a Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen; b Department of Chemical Technology and Institute of Biomedical Technology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands
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Abstract |
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Introduction |
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The aim of this study was to determine possible antimicrobial effects on different Gram-positive and Gram-negative bacteria of a homologous series of three methacrylate polymers and copolymers varying in surface charge. To this end, initial adhesion and subsequent surface growth of Staphylococcus aureus, S. epidermidis, E. coli and P. aeruginosa were measured in a parallel plate flow chamber.
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Materials and methods |
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S. aureus ATCC 12600, S. epidermidis HBH2 102, P. aeruginosa AK1 and E. coli O2K2 were used in this study. Each strain was grown up from a frozen stock by incubation overnight at 37°C on blood agar. The plate was then kept at 4°C for up to a week. Several colonies were used to inoculate 5 mL of the following broths: tryptone soya (TSB; Oxoid, Basingstoke, UK) for the staphylococci, nutrient (NB; Oxoid) for P. aeruginosa and brainheart infusion (BHI; Oxoid) for E. coli. Each broth was prepared in phosphate-buffered saline (PBS). This pre-culture was incubated at 37°C in ambient air for 24 h and used to inoculate a second culture (150 mL TSB in PBS for the staphylococci, 100 mL NB in PBS for P. aeruginosa or 100 mL BHI in PBS for E. coli), which was grown for 18 h. The bacteria from the second culture were harvested by centrifugation (5 min, 5000g) and washed twice with sterile Millipore-Q water. Bacteria were then suspended in sterile PBS to a concentration of 3 x 108 cells/mL. S. epidermidis was first sonicated on ice (3 x 10 s) to aid dispersal.
In order to determine the generation time of the bacteria in suspension, 1 mL of the preculture was suspended in 200 mL of the appropriate growth medium and growth curves were recorded at 37°C using optical density measurements at 600 nm. Generation times were derived from the doubling times of the optical density values.
Polymer synthesis
For this study, homopolymers of methyl methacrylate (MMA; Merck, Darmstadt, Germany) (PMMA) and copolymers of MMA with either 15 mol% methacrylic acid (MAA; Aldrich, Milwaukee, WI, USA) (PMMA/MAA 85/15) or 15 mol% trimethylaminoethyl methacrylate chloride (TMAEMA-Cl, 75%, Aldrich) (PMMA/ TMAEMA-Cl 85/15) were used (see Figure 1 for structural formulae). Polymers were synthesized as described previously5,12 by radical polymerization of the monomers using 2,2'-azobis(methyl isobutyrate) as an initiator, synthesized from 2,2'-azobisbutyronitrile (Merck) as described by Mortimer.13 Solvents and MMA were distilled before use. The purity of the polymers was checked by nuclear magnetic resonance.
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The chemical composition of the films was determined by X-ray photoelectron spectroscopy (XPS) using a S-Probe spectrometer (Surface Science Instruments, Mountain View, CA, USA). The elemental surface compositions were expressed in atomic %, setting %C + %O + %N + %Si + %Cl to 100%. Zeta () potentials of the film surfaces were derived from the pressure dependence of the streaming potentials using rectangular platinum electrodes (5.0 x 25.0 mm) located at both ends of a parallel plate flow chamber,14 which was made up of the glass slides separated by a 0.2 mm Teflon gasket. Streaming potentials were measured over 7 h in PBS (pH 7.0) at 10 different pressures ranging from 37.5 to 150 Torr. Each pressure was applied for 10 s in both directions. Water contact angles were measured at room temperature with an image analysing system, using the sessile drop technique. Each value was obtained by averaging results of at least three droplets on one sample.
Bacterial potentials
Bacterial potentials were derived from particulate microelectrophoresis.15,16 Three separate cultures of each strain were harvested and washed as described above. Immediately after the bacteria were resuspended (5 x 107 cells/ mL) in sterile PBS (pH 7.0), measurements were taken at 150 V using a Lazer Zee Meter 501 (PenKem, Bedford Hills, NY, USA). These were converted into apparent
potentials assuming the HelmholtzSmoluchowski equation.
