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This study concerns the use of low-energy pulsed ultrasound as nondestructive photodynamic antimicrobial therapy for controlling dental plaque. We examined the antibacterial and bactericidal effects of low-energy pulsed ultrasound on mutans streptococci and its inhibitory effects on bacterial cell adhesion of Streptococcus mutans. The results indicated weak antibacterial and bactericidal effects. However, ultrasonic stimulation for less than 20 min markedly decreased bacterial cell adhesion. To analyze the mechanism underlying the inhibitory effect, we examined cell surface protein antigen C (PAc) and glucosyltransferase I (GTF-I) expression in S. mutans. The levels of PAc gene and protein expression were markedly decreased by ultrasonic stimulation for 20 min. However, no change in GTF-I expression was observed. The expression of stress response heat shock proteins GroEL and DnaK was also examined. GroEL and DnaK levels were significantly decreased by ultrasonic stimulation, and the expression of the PAc protein was also diminished upon the addition of GroEL or DnaK inhibitors without ultrasonic stimulation. These observations suggest that the expression of the PAc protein in S. mutans may be dependent on heat shock proteins. Thus, low-energy pulsed ultrasound decreases bacterial adhesion by the inhibitory effect on the PAc protein and heat shock protein expression and may be useful as photodynamic antimicrobial chemotherapy in controlling dental plaque.
The mutans streptococci Streptococcus mutans and Streptococcus sobrinus are believed to be the primary etiological agents of human dental caries, as many studies have demonstrated correlations between the presence of caries and elevated numbers of these organisms in dental plaque (25). In addition, experimental studies of animals have indicated the extreme cariogenic nature of these organisms (43, 47). Therefore, both species are believed to be highly cariogenic in dental plaque. Colonization of tooth surfaces by these microorganisms is the first step in the induction of dental caries. The colonization process is mediated by sucrose-independent and sucrose-dependent mechanisms (18, 19). The former mechanisms involve an interaction between bacterial cells and acquired pellicles on the tooth surfaces via the cell surface protein antigen C (PAc) or protein antigen G (PAg) in S. mutans and S. sobrinus, respectively (19, 30). The latter mechanisms are attributable to the synthesis of water-insoluble glucan from sucrose, catalyzed by glucosyltransferase (GTF) (22). To prevent dental caries, one must remove plaque containing mutans streptococci. However, it is difficult to remove plaque completely using conventional methods. Novel methods, such as the use of some chemical agents, laser irradiation, and both sonic and ultrasonic treatments, may be useful for controlling plaque (1, 2, 3, 20, 45, 46).
Therapeutic ultrasound, which has a long history of use as a therapeutic, diagnostic, and surgical tool (4, 5, 7, 38), uses sound waves to transfer mechanical energy to tissues and cells. The application of therapeutic and surgical ultrasound (1 to 300 W/cm2) generates considerable heat in living tissue and can homogenize tissues. In addition, the cells in the tissues are destroyed, proteins are denatured, and random fragmentation of DNA and RNA may occur (39). Low-intensity pulsed ultrasound (<100 mW/cm2), which is nonthermogenic and nondestructive, is widely used to accelerate bone growth during fracture healing and distraction osteogenesis (9, 33). Low-intensity pulsed ultrasound can accelerate osteogenic differentiation and the differentiation from progenitor cells of myoblasts to osteoblasts (12, 26, 40, 41, 42). The possible effects of low- and high-intensity ultrasound on tissues and cells include mechanical stress or production of free radicals due to ultrasound irradiation, which may be recognized as oxidative stress (14, 15, 32). Recently, the inactivation of pathogens through the production of the free radicals, termed photodynamic therapy, has been used in anticancer therapy (10, 29). Photodynamic antimicrobial chemotherapy on pathogenic microbes has also been reported (13, 21), and ultrasonic stimulation may be considered an appropriate photosensitizer (27). However, the appropriate parameters for ultrasonic stimulation in terms of power and applicable devices have not been determined.
Living microorganisms can adapt to diverse environmental conditions, such as carbon starvation or pH, mechanical, osmotic, oxidative, or heat shock stress, enabling survival under physiological stress. Heat shock proteins (HSPs), which act as stress proteins, are among the most highly conserved proteins in nature. First discovered in studies of thermal stress, HSPs can respond to other types of stress and have a number of important biological roles, e.g., as molecular chaperones and in protein homeostasis. HSPs are divided into families: HSP60 (approximately 60 kDa; GroEL) and HSP70 (approximately 70 kDa; DnaK) are identified mainly in bacteria (8, 11, 17). Ultrasonic stimulation is a form of environmental stress and may influence HSPs in photodynamic antimicrobial chemotherapy. However, there have been no reports to date on the association between ultrasonic stimulation and HSP expression.
