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1 Department of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
2 Department of Medical Microbiology, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Correspondence
Bastiaan P. Krom
b.p.krom{at}med.umcg.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Additional tables of zeta potential distributions, and a figure showing that sonication had no effect on zeta potential distributions, are provided as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Several traits of E. faecalis are described as virulence factors (reviewed by Kayaoglu & Østavik, 2004), and adhesion to biotic and abiotic surfaces is one of them. E. faecalis is one of the predominant organisms found in biofilms in clogged biliary stents, used to palliate obstructions of the biliary and pancreatic ducts (Dowidar et al., 1991). E. faecalis can translocate from the duodenum to colonize the stent (Di Rosa et al., 1999), where it encounters a high concentration of bile.
Factors specific for E. faecalis involved in adhesion include adhesins (e.g. the enterococcal surface protein, Esp) and aggregation substances (Aggs) (reviewed by Kayaoglu & Østavik, 2004). Esp was found to be enriched in infection-derived enterococcal strains (Shankar et al., 1999) and it is associated with enhanced adhesion to abiotic surfaces and biofilm formation by E. faecalis (Toledo-Arana et al., 2001; Tendolkar et al., 2004). Aggregation substances (Aggs) are plasmid-encoded surface proteins involved in conjugation, although a gene encoding an Agg was identified on a pathogenicity island of E. faecalis (Shankar et al., 2001a). Several different Aggs have been identified, and all are expressed in response to pheromones, resulting in formation of aggregates of donor and recipient cells and transfer of plasmids (Clewell, 1993). Waar et al. (2002c) found that E. faecalis strains expressing Esp and Agg adhered better to hydrophobic bile drain materials.
The favourable interactions between specific adhesion sites are confined to localized sites on the cell surface. The interactions originating from the entire cell body are usually referred to as non-specific interactions, although both specific and non-specific interactions are mediated by the same fundamental physico-chemical forces (Van Oss, 2003), including Lifshitz–Van der Waals, electrostatic and acid–base interactions (Van Loosdrecht et al., 1987). In general, investigation of bacterial cell surface properties determines average properties of an entire culture rather than distinguishing between individual cells. Specific interactions are short-ranged, but strong as compared with the non-specific interactions (Van Oss, 2003). Hence the latter are responsible for initial, reversible adhesion. Since all surfaces occurring in nature carry a net negative charge, electrostatic interactions in bacterial adhesion are nearly always repulsive and have to be overcome by attractive Lifshitz–Van der Waals, hydrophobic and specific interaction forces. E. faecalis strains grown in the presence of ox bile, for instance, were generally more hydrophobic and more negatively charged than after growth in the absence of bile, and accordingly adhered better to abiotic surfaces (Waar et al., 2002b). In the measurement of bacterial cell surface charge or hydrophobicity, or any other property, bacterial cultures are usually considered as populations of identical organisms, although it is known that several strains display distinct subpopulations even in pure cultures. In these cultures, the distribution of properties of the cells is more heterogeneous than normally assumed, which goes beyond differences in, for example, cell size. Different subpopulations within one culture can differ in flagellation (Streger et al., 2002), natural competence (Dubnau, 1991), autofluorescence (Kell et al., 1991), or cell surface charge (Cowan et al., 1992).
With microelectrophoresis it is possible to study individual cells with respect to their surface charge or zeta potential (Glynn et al., 1998; Van der Mei & Busscher, 2001; Wilson et al., 2001). The zeta potential is correlated with the charge on the bacterial cell surface by the nature and number of ionizable groups exposed on the surface and depends amongst other factors on pH and ionic strength. In the case of a heterogeneous population, the zeta potential distribution measured may either be an extremely wide, usually Gaussian distribution or display separate Gaussian distributions (Geertsema-Doornbusch et al., 1994; Noordmans et al., 1993; Streger et al., 2002; Glynn et al., 1998). Often fresh clinical isolates display greater culture heterogeneity than laboratory strains kept in stock for many years.
