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Applied and Environmental Microbiology, May 2005, p. 2705-2712, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2705-2712.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of a Quartz Crystal Microbalance To Investigate the Antiadhesive Potential of N-Acetyl-L-Cysteine

Ann-Cathrin Olofsson,^1* Malte Hermansson,² and Hans Elwing¹

Department of Cell and Molecular Biology-Interface Biophysics, Göteborg University, Box 462, 405 30 Göteborg, Sweden,¹ Department of Cell and Molecular Biology-Microbiology, Göteborg University, Box 462, 405 30 Göteborg, Sweden²

Received 20 August 2004/ Accepted 2 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The reduction of bacterial biofilm formation on stainless steel surfaces by N-acetyl-L-cysteine (NAC) is attributed to effects on bacterial growth and polysaccharide production, as well as an increase in the wettability of steel surfaces. In this report, we show that NAC-coated stainless steel and polystyrene surfaces affect both the initial adhesion of Bacillus cereus and Bacillus subtilis and the viscoelastic properties of the interaction between the adhered bacteria and the surface. A quartz crystal microbalance with dissipation was shown to be a powerful and sensitive technique for investigating changes in the applied NAC coating for initial cell surface interactions of bacteria. The kinetics of frequency and dissipation shifts were dependent on the bacteria, the life cycle stage of the bacteria, and the surface. We found that exponentially grown cells gave rise to a positive frequency shift as long as their cell surface hydrophobicity was zero. Furthermore, when the characteristics of binding between the cell and the surface for different growth phases were compared, the rigidity increased from exponentially grown cells to starved cells. There was a trend in which an increase in the viscoelastic properties of the interaction, caused by the NAC coating on stainless steel, resulted in a reduction in irreversibly adhered cells. Interestingly, for B. cereus that adhered to polystyrene, the viscoelastic properties decreased, while there was a reduction in adhered cells, regardless of the life cycle stage. Altogether, NAC coating on surfaces was often effective and could both decrease the initial adhesion and increase the detachment of adhered cells and spores. The most effective reduction was found for B. cereus spores, for which the decrease was caused by a combination of these two parameters.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria commonly adhere to a surface and are part of a complex biofilm. Since the growth of biofilms causes enormous economic losses and health problems in industrial systems like food and paper production, huge efforts are made to solve this problem. The adhesion of bacteria to surfaces is a complex interplay of physical, chemical, and biological factors (5), and there are large variations in the adhesion behaviors of different bacteria and also for different life cycle stages of one strain (41). It is very important to consider these differences when antiadhesion coatings are developed. It is generally thought that bacterial adhesion is preceded by the adsorption of a conditioning layer of molecules that influence adhesion of bacteria to surfaces (43). A good antiadhesion coating should form a conditioning film that prevents adhesion and has a low cohesive strength, making a weak bond between the biofilm and the surface (6). One such agent may be N-acetyl-L-cysteine (NAC). NAC is commonly used in medical treatment of chronic bronchitis and cancer (38, 46) and is one of the smallest drug molecules in use (30). Moreover, we recently demonstrated that NAC affects several processes that are important for bacterial biofilm formation on stainless steel surfaces, including a drastic reduction in extracellular polysaccharide production, and thus acts as an antibiofilm substance when it is present in LB or process water. Furthermore, NAC was also shown to increase the wettability of stainless steel surfaces (31), but the exact mechanism of action of NAC for bacterial adhesion and biofilm formation is still relatively unknown.

Detection of the effects of antiadhesive coatings on bacterial adhesion on equipment and devices is conventionally performed by methods that measure the number of culturable cells and/or the total number of cells adhering to a surface. None of these techniques reveal the actual interaction of bacteria with the surface. Quartz crystal microbalance (QCM) has previously been used as a technique to follow adsorption processes at solid-liquid interfaces in chemical and biological research (50), but only a few studies have been performed with bacteria and with the energy dissipation (D) factor as a parameter (33, 34). QCM with dissipation (QCM-D) allows simultaneous measurement of frequency () and energy dissipation. This technique makes it possible to detect very small mass changes as well as viscoelastic properties during the adhesion of cells to a crystal surface. Plotting changes in energy dissipation (D) versus changes in frequency () reveals the adhesion behavior of bacteria, which makes it possible to actually follow the interaction and adhesion process over time.

