Adhesion to Bile Drain Materials and Physicochemical Surface Properties of Enterococcus faecalis Strains Grown in the Presence of Bile

Abstract

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.

Biliary drains play an important role in biliary surgery and drainage, but infections associated with biliary drains constitute a major complication (21). One of the predominant microbial species isolated from biliary drain devices is Enterococcus faecalis (21).

Biomaterial-centered infections are initiated by bacterial adhesion and biofilm formation on the indwelling device (e.g., bile drain) (4). Initial microbial adhesion is generally believed to depend on the physicochemical properties of the microbial and biomaterial surfaces, such as hydrophobicity or electrostatic charge (1). These physicochemical properties can be linked to the chemical composition of the bacterial cell surface as measured by X-ray photoelectron spectroscopy (XPS) (17). Some reports indicate that bile plays a role in the adhesion of bacteria. For example, the presence of bile in the gut might be beneficial, as it inhibits bacterial invasion of enterocytes and bacterial translocation through the gut wall (20). E. faecalis is resistant to the bactericidal effects of bile, and this resistance is induced by expression of a large number of stress proteins (6).

Two surface proteins of E. faecalis, the aggregation substance (Agg) and enterococcal surface protein (Esp), are reported to be associated with infections, suggesting that these proteins may increase the ability of this microorganism to adhere (9, 15). Recently, it was also shown that the presence of the esp gene was associated with the capacity of E. faecalis to form a biofilm on a polystyrene surface (16).

The aim of this study is to determine whether growth in the presence of (ox) bile of E. faecalis strains expressing the surface proteins Agg (Asa1 and Asa373) and Esp influences the numbers of bacteria adhering to different bile drain materials. Also, the influence of growth in the presence of bile on cell surface properties involved in initial adhesion (hydrophobicity, electrostatic charge, and chemical composition) was determined.

MATERIALS AND METHODS

Strains and growth conditions.

Three isogenic E. faecalis strains were used in this study: the plasmid-free strain OG1X (7); OG1X carrying the sex pheromone-responsive plasmid pAD1 [OG1XE(pAD1)] encoding Agg Asa1, with a positive regulator gene inserted which induces constitutive expression of this plasmid (11); and OG1X carrying plasmid pAM373 [OG1X(pAM373)], which expresses Asa373 (12). The three isogenic OG1X-derived strains did not contain the esp gene, as confirmed by PCR. Expression of Agg was checked using a fluorescent-antibody technique with polyclonal antibodies against Asa1 or Asa373, and the presence of the gene encoding Asa1 or Asa373 was checked with Southern blot hybridization. E. faecalis MMH594 expresses Esp but not Agg, as confirmed by immunological fluorescence microscopy. The presence or absence of these characteristics was checked at regular time intervals.

The strains from a frozen stock were streaked on blood agar plates (blood agar base CM55 supplemented with 5% sterile sheep blood; Oxoid) and grown overnight at 37°C. Several colonies were used to inoculate 3 ml of Todd-Hewitt broth (THB) (Oxoid) that was incubated at 37°C for 24 h. From this preculture, 2 ml was used to inoculate 200 ml of THB with or without 50 mg of ox bile ml⁻¹ (Bacto Oxgall; Difco). This second culture was incubated for 20 h at 37°C without shaking. Ox bile was chosen instead of human bile, because it was easier to obtain and its composition was more consistent. The powdered bile was dissolved in demineralized water and filter sterilized immediately before use. The final concentration of ox bile was similar to the physiological concentration of ox bile. E. faecalis MMH594 was grown in THB with 500 μg of gentamicin ml⁻¹. E. faecalis OG1X(pAM373) was grown in the presence of pheromone as described previously (8). Bacteria from the second culture were harvested by centrifugation at 10,000 × g for 5 min at 10°C and washed twice with demineralized water. Subsequently, bacteria were sonicated on ice twice for 10 s each to separate cell clusters, and a fraction was counted in a Bürker-Türk counting chamber. The bacteria were diluted in phosphate-buffered saline (PBS) (10 mM KPi and 0.15 M NaCl at pH 7) to a concentration of 3 × 10⁸ cells ml⁻¹.

To check the growth phase during adhesion experiments, growth curves with and without ox bile were compared. Bacteria were suspended in THB with or without 50 mg of ox bile ml⁻¹ at a concentration of 10⁶ cells ml⁻¹ and grown for 24 h at 37°C. The optical density at 540 nm was measured at regular time intervals.

Microbial cell surface characterization.

The surface properties of the E. faecalis strains were characterized after growth in THB with or without ox bile added.

