed of β- -linked n-acetylglucosamine residues, synthesized by enzymes encoded in icaadbc is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated s. epidermidis infection. extracellular mucous layer extracts of s. epidermidis contain another major polysaccharide, referred to as 20-kda polysaccharide, composed mainly out of glucose, n-acetylglucosamine, and being partially sulfated. 20-kdaps antiserum prevents adhesion of s. epidermidis on endothelial cells and development of experimental keratitis in rabbits. here we provide experimental evidence that 20-kdaps and pia represent distinct molecules and that 20-kdaps is implicated in endocytosis of s. epidermidis bacterial cells by human monocyte-derived macrophages.  analysis of clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kdaps is expressed exclusively in s. epidermidis but not in other coagulase-negative staphylococcal species. tn917-insertion in various locations in icaadbc in mutants m m m and m of s. epidermidis are abolished for pia synthesis, while 20-kdaps expression appears unaltered as compared to wild-type strains using specific anti-pia and anti-20-kdaps antisera. while periodate oxidation and dispersin b treatments abolish immuno-reactivity and intercellular adhesive properties of pia, no abrogative activity is exerted towards 20-kdaps immunochemical reactivity following these treatments. pia polysaccharide i-containing fractions eluting from q-sepharose were devoid of detectable 20-kdaps using specific elisa. preincubation of non-20-kdaps-producing clinical strain with increasing amounts of 20-kdaps inhibits endocytosis by human macrophages, whereas, preincubation of 20-kdaps-producing strain atcc with 20-kdaps antiserum enhances bacterial endocytosis by human macrophages.  in conclusion, icaadbc is not involved in 20-kdaps synthesis, while the chemical and chromatographic properties of pia and 20-kdaps are distinct. 20-kdaps exhibits anti-phagocytic properties, whereas, 20-kdaps antiserum may have a beneficial effect on combating infection by 20-kdaps-producing s. epidermidis. 