The objectives of this study were, in general, to obtain to a better understanding of how NAC influences initial adhesion processes and to investigate the potential of a quartz crystal microbalance with dissipation as a tool for monitoring the effect that NAC has on adhesion when it is applied as a coating. More specifically, we wanted to study the effect of NAC on the interaction between bacteria in different growth phases and surfaces and to test its potential as an antiadhesion coating. Since the initial adhesion is dependent on the substratum wettability, as well as the cell surface hydrophobicity (CSH) of bacteria, the contact angles of stainless steel and polystyrene surfaces were determined when they were not coated and coated with NAC, as well as the CSH of bacteria in different stages of their life cycle. Furthermore, we investigated the effect of NAC as a coating on stainless steel and polystyrene surfaces on (i) the adhesion of bacteria in different stages of their life cycle and (ii) the viscoelastic properties of the bond between the cell and the surface. Moreover, we attempted to elucidate the measured positive shift for exponentially grown cells during attachment. We focused on Bacillus subtilis and Bacillus cereus cells, since they are common contaminants in the processing of food and hygienic paper. Bacillus cells, and in particular Bacillus spores, have been found to have a pronounced ability to adhere to stainless steel surfaces, which is the most widespread material used in processing equipment in a wide variety of industries (21, 26). In conclusion, in this work we gained a better understanding of initial bacterial adhesion interactions with surfaces coated with NAC, and below we discuss possibilities that may facilitate efficient use of this compound as an antiadhesion/biofilm coating agent.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of exponentially grown cells, starved cells, and spores.
The bacterial strains used in this study were B. cereus ATTC 14579 and B. subtilis wild-type strain 168 (trpC2) (31). Cell suspensions were prepared after growth at 37°C in minimal medium M9 containing (per liter): Na₂ HPO₄, 3.0 g; KH₂PO₄, 1.5 g; NH₄Cl, 0.5 g; NaCl, 0.25 g; CaCl₂, 3.0 g; MgSO₄ · 7H₂O, 0.246 g; glucose, 2%; thiamine, 0.5%; and tryptophan, 20 mg. Exponentially grown cells were grown to an optical density at 600 nm corresponding to a concentration of approximately 1.4 x 10⁸ cells/ml^–1, as determined by measuring the CFU/ml^–1. The cells were harvested in the exponential growth phase by centrifugation (6,000 rpm, 10 min), and after this they were washed two times and resuspended in a degassed physiological saline solution (PS) (0.9% [wt/vol] NaCl). Exponentially grown cells were used immediately, while the rest of the cells were allowed to starve for 24 h, after which the washing in degassed PS was repeated. Spore suspensions were prepared by growing the cells overnight in sporulation broth (15 g tryptone per liter, 1.5 g yeast extract per liter, 0.15 g CaCl₂ per liter, 0.25 g MgSO₄ per liter), after which sporulation agar (8 g nutrient broth per liter, 0.25 g MgSO₄ per liter, 0.97 g KCl per liter, 0.15 g CaCl₂ per liter, 2 mg MnCl₂ per liter, 0.3 mg FeSO₄ per liter, 3% agar) was inoculated with vegetative cells and incubated for 10 days at 30°C (2). The spores were harvested from the agar surface and washed five times in PS. The spore concentration in the experiments was determined by counting in a Bürker chamber (FORTUNA, Germany) with visible light. Good spore yields for both strains (93 to 97%) were obtained. Spores of B. cereus have appendages on the surface (24, 49).

HIC.
Hydrophobic interaction chromatography (HIC) was used to test cell adhesion to a hydrophobic surface and was performed as described previously (32). First, an octyl-Sepharose CL-4B gel (2 ml) (Pharmacia, Sweden) was washed and equilibrated by passing eluate (5 ml of PS) through the Pasteur pipette containing the gel. Approximately 1 x 10⁹ cells (1 ml) were applied to the gel, and after this the cells were eluted with 12 ml of PS. Cells (1 x 10⁹) were added to 12 ml of PS, and the initial optical density (OD_i) was measured at 420 nm (Novaspec, LKB). The optical density of the eluate (OD_e) was measured as well, and the CSH was expressed as (OD_i – OD_e)/OD_e.

Quartz crystal microbalance technique with dissipation monitoring.
QCM-D (Q-Sense AB, Göteborg, Sweden) is a flow system that simultaneously measures and D as described previously (33). The technique is surface sensitive, with a sensitivity of less than 1 ng/cm². The quartz crystal, consisting of a sensor with electrodes on each side, is mounted in a detection cell. When an AC voltage is applied across the electrodes, the crystal oscillates at its resonant frequency. Changes in the resonant frequency and the dissipation factor were measured for 90 or 24 h. The data collected were transferred to a personal computer and analyzed by KaleidaGraph 3.04. The temperature of the detection cell was stabilized at 22°C.

