Heparin stimulates S. aureus biofilm formation in vitro.
The widespread use of sodium heparin as a catheter-lock solution led us to test whether this compound has an impact upon the adherence of S. aureus to abiotic surfaces. S. aureus strain MZ100, a laboratory wild-type strain, was grown overnight in TSB, then diluted into TSB plus 0.2% glucose with the addition of serial dilutions of sodium heparin, and allowed to form a biofilm for 16 h in a 96-well polystyrene microtiter plate. Nonadherent cells were removed, and biofilms were stained with crystal violet. The relative amount of biofilm formation was determined by solubilizing crystal violet in acetic acid and determining optical density with a spectrophotometer.
Stimulation of bacterial adhesion to plastic by heparin was observed over a range from 0.1 unit/ml to the maximum tested dose of 1,000 units/ml (Fig. ). These are relevant levels as the concentration of heparin used in catheter lock solution is commonly up to 10,000 units/ml. Heparin, in the absence of cells did not stimulate crystal violet staining of wells (data not shown). We tested other heparin-like glycosaminoglycans and anionic polysaccharides to determine if the effect is specific to heparin and observed statistically significant biofilm stimulation with the addition of sodium heparin, ammonium heparin (P
< 0.01), and to a lesser degree heparan sulfate, chondroitin sulfate B, and dextran sulfate, but not chondroitin sulfate C (Fig. ). We also found that heparin-stimulated biofilms displayed antibiotic resistance levels indistinguishable from nonstimulated biofilms, including highly elevated levels of vancomycin resistance compared to planktonic cells (data not shown) (42
FIG. 1. Sodium heparin increases the adherence of S. aureus to polystyrene. A. Dose response. Serial dilutions of sodium heparin were added to cultures and biofilms were allowed to form for 16 h, at which time nonadherent cells were removed by vigorous washing. (more ...)
Biofilm formation in the presence and absence of heparin was assessed over time using the microtiter plate assay. A twofold increase in crystal violet staining was seen as early as 3.5 h; at 6 to 9 h approximately three times as much staining was observed (see Fig. ).
The adherence of S. aureus to silicone elastomer coupons was also determined. Silicone elastomer is a common component of catheters. At 24 h, a twofold increase in adherence to silicone elastomer coupons was recorded (0.11 ± 0.04 A550 units for saline versus 0.25 ± 0.09 A550 units for heparin, P < 0.05).
Microscopic observation of heparin-stimulated biofilms.
S. aureus biofilms were allowed to form overnight on polyvinylchloride under static conditions and then washed repeatedly and prepared for scanning electron microscopy. Scanning electron microscopy images suggest that surface coverage and the three-dimensional structure of biofilms were stimulated by heparin at 1,000 U/ml (Fig. , panels A versus B).
FIG. 2. Sodium heparin enhances S. aureus biofilm formation. The effect of heparin on the formation of S. aureus MZ100 biofilms on abiotic surfaces was assessed microscopically. Scanning electron micrographs of 12-hour-old S. aureus biofilms on polyvinylchloride (more ...)
Phase microscopy of heparin-treated biofilms formed on polystyrene similarly reveal that heparin promotes biofilm formation and that heparin-grown biofilms subject to shear forces through repeated washes with PBS appeared to be more tenacious than those formed with control medium (Fig. , panels C and D).
Epifluorescent staining of biofilms was also utilized to assess the effect of heparin on biofilms. Syto-9, a quantitative DNA-binding fluorescent dye, was used to determine relative amounts of biofilm. Figure , panels C and D, depicts digital micrographs of Syto-9-stained 4-hour biofilms taken with the same exposure time. Heparin-treated biofilms were more brightly stained and exhibited larger but not more numerous microcolonies. When relative fluorescence was measured, we reproducibly found a 2- to 10-fold increase in the heparin-treated sample. In a sample experiment taken 5 hours after the inoculation, we found a 3.5-fold increase in biofilm fluorescence in the heparin-treated sample [7.5 × 1010± 1.1 × 1010 relative fluorescent units (RFU) for the heparin-treated strain and 2.1 × 109± 1.4 × 109 RFU for the control, n ≥ 5 fields, P < 0.01].
Biofilms are often characterized by the presence of an extracellular matrix. We used calcofluor, a fluorescent dye that has been used to stain the extracellular matrix of biofilms in other species, to determine whether heparin-treated biofilms exhibit this hallmark of biofilm formation (Fig. ) (24
). At 5 hours, biofilms formed with and without heparin both consisted of monolayers with little to no calcofluor staining interspersed with large phase-bright microcolonies that stained brightly with calcofluor (Fig. ). The relative fluorescence of several fields was quantified using Open Lab software. A significant difference in brightness was observed from heparin-treated biofilms at both 5 and at 21 hours; at 5 h the average RFU/visual field was found to be 1.9 × 1010
± 3.3 × 109
for heparin-treated biofilms versus 7.4 × 109
± 5.5 × 109
for control biofilms (P
< 0.001, n
≥ 8 fields); data are not shown for 21 h. Similar results were observed for Styo-9 at 4 h.
