BioChip J. (2010) 4(4): 257-263 DOI 10.1007/s13206-010-4401-8
Original Research
Environmental factors that affect Streptococcus mutans biofilm formation in a microfluidic device mimicking teeth Wahhida Shumi1, Jeesun Lim2, Seong-Won Nam2, Kangmu Lee2, So Hyun Kim2, Mi-Hyun Kim3, Kyung-Suk Cho1 & Sungsu Park2
Received: 29 July 2010 / Accepted: 27 September 2010 / Published online: 20 November 2010 � The Korean BioChip Society and Springer 2010
Abstract Streptococcus mutans is the primary etiological agent responsible for dental caries. Microfluidic devices have been used to provide a level of control over bacterial microenvironments under laminar flow conditions. In this study, we used a microfluidic device packed with glass beads to simulate the interproximal space, which is the space between the teeth. In the device, the effects of environmental factors, such as sucrose and metal ions, on S. mutans attachment and biofilm formation were quantitatively measured using confocal laser scanning microscopy and atomic force microscopy. We determined that sucrose was required for both bacterial attachment and exopolysaccharide (EPS) production in S. mutans. These results suggest that the in vivo condition between the teeth was successfully mimicked and that the device is highly suitable for in situ monitoring of oral biofilms. Keywords: Streptococcus mutans, Microfluidic device, Teeth, Biofilm, Environmental factors
Introduction Oral biofilm is a dental plaque that consists of bacterial populations and insoluble glucans. The adherence of Streptococcus mutans to the tooth surface is a major factor in the development of dental plaque because S. 1
Department of Environmental Science and Engineering, Ewha Womans University, Seoul 120-750, Korea 2 Department of Chemistry and Nano Science (BK21), Ewha Womans University, Seoul 120-750, Korea 3 Department of Biological & Chemical Engineering, Yanbian University of Science & Technology, Yanji City 133000, China Correspondence and requests for materials should be addressed to S. Park (
[email protected])
mutans initiates the formation of plaques1-5. Once S. mutans adheres to the surface, it follows the typical developmental stages of biofilms; the accumulation of exopolysaccrides (EPS), the maturation of the biofilm and the dispersion of cells to form new colonies6,7. The formation and maturation of S. mutans biofilms are greatly affected by environmental factors, such as nutrients, pH levels, shear rate, etc8-10. For example, S. mutans in the presence of sugar promotes the biosynthesis of insoluble glucans, which causes the bacteria to firmly adhere to the tooth surface11,12. The intake of fermentable sugars also induces a rapid decrease in the pH of dental plaques from neutrality to pH 5.0 or below13,14. Due to the medical importance of dental plaques, several biofilm assays have been developed to monitor the effects of environmental factors on the formation of S. mutans biofilms on the tooth surface15-18. Although the microliter plate-based assay is an accurate and convenient method for the quantification of EPS produced by bacteria attached to the surface, it is only useful for the quantification of EPS produced by bacteria under static or semi-static conditions. To solve this problem, biofilm assays using flow systems have been developed to simulate the dynamic flow conditions in the oral cavity. Most of these flow systems use stimulated saliva flow rates (1 to 2 mL/min), while biofilm formation occurs mostly at un-stimulated saliva flow rates (0 to 0.6 mL/min)19. Microfabricated microfluidic systems have the ability to provide a level of control over the biological cell culture microenvironment that cannot be achieved under traditional culture conditions such as plastic plates20,21. Microfluidic systems can produce confined and well-defined systems on the cellular length
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scale22,23, which can incorporate complex designed topographies, densities of extracellular matrix signaling molecules, nonrandom organization of cells of different types, and the ability to mimic in vivo flow18,23,24. Previously, we reported on a glass bead-packed microfluidic system for the nanoscale investigation of biofilms formed under laminar flows. This previous study was conducted because biofilm formation on glass beads packed in the microfluidic device can be easily removed and located for imaging under an atomic force microscope (AFM) and scanning electron microscope (SEM)18. In this study, we used a microfluidic device packed with glass beads to simulate the proximal surfaces of adjoining teeth. The effects of incubation time, sucrose concentration and divalent metal ion concentration on the formation of S. mutans bio2
Biofilm absorbance (595 nm)
24 h 48 h
1.6
films attached to the glass beads were monitored at a flow rate of 10 μL/min, which simulates the un-stimulated flow rates found in the human mouth.