Preparation of hydrophilic quartz and hydrophobic crystal surfaces.
Stainless steel 5-MHz crystals (QSX 304; Maxtek Inc., Torrance, Calif.) were used as a hydrophilic surface in QCM experiments. The crystals were cleaned by keeping them in 1% Hellmanex II (Hellma GMBH & Co. KG, Müllheim, Germany) overnight. Immediately prior to use the surfaces were sonicated for 10 min and rinsed with 70% ethanol and MilliQ water (MQ) during sonication (10 min). The crystals were rinsed with MQ and dried in nitrogen. Finally, the surfaces were oxidized for 10 min in a UV-ozone chamber, resulting in hydrophilic surfaces. Hydrophobic surfaces were obtained as follows. AT-cut 5-MHz crystals (Maxtek Inc., Torrance, Calif.) coated with an evaporated gold film were cleaned by treating the surfaces with a mixture of H₂O, NH₄OH, and H₂O₂ (ratio, 4.7:1:1 [vol/vol/vol]) heated to 80°C. After 10 min, the crystals were rinsed with MQ and dried in nitrogen. After this, the surfaces were methyl terminated by immersing crystals in a saturated solution of octadecylmercaptan (Aldrich Chemical Co. Ltd.) in hexane for at least 24 h at room temperature. Immediately prior to use the surfaces were coated with a polystyrene film. Crystals were transferred to a spin coater, and a 50-µl drop of polystyrene dissolved in 0.5% toluene was added to the surface. The surface was spun at 2,000 rpm for 2 min.

Contact angle measurements for solid surfaces.
The relative surface hydrophobicity was determined by measuring the water contact angle on stainless steel crystals (42). A water droplet was applied to the crystals, and then the diameter of the droplet was determined with a measuring ocular and the contact angle was calculated (9).

Adhesion of exponentially grown cells, starved cells, and spores using QCM-D.
Immediately prior to use the PS was degassed by sonication in a vacuum for 10 min to prevent the formation of air bubbles on the electrode. Crystal surfaces were coated or not coated with 0.5 mg/ml^–1 of NAC for 15 min, and then the detection chamber was washed thoroughly with PS. Frequency and dissipation were measured until a constant baseline was acquired, and then monitoring was performed by changing the flow of PS to an identical buffer containing bacterial cells or spores.

Bacterial exponentially grown cells, starved cells, or spores were added to a final concentration of 1.4 x 10⁸ cells/ml^–1 in PS. The frequency and dissipation shifts caused by adhesion of cells or spores were measured continuously for 90 min or 24 h. Crystals were removed from the QCM detection chamber, rinsed, and stained for 10 min in acridine orange (100 µg/ml)-2% formaldehyde (22) in phosphate-buffered saline. Attached cells or spores were counted directly on the crystal surfaces. Surfaces were examined with an Olympus epifluorescence microscope at a magnification of x1,250 using blue light. At least 30 fields of view were counted for each surface.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Assays to study surface hydrophobicity.
The degree of CSH of bacteria and the wettability of surfaces have been reported to influence bacterial adhesion to different surfaces in various environments (1, 5, 15-17, 21, 32, 42, 47, 53).

To obtain insight into the adhesion behavior of B. cereus and B. subtilis cells in different stages in their life cycles, the CSH of the cells was investigated. CSH was measured for exponentially grown cells, starved cells, and spores of B. cereus and B. subtilis by HIC, in which an octyl-Sepharose CL-4B gel was used as a surface. Spores of B. cereus had a high affinity for the octyl-Sepharose, whereas exponentially grown and starved cells of B. cereus, as well as all three life cycle stages of B. subtilis, displayed varied but low affinity for the hydrophobic gel (Table 1). The high CSH of B. cereus spores detected using HIC is in line with previously published data which showed that the long appendages found on the spore surface make the spores very hydrophobic (21) and thus result in high affinity for hydrophobic surfaces, such as octyl-Sepharose.