To develop the use of low-energy ultrasound as nondestructive photodynamic antimicrobial chemotherapy for controlling dental plaque (28, 35), we tested the antibacterial effects of ultrasonic stimulation and inhibitory effects on bacterial adhesion and the colonization process. Furthermore, inhibitory effects on HSPs were also determined in mutans streptococci.
The intensity of ultrasonic stimulation was measured with a hydrophone (Toray Engineering), according to the American Institute of Ultrasound in Medicine and National Electrical Manufacturers Association standard (37). The intensity was determined as spatial-peak, temporal-average intensity (Ispta).
Streptococcus oralis ATCC 9811, S. mutans ATCC 25175, and S. sobrinus MT8145 were used. S. oralis and S. mutans were gifts from the Department of Microbiology, Nihon University School of Dentistry, Tokyo, Japan; S. sobrinus was a lab stock. Todd-Hewitt broth (Difco) was the medium used for all strains.
Bacteria were cultured aerobically in Todd-Hewitt broth at 37°C until late logarithmic phase. Each cell suspension was adjusted to obtain an optical density at a wavelength of 550 nm (OD550) of 0.1 in the broth. The adjusted cell suspension (2.7 ml) was stimulated with low-energy pulsed ultrasound using an ultrasonic transducer (prototype; Asahi Irika) at 1.6 MHz and 3.0 to 12.0 V in pulsed-wave mode. The ultrasonic probe was a flat and rectangular solid (8 by 3 by 1 mm) to allow for diffusion of the ultrasonic energy in the medium. The bacteria were incubated aerobically at 37°C for 6 h, and growth was determined by measuring the absorbance at 550 nm. The cells were stimulated continuously with the ultrasound during the incubation period. All test runs were performed in triplicate.
Next, the bactericidal effects of ultrasonic stimulation on streptococci were determined. After stimulation of the cells with low-energy pulsed ultrasound at 9.0 or 12.0 V, 10-fold dilution series of the bacterial suspensions were prepared and then immediately plated on brain heart infusion agar (Difco) plates. The plates were incubated aerobically at 37°C for 48 h, and the bactericidal effect was evaluated based on colony formation. Tests were performed in triplicate. Statistical significance was determined using Scheffe's method, and a P value of <0.05 was considered statistically significant.
S. mutans and S. sobrinus were precultured until late logarithmic phase and adjusted to an OD550 of 1.0 using fresh medium. Aliquots of 100 μl of the bacterial suspension of each strain were transferred to 96-well sterile polystyrene U-bottomed microtiter plates and incubated aerobically at 37°C for 12 h. After being washed twice with phosphate-buffered saline (PBS), the adherent bacteria in 200 μl of PBS were stimulated with low-energy pulsed ultrasound and a small circular probe (diameter, 3 mm; height, 1 mm; Asahi Irika) (40, 41) at 1.6 MHz and 9.0 V for 30 min. The bacteria were stained with 1% crystal violet for 1 min and washed three times with PBS. The absorbance was determined with a plate reader at 575 nm (n = 7) (44). Statistical significance was determined using Scheffe's method, as described previously.
After S. mutans and S. sobrinus were cultured until late logarithmic phase and then adjusted to an OD550 of 1.0, the bacterial suspensions (2.7 ml) were treated with ultrasonic stimulation at 1.6 MHz and 9.0 V for 20 min using the ultrasonic stimulator. The bacteria were rinsed with PBS and then exposed to lysis buffer consisting of 50 mM Tris-HCl, 0.1% Triton X-100, 0.1 mM EDTA, and a protease inhibitor cocktail. Cells in the lysis buffer were sonicated three times for 20 s. The concentration of total protein was constant by the Bradford method (data not shown). Aliquots containing equal amounts of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer were subjected to SDS-PAGE and Western blotting. Silver staining confirmed the equal loading of protein on the gels (data not shown). Western blotting was performed using rabbit polyclonal anti-PAg protein and anti-GTF-I (glucosyltransferase I) antibodies derived from S. sobrinus (16) and horseradish peroxidase-conjugated secondary antibody (GE Healthcare). Immunoreactive proteins were visualized using a chemiluminescence kit (GE Healthcare) and exposed to X-ray film. To confirm the equal loading of total protein, we stained the gels with a silver staining kit (Wako Chemical).