In the current study, E. faecalis strains isolated from clogged biliary stents were investigated for the presence of factors involved in adhesion to abiotic surfaces. It was found that four of six isolates were heterogeneous with respect to zeta potential and that this heterogeneity was not related to the presence of esp or Agg, to quorum sensing, or to growth medium and growth phase. Furthermore, the data suggested that culture heterogeneity in zeta potential enhances adhesion to an abiotic surface. Analysis of the zeta potential distribution of 46 clinical isolates indicated that culture heterogeneity in zeta potential is a common phenomenon and might serve an important role during virulence and pathogenesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The enterococcal laboratory strains used in this study were: E. faecalis JH2-2 (Jacob & Hobbs, 1974), OG1X (Ike et al., 1983), OG1X(pAD1) (Clewell et al., 1982), OG1XE(pAD1) (Muscholl et al., 1993), OG1X(pAM373) (Clewell et al., 1985), FA2-2 (Clewell et al., 1982), FA2-2(pAD1) (Chow et al., 1993), MMH594 (Shankar et al., 1999), MMH594b (Shankar et al., 2001b), OG1RF (Dunny et al., 1978) and OG1RF(pCF10) (Dunny et al., 1981). All isolates were cultured from frozen stock on blood agar plates. Precultures were grown in 3 ml brain heart infusion (BHI, Oxoid) overnight aerobically at 37 °C. Cultures were grown from precultures in 200 ml BHI with or without 50 mg ml–1 ox bile (Merck) overnight at 37 °C.
Genetic analysis.
E. faecalis strains isolated from clogged biliary stents were evaluated for genetic relatedness using PFGE and random amplified polymorphic DNA (RAPD). PFGE was performed as described by Murray et al. (1990). Chromosomal DNA was digested with SmaI. RAPD was performed as described by Barbier et al. (1996) using the following primers: ERIC-1, 5'-ATGTAAGCTCCTGGGGATTCAC-3'; ERIC-2, 5'-AAGTAAGTGACTGGGGTGAGCG-3'; 23SR, 5'-GGTACCTTAGATGTTTCAGTTC-3'; and 16SR, 5'-TTGTACACACCGCCCGTCA-3'.
Detection of esp and Agg.
The presence of the esp gene in the E. faecalis strains isolated from clogged biliary stents was determined by PCR amplification using the primers esp11 and esp12 as described by Shankar et al. (1999).
The presence of Aggs was detected in a clumping assay as described elsewhere (Dunny et al., 1978) with some modifications. The supernatant of an overnight culture of the pheromone-producing strain E. faecalis JH2-2 (Jacob & Hobbs, 1974) grown in BHI was autoclaved and used as a source of pheromones. A 2 ml volume of fresh BHI was mixed with 0·5 ml of the supernatant, and 25 µl of overnight cultured enterococci was added. The mixture was grown for 4 h at 37 °C in a rotating device at 65 r.p.m. and examined for the formation of aggregates.
Physico-chemical cell surface properties.
The hydrophobicity of the E. faecalis strains isolated from clogged biliary stents were assessed by water contact angle measurements by employing the sessile drop technique on bacteria deposited on membrane filters. Briefly, the bacteria from a 200 ml overnight culture were harvested by centrifugation (6500 g, 5 min, 10 °C), washed twice and resuspended in demineralized water, and deposited on 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 dried for 30 min in order to measure plateau water contact angles. Measurements were performed in triplicate with separately cultured bacteria, with six water droplets measured on each filter.
Zeta potential distributions of all strains were determined by microelectrophoresis. The strains were grown overnight, harvested by centrifugation (6500 g, 5 min, 10 °C) and washed twice with 10 mM potassium phosphate pH 7·0. Zeta potential distributions were measured in 10 mM potassium phosphate pH 2, 3, 4, 5, 7 and 9 with a Lazer Zee Meter 501 (PenKem), equipped with image analysis options for zeta sizing. Briefly, the microelectrophoresis chamber was filled with a bacterial suspension with a density of 107–108 cells ml–1 and a voltage difference of 150 V was applied over the chamber. The velocity of each individual bacterium was determined by image sequence analysis and from this its zeta potential was calculated, assuming that the Helmholtz–Smoluchowski equation held. Zeta potential distributions were measured in triplicate with separately cultured bacteria.