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TABLE 1. Bacillus strains used in this study and CSH of their different growth stages

To further examine the adhesion mechanism of cells in different growth phases, we used QCM-D. Commonly, when bacteria attach to the surface of a stainless steel or polystyrene crystal, the total oscillating mass increases, which results in a decrease in the frequency and an increase in the energy dissipation. Thus, negative frequency shifts () (39, 44) and positive dissipation shifts (D) result from cells attaching to the surface. Moreover, frequency and dissipation measurements are obtained simultaneously by switching the electric field applied to the crystal on and off. The crystal starts to oscillate at its resonant frequency, and when the electric field is switched off, the oscillation decay is recorded. The decay of the signal may be influenced by adhesion of bacteria to the surface. However, the frequency shift is related to mass loading on the crystal surface, but when monitoring is done in liquid, the frequency also depends on the viscosity and density of the liquid in contact with the resonator (25, 40) and is not necessarily directly proportional to the change in (3, 7, 11, 19, 29, 37). The frequency shift is proportional to the attached mass only when the mass is rigid and tightly coupled to the surface (20), which is not the case for bacterial cells. Furthermore, nonrigid binding involves energy dissipation due to internal friction or trapping of water by the cells, which causes damping of the oscillation of the crystal (40). Since there are great differences in morphology between cells at different stages of the life cycle and thus in the contact area when the cells are interacting with the surface, cells give different shift responses (Fig. 1).

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FIG. 1. Representative graphs showing frequency and dissipation measured by QCM-D versus time for B. cereus and B. subtilis. The effects of NAC on mass () and dissipation (D) during 90 min of adhesion of exponentially grown cells, starved cells, and spores to stainless steel (a) and polystyrene (b) were determined. Black lines, for surfaces not coated with NAC; blue lines, D for surfaces not coated with NAC; red lines, for surfaces coated with NAC (0.5 mg/ml–1); green lines, D for surfaces coated with NAC (0.5 mg/ml–1). The experiment was run four times, and the values obtained varied by <10%.

Interestingly, when shifts for exponentially grown cells during attachment were measured, the shift was positive instead of negative on every occasion (Fig. 1 and 2). A positive shift in theory means less interacting mass. Since the shift was positive from time zero, this could not be the case. To elucidate this phenomenon, we first examined the possibility that biosurfactants excreted by cells into the media could influence the cell-surface interaction. However, no shift was detected for either frequency or dissipation when measurements were obtained for supernatants without cells, suggesting that the cells have to be present to give a positive shift (data not shown). Second, we examined whether the change in CSH of cells over time corresponded to measured changes in . Frequency and dissipation shifts were simultaneously measured for 24 h for exponentially grown B. subtilis and B. cereus cells on stainless steel and polystyrene crystal surfaces (Fig. 2). During this time CSH was measured by HIC. We found that as long as the CSH of the cells was zero (hydrophilic), the shifts were positive. As the CSH increased with time of starvation, the frequency shift became negative.

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FIG. 2. Relationship between measured CSH of bacteria and recorded shift. Exponentially grown cells were transferred from nutrition media to PS, inducing starvation. The data are a summary of the results of monitoring the frequency of cells during 24 h of adhesion. The values are mean CSH data for four different cultures measured by HIC for B. subtilis and B. cereus. The standard deviations for CSH were <20%. QCM-D measurements were repeated three times, and the data are representative of measurements for both stainless steel and polystyrene crystal surfaces for both bacteria. The values obtained varied by <10%.

Effect of NAC on adhesion and viscoelastic properties.
The initial bacterial adhesion and the ease with which the initially adhered bacteria can be detached have been reported to influence the effect of a coating (6). Since NAC is known to act as an antiadhesion/biofilm agent (31), we wanted to study the effect of NAC on these parameters when exponentially grown cells, starved cells, and spores of bacteria were interacting with stainless steel and polystyrene surfaces. These surfaces represented a hydrophilic surface and a hydrophobic surface, respectively. To confirm that the stainless steel and polystyrene crystal surfaces were coated with NAC, surface contact angle measurements were obtained. NAC significantly (P < 0.0001, as determined by Student's t test) decreased the contact angle (i.e., increased the wettability) of the stainless steel crystal surfaces from 26° to 18°. The contact angles of polystyrene surfaces were >85° and decreased to 82° (P < 0.005, as determined by Student's t test) after treatment with NAC. Coating with NAC made both the surfaces less hydrophobic, although the effect was larger for stainless steel surfaces.

As Fig. 1 shows, interactions of bacteria in different growth phases with surfaces that were coated or not coated with NAC had quite different patterns.

To understand these results, we counted the number of irreversibly adhered bacteria (Fig. 3) and calculated the D_{90 min}/_{90 min} values (viscoelastic properties) (Fig. 4) for B. subtilis and B. cereus adhered to NAC-coated or uncoated stainless steel and polystyrene surfaces.

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FIG. 3. Effect of NAC on the adhesion of exponentially grown cells, starved cells, and spores of B. cereus and B. subtilis to stainless steel (dark gray bars, surfaces not coated with NAC; open bars, surfaces coated with NAC [0.5 mg/ml–1]) and to polystyrene (black bars, surfaces not coated with NAC; light gray bars, surfaces coated with NAC [0.5 mg/ml–1]). Numbers of irreversibly adhered bacteria on surfaces were determined after 90 min of adhesion (after rinsing of the surface) directly on the crystal surfaces by acridine orange direct counting with an epifluorescence microscope. The data shown are means ± standard deviations from four experiments.

