Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands1
Division of Aquatical Microbiology, Faculty of Chemistry, Gerhard Mercator-University of Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany2
Author for correspondence: Henny C. van der Mei. Tel: +31 50 3633140. Fax: +31 50 3633159. e-mail: h.c.van.der.mei{at}med.rug.nl
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ABSTRACT |
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Keywords: proteins, polysaccharides, bacterial adhesion, footprints, atomic force microscopy
Abbreviations: ADSA-P, axisymmetric drop shape analysis by profile; AFM, atomic force microscopy; DDS, dimethyldichlorosilane; EPS, extracellular polymeric substance(s); XPS, X-ray photoelectron spectroscopy
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INTRODUCTION |
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More than 90% of the EPS volume consists of water (Schmitt & Flemming, 1999 ; Sutherland, 1997
), located in pores and with a minor portion bound to the EPS molecules (Schmitt et al., 1997
; Christensen & Characklis, 1990
). EPS has long been considered to consist only of polysaccharides, but recently considerable amounts of proteins, as well as humic substances, nucleic acids and lipids (Cooksey, 1992
; Nielsen et al., 1997
; Flemming & Wingender, 2001
) have been identified as EPS constituents. However, differences in chemical composition as well as in function of the different kinds of EPS have been reported (Omar et al., 1983
; Beech et al., 1999
).
A role of EPS in initial adhesion of Pseudomonas to surfaces has not yet been established. However, it has been found that attachment to an inert substratum stimulates bacterial EPS synthesis (Vandevivere & Kirchman, 1993 ; Allison & Sutherland, 1987
). EPS components, like polysaccharides (Fletcher & Floodgate, 1973
; Costerton et al., 1985
; Azeredo et al., 1999
; Azeredo & Oliveira, 2000
) and proteins (Danielsson et al., 1977
; Dûfrene et al., 1996b
), enhance bacterial adhesion, while lipopolysaccharides (Williams & Fletcher, 1996
), uronic acids (Pringle et al., 1983
) and biosurfactants (Velraeds et al., 1998a
; Van Hoogmoed et al., 2000
; Heinemann et al., 2000
) discourage adhesion.
In most natural environments, cyclic detachment and redeposition of organisms occurs more readily than a single round of deposition and adhesion. Passing airliquid interfaces have been demonstrated to yield extremely efficient detachment forces, as in the oral cavity during eating, speaking, drinking and swallowing (Busscher et al., 1992 ), on the eye and on contact lenses caused by blinking (Landa et al., 1998
), on rocks and ship hulls in marine environments (Cooksey & Wigglesworth-Cooksey, 1995
) and in biodeterioration of monumental buildings by falling rain drops (Urzi et al., 1991
). Detached organisms may leave footprints (Neu & Marshall, 1991
) behind, that either consist of cell surface fragments left behind (Neu, 1992
), excreted biosurfactants (Velraeds et al., 1998b
) or EPS. These footprints form a new surface in the redeposition of a new generation of depositing organisms.
The aim of the present study is to investigate the role of EPS in the initial adhesion of Pseudomonas aeruginosa to substratum surfaces with different hydrophobicities. To this end, the deposition and redeposition after detachment by the passage of an airliquid interface of an EPS-producing and a putatively EPS-non-producing strain of P. aeruginosa was studied in a parallel plate flow chamber.
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METHODS |
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EPS production and isolation.
Strains were examined for EPS production using axisymmetric drop shape analysis by profile (ADSA-P; Cheng et al., 1990 ) through effects of EPS production on the surface tensions of bacterial suspensions. Droplets (0·1 ml) containing 1x109 cells of both strains ml-1, suspended in PBS (pH 7·0), were placed on a clean piece of fluoroethylenepropylene and the circumference of the suspension droplet was monitored during 2 h in an enclosed chamber at room temperature. Droplet circumferences were recorded twice with a minimal time interval (<0·5 s) between measurements and fitted to the Laplace equation of capillarity, yielding the surface tension of the bacterial suspensions. EPS production was inferred from a decrease in surface tension of the bacterial suspension.
EPS-producing P. aeruginosa SG81 cells from a 250 ml culture (containing approximately 2·5x1011 cells) were washed three times with 60 ml Millipore-Q water and the supernatants containing the EPS were collected. The supernatants were centrifuged again (5 min, 10000 g), decanted and the EPS-containing supernatant was dialysed for 24 h against Millipore-Q water, using a cellulose membrane with a 14 kDa cut-off (Spectrum). EPS (150 ml) isolated in this manner was mixed with 150 ml PBS for substratum coating in deposition assays.