FIG. 3. Sodium heparin enhances S. aureus biofilm formation. The effect of heparin formation of S. aureus (MZ100) biofilms on abiotic surfaces was assessed microscopically. S. aureus biofilms (4 hours) formed on polystyrene were viewed with phase 2 microscopy (more ...)
Observation of biofilms formed in the presence of heparin (Fig. ) suggests that they have more three-dimensional architecture than do biofilms grown with control medium (Fig. and data not shown) and are consistent with the microtiter plate analysis results that heparin promotes biofilm formation.
Heparin does not promote S. aureus biofilm formation by accelerating growth.
Increased growth in the presence of the polysaccharide heparin could account for the more robust biofilms we observed. To determine whether sodium heparin stimulates or impedes the growth of a S. aureus population, the optical density of MZ100 cultures grown in TSB plus 0.2% glucose in the presence of either saline or sodium heparin (1,000 U/ml) was assessed over 24 h. Sonication was used to disrupt any cell-cell interactions that could alter optical density readings. Heparin at 1,000 U/ml had no discernible impact upon the growth rate of S. aureus strain MZ100 (see Fig. ), suggesting that it does not stimulate biofilm formation by accelerating growth. The same results were found for cultures grown without the addition of 0.2% glucose (data not shown).
Sodium heparin promotes cell-cell interactions but not primary attachment.
Staphylococcal biofilm formation has been divided into two developmental stages. Primary attachment is the first stage, wherein cells form stable interactions with a surface (22
). This is followed by a growth and cell-cell interaction-dependent accumulation phase. We examined the effect of sodium heparin on each stage of S. aureus
Cells adherent to plastic were observed with phase-contrast microscopy, and the foci were counted, with a single cell or a group of associated cells counted as one focus. Heparin did not affect primary attachment at 10 or 30 min (see Table ).
Heparin does not promote primary cell surface attachment
Cell-cell interactions were assessed both in the planktonic phase and on a plastic surface. Overnight planktonic cultures were sonicated to disrupt the majority of existing cell-cell interactions and then subcultured into TSB plus 0.2% glucose with either heparin or saline added to 10% (vol/vol). Samples were placed in 24-well dishes and incubated at 37°C without shaking. Aliquots of cells were removed from the planktonic phase of nonshaking cultures, observed microscopically, and assessed for cell clustering over the course of 3 h. The frequency of cell clusters of three or more bacteria was up to fourfold greater for cells in the presence of heparin (Fig. ). By 1 hour, 41% of microscopic foci from heparin-containing cultures consisted of clusters of three or more cells, compared to only 14% foci for cells grown without heparin. By 2 hours heparin cultures exhibited 70% clustered foci compared to 19% foci in the saline control. Planktonic cells grown overnight in heparin and incubated on a rapidly rotating wheel at 37°C also exhibit elevated levels of clustered foci compared to a no-heparin control (data not shown).
FIG. 4. Heparin indirectly stimulates S. aureus cell-cell interactions. The effect of heparin on cell-cell interactions was assessed using phase-contrast microscopy with strain MZ100. Foci were counted and classified as either clusters (≥3 cells/focus) (more ...)
One potential caveat to the cell-cell interaction experiments is the possibility that the clustered foci we observed were from cells that failed to separate after division rather than derived from cells that came in contact with each other and then adhered. To address this possibility, two strains were mixed in TSB plus 0.2% glucose with heparin (1,000 U/ml) and plasmid-selective antibiotic, one with a plasmid-borne copy of dsred (which codes for a red fluorescent protein) under the control of a strong promoter and the other with a vector control. Using epifluorescent microscopy, we observed at 3 h that more than 40% of clusters of three cells or more contained both red-fluorescent and nonfluorescent cells, indicating that newly formed cell-cell interactions do occur under these conditions, in clusters of six or more cells more than 60% of clusters were mixed (n > 100, data not shown).
The cell-clustering phenotype was assessed in similar experiments on a polystyrene surface. We found that heparin mediates cell-cell adherence on the surface in a manner similar to that observed for planktonic cells (Fig. ). We also determined surface coverage by S. aureus in the presence and absence of heparin as described in Materials and Methods and found that heparin was associated with more surface coverage at 2 h (22% coverage with heparin, 11% without, P < 0.05) and 3 h (29% with heparin, 19% without, P < 0.05) but not at 30 min (2.7% with heparin, 3.6% without, P > 0.05).