S. mutans and S. sobrinus were treated with ultrasonic stimulation as described previously. Bacterial total RNA was isolated using an easy-Red bacterium/yeast/fungus (BYF) total RNA extraction kit (iNtRON Biotechnology), according to the manufacturer's instructions. The concentration of total RNA was measured with a spectrophotometer (260/280 nm). Aliquots containing equal amounts of total RNA were subjected to reverse transcription and real-time PCR analysis. RNA (0.5 μg/reaction) was reverse transcribed using a You-Prime first-strand kit and random hexamers (GE Healthcare) at 42°C for 60 min. Real-time PCR was performed using 1.0 μl cDNA template, TaqMan universal PCR master mix (Roche), and the appropriate primer/probe set (Table (Table1)1) for the target genes pac and gtfI for S. mutans or pag for S. sobrinus. Primer/probe sets were designed using Primer Express software (Applied Biosystems). Finally, 16S rRNA was used as an internal control. PCR amplification was performed in capped 96-well optical plates. The reaction conditions were as follows: 5 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The gene-specific PCR products were measured continuously using the ABI Prism 7700 detection system (Applied Biosystems). Samples were normalized against the internal control, and data represent the means ± standard deviations from three experiments. Statistical significance was determined using Scheffe's method, and a P value of <0.05 was considered statistically significant.
To determine whether the inhibition of the PAc protein was dependent on HSP expression, the PAc protein level and the bacterial cell adhesion in S. mutans after treatment with 0 to 8 mM ETB (C24H33NO6, GroEL inhibitor; Wako Chemical) for 60 min or 0 to 600 mM N-formyl-3,4-methylenedioxy-benzylidine-γ-butyrolactam (DnaK inhibitor; Calbiochem-Novabiochem International) for 60 min were examined. SDS-PAGE, Western blotting, and the experiment for the bacterial adhesion were followed as mentioned above.
The sound intensity (Ispta) of the ultrasound used in this study was 64 to 1,100 mW/cm2 (Table (Table2).2). The Ispta at 6.0, 9.0, and 12.0 V was higher than 100 mW/cm2 and exceeded the power of low-intensity pulsed ultrasound (40, 41, 42).
After being stimulated with ultrasound at 610 or 1,100 mW/cm2 (9.0 or 12.0 V), the streptococci did not grow during incubation (P of <0.05 compared with control) (Fig. (Fig.1A).1A). The growth of the bacteria exposed to ultrasonic stimulation at 64 or 270 mW/cm2 (3.0 or 6.0 V) and the controls reached stationary phase, and there were no significant differences among the groups at any time point (P > 0.05). S. mutans in medium containing 1% sucrose was stimulated at 610 mW/cm2 (9.0 V) and did not grow during incubation (Fig. (Fig.1B),1B), whereas the controls grew until stationary phase. The bactericidal effects of low-energy pulsed ultrasound at 610 or 1,100 mW/cm2 (9.0 or 12.0 V) were significantly (P < 0.01) greater than those in control cultures, as follows: 99.0% for S. oralis and S. mutans stimulated for more than 3 h and S. sobrinus stimulated for 6 h (Fig. (Fig.2).2). There were no differences in the effect between ultrasound at 610 and 1,100 mW/cm2 (P > 0.05). These data suggest that low-energy pulsed ultrasound treatment would be needed for a long time to reduce bacterial growth.
The bacterial adhesion of S. mutans and S. sobrinus decreased in a time-dependent manner (Fig. (Fig.3A).3A). Low-energy pulsed ultrasound treatment at 610 mW/cm2 (9.0 V) for 20 min inhibited the adhesion of S. mutans and S. sobrinus by approximately 60% and 50%, respectively (P < 0.01). Next, the gene and protein expression levels of PAc and GTF-I and those of PAg were determined in S. mutans and S. sobrinus, respectively. The gene and protein expression levels of PAc and PAg were markedly reduced in a time-dependent manner by low-energy pulsed ultrasound stimulation (Fig. 3B and C). However, the GTF-I protein and gene expression levels were unchanged by ultrasonic stimulation (P > 0.05). These observations suggest that low-energy pulsed ultrasonic stimulation for less than 20 min can decrease bacterial adhesion and that both protein and gene expression levels of PAc and PAg are related to bacterial adhesion.
HSP60 and HSP70, GroEL and DnaK in S. mutans, are stress response proteins (23). We hypothesized that low-energy pulsed ultrasonic stimulation represents an environmental stress against bacteria. GroEL and DnaK protein and gene expression levels were decreased in a time-dependent manner by low-energy ultrasound stimulation (Fig. 4A and B), and the temperature of the medium stimulated with ultrasound did not change (data not shown). These observations suggest that low-energy pulsed ultrasonic stimulation—and not heat shock stress—may be an environmental stress in S. mutans.