Physico-chemical characterization of bacterial cell surfaces.
Water contact angles (w) on lawns of both P. aeruginosa strains were determined using the sessile drop technique (Van Oss & Gillman, 1972
; Busscher et al., 1984
). Briefly, bacterial cells were layered from Millipore-Q water onto 0·45 µm pore filters (Millipore) using negative pressure. The filters were left to air dry in ambient air until so-called plateau contact angles could be measured.
Electrophoretic mobilities of P. aeruginosa strains were measured in PBS supplemented with 5% NB medium with a Lazer Zee Meter 501 (PenKem), which uses scattering of incident laser light to enable detection of bacteria at relatively low magnifications. Aliquots of the bacterial suspensions were diluted to a density of approximately 1x108 cells ml-1 in PBS (pH 7·0) prior to the measurements. The voltage difference across the electrophoresis chamber was set to 150 V and the electrophoretic mobilities measured were converted to apparent zeta potentials () through the use of the Von Smoluchowski equation (Hiemenz, 1977
).
X-ray photoelectron spectroscopy (XPS) was applied to obtain the overall chemical composition of the microbial cells. After culturing and washing, the bacterial pellets were transferred to stainless steel troughs and frozen in liquid nitrogen. The samples were subsequently freeze-dried and the resulting bacterial powder was pressed in small stainless steel cups and inserted into the XPS chamber (S-Probe; Surface Science Instruments). X-rays (10 kV, 22 mA) at a spot size of 250x1000 µm were produced using an aluminium anode. A scan of the overall spectrum in the binding energy range of 11200 eV at low resolution (pass energy 150 V) was recorded, followed by scans over a 20 eV binding energy range at high resolution (pass energy 50 eV) for C1s (carbon), O1s (oxygen), N1s (nitrogen) and P2p (phosphorus). The area under a peak, after linear background subtraction, was used to calculate peak intensities, yielding elemental surface concentration ratios for nitrogen, oxygen and phosphorus to carbon, after correction with sensitivity factors provided by the manufacturer. Bacterial cell surface characterizations were carried out in triplicate on three different bacterial cultures.
Substratum surfaces.
Glass microscope slides (76x25x1·5 mm) were used as substratum surfaces. Glass surfaces were cleaned with 2% (w/v) RBS 35 detergent (Omnilabo International) and alternately rinsed in water, methanol, water again and finally demineralized water. Cleaned glass plates were used as a negatively charged, hydrophilic substratum surface, while hydrophobic, negatively charged surfaces were prepared from cleaned glass by first drying in an oven at 80 °C, followed by silanization in 0·05% (w/v) dimethyldichlorosilane (DDS) in trichloroethylene. After silanization for 15 min, the surfaces were rinsed with trichloroethylene, methanol and Millipore-Q water.
Physico-chemical characterization of substratum surfaces.
Water contact angles (w) of glass and DDS-coated glass, as well as of EPS-coated glass were measured by the sessile drop technique, as described above. Zeta potentials (
) of bare glass and DDS-coated glass surfaces in PBS (pH 7·0) were measured by streaming potentials in a parallel plate flow chamber (Van Wagenen & Andrade, 1980
).
In redeposition experiments, the chemical composition of the substrata after air-bubble-induced detachment of adhering bacteria was determined by XPS after the first and second deposition cycle to detect potential footprints. However, since both glass surfaces and EPS are rich in oxygen, complicating the interpretation of XPS data, redeposition experiments were also carried out on gold-coated glass slides to uniquely identify potential EPS material left behind after bacterial detachment. In addition, potential footprints on glass were sought by atomic force microscopy (AFM). AFM images and forcedistance measurements were taken under water at room temperature using an optical lever microscope (Nanoscope IIIa Dimersion 3100; Digital Instruments). Contact mode topographic images were recorded in both height and deflection modes. Height images provided quantitative topographic information from which the mean roughness (Ra) of the surfaces was calculated, while deflection images revealed finer surface details. The imaging force was kept below 1 nN and the scan rate was in the range of 24 Hz. A Si3N4 cantilever tip of 0·06 N m-1 spring constant was used.
Parallel plate flow chamber and image analysis.
Adhesion experiments were carried out in a parallel plate flow chamber containing three basic modules: a flow loop, a syringe pump and image analysis components (Gómez-Suárez et al., 1999 ). The chamber is made of poly(methyl methacrylate) with channel dimensions 76x38x0·6 mm (lxwxh) for deposition assays. For detachment and redeposition experiments the channel of the flow chamber was modified (76x5x0·6mm, lxwxh) to control the passage and velocity of the air bubbles introduced. The top plate was made out of glass and separated from the bottom plate by two Teflon spacers. The bottom plate was made of glass or DDS-coated glass, depending of the substratum under study. A pulse-free liquid flow was achieved by hydrostatic pressure and recirculated by a roller pump. The volumetric flow was 0·025 ml s-1, yielding to a laminar flow (Reynolds number 0·6).