Effect of a protein synthesis inhibitor on heparin-dependent cell-cell interactions.
The observed delay in elevated levels of cell-cell interactions and surface coverage in the presence of heparin raised the question of whether the effect of heparin in this system is dependent on protein synthesis. Chloramphenicol at 30 μg/ml was added to cells in the presence or absence of heparin (1,000 U/ml heparin or saline in TSB plus 0.2% glucose) to inhibit protein synthesis. It is formally possible that the kinetics of cell-cell interactions would be different in the experimental samples because cell number would not increase over time. Therefore, a double inoculum of cells (≈4 × 107 cells) was added to the samples containing chloramphenicol as a control for final cell number, because we had previously determined this was the approximate change in cell number at 3 h when samples were taken to assess cell clustering. We found that cells in which protein synthesis was inhibited by chloramphenicol did not demonstrate a heparin-dependent increase in cell-cell interactions at 180 min (Fig. ). The lack of this increase in cell-cell interactions suggests that heparin does not directly mediate cell-cell interactions.
Influence of sodium heparin on sigma B activity.
One possible mechanism for the effect of heparin in stimulating biofilm formation is that cells treated with heparin could be stressed without an obvious change in growth rate. Such stress might lead to stimulation of biofilm formation as has been demonstrated for high levels of ethanol and sodium chloride. Sigma B is a sigma factor that activates an array of genes in response to cellular stress (6
). Sigma B activity correlates with cellular stress and has been implicated in staphylococcal biofilm formation (34
A transcriptional fusion of gfp
to a sigma B-dependent promoter (asp23
) was employed to investigate whether heparin stimulates a stress response (20
). For this experiment we utilized strain SH1000, known to be wild type for sigB
). Strain MZ100 is known to have a deletion in the sigB
regulatory gene rsbU
so it is inappropriate for studying sigB
expression and activity (20
). SH1000 and an isogenic sigB
mutant strain were transformed with a plasmid-borne asp23
reporter construct (27
). Cells were grown in TSB plus 0.2% glucose with sodium heparin (1 to 1,000 U/ml) or saline. Expression of asp23
was then determined using a fluorometer. No change in asp23
transcription with respect to heparin was observed in SH1000 (3,857 RFU without heparin and 3,889 RFU with heparin at 100 U/ml, data not shown). Similarly, heparin caused no change in sigma B activity in two clinical isolates transformed with the asp23
reporter construct (data not shown). sigB
mutants of SH1000 displayed low GFP levels compared to the wild-type SH1000 (for example, at 1 U/ml of heparin, the sigB
mutant had 61 RFU and the wild type had 3,533 RFU).
A role for sigma B in heparin-dependent biofilm formation was further tested genetically. We hypothesized that if components of the sigma B regulon were responsible for increased bacterial attachment in the presence of heparin, then sigB
mutants would not display increased attachment with the addition of heparin. Consistent with another report (63
), a sigB
mutation in MZ100 did not adversely affect biofilm formation, nor did it reduce PIA levels (data not shown). However, we observed that a sigB
mutation in the SH1000 background conferred a 70 to 80% reduction in biofilm formation that was partially rescued by heparin. In one representative experiment where biofilms were allowed to form for 8 h, heparin stimulated a 92% increase in biofilm formation in the sigB
mutant and a similar increase for SH1000 (68%, P
Sodium heparin enhances biofilm formation of known biofilm mutants.
We hypothesized that if known biofilm-related factors were responsible for increased bacterial attachment in the presence of heparin, strains with mutations in these factors would not display increased attachment with the addition of heparin. To this end we tested agr
, and sarA
mutants for biofilm formation in the presence and absence of heparin using the microtiter dish assay (Fig. ). In the absence of heparin, these mutants behaved as previously described when we tested them in the MZ100 background (3
). Mutation of the gene that codes for the staphylococcal accessory regulator, sarA
), severely reduced biofilm formation (P
< 0.01) and was almost completely rescued by the addition of exogenous heparin at 1,000 U/ml to wild-type levels without heparin (P
< 0.01, Fig. ). We found that an agr
mutation in the MZ100 background confers a hyperbiofilm phenotype that was further enhanced by heparin (P
< 0.01). Mutation of the hla
gene, which codes for alpha-toxin, confers a biofilm-defective phenotype (P
< 0.01) in MZ100 that was completely rescued by heparin (P
FIG. 5. Effect of heparin on known biofilm formation mutants. A. Mutants (gene names noted) were assessed for biofilm formation in the microtiter dish assay (8-h biofilms) with heparin at 1,000 U/ml (shaded bars) or with saline (white bars). B. Relative PIA levels (more ...)