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FIG. 4. Effect of NAC on the viscoelastic properties of the interaction of cells in different growth stages during adhesion to stainless steel and polystyrene. The viscoelastic properties of attached bacteria were expressed by relating the total energy dissipation to the total attached mass on the surface at every time (D/f). Dark gray bars, stainless steel surfaces not coated with NAC; open bars, stainless steel surfaces coated with NAC (0.5 mg/ml–1); black bars, polystyrene surfaces not coated with NAC; light gray bars, polystyrene surfaces coated with NAC (0.5 mg/ml–1). The data are means ± standard deviations from four experiments.

The viscoelastic properties of attached bacteria are displayed by relating D, interpreted as the total energy dissipation, to the total attached mass on the surface at every time (D/). This eliminates time and number of attached bacteria as explicit parameters. When cells are attached to the surface of a crystal, the energy dissipation increases, more or less depending on the structure of the film formed. The magnitude of the increase is greater for flexible and viscoelastic films than for thin rigid films (13, 19). This implies that rigid binding gives a low D/ value, while increasing viscosity is accompanied by an increased D/ value. The viscoelastic properties together with frequency measurements were found to be a predictor of the antiadhesive effect of the NAC coating. This demands careful study of the interaction between bacteria and coated and noncoated surfaces, since the interaction was shown to be both bacterium and surface dependent.

Accordingly, the antiadhesive effect of NAC can be seen in the following two ways. First, fewer cells interact with the surfaces, as shown by a lower shift simultaneous with no change in the viscoelastic properties (D/) detected, meaning that the antiadhesive effect is all due to the coating's ability to make the surface less favorable for adherence. Second, the shift is the same for non-NAC-coated and NAC-coated surfaces (or even larger for NAC-coated surfaces), while there are changes in the viscoelastic properties which weaken the bond between the cell and the surface, resulting in detachment. The coating is most effective when there is a combination of antiadhesion and detachment (for example, the effect of NAC on the adhesion of spores of B. cereus to stainless steel and polystyrene) (Fig. 1a and b and 3).

(i) Exponentially grown cells.
As shown in Fig. 3, the presence of NAC had a very good antiadhesive effect on exponentially grown cells of B. cereus and B. subtilis on both stainless steel and polystyrene surfaces (average, 64% ± 36%) (P < 0.001, as determined by Student's t test).

The positive shifts for B. cereus were approximately the same at the end of the measured period for non-NAC-coated and NAC-coated stainless steel surfaces (Fig. 1a). However, the D shift increased when NAC-coated surfaces were used (giving a higher D_{90 min}/_{90 min} value; P < 0.005, as determined by Student's t test) (Fig. 4), thus resulting in an overall decrease in adhesion, since more B. cereus cells were washed off the NAC-coated surface. In contrast, B. subtilis showed a much lower positive shift on NAC-coated steel surfaces than on noncoated surfaces (Fig. 1a) and no change in viscoelastic properties (Fig. 4), indicating that there was lower initial adhesion of bacteria to the surface.

Both bacteria showed a larger positive shift when they were adhering to NAC-coated polystyrene surfaces than when they were adhering to noncoated polystyrene surfaces, which indicates that more cells interacted with surfaces conditioned with NAC (Fig. 1b). The difference was larger for B. cereus cells. Furthermore, the interacting B. subtilis cells showed a large increase in viscoelastic properties (increase in the D_{90 min}/_{90 min} value; P < 0.001, as determined by Student's t test), which explains the decrease in adhesion. Surprisingly, the viscoelastic properties of the bonding between B. cereus and polystyrene decreased, while there was a reduction in adhered cells (Fig. 4). Thus, NAC acts as an antiadhesive substance when exponentially grown cells adhere to both stainless steel and polystyrene, although it has different effects on the viscoelastic properties, giving decreased adhesion of B. cereus when it interacts with polystyrene surfaces compared to stainless steel surfaces. This was found to be the case regardless of the life cycle stage.

(ii) Starved cells.
Pretreatment with NAC had a pronounced antiadhesive effect (average, 73% ± 21%) (P < 0.001, as determined by Student's t test) for starved B. cereus cells on both surfaces (Fig. 3).