For zeta potential measurements, each bacterial strain was resuspended in 50 ml of PBS to a density of approximately 10⁷ cells ml⁻¹. The electrophoretic mobility of each sample was measured at 150 V using a Lazer Zee Meter 501 (PenKem). The mobility of the bacteria under the applied voltage was converted to an apparent zeta potential using the Helmholtz-Smoluchowski equation (13). The zeta potentials were measured in triplicate with separately cultured bacteria.

Water contact angles were determined by the sessile drop technique as previously described (3). Briefly, bacteria were resuspended in demineralized water and deposited onto a 0.45-μm-pore-size filter (Millipore) using negative pressure. A lawn of approximately 50 stacked layers of bacteria was produced on the filter. The filters were left to air dry until so-called plateau water contact angles could be measured (approximately 30 min) using an automated contour monitor. For each strain, contact angles were measured in triplicate with separately cultured bacteria.

The chemical composition of the bacterial surfaces was determined by XPS. The washed pellets were transferred to stainless steel troughs, frozen in liquid nitrogen, and subsequently freeze-dried in a Leybold Heraus Combitron CM30 freeze drier. The samples were pressed into small stainless steel cups, put into the XPS chamber (S-Probe; Surface Science Instruments) and analyzed as described by Rouxhet and Genet (14). X-ray production (10 kV, 22 mA) with a spot size of 250 by 1,000 μm occurred using an aluminum anode. Scans were made of the overall spectrum in the binding energy range of 0 to 1,100 eV at low resolution (pass energy, 150 eV). The area under each peak, after Shirley background subtraction, was used to calculate peak intensities, yielding elemental surface concentration ratios for nitrogen (N), oxygen (O), and phosphorus (P) to carbon (C). Two samples from separate cultures of each strain were examined.

Substratum surfaces.

Implant-grade silicone rubber (SR) was obtained from Medin, and poly(tetrafluoroethylene-co-hexafluoropropylene) (fluoro-ethylene-propylene; FEP) was supplied by Fluorplast. For cleaning, substrata were sonicated for 3 min in a surfactant solution (2% RBS 35 in water; Omniclean), rinsed thoroughly with water, and then washed with methanol and demineralized water before use, yielding two hydrophobic surfaces with water contact angles of 115 and 108° for SR and FEP, respectively.

Parallel-plate flow chamber, image analysis, and adhesion.

The flow chamber (internal dimensions, 76 mm long by 38 mm wide by 0.6 mm high) and image analysis system have been described in detail previously (2). Images were taken from the bottom plate (58 by 38 mm) of the parallel-plate flow chamber with the material under study attached. The flow chamber was cleaned with the detergent Extran (Merck) and thoroughly rinsed with water and demineralized water. A bacterial suspension of 3 × 10⁸ cells ml⁻¹ in PBS was allowed to flow through the system at a flow rate of 1.44 ml min⁻¹ for 4 h with recirculation at room temperature, and images were taken at different time intervals and analyzed. The shear rate was 10.6 s⁻¹, which corresponds to the shear rate in a bile drain with a diameter of 2 mm at a bile production rate of 30 ml h⁻¹. The adhesion of the different E. faecalis strains after growth in the absence and presence of ox bile was compared. All adhesion experiments were performed in triplicate with separately cultured bacteria. E. faecalis MMH594 is highly resistant to gentamicin and was grown in the presence of gentamicin to keep the selective pressure. Growth in the presence of gentamicin did not influence adhesion because the ranges of values for adhesion of strain MMH594 after growth in medium with and without antibiotics were the same (data not shown).

Statistical analysis.

Data with and without growth in ox bile were compared with the Student's t test assuming normal distribution of the data. Significance was defined at P ≤ 0.05.

RESULTS

Growth in ox bile.

Growth curves showed that the E. faecalis strains reach the logarithmic growth phase after about the same time period in medium with and without bile and were harvested in early log phase. However, total numbers of bacteria were lower when bacteria were grown in the presence of ox bile.

Microbial cell surface characterization.

Table 1 shows that all E. faecalis strains became significantly more hydrophobic when grown in the presence of ox bile, except for OG1XE(pAD1) (difference not statistically significant). The zeta potentials of all E. faecalis strains in PBS are negative and become even more negative for most strains grown in the presence of ox bile (Table 1). The chemical cell surface compositions determined by XPS are also shown in Table 1. The N/C and P/C elemental surface concentration ratios decreased when the strains were grown in the presence of ox bile, while the O/C surface concentration ratios increased, except for OG1X(pAM373), which had a lower O/C surface concentration ratio in the presence of ox bile.

TABLE 1.