Results and Discussion Biofilm formation under static conditions
To determine whether S. mutans can form biofilms under a static condition, a biofilm assay was performed in 96 well microtiter plates. Our results demonstrated that S. mutans was able to form biofilm only in the presence of sucrose (Figure 1). We also found that the biofilm forming capability of the microorganism was greatly enhanced by sucrose. At 10 mM sucrose, maximum growth of biofilm was observed. Since there was no significant difference in EPS production between 24 hr-incubated and 48 hr-incubated bacteria in the microtiter plates with or without sucrose, S. mutans appeared to form mature biofilms within 24 hrs under a static condition, irrespective of sucrose concentration.
1.2
Effects of sucrose on bacterial attachment and EPS production under the laminar flow condition
0.8
0.4
0
0
10
100
1000
Sucrose concentration (μM)
Figure 1. Biofilm formation by S. mutans under the static condition in the absence or presence of various concentrations (10 to 1,000 μM) of sucrose. Each bar in the graph represents a mean value with the standard deviation.
A
B
The adherence of S. mutans to the tooth surface and the formation of dental plaque are of significanceto the development of dental caries1,2. To determine the effects of sucrose on the initial attachment of S. mutans to the glass surface, the bacterial culture in TSB (trypticas soy broth) was mixed with various concentrations (0 to 1 mM) of sucrose and the mixtures were flown at 10 μL/min for 3 hr in the microfluidic channels packed with glass beads. AFM was then used to obtain images of the cells attached to the beads rather than
C
Figure 2. AFM images showing the surface of the glass beads that were in the microchannel in which S. mutans cells were flown in TSB at 10 μL/min with or without sucrose for 3 hr. (A) The surface of a glass bead with no bacterial attachment in TSB with no sucrose. The particles shown here are crystallized salt. (B), (C) The surfaces of the glass beads with bacterial attachment in TSB supplemented with 100 μM and 1 mM sucrose, respectively.
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CLSM because the later is not suitable for obtaining images of a small number of bacteria cells. S. mutans cells were not able to adhere to the surface of glass beads in the absence of sucrose (Figure 2A); however, they were able to adhere to the beads in the presence of sucrose (Figure 2B and 2C). This indicates that the attachment of S. mutans to the beads requires sucrose.
A
B
50 μm 50 μm
C
D
S. mutans uses several carbohydrates, including sucrose, for the production for EPS, which facilitates bacterial adhesion7,25. In addition, sucrose promotes bacterial adhesion as well as EPS production in S. mutans. After 24 hr-incubation in the microchannel, the adherent cells did not produce any detectable EPS in the absence of sucrose (Figure 3A). However, the cells on the beads started to produce a detectable amount of EPS at 10 μM sucrose (Figure 3B), demonstrating that sucrose is necessary for S. mutans to produce EPS. EPS production increased with an increase in sucrose concentration in TSB, as shown in Figure 3C and 3D. EPS initiates the development of caries in the teeth by lowering the local pH during fasting. Collectively, our results suggest that sucrose is required for EPS production in S. mutans under laminar flow conditions and should be removed from the oral cavity in order to inhibit the formation of caries. Effect of divalent metal ions on S. mutans biofilm under laminar flow condition
Figure 3. Effect of sucrose on EPS production in S. mutans cells attached to the glass beads. CLSM images show EPS stained with TMR-ConA (red). (A) The glass beads were from the microchannel that was subjected to a flow of S. mutans in TSB with no sucrose supplement for 24 hr. (B-D) Glass beads from the microchannel that was subjected to a flow of S. mutans in TSB with 10, 100 and 1,000 μM sucrose for 24 hr, respectively.
Previously, we showed that under iron-repleted conditions, S. mutans produced a lower amount of EPS and thus formed smaller biofilms compared to iron-depleted conditions26. In this study, we determined that other divalent metal ions also have inhibitory effects against biofilm formation in S. mutans. As shown in Figure 4, the divalent metal ions inhibited biofilm formation as well. These results suggest that divalent metal ions, including ferric ions, can be used to inhibit the development of caries. Long-term monitoring of S. mutans biofilm under the laminar flow condition
To determine the effects of incubation time on S. mu-
35
Light intensity (595 nm)
30 25 20 15 10 5 0 C
10
100 ZnCl2
200
10
100 CaCl2
200
10
100 MgCl2
200
10
100
200
MnCl2
Divalent metal ion concentration (μM)
Figure 4. Effect of divalent metal ions on S. mutans biofilm. Control condition in which there was no divalent metal ions in TSB.