Stability of the subpopulations
Serial passaging.
Four biliary stent isolates were serially passaged up to 50 times in liquid medium to test the stability of the subpopulations. Passaging of the strains consisted of a daily repetitive inoculation of 100 µl of an overnight 3 ml culture in 3 ml fresh BHI for a total of 50 days (50x culture). The 50x cultures were stored in 7 % dimethyl sulfoxide at –80 °C and their zeta potential distributions were measured as described above.
Anion-exchange chromatography.
To enrich a bacterial culture for one subpopulation, a heterogeneous biliary stent isolate was passed through an anion-exchange column. The DEAE-Sephadex resin (Pharmacia) was preswollen in sterile 10 mM potassium phosphate pH 7·0 and packed in a column (5·5x1·5 cm i.d.). The column was equilibrated with sterile 10 mM potassium phosphate pH 7·0 and checked for sterility by addition of column material to fresh BHI; no growth appeared. A bacterial suspension in 30 ml 10 mM potassium phosphate pH 7·0 of OD600 1·0 was applied to the column, allowed to pass through by gravity and collected in 5 ml fractions. A 100 µl sample of each fraction was inoculated on a blood agar plate, after which the remainder of the fractions was added to 200 ml fresh BHI and grown overnight at 37 °C. The zeta potential distributions were determined from the resulting cultures.
Quorum sensing.
In order to analyse the effect of quorum sensing on heterogeneity, E. faecalis BS385 was grown in fresh and spent BHI. The supernatant of an overnight culture in BHI was passed through a 0·22 µm pore-size syringe filter (Millipore) to yield a sterile conditioned medium that was subsequently added to fresh BHI to produce a final concentration of 50 % fresh and 50 % conditioned BHI (spent medium). The fresh BHI was twice concentrated to compensate for the loss of nutrients in the conditioned BHI. During the growth, 5 ml aliquots were taken, cells washed twice with 10 mM potassium phosphate pH 7·0 and the zeta potential distribution determined.
Effect of incubation in the presence of ox bile.
Incubation in the presence of ox bile was performed with bacteria grown in BHI. The cells were incubated in 10 mM potassium phosphate pH 7·0 with or without 50 mg ox bile ml–1 for 24 h at 37 °C and 4 °C. After washing the bacteria with 10 mM potassium phosphate pH 7·0, the zeta potential distributions and water contact angles were measured, as described above.
Bacterial adhesion.
A parallel-plate flow chamber (175x17x0·75 mm) was used to monitor bacterial adhesion as described previously (Waar et al., 2002c). Images were taken from the glass bottom plate. Bacteria from a 200 ml overnight culture in BHI were harvested by centrifugation (6500 g, 5 min, 10 °C) and washed twice and resuspended in 10 mM potassium phosphate pH 7·0, to a concentration of 3x108 cells ml–1. The bacterial suspension was allowed to flow through the system at a flow rate of 0·02 ml s–1 (shear rate 10 s–1) for 4 h with recirculation at room temperature. The adhesion to a negatively charged glass surface of a homogeneous strain, a heterogeneous strain and its enriched subpopulation obtained by anion-exchange chromatography were compared.
The initial increase in number of adhering bacteria over time was expressed as the initial deposition rate j0, while the number of bacteria adhering after 4 h, n4h, was taken as an estimate of microbial adhesion in a more advanced state of the process. All adhesion experiments were performed in triplicate with separately cultured bacteria.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The expression of Aggs was determined with a clumping assay. Only E. faecalis BS12297 reacted to pheromones produced by E. faecalis JH2-2 and formed large aggregates indicating the expression of an Agg (Table 1). Interestingly, although PFGE and RAPD revealed that E. faecalis BS4126 and BS12297 showed close genetic relatedness, BS4126 did not aggregate, in contrast to BS12297, indicating that these two strains at least differ in the presence of an Agg in BS12297. Therefore, both E. faecalis BS4126 and BS12297 were included in this study.