The addition of either of the HSP inhibitors markedly decreased PAc protein expression in a dose-dependent manner (Fig. (Fig.5A)5A) but did not affect the total protein level of S. mutans (data not shown). The bacterial adhesion of S. mutans was decreased in a dose-dependent manner by approximately 70% (P < 0.01) by the addition of either of the HSP inhibitors (Fig. (Fig.5B),5B), suggesting that the adhesion of S. mutans via PAc protein expression may be dependent on the HSPs GroEL and DnaK.
We examined the use of low-energy pulsed ultrasound as photodynamic antimicrobial chemotherapy. Our results demonstrate that ultrasonic stimulation has antibacterial effects, inhibitory effects on bacterial adhesion, and effects on HSP expression in mutans streptococci. Low-energy pulsed ultrasound had weak antibacterial and bactericidal effects on S. oralis, S. mutans, and S. sobrinus. However, ultrasound exposure markedly inhibited bacterial adhesion and downregulated not only PAc and PAg protein expression levels, which are related to bacterial adhesion, but also the protein levels of the HSPs GroEL and DnaK. Furthermore, PAc levels were decreased by the addition of HSP inhibitors, suggesting that PAc expression may be dependent on GroEL and DnaK. Thus, low-energy pulsed ultrasound may decrease bacterial adhesion via its inhibitory effects on PAc and HSP expression. This is the first study to demonstrate the effects of low-energy pulsed ultrasound as photodynamic antimicrobial chemotherapy and to elucidate part of the biological mechanisms of bacterial adhesion in S. mutans.
Controlling dental plaque involves removing microbial plaque and preventing its accumulation on the teeth and adjacent gingival surfaces (28, 35). The removal of dental plaque is required for the treatment and prevention of dental caries and gingival inflammation. Stimulation with low-energy pulsed ultrasound may reduce the accumulation of dental plaque through the colonization of mutans streptococci because of its inhibitory effects on bacterial adhesion. Low-energy ultrasound inhibits plaque accumulation, although it has weak antibacterial effects.
Low-energy pulsed ultrasound stimulation decreased PAc and PAg expression at both the gene and protein levels, but no change was observed in gtfI gene expression. The PAc and PAg proteins play important roles in the interactions between bacteria and acquired pellicles on the tooth surface in a sucrose-independent manner; they are needed in the first phase of colonization (18, 19). In contrast, GTF-I is related to the synthesis of water-insoluble glucan from sucrose, and the next phase involves the maturation of dental plaque by its catalytic ability. Although further studies are required, our observations suggest that ultrasound stimulation may be more effective in the first phase of colonization.
Ultrasonic stimulation is one form of mechanical stress, and the sensors at the surface of mammalian cells are called mechanosensors (31, 34). However, there have been no previous molecular biological reports regarding mechanosensors, and we did not detect such sensors in S. mutans. We hypothesized that ultrasonic stimulation may influence the expression of HSPs, which play a central role in tolerance of environmental stresses. HSPs also participate in a variety of cellular processes, including protein folding, protein translocation, and both the assembly and disassembly of protein complexes in Escherichia coli (6, 36). Moreover, they act as stress response proteins and work to regulate various cellular processes in S. mutans (23, 24). The levels of GroEL and DnaK expression were significantly decreased; thus, the expression of stress response proteins was disrupted by ultrasonic stimulation, and a variety of cellular processes, including bacterial adhesion, may have been downregulated. To confirm the relationship between HSPs and the PAc protein, we examined the protein levels of PAc with the addition of GroEL and DnaK inhibitors. Bacterial adhesion and the expression levels of PAc were significantly decreased by these inhibitors; thus, the GroEL and DnaK proteins might be associated with the PAc protein and may be involved in upstream signal transduction. The PAc protein level should be examined in a HSP mutant or disrupted strain of S. mutans to confirm the relationship between HSP and the PAc protein, and further studies are required to determine the signal transduction pathway related to bacterial cell adhesion.
In summary, the present study suggests that low-energy pulsed ultrasound inhibits bacterial cell adhesion and downregulates PAc protein levels in S. mutans via the HSPs GroEL and DnaK. Therefore, ultrasonic stimulation may decrease the colonization and maturation of dental plaque and may be a useful method of photodynamic antimicrobial chemotherapy for controlling dental plaque. Further biological and clinical studies are needed.
This study was supported by the Promotion and Mutual Aid Corporation for Private Schools of Japan, the Uemura Fund, and a grant from the Dental Research Center of Nihon University School of Dentistry.
Published ahead of print on 18 December 2009.