In addition to the microtiter dish assay we directly observed 3- to 5-hour biofilms with phase microscopy and fluorescent microscopy. To quantify the microscopic data we stained cells with Syto-9, a quantitative fluorescent stain that binds to DNA, and measured the relative fluorescence of several fields of view for each strain. These results closely mirror these found with the microtiter plate assay (data not shown).
has adhesins on its surface that enable it to bind to a number of proteins, such as fibrinogen and fibronectin. These adhesins may have a role in heparin-stimulated biofilm formation. We tested whether some of these surface adhesins are necessary for heparin-dependent biofilm stimulation after 24 h of incubation. ClfA and ClfB are surface molecules that help S. aureus
bind to fibrinogen (40
). We determined whether clfA clfB
double mutants were stimulated in biofilm formation by heparin using the microtiter dish assay. In the 8325-4 background, the wild type had an A550
reading at 24 h of 0.26 ± 0.017 with saline and 2.67 ± 0.225 with heparin, and the clfA clfB
double mutant was also stimulated by heparin (0.15 ± 0.21 with saline and 1.58 ± 0.18 with heparin). We observed a similar pattern with a clfA clfB
double mutant in the Newman strain background (0.16 ± 0.22 with saline and 1.34 ± 0.22 with heparin).
binds to fibronectin via the large surface proteins FnbA and FnbB (18
). We found that fnbA
mutants were stimulated in adherence by heparin. SH1000 strains with fnbA
mutations exhibited an increase in A550
reading from 0.18 ± 0.02 with saline to 2.16 ± 0.2 with heparin for fnbA
and from 0.2 ± 0.03 in saline to 1.4 ± 0.15 with heparin for fnbB
Heparin stimulates biofilms independently of polysaccharide intercellular adhesin.
Polysaccharide intercellular adhesin is produced by the icaADBC
gene products and is a well-characterized factor involved in staphylococcal biofilm formation (22
). Staphylococcal strains with ica
mutations are reported to form poor biofilms in which primary attachment is unaffected but later stages of biofilm formation are impeded (39
). In addition, cultures of S. aureus
containing episomal copies of the ica
locus exhibit enhanced biofilm formation, suggesting that elevated levels of PIA are sufficient to stimulate biofilm formation (12
). Therefore, we tested whether heparin enhances biofilm formation through increased PIA levels.
Levels of PIA from cells grown to the stationary phase in the presence or absence of heparin (1,000 U/ml) were assayed biochemically (Fig. ). PIA antibodies were used to probe a dilution series of cell lysates. An isogenic ica deletion strain was generated for use as a negative control. PIA levels were indistinguishable for cells grown in the presence and absence of heparin (Fig. ).
The dependence of biofilm formation on PIA was also tested genetically. Biofilm formation of isogenic ica
Δ and wild-type strains was tested in the presence and absence of heparin. We found that deletion of the ica
operon in MZ100 conferred no significant reduction in biofilm formation (P
= 0.43) as has been reported in some strain backgrounds (4
) (Fig. ). Heparin stimulated biofilm formation by both MZ100 and its ica
derivative. Therefore, we performed the analysis in strain Sa113, where the ica
locus has been shown to be important in biofilm formation (12
Δ mutants in the Sa113 background were significantly reduced in biofilm formation (P
< 0.01) and were completely rescued by heparin (Fig. ). The observations that an ica
Δ mutant strain can form robust biofilms in the presence of heparin and that PIA levels are not altered are consistent with heparin stimulating biofilm formation independently of PIA.
Heparin affects biofilms formation of other S. aureus strains and other staphylococcal species.
Since there can be major phenotypic differences between S. aureus strains, the effect of heparin on several strains was tested. We found that heparin stimulated biofilm formation in seven of seven laboratory strains tested (Col, Newman, MZ100, RN4220, SH1000, 8325-4, and Sa113; the last five are closely related). The increase in biofilm formed in the presence of heparin versus an equal volume of saline ranged from 80 to 800% when assessed by the 96-well plate biofilm assay (data not shown). Eight out of eight S. aureus clinical isolates also exhibited increased biofilm formation in the presence of heparin (data not shown).
is a source of urinary tract infections. Heparin had no discernible effect upon biofilm formation in the one strain of S. saprophyticus
tested. One of three Staphylococcus epidermidis
strains tested (strain O-47) (25
) had a biofilm more than 200% greater with heparin than with saline when assessed by the microtiter plate biofilm assay. Strain ATCC R97-03 exhibited less than half of the biofilm formed in saline, and ATCC R94-10 had a slight reduction in biofilm formation in the presence of heparin (data not shown).