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. Five out of the six isolates can be classified as hydrophilic, while BS937 possessed an intermediate hydrophobicity, with a water contact angle of 41 degrees.
Zeta potential distributions
The pH-dependent zeta potential distributions of the six E. faecalis cultures are presented in Fig. 1. Zeta potentials were negative for all strains over the entire pH range and four out of the six strains showed bimodal zeta potential distributions. Cultures of strains BS4126, BS12297, BS385 and BS1037 were heterogeneous with respect to their zeta potentials and at physiological pH value; these strains displayed two different zeta potentials.
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Because one of the isolates was shown to possess an agg gene, the effect of Agg expression on the zeta potential distribution was analysed. When strain BS12297 was grown in the absence or presence of pheromone the zeta potential distribution was comparable. Identical results were obtained using E. faecalis OG1RF and OG1RF(pCF10), OG1X, OG1X(pAD1) and OG1X(pAM373), and FA2-2 and FA2-2(pAD1), indicating that Agg expression does not influence zeta potential distribution (see supplementary Table S1, which lists zeta potential distributions of enterococcal laboratory strains grown in the absence or presence of pheromone).
Stability of heterogeneous subpopulations
Serial passaging.
To investigate whether culture heterogeneity was a stable trait, three heterogeneous strains and one homogeneous strain were serially passaged up to 50 times. Fig. 2 shows that the distributions of bacteria with a more or less negative zeta potential did not change significantly for the heterogeneous strains BS385, BS4126 and BS1037. The homogeneous strain BS937 did not change by serial passaging and continued to display a single population.
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). Whereas the more negative subpopulation was in the minority prior to anion-exchange chromatography, this subpopulation presented a clear majority after anion-exchange chromatography. The fractions from the anion-exchange column were plated and individual colonies were subsequently cultured. However, no homogeneous cultures could be grown from up to 30 single colonies and the zeta potential distribution of all colonies invariably returned to the original distribution.
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Growth and incubation in the presence of ox bile.
The maximum growth rates of the E. faecalis strains grown in the presence of ox bile were reduced by a factor of 1·7 (BS11297) to 1·1 (BS385) compared to the maximum growth rates of the strains grown without ox bile (Table 1). The total numbers of bacteria in the stationary phase were lower when bacteria were grown in the presence of ox bile. Three strains became slightly more hydrophobic when grown in the presence of ox bile, while the other three strains became more hydrophilic (see Table 1). After growth in the presence of ox bile, however, culture heterogeneity disappeared and all strains displayed one population (see Table 2).
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Bacterial adhesion
Table 3 compares the adhesion to a glass surface of the heterogeneous strain E. faecalis BS385, its subpopulation enriched in the more negatively charged bacteria and homogeneous strain E. faecalis BS11297. Although the initial deposition rate was highest for the homogeneous strain, 16 times less bacteria adhered after 4 h from the homogeneous strain than from the heterogeneous strain. The initial deposition rate and the number of adhering bacteria after 4 h were both significantly higher for the heterogeneous strain than for the more negatively charged subpopulation.
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). Frequently used E. faecalis laboratory strains also showed heterogeneity in zeta potential (see Table 4). Thus, culture heterogeneity appears to be a common trait among E. faecalis strains, irrespective of the presence of esp or Agg (see also Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Simple exposure by incubation in ox bile without growth did not affect the hydrophobicity or the zeta potential distribution. This shows that the changes in surface properties after growth in the presence of ox bile are not caused by adsorption of bile salts to the cell surface, but by a change in physiology of the bacteria in response to bile.