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Table 1. Production of live cells, EPS, total biomass, thickness, and surface coverage of the biofilm developed by S. mutans during the various incubation periods. Light intensity (A.U.)
Incubation periods (h)
Live cells
EPS
Total biomass
Thickness (μm)
Surface coverage (%)
0 12 24 36 48 60 72
0.9±0.6 6.3±0.2 31.6±0.2 68.4±3.1 68.5±0.1 67.2±2.1 84.2±4.9
0.1±0.1 7.5±0.4 22.1±1.8 41.2±3.9 57.6±2.4 78.2±7.1 97.4±2.3
1.0±0.6 3.9±2.8 5.7±1.3 9.4±6.4 14.5±4.7 15.8±2.6 31.3±2.8
0±0.2 0.1±0.6 3.7±2.5 41.9±2.1 76.8±5.1 85.9±3.2 97.3±2.2
2.0±0.2 8.2±0.7 10.8±0.9 22.5±0.7 37.2±0.2 55.9±3.5 91.5±2.8
A
B
C
D
G
H
50 μm
E
F
Figure 5. Long-term monitoring of the development of S. mutans biofilms under laminar flow conditions. (A) Control glass beads (optical image). (B-H) Live cells attached to the glass beads after 0, 12, 24, 36, 48, 60 and 72 hours, respectively. Live cells stained with SYTO16 were observed under CLSM.
tans biofilm formation, TSB containing 1 mM of sucrose was flown into the microchannel containing glass beads with S. mutans for various lengths of time (072 hr). As presented in Figure 4, the number of live cells attached to the glass beads increased with an increase in incubation time, indicating that S. mutans biofilms were well adapted to the laminar flow condition. This is in contrast with the results (Figure 5) observed in the microtiter plates, in which incubation times over 24 hr did not affect S. mutans biofilm formation. The biofilm data (Table 1) supports the notion that S. mutans is able to form and develop biofilms under laminar flow conditions that mimic in vivo conditions. Taken together, these results suggest that the microfluidic device packed with glass beads is an
appropriate model to study biofilm formation by S. mutans on a tooth surface. In addition, this device can be utilized to study the transitional processes of oral biofilm development in situ.
Conclusions Since S. mutans is the primary etiological agent of dental caries, we used a microfluidic device packed with glass beads to study S. mutans attachment and biofilm formation under conditions that mimic in vivo conditions. Unlike under static conditions, S. mutans produced significant amount of EPS and readily formed biofilms under the laminar flow condition. Thus,
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Outlet
Inlet
S. mutans Glass bead
Figure 6. Microfluidic device packed with glass beads for oral biofilm formation.
the microfluidic device developed in this study is a suitable laboratory tool to study biofilm organisms.
Materials and Methods Bacterial strain and culture condition
The bacterial strain used in this study was S. mutans 3065, which was obtained from ATCC (Manassas, VA, USA). The strain was preserved in a brain heart inclusion (BHI) agar plate to maintain its activity. The preculture of S. mutans was prepared by inoculating the single colony from the BHI agar plate into 5 mL TSB (trypticase soy broth) and incubating at 37� C and 220 rpm overnight. The planktonic growth rates of the strain were measured by calculating the optical density at 600 nm. Changes in pH were monitored during both the planktonic and biofilm stages using a pH meter (pH 56, Milwaukee Instruments, Inc., Rocky Mount, NC, USA). Biofilm formation under a static condition
The biofilm assay was performed following a previously reported method18 with some modifications. Briefly, the pre-culture was mixed with fresh TSB with or without sucrose at a 1 : 20 ratio. Two hundred microliters of the diluted culture were inoculated into each well of polystyrene 96 well microtiter plates and sealed with parafilm. The plates were then incubated at 25� C for 24 or 48 hr. After this incubation step, the plates were washed three times with 200 μL of triple distilled water (TDW) and dried in air for 20 min. Next, the biofilm was stained for 20 min with 0.1% crystal violet (wt/vol). Excess dye was washed with TDW by suction and dried in air for 20 min. 200 μL of 95% ethyl alcohol was added to the plates and the plates were incubated for 30 min. The absorbance of each well was then measured at 595 nm. Each data bar represents the mean of six replicate wells, and the standard deviations were calculated.