The entire flow chamber was mounted on the stage of a phase-contrast microscope (Olympus BH-2) equipped with a x40 ultra-long working distance objective (Olympus ULWD-CD Plan 40 PL). A charged-coupled device camera (CCD-MXR/5010, Adimec Advanced Image Systems) was mounted on the phase-contrast microscope and connected to a PCVision+ video frame grabber interface card (Imaging Technology Inc.) to digitize the video signal. For enumeration of the number of bacteria adhering to the bottom plate before and after the passage of an air bubble, a cell-finder slide (76x26 mm) attached to the outside of the bottom plate was focused for accurate repositioning (±2 µm) of the flow chamber in the enumeration process.
Experimental protocol.
All experiments were performed at room temperature in triplicate with suspensions of separate bacterial cultures and newly prepared collector surfaces. Before each experiment, all tubes, flow chamber and syringe pump were filled with PBS and care was taken to remove all air bubbles from the system. The PBS solution was first perfused through the chamber for 30 min. At this stage and when appropriate, the flow was switched to PBS supplemented with isolated EPS for 1 h after which the flow was switched for 30 min to PBS to remove all remnants of EPS components from the tubing and the flow chamber.
In deposition experiments, after perfusion with PBS, bacteria were allowed to adhere on the different substrata up to 5 h from a suspension containing 3x108 bacteria ml-1 in PBS supplemented with 5% (v/v) NB. Initial deposition rates and numbers of adhering cells after 5 h of both P. aeruginosa strains on bare, EPS pre-coated and DDS-coated glass were determined.
In detachment experiments, 4x106 bacteria cm-2 were allowed to adhere to the substrata from PBS supplemented with 5% (v/v) NB, as enumerated by the image analysis system on ten arbitrarily chosen areas of 0·017 mm2 distributed over the length of the substratum surface. Subsequently, flow was switched for 30 min to PBS, again to remove all non-adhering bacteria from the flow chamber and the tubing system. At this stage, a 0·1±0·02 ml air bubble fully spanning the width of the flow chamber was introduced in the tubing and passed over the substratum surface at a controlled velocity of 5·6 mm s-1 with the aid of a syringe pump (Terumo SC512). The syringe pump was connected to the flow chamber by a three-way valve and filled with PBS. The number of bacteria that remained adhering after air bubble passage was enumerated after retrieving the positions used before with the aid of the cell-finder.
In redeposition experiments, P. aeruginosa was allowed to adhere to glass from a suspension with 1x109 cells ml-1 in PBS supplemented with 5% (v/v) NB medium up to 40 min. Thereafter, six air bubbles were allowed to pass over the substratum surface at 5·6 mm s-1, removing all adhering bacteria. Finally, a fresh bacterial suspension was allowed to adhere again to this substratum surface and the number of adhering bacteria enumerated. This process of detachment, deposition and adhesion was repeated for up to six cycles.
The initial increase in the number of adhering bacteria with time was expressed as an initial deposition rate (j0). This rate was calculated in deposition and redeposition experiments and represents the number of initially adhered bacterial cells per unit area and time. The number of bacterial cells detached after a passing an air bubble was expressed as the percentage of bacteria caused to detach, relative to the number of adhering bacteria prior to air bubble passage.
Data analysis.
Detachment data were analysed by a one-way analysis of variance (ANOVA). ANOVA tests the ratio of the mean squares between groups and within groups, and provides an F-ratio (i.e. Fisher distribution) and its corresponding P value. If the null hypothesis is true, the F ratio should be approximately 1, since both mean squares between and within groups should be about the same. If the ratio is much larger than one, the null hypothesis is false. The shape of the F distribution depends on the degrees of freedom within and between groups: the lower the degrees of freedom, the larger the value of F needed for significance. A P value of less than 5% was considered to indicate a significant difference between groups. The subscript of F indicates the value of F needed for a hypothesis to be significant at the 5% level. When differences among means were determined, post-hoc Dunnetts range tests and pairwise multiple comparisons were employed.