Starved B. cereus cells exhibited a large difference in their adhesion to stainless steel. Noncoated surfaces were not saturated, while NAC-coated surfaces were saturated at the end of the experimental period. NAC-coated steel surfaces showed only about one-half the negative frequency shift of the noncoated surfaces, and the dissipation shifts were higher. This and a more viscoelastic interaction between the cell and the surface resulted in a large decrease in the irreversibly adhered B. cereus cells on steel surfaces. The antiadhesive effect of NAC on B. cereus on polystyrene surfaces, although a larger mass was interacting with the surface, was due to a decrease in viscoelastic interactions.

Moreover, NAC had no significant effect on the adhesion of starved cells of B. subtilis to either stainless steel or polystyrene surfaces (Fig. 3). The negative frequency shifts showed a small increase when surfaces were coated with NAC, while the dissipation shifts were approximately the same on both surfaces for noncoated and NAC-coated surfaces when starved cells of B. subtilis adhered. As shown by the unchanged D_{90 min}/_{90 min} values, NAC had no effect on the viscoelastic properties.

(iii) Spores.
Spores exhibit passive adhesion to solid surfaces since they are metabolically inactive and do not contribute actively to the process, either by taxis or by excretion of polymer products (22). The structure of the spore differs greatly from that of the vegetative cell since it is multilayered. Spore morphology and chemical composition also differ among Bacillus species.

NAC had a good antiadhesive effect on both B. cereus and B. subtilis spores adhering to stainless steel crystals (82% ± 26%) (P < 0.001, as determined by Student's t test).

NAC affected the hydrophobic spores of B. cereus by lowering the frequency shift (Fig. 1), and in combination with a D_{90 min}/_{90 min} value that was twice as high (Fig. 4) the interaction became more viscoelastic and the number of attached spores decreased (Fig. 3). Coating with NAC had no significant effect on the frequency shift when B. subtilis spores were adhering to the surface. Instead, a dramatic increase in dissipation was seen, which resulted in a D_{90 min}/_{90 min} value that was six times larger for NAC-coated surfaces than for noncoated surfaces, indicating that there were loosely attached spores.

For polystyrene the number of adhered B. cereus spores decreased by as much as 90% ± 38% (P < 0.001, as determined by Student's t test), while the adhesion of spores of B. subtilis increased (Fig. 3). When B. cereus spores adhered to NAC-coated surfaces, lower shifts (Fig. 1) and viscoelastic properties were observed (Fig. 4), resulting in a large decrease in the number of adhered spores. B. subtilis spores interacting with NAC-coated polystyrene surfaces showed a large negative shift and unchanged D_{90 min}/_{90 min} value compared to the data for nontreated surfaces, indicating that the increase in adhesion was caused by an increase in irreversibly bound spores.

We concluded that NAC influenced the initial adhesion as well as the viscoelastic properties of adhering cells. The magnitude and the kinetics of frequency and dissipation shifts are dependent on the bacteria, the life cycle stage of the bacteria, and the surface. The viscoelastic properties together with frequency measurements were found to be predictors of the antiadhesive effect of the NAC coating. This demands careful study of the interaction between bacteria and coated and noncoated surfaces, since the interaction was shown to be both bacterium and surface dependent. Furthermore, when the binding between the cell and the surface for different growth phases was studied, the rigidity increased from exponentially grown cells to starved cells. Surfaces coated with NAC had a general antiadhesive effect on exponentially grown cells, starved cells, and spores of B. cereus on both stainless steel and polystyrene surfaces. For B. subtilis there was a pronounced effect on exponentially grown cells on both surfaces, while there was no effect on starved cells on the same surfaces. The amount of adhered spores decreased on stainless steel, while the amount increased on polystyrene surfaces. Furthermore, for noncoated surfaces, larger numbers of exponentially grown and starved cells adhered to stainless steel surfaces, while larger numbers of B. subtilis spores adhered to polystyrene surfaces and equal numbers of B. cereus spores adhered to both surfaces.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many problems are associated with biofilm formation, and once biofilms have become established, they are difficult to abolish. The methods for prevention and elimination of biofilms include the use of antiadhesive coatings, antimicrobial agents, and biocides (4, 8, 10, 23, 27, 35).

In this study we tested the effect of NAC when it was used to coat a surface in order to obtain a better understanding of how NAC influences initial adhesion processes and to investigate the potential of QCM-D as a tool for monitoring the effect that NAC has on adhesion when it is applied as a coating.