Fabrication of a microfluidic device and immobilization of glass beads
The microfluidic device18 consisted of a microchannel and a 1 cm square microchamber with an array of micropillars for the immobilization of glass beads (as shown in Figure 6). The microchamber contained an array of 200 μm square micropillars with a 100 μm gap size. The SU-8 master containing microfluidic device pattern was fabricated using a standard soft lithographic technique27. Once the master was constructed, the surface of the SU-8 replica master was treated with the vapor of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-11-trichlorosilane for 15 min to passivate the surface. A mixture of PDMS (polydimethylsiloxiane, polymer) and Sylgard 184 (Dow-corning, MI, USA) at a 10 : 1 ratio was prepared by mixing thoroughly with stirring and poured onto the SU-8 master. The master containing the mixture was put in a vacuum chamber for 15 min. After removal, the master was kept in air at room temperature for 15 to 20 min for degassing. Finally, the device was cured for 1 h at 80� C in an oven. The PDMS replica was peeled off and holes were punched out for fluidic connections using a steel punch. The interfacing surfaces (hardened surfaces of the PDMS replica and the slide glass) were treated with an oxygen plasma treatment (Cute model, FEMTO Science, South Korea) at 50 W for 40 sec prior to assembly to facilitate permanent covalent bonding. Silicon tubes were inserted into the ports to introduce the samples. A syringe pump (KD scientific, Holliston, MA, USA) was connected through the tubing, which could continuously provide media into the channel. Packing of glass beads into the microchannel was done by manually flowing a slurry of glass beads (diameter: 150212 μm, SIGMA) into the channel using a syringe via the inlet. Bacterial inoculation in the microchannel and studying the effects of environmental factors on biofilm formation
The microchannel was washed twice with fresh TSB and then filled with the pre-culture. The device was incubated for 30 min at room temperature to allow some cells in the microchannel to attach to the glass surface. After the cells attached, an air tight syringe pump was connected to the device through the inlet. Once S. mutans cells were attached to the glass beads, the effects of environmental factors, such as sucrose concentration and incubation time, were examined as described below. To investigate the effect of sucrose on biofilm formation, fresh TBS with or without sucrose was flown into the microchannel at 10 μL/ min and maintained at room temperature for various
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hours. To explore the effect of the concentration of divalent ions on biofilm formation, each divalent ion (ZnCl2, CaCl2, MgCl2 and MnCl2) at various concentrations (10, 100 and 200 μM) was added to fresh TSB and TSB containing the metal ions were flown into the microchannel at 10 μL/min and kept at room temperature for various time periods. Imaging of S. mutans biofilms in the microchannel
To stain EPS produced by S. mutans cells in the microchannel, unbound bacterial cells were first removed by flowing PBS (pH 7.4) at 10 μL/min into the channel for 10 min. EPS was then stained by filling the microchannel with tetramethylrhodamine-conjugates (20 μg/mL) of conconavalin A (TMR-ConA: excitation and emission at 557 and 576 nm) obtained from Invitrogen (Carlsbad, CA, USA) and by incubating for 30 min in the dark at room temperature. Excess dye was washed out by flowing PBS into the channel for 30 min. To stain live S. mutans cells in the channel, unbound cells were removed by the method described above and 20 μM SYTO16 green fluorescence nucleic acid stain (excitation and emission at 488 and 518 nm) was introduced into the channel and incubated for 30 min in the dark at room temperature. Excess stain was removed as described above. Fluorescent images of the biofilms were obtained using confocal laser scanning microscopy (CLSM) (LSM 510, Zeiss). Digital image analysis of the CLSM thin sections was performed using the Zeiss LSM software. To quantify the number of viable cells, fluorescence intensities from the confocal images were analyzed using the Image J program (NIH, USA). The fluorescence intensity of light was measured in arbitrary units (A.U.). Prior to AFM imaging, TSB and unbounded cells in the microchannel were washed by flowing PBS into the channel using the syringe pump. After the top PDMS layer was cut out using a razor blade, the beads were carefully taken out and dried for a few hours in air. AFM images were obtained in air with a Nanoscope IV Bioscope (Veeco Digital Instruments, Plainview, N.Y., USA). All images were collected in contact mode using sharpened silicon nitride cantilevers with spring constants of 0.02 N m-1 and a tip radius of ⁄20 nm. Height and deflection images were simultaneously acquired at a scan rate of 1 Hz. To accurately represent the topography of the sample surface, all quantitative cellular measurements were taken from the height data corresponding to the deflection mode images. Acknowledgements
This research was supported equally by the programs (2010-0020772 and R01-2008-000-
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20593-0) through the National Research Foundation (NRF) in Korea and the grant (10816) from the Seoul Research and Business Development Program. W. Shumi was supported by EGPP (Ewha Global Partnership Program).
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