Water contact angles, zeta potentials, and elemental surface composition ratios as determined by XPS for E. faecalis strains expressing Agg or Esp grown in the absence or presence of ox bile

Strain	Ox bilea	Phenotype	θWb	ζc	Elemental surface compositiond
					N/C	O/C	P/C
OG1X	−	Agg− Esp−	35	−17	0.11	0.46	0.03
	+		40e	−17	0.10	0.56	0.03
OG1XE(pAD1)	−	Agg+ Esp−	47	−12	0.12	0.47	0.02
	+		48	−19e	0.12	0.49	0.01
OG1X(pAM373)	−	Agg+ Esp−	33	−14	0.13	0.58	0.03
	+		41e	−18e	0.09	0.44	0.01
MMH594	−	Agg− Esp+	33	−20	0.10	0.50	0.02
	+		37e	−28e	0.10	0.53	0.02

^aE. faecalis strains were grown in the absence (−) or presence (+) of ox bile.

^bθ_W, water contact angles (in degrees). The water contact angles were measured on three separate cultures yielding a standard deviation of less than 15%.

^cζ, zeta potentials (in millivolts). The zeta potentials were measured in PBS on three separate cultures yielding a standard deviation of less than 20%.

^dThe elemental surface compositions of carbon, oxygen, nitrogen, and phosphorus were measured on two separate cultures yielding a standard deviation of <30%.

^eSignificantly different from the value for the corresponding strain grown in the absence of ox bile (P ≤ 0.05).

Initial deposition rates and numbers of bacteria adhering in a stationary end point.

The numbers of bacteria in the images taken were transformed to numbers of bacteria adhering per unit area and fitted to an exponential curve by the least-squares method. The number of bacteria adhering at stationary end point of the adhesion process could be estimated from this exponential curve (SigmaPlot for Windows, version 5.00; SPSS Inc.). The initial increase in the number of adhering bacteria over time was expressed in an initial deposition rate. Table 2 shows that upon growth in the presence of bile, initial deposition rates of OG1X and OG1XE(pAD1) on SR became significantly higher, while on FEP, the increases for these strains were not statistically significant. After growth in ox bile, numbers of bacteria adhering at the stationary end point of adhesion were significantly higher for E. faecalis OG1X and MMH594, both on FEP and SR.

TABLE 2.

Initial deposition rate, number of bacteria at stationary end point of adhesion, and degree of positive cooperativity, as derived from radial distribution functions, involved in the adhesion of E. faecalis strains to FEP and SR^a

Strain	Ox bileb	Phenotype	j0c		n at t∞d		g(rp)e
			FEP	SR	FEP	SR	FEP	SR
OG1X	−	Agg− Esp−	1,646	839	6.1	2.6	1.4	2.3
	+		1,819	1,669f	8.2f	5.0f	1.2f	1.6f
OG1XE(pAD1)	−	Agg+ Esp−	1,703	990	14.0	11.2	3.2	2.8
	+		1,775	1,637f	14.0	11.8	2.0f	1.8f
OG1X(pAM373)	−	Agg+ Esp−	1,619	1,260	11.5	7.9	2.0	2.6
	+		1,639	1,285	9.9	6.9	2.3f	2.5
MMH594	−	Agg− Esp+	1,719	1,806	10.3	7.1	1.0	1.1
	+		1,843	1,818	22.3f	10.6f	1.0	1.1

^aData were measured for different E. faecalis strains after growth in the absence or presence of ox bile. Experiments were performed in triplicate with separate bacterial cultures and yielded a standard deviation of less than <30%.

^bE. faecalis strains were grown in the absence (−) or presence (+) of ox bile.

^cj₀, initial deposition rate (in centimeters⁻² second⁻¹).

^dn at t_∞, number of bacteria at the stationary end point of adhesion (in 10⁶ centimeters⁻²).

^eg(r_p), degree of positive cooperativity.

^fSignificantly different from the value for the corresponding strain grown in the absence of ox bile (P ≤ 0.05).

Positive cooperativity.

Positive cooperativity in microbial adhesion to surfaces is defined as the ability of one adhering organism to stimulate the adhesion of other organisms in its immediate vicinity and reflected by the spatial arrangement of adhering organisms as analyzed by radial pair distribution functions g(r) (19). When enterococci are randomly distributed over the entire substratum surface, g(r) = 1. However, if there is preferential adhesion at a given separation distance r between adhering bacteria, then g(r) > 1. Table 2 summarizes the degree of positive cooperativity. OG1XE(pAD1) adhered mostly through positive cooperative mechanisms, and its degree of positive cooperativity became significantly lower after growth in ox bile. The weak positive cooperativity of strain OG1X also became significantly lower after growth in ox bile.