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RESULTS |
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As proteins contain negligible amounts of carbon singly bound to oxygen (Dûfrene & Rouxhet, 1996 ), the ratio of carbon due to polysaccharides, CPS, can be calculated from:
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A residual content due to hydrocarbon-like compounds, CHC, subsequently results according to:
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Based on this analysis, it can be concluded (see also Table 3) that the EPS left after detachment contains up to 68% protein. The amounts of protein (CPR) and polysaccharides (CPS) decreased significantly (F7·71=158·4, P=0·002) after the second deposition cycle for both strains, leaving significantly higher (F7·71=162·4, P=0·02) amounts of uronic acids. However, the increase in CUA was significantly higher for the EPS-producing strain SG81 than for the non-EPS-producing strain SG81R1.
Topographic height AFM images of EPS left on glass after the first air-bubble-induced detachment of adhering P. aeruginosa are shown in Fig. 3. Streaks oriented in the direction of the scan can be observed for both SG81 and SG81R1. However, those from the EPS-producing strain were more numerous. A cross-section of those streaks revealed an irregular film of thickness ranging from 3 to 32 nm for the EPS-producing SG81, and from 1·5 to 9 nm for the non-EPS-producing strain SG81R1. The mean surface roughness, Ra, of the substrata after the first deposition cycle of the EPS-producing strain increased significantly (F7·71=10·36, P=0·032), as compared to bare glass substratum (Table 4
). No significant difference was found between the roughness of glass prior to and after adhesion and detachment of the non-EPS-producing strain SG81R1, but after the second cycle of deposition and detachment, a significantly elevated (F7·71=10·3, P=0·008) mean roughness was observed for both strains.
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DISCUSSION |
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Most often and contrary to the present findings, EPS is reported to enhance bacterial adhesion to inert substrata (Costerton et al., 1985 ; Van Loosdrecht et al., 1990
). For instance, Dûfrene et al. (1996a
) found that adhesion of Azospirillum brasilense to both hydrophilic and hydrophobic substrata occurred only after stimulated EPS production and correlated it to an increase of protein content of the EPS over time. A similar increase in adhesion of Pseudomonas fluorescens associated with high protein contents in EPS has been reported by DeFlaun et al. (1990)
. Recently, Azeredo & Oliveira (2000)
found that the presence of surface-active compounds increased the adhesion of Sphingomonas paucimobilis to glass.
It is interesting that the non-EPS-producing strain, as shown in this study by a lack of a decrease in surface tension of a suspension of strain SG81R1, has cell surface characteristics similar to those of the EPS-producer SG81. Moreover, after repeated cycles of deposition and detachment, SG81R1 shows similar adhesion behaviour to SG81, and also the substratum characteristics after detachment become alike. This indicates that SG81R1 excretes minor amounts of EPS, undetectable by ADSA-P. The role of this EPS in discouraging bacterial adhesion only becomes evident after several cycles of detachment and deposition. Recently, production of a minor amount of EPS by P. aeruginosa SG81R1, growing in a biofilm, has been described by Strathmann et al. (2002) . Alternatively, Vandevivere & Kirchman (1993)
as well as Allison & Sutherland (1987)
suggested that attachment stimulates bacterial EPS synthesis, and putatively non-EPS-producing P. aeruginosa strains related to cystic fibrosis could through mutation or selection turn into EPS-producing ones during adaptation to different ecological sites, such as stress and growth conditions (Boucher et al., 2000
; Grobe et al., 1995
).
The EPS excreted by P. aeruginosa SG81 is highly surface active, which is probably due to a uronic-acid-containing polymer. Grobe et al. (1995) have analysed the chemical composition of EPS excreted by P. aeruginosa SG81 grown on Pseudomonas Isolation Agar and found that the crude extracellular material of this strain contained polysaccharides (16·4%), uronic acids (79·3%) and small amounts of proteins (4·3%). The chemical composition obtained by XPS of the footprints left behind by P. aeruginosa after air-bubble-induced detachment showed a greater content of uronic acid in the footprints of the EPS-producing strain than of the putatively non-EPS-producing strain. The uronic acid content calculated increased with the number of detachment and redeposition cycles. Uronic acids (i.e. mannuronic and glucuronic acids) excreted by P. fluorescens strains have been reported to have surface-active properties, preventing their adhesion to surfaces (Pringle et al., 1983
), an effect similar to that of the uronic acids excreted by the P. aeruginosa strain in this study. These findings agree with earlier studies that showed how the excretion of surface-active material by lactobacilli (McGroarty & Reid, 1988
; Velraeds et al., 1998a
), Streptococcus thermophilus (Busscher et al., 1997
) or Streptococcus mutans (Van Hoogmoed et al., 2000
) either discourage their own adhesion or adhesion of competitors to substrata.
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Received 24 September 2001;
revised 17 December 2001;
accepted 21 December 2001.
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