The parallel plate flow chamber, image analysis, adhesion and surface growth assay
The flow chamber (dimensions: l x w x h = 76 x 38 x 0.6 mm), image analysis system and adhesion and surface-growth assays have all been described in detail.17,18 Images were taken from the bottom plate (58 x 38 mm) of the parallel plate flow chamber, which consisted of the spin-coated microscope coverslip affixed centrally with double-sided tape (0.06 mm thick) in a groove (18 x 18 x 0.16 mm) made in a thicker (2.0 mm) PMMA plate. The top plate of the chamber was made of glass. The chamber was heat sterilized as a whole, except for the PMMA plate, which was disinfected in 70% ethanol. The coated coverslip had been kept sterile ready for use. The flow chamber was equipped with heating elements and kept at 37°C throughout an experiment. Deposition and surface growth was observed with a CCD-MXRi camera (High Technology, Eindhoven, The Netherlands) mounted on a phasecontrast microscope (Olympus BH-2) equipped with a 40x ultra-long working distance lens (Olympus ULWD-CD Plan 40 PL). The camera was coupled to an image analyser (TEA; Difa, Breda, The Netherlands).
Before each experiment, all tubes and the flow chamber were filled with sterile PBS, taking care to remove all air bubbles from the system. PBS was allowed to flow through the system for 1 h at a flow rate of 0.025 mL/s (corresponding to a shear rate of 10/s), while the flow chamber was heated to 37°C. Flow was then switched to a bacterial suspension of 3 x 108 cells/mL in PBS, which was perfused for 1 h at the same flow rate without re-circulation. The flow was then switched back to buffer alone for 15 min at the same flow rate to remove unbound organisms from the tubes and the flow chamber. Finally, flow was switched to a growth medium, TSB for the staphylococci, NB for P. aeruginosa and BHI for E. coli. The medium was perfused through the system for 6 h at the same flow rate without re-circulation. The experiments were performed in duplicate.
During the experiment, images were recorded and analysed automatically to give the number of adhering bacteria as a function of time. The initial deposition rate was expressed as the increase in the number of adhering bacteria per unit area and time. The division time of adhering bacteria was also monitored to measure a generation time. The numbers of growing and non-growing bacteria were determined, starting with the image taken after 2 h of flow with growth medium, and following the individual bacteria for the next 4 h. Subsequently, a percentage of growing bacteria relative to the number of adhering bacteria at 2 h was calculated. A desorption rate constant (kdes) was obtained by performing a non-linear least squares fit of the increasing part of the growth curve using the model of Barton et al.:19
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In this equation, ni is the number of bacteria after the ith time increment (t), nng is the determined number of non-growing bacteria, ng0 is the number of growing bacteria at the beginning of the logarithmic growth phase and g is the generation time. Desorption of non-growing bacteria was negligible compared with desorption of growing bacteria. When no growing bacteria were present, kdes was determined by dividing the number of desorbed bacteria by the number of adhering bacteria for each time increment after 2 h of flow with growth medium and calculating the average.
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Results |
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Table I gives
potentials of the polymer films in PBS.
potentials ranged from 18 mV (PMMA/MMA) to +12 mV (PMMA/TMAEMA-Cl) and were stable over 7 h. Water contact angles on the polymer films were typical of an intermediately hydrophobic surface and showed no major variation with
potential. This indicated that all results could be interpreted without the interfering influences of substratum hydrophobicity. XPS analyses indicated that the positive charge originated from nitrogen-containing groups, while the increased negative charge was caused by oxygen-containing groups on the modified acrylate surfaces.
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All bacterial strains studied here were negatively charged in PBS and their potentials were 10 mV for S. aureus ATCC 12600, 8 mV for S. epidermidis HBH2 102, 16 mV for E. coli O2K2 and 7 mV for P. aeruginosa AK1.