The effects of NAC on adhesion and biofilm processes on solid surfaces have not been investigated in depth. Previously, we investigated the possible use of NAC to control the buildup of bacterial biofilms in paper mills. We concluded that NAC decreases the production of extracellular polysaccharide by B. cereus and B. subtilis, as well as a spectrum of other gram-positive and gram-negative bacteria, when it is present during growth (31). Extracellular polysaccharide is one of the major components in biofilms, and when it was reduced, it was shown to change the texture of the biofilm formed. We proposed that the reduction of bacterial adhesion by NAC is a chemical and biological effect.

Information concerning the effect of antiadhesive coatings on the bacterial status of equipment and devices is conventionally obtained by direct methods that remove bacteria from the surface by swabbing or sonication and determine bacterial numbers by agar plating (45) or direct counting using epifluorescence microscopy (18, 51). Since none of these techniques reveal the actual interaction of bacteria with the surface, it has been difficult, if not impossible, to learn more about how the coatings affect the binding between bacteria and surfaces.

The QCM-D technique allows us to study the kinetics of adsorption of bacteria, in different life cycle stages, to solid substrata and to determine viscoelastic properties of the interface between the bacteria and the substratum. By coating the surface with NAC, we studied the effect on the adhesion properties over time. There is also the possibility of performing studies over a longer time, with growing biofilms.

In applications comprised of living cells, including bacteria, the relationship between measured changes in frequency shifts and attached biomass is not linear (29). The effectively coupled mass depends on how the oscillatory motion of the crystal propagates into and through an adsorbed viscoelastic film (40). The direct contact area between the cell and the surface is very small compared to the area of whole bacterial cells and may vary depending on the surface, the growth phase of the bacteria, and the adhesion mechanism involved (28).

Stainless steel and polystyrene surfaces differ in their chemical aspects, one of which is wettability. Different bacteria and different life cycle stages of bacteria differ in morphology and physiology and express different cell surface structures. The largest difference between B. cereus and B. subtilis is the spores; B. cereus spores are very hydrophobic compared to B. subtilis spores due to appendages on the surface (24, 49). This difference is also reflected in differences in frequency and dissipation shifts (Fig. 1).

Exponentially grown B. cereus and B. subtilis cells transferred from nutrition media into physiological saline, inducing starvation, exhibited a positive frequency shift during interaction with surfaces (Fig. 1 and 2). We found that as long as the CSH of the cells was zero (hydrophilic), the shifts were positive (Fig. 2). As the CSH increased with time of starvation, the frequency shift became negative. The viscoelastic ratio for the exponentially grown cells was much larger than that for starved cells and spores, which suggests that there was more flexible binding to the surfaces. The possibility that a high viscoelastic ratio could induce a positive frequency shift is supported by a study of Marxer et al. (25), which showed that for viscoelastic systems, such as cells (African green monkey kidney epithelial cells and HeLa cells), an increase in viscosity and the viscoelasticity ratio can induce an increase in positive frequency, likely due to alteration of cytosolic viscosity (25). Moreover, Kanazawa's equation indicates that frequency shifts can occur by modification of the liquid properties, such as the viscosity and density, in the absence of cell desorption (12). In our experiments, no shift was detected for either frequency or dissipation when measurements were obtained for supernatants without cells, suggesting that the cells have to be present and in contact with the surface to give a positive shift. After 90 min of interaction, fluorescence microscopy indicated that the cells were attached to the steel and polystyrene surfaces, although the number of cells was less than the numbers of starved cells and spores (Fig. 3). Previous studies have shown that exponentially grown cells are less adhesive (14, 36, 41). Furthermore, there is a bondaging or rigidification of the viscoelastic properties of the binding of exponentially grown cells to surfaces, probably due to an increase in CSH with time as the cells starve (Fig. 2). The large decrease in viscoelastic properties seen for the starved cells indicates more rigid binding (Fig. 4). This is in line with the increased number of adhered starved cells detected (Fig. 3). There was a large difference in the numbers of cells adhered to the surfaces for the different growth phases.

Alternatively, the positive shift could be explained by the presence of biosurfactants on the surface of the cell. Biosurfactants are microbial compounds that exhibit pronounced surface activity, such as surface and interfacial tension reductions, which could have major consequences for bacterial adhesion. The cell itself, which is composed of a mosaic of hydrophobic and hydrophilic moieties, can be considered to be a biosurfactant (52). If cells slip on the electrode due to biosurfactants, a decrease in decay length results in an increase in frequency and therefore a positive shift (48). The presence of biosurfactants and high viscoelasticity of the binding between the cells and the surfaces may explain the low number of cells irreversibly adhered to the surfaces.