Five of the six E. faecalis strains isolated from clogged biliary stents were esp-positive, which is in accordance with a previous study where it was shown that esp was significantly enriched in infection-derived enterococcal strains (Shankar et al., 1999). There was no correlation between the presence of esp and heterogeneity in zeta potential, which is confirmed by the fact that the zeta potential distribution of MMH594 (Esp+) and MMH594b (Esp–) is not significantly different. Donelli et al. (2004) found that Agg might be one of the most important causative factors in the clogging of biliary stents. Six out of twelve E. faecalis isolated from clogged biliary stents were Agg-positive (Donelli et al., 2004). Expression of Agg enhanced the adhesion of E. faecalis strains to bile drain materials through positive cooperativity (Waar et al., 2002c) and it has been shown that Agg is expressed in vivo (Hirt et al., 2002). However, in the present study, only one of the six isolates reacted to pheromones indicating the presence of an Agg, and no correlation between the presence or absence of Agg and heterogeneity in zeta potential was present.
Culture heterogeneity in zeta potential is seen for bacteria isolated from different environments, ranging from the human oral cavity (Cowan et al., 1992) to freshwater reservoirs (Streger et al., 2002). The results presented here indicate that heterogeneity in zeta potential is a common trait of E. faecalis and independent of the origin of the isolate (see Table 4). Two discrete electrophoretic mobilities in a pure culture of E. faecalis have been previously found (Ebersole & McCormick, 1993) and were suggested to be due to differences in chain assemblage, i.e. single cells and dimers versus longer chains. However, in the present study the cell suspensions were not routinely sonicated to break chains into single cells or dimers, because experiments had indicated that sonication had no effect on the zeta potential distributions (see supplementary Fig. S1 for the effect of sonication on the chain length and supplementary Table S3 for the effect of sonication on the zeta potential distributions). Therefore we conclude that culture heterogeneity is due to intrapopulation variance in cell surface charge and not due to possible variations in chain length.
It was possible to enrich one subpopulation using affinity chromatography. The zeta potential distributions of single colonies from the enriched subpopulation invariably returned to the original distribution after growth in liquid medium, indicating that the difference in zeta potential is not caused by a mutation in part of the culture. It is our hypothesis that differential gene expression during culture is the basis for the observed heterogeneity, in analogy to the development of competence in Bacillus subtilis (reviewed by Dubnau, 1991; Hamoen et al., 2003). It is interesting that only one-third of the faeces-derived isolates showed heterogeneity, while two-thirds of either blood- or stent-derived isolates that had been involved in infection showed culture heterogeneity in their zeta potentials. Further studies are necessary to establish the putative role of culture heterogeneity in the pathogenicity of E. faecalis.
An important enterococcal virulence factor is adhesion. In this study we found that heterogeneous strains adhered significantly better to glass surfaces than homogeneous strains. Note that glass was used, as it has a negative surface charge that is comparable to polyethylene (a biliary stent material) (–75 mV and –70 mV, respectively, in 1 mM NaCl pH 7·0: Voigt et al., 1983), and due to its transparency it is readily applicable in the parallel-plate flow chamber. In addition, heterogeneous strains present two different surface charges to their environment, allowing them to adhere to surfaces with different surface properties, thus increasing their chances of successful colonization of different biomedical surfaces.
In conclusion, two-thirds of the biliary stent isolates showed culture heterogeneity in cell surface charge, which is a common phenomenon among E. faecalis strains. Culture heterogeneity offers the organism an advantage in adhesion to a negatively charged surface. Interestingly, two-thirds of the pathogenic isolates of E. faecalis examined showed culture heterogeneity in zeta potential, while this was true for only one-third of the faecal isolates. Future research will focus on identification of processes regulating the observed heterogeneity in zeta potential and further investigate the role of this phenomenon in virulence and pathogenicity.
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ACKNOWLEDGEMENTS |
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Received 23 August 2005;
revised 16 November 2005;
accepted 16 November 2005.
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