DISCUSSION

The main finding of this study is that growth in the presence of ox bile stimulates the adhesion of some E. faecalis strains, as up to twofold-more bacteria adhered at the stationary end point of adhesion for strain OG1X not expressing Agg or Esp and strain MMH594 expressing Esp on its surface. These results indicate that growth in bile changes the adhesion properties of E. faecalis and thereby possibly increases its capability to adhere and infect. Analysis of the cell surface properties revealed that the strains were generally more hydrophobic and zeta potentials were more negative after growth in the presence of ox bile, concurrent with decreases in N/C and P/C elemental surface concentration ratios and an increase in O/C surface concentration ratio [except for OG1X(pAM373)].

Hydrophobic and charge interactions contribute to the initial adhesion to surfaces of most pathogens, and a relationship between hydrophobicity and infection has been described for different microorganisms (5). Different growth conditions can alter the hydrophobicity and charge of a microorganism (10). In this study, we found an increase in hydrophobicity when bacteria were grown in the presence of ox bile and a more negative zeta potential. These changes may be the effect of the expression of different surface proteins in response to bile salts present in the growth medium (6). A negative surface charge is often associated with the presence of oxygen-rich groups, whereas a more positive charge is associated with the presence of nitrogen-rich groups (17). In this respect, it is interesting that all enterococcal strains possessing a higher number of oxygen-rich groups (and a similar or slightly lower number of nitrogen-rich groups) after growth in the presence of ox bile have a more negative zeta potential. Alternatively, strain OG1X(pAM373) has a lower number of oxygen-rich groups upon growth in the presence of ox bile, but this strain gains negative charge through a relatively large loss of positively charged, nitrogen-rich groups. In oral streptococci, lower N/C elemental surface concentration ratios and higher O/C surface concentration ratios have been associated with decreased expression of proteinaceous, fibrillar structures on the surface and a loss of cell surface hydrophobicity (17). Despite the loss of nitrogen-rich surface groups upon growth in the presence of ox bile, strains did not become more hydrophilic but showed minor increases in cell surface hydrophobicity, except for E. faecalis OG1XE(pAD1), which was most hydrophobic when grown in the absence of ox bile.

In this study, we found an increase in deposition rates and/or numbers of bacteria adhering at the stationary end point of adhesion of the E. faecalis strains after growth in ox bile. This is in line with the generally higher cell surface hydrophobicity after growth in bile, as hydrophobic strains adhere preferentially to hydrophobic substrata. Evidently, the stronger electrostatic repulsion between the strains and the negatively charged substrata caused by their more negative zeta potentials is overruled by the increased cell surface hydrophobicity.

In a previous study, we reported that enterococci expressing the sex pheromone plasmid encoding Asa1 or Asa373 adhere in high numbers to FEP and SR through positive cooperative mechanisms via specific interactions between the bacteria mediated by Agg. E. faecalis strains MMH594 and OG1X did not adhere to the substratum through strong specific positive cooperativity (19). These differences in adhesion mechanism also explain the difference in reaction to growth in ox bile as reported in this study. The positive cooperativity described for the strains expressing Agg probably diminishes the influence of growth in the presence of ox bile on adhesion. However, the positive cooperativity exhibited by OG1XE(pAD1) is partly inhibited by growth in ox bile, possibly due to interference of components of the ox bile with receptors involved in the specific interaction between bacteria (Table 2). The positive cooperativity exhibited by OG1X(pAM373) is not inhibited by growth in ox bile. The difference in loss of positive cooperativity between the two strains expressing Agg can be explained by the different changes in elemental surface composition and water contact angles after growth in ox bile (Table 1). Finally, the weak positive cooperativity exhibited by OG1X was also inhibited by bile. This inhibition might be due to changes in physicochemical surface properties after growing E. faecalis OG1X in ox bile (Table 1).

Several reports described a possible association between the presence of Agg and infection. This might not be in line with our findings that growth in bile increases adhesion only for the strains not expressing Agg. However, in a previous study of the association between Agg of E. faecalis and infection in liver transplant patients, we found an association between Agg and infection only if the E. faecalis isolates were divided into different genogroups specific for liver transplant patients (18). Therefore, we think that not only the presence of surface proteins but also the environment, growth conditions, and selection of specific genogroups may contribute to virulence of E. faecalis.

In conclusion, growth in the presence of (ox) bile changes the surface properties of the E. faecalis strains and increases adhesion of the strain expressing Esp (MMH594) and the strain not expressing Esp or Agg (OG1X) but not of the strains expressing Agg (Asa1 and Asa373). The findings of this study will stimulate further research on the effect of bile on E. faecalis. Questions that need to be answered are whether and which surface proteins are regulated or modified in reaction to the presence of bile, since bile components may bind to the surface of E. faecalis.