Adhesion and surface growth
Figures 2 and 3 show examples of images taken during surface growth of S. aureus and P. aeruginosa, respectively. Note that more bacteria adhered to the positively charged PMMA/TMAEMA-Cl surface after 2 h, but after additional growth (4 h) the most S. aureus microcolonies were found on the PMMA () surface. Proliferating P. aeruginosa were seen only on the negatively charged surfaces and growth appears absent by comparison with the images taken after 2 and 4 h on the positively charged PMMA/ TMAEMA-Cl surface. The numbers of adhering bacteria during adhesion and surface growth on the charged methacrylates are shown graphically in Figure 4
. During the growth phase, proliferating staphylococci were present on all surfaces from 1 h after the introduction of growth medium. On the negatively charged surfaces, most of the E. coli and P. aeruginosa cells were proliferating within 30 min. The numbers of E. coli increased slowly, however, because most newly formed bacteria desorbed directly from these surfaces. Table II
gives initial deposition rates (j0), percentages of growing bacteria after 2 h, generation times (g) and desorption rate constants (kdes) of adhering bacteria. Initial deposition rates were highest for the staphylococci and generally increased as the substrata became less negatively charged. Under conditions of electrostatic attraction, as on PMMA/TMAEMA-Cl, initial adhesion rates were maximal. Initial adhesion rates of P. aeruginosa AK1 were the lowest of all four strains, but also increased as the electrostatic repulsion between the bacteria and the substratum surface disappeared on PMMA/TMAEMA-Cl. Staphylococci grew on all substratum surfaces, although the addition of negative and positive charge to PMMA decreased the relative number of growing staphylococci by a factor of 4 and 2, respectively. The generation times of adherent staphylococci were comparable with those measured for planktonic S. aureus ATCC 12600 (31 min) and S. epidermidis HBH2 102 (46 min). Gram-negative bacilli grew only on negatively charged surfaces and their generation times also compared with the generation times of planktonic bacteria, viz. 23 min for E. coli O2K2 and 43 min for P. aeruginosa AK1. All strains showed desorption of adhering bacteria. For the staphylococci, desorption rate constants on the positively charged material were similar to those on the negatively charged materials. As noted, Gram-negative bacilli did not grow on the positively charged surface and they had a very low desorption rate. Desorption was higher from the negatively charged surfaces.
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Discussion |
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Previously, it has been demonstrated that when the binding strength of adhering P. aeruginosa AK1 to substrata increases, the surface growth reduces.11 Complete inhibition of growth, as found here for Gram-negative bacilli, possibly indicates that elongation of adhering bacteria, necessary for cell division, is impeded by strong binding through attractive electrostatic interactions. Soluble quaternary ammonium salts have been known for a long time to exhibit antimicrobial activity20 through interaction with the bacterial cell membrane.21 The quaternary ammonium groups of the positively charged polymer, although insoluble, may disrupt the cell membrane of the Gramnegative organisms. Gram-positive bacterial strains have a comparatively thicker and more rigid peptidoglycan layer and extensive contact of the membrane with the immobilized quaternary ammonium groups is less likely to occur, even under conditions of electrostatic attraction. This might explain why the surface growth of Gram-positive bacteria is less affected by the substratum charge. Several groups have also reported a reduction in viable count, greatest for Gram-negative bacteria, when adding positively charged insoluble powders to bacterial suspensions.22,23 From these experiments, however, it is not clear whether this reduction is the result of strong bacterial binding to the particles or of reduced viability of planktonic organisms. Our results clearly show that a positively charged surface can totally inhibit growth of some adhering bacteria.
Initial bacterial adhesion has always been recognized as an essential step in biofilm formation. This study shows that when a biomaterial surface is more negatively charged, this may reduce the chance of bacterial adhesion, and delay the formation of a biofilm. Positively charged surfaces are more adhesive, but the strong electrostatic attraction of the organisms impedes surface growth of Gram-negative bacilli. This study therefore points to a new pathway for the development of biomaterials with a low risk of infection and complements current approaches based on preparing non-adhesive surfaces. This may be important in clinical situations where adsorbing proteins are not abundantly present to mask the initial surface, for example in urinary catheters or voice prostheses for laryngectomized patients with hampered salivary flow due to irradiation. As biomaterials are often infected during implantation surgery, a positively charged surface could prevent bacterial proliferation.
As it has also been argued that adsorbed protein films convey the properties of the underlying surface to the interface with adhering bacteria,8 the present results pose an interesting dilemma. Adhesion and growth appear to be oppositely affected by the surface characteristics of a biomaterial. This may explain why several biomaterials have been found to be non-adhesive in vitro, while showing huge biofilm formation once implanted in the human body. Everaert et al.,24 for instance, found that hydrophilized silicone rubber in laryngectomized patients with reduced salivary flow attracted lower numbers of yeasts and bacteria under laboratory conditions in a parallel plate flow chamber than authentic, hydrophobic silicone rubber, but during use as a voice prosthesis a much thicker biofilm formed on the hydrophilized silicone rubber.
In conclusion, in order to develop biomaterial surfaces with a low risk of infection, in vitro studies should not only take into account initial adhesion, but also look at surface growth of the adhering bacteria.
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Notes |
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References |
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Received 25 September 2000; returned 31 January 2001; revised 6 February 2001; accepted 12 March 2001