Adhesion of bacteria to surfaces in general involves a complex interplay of physical, chemical, and biological factors (5, 16). NAC adsorbs to steel and polystyrene surfaces, as shown by the increased wettability of the surfaces, creating a coating of NAC molecules. The results show that the antiadhesive effect of the NAC coating can be different for different bacteria, growth phases, and surfaces. Here we show that an effective NAC coating both decreased the initial adhesion and changed the viscoelastic properties of interacting cells and spores, which resulted in an increased number of adhered cells that washed off the surfaces, leaving fewer irreversibly adhered bacteria on the surface. There was quite a difference between a noncoated surface and the effect of the NAC coating on cell-surface interactions for B. cereus and B. subtilis. Although the exponentially grown cells of both strains were hydrophilic, the adhesion patterns and viscoelastic ratios were different. Electrostatic and morphological differences, as well as substances excreted on the cell surface, may cause these differences in the adhesion pattern.

The effect of the NAC coating was increased by increasing the CSH of the B. cereus cells, and the largest effect was on the adhering spores on polystyrene and stainless steel. This may be explained by the increased wettability of the surfaces caused by coating the surface with NAC, as well as a cohesive failure in binding. It is interesting that surfaces coated with NAC had a general antiadhesive effect on all growth phases of B. cereus on both surfaces, although the CSH of the different life cycle stages, as well as the wettabilities of the two surfaces, were so different (Table 1 and Fig. 3).

We saw a trend in which NAC-coated stainless steel surfaces increased the viscoelastic properties, resulting in more flexible binding between cells or spores and the surfaces, which resulted in a smaller number of irreversibly adhered bacteria. This makes the viscoelastic properties a predictor of the effect of the NAC coating on adhesion to stainless steel surfaces for the bacteria studied. This trend was not seen for polystyrene, which means that the change in viscoelastic properties caused by the coating is not a general predictor unless the specific interactions caused by different bacteria have been studied. The same increase in viscoelastic properties that was seen for stainless steel surfaces was detected for B. subtilis cells and spores. On the other hand, the NAC coating generally decreased the viscoelastic properties of the binding, indicating that there was a more rigid interaction between B. cereus and polystyrene, regardless of the growth phase, which, however, resulted in fewer irreversibly adhered cells. This could be explained by the mechanisms involved in adhesion between B. cereus and the hydrophobic polystyrene. The interaction of spores with noncoated polystyrene surfaces may be mediated by appendages, which explains the high dissipation shift. Coupling of appendages to the polystyrene surface suggests that there is very flexible binding of attached spores, in which the cell is not so closely coupled to the surface. Coating with NAC made the surface less hydrophobic while it decreased the negative frequency shift and dissipation, which implies that different structures of the spore surface may interact with the surface, resulting in closer coupling to the surface, although the number of irreversibly adhered spores is decreased by 90% ± 16% (P < 0.001, as determined by Student's t test). NAC forms a conditioning film through which adhesion of the bacteria is mediated. Cohesive failure in the NAC conditioning film is probably responsible for the detachment of cells from surfaces.

In the design of new antiadhesive coatings it is very important to consider both initial bacterial adhesion and the ease with which the initially adhered bacteria can be detached (6). Our results indicate that it might be hard to find a coating that has antiadhesive effects for all kinds of bacteria in their different growth phases. Due to this it is very important to be familiar with the system in which the coating or agents are applied.

We propose that the magnitude and the kinetics of frequency and dissipation shifts are dependent on the bacteria, the life cycle stage of the bacteria, and the surface. Apparently, the surface-sensitive QCM-D technique is an excellent tool for studying interactions and adhesion processes of bacteria. Exponentially grown cells induced a positive frequency shift as long as their CSH was zero. Moreover, for noncoated surfaces, larger numbers of exponentially grown and starved cells adhered to stainless steel surfaces, while larger numbers of B. subtilis spores adhered to polystyrene surfaces and equal numbers of B. cereus spores adhered to both surfaces. Coating with NAC increases the wettability of both stainless steel and polystyrene surfaces. Altogether, NAC-coated surfaces could decrease initial adhesion and change the viscoelastic properties of the binding between the cell or spore and the surface and increase the detachment of adhered cells and spores.

 
The Association of Swedish Chemical Industries financially supported this project, which is gratefully acknowledged.

Kristian Kvint is gratefully acknowledged for valuable comments during preparation of this paper.

 
* Corresponding author. Mailing address: Department of Cell and Molecular Biology-Interface Biophysics, Göteborg University, Box 462, 405 30 Göteborg, Sweden. Phone: 46 31 7732593. Fax: 46 31 7732599. E-mail: anki.olofsson{at}gmm.gu.se.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, May 2005, p. 2705-2712, Vol. 71, No. 5
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