Antimicrobial Activity and Biofilm Formation Inhibition of Green Tea ...

Biotechnology and Bioprocess Engineering 15: 359-364 (2010) DOI 10.1007/s12257-009-0195-8

RESEARCH PAPER

Antimicrobial Activity and Biofilm Formation Inhibition of Green Tea Polyphenols on Human Teeth Yun-Seok Cho, Jay Jooyoung Oh, and Kye-Heon Oh

Received: 3 August 2009 / Revised: 5 October 2009 / Accepted: 8 October 2009 © The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract The antimicrobial effects and biofilm formation Keywords: green tea polyphenols, antimicrobial activity,

inhibition of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L) were evaluated against 12 oral microorganisms. Effective antimicrobial activity against all microorganisms tested, including Lactobacillus spp. (Lactobacillus acidophilus and Lactobacillus plantarum), Streptococcus spp. (Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus, Streptococcus mitis, and Streptococcus salivarius), Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, and Candida albicans, was shown at 2,000 µg/mL TPP within 5 min of incubation. Scanning electron microscopy (SEM) analysis revealed various morphological changes, such as the presence of perforations, the formation of cell aggregates, and the leakage of cytoplasmic materials from cells treated with TPP, depending on the bacteria. The potential role of TPP in biofilm formation inhibition on human teeth was evaluated in BHI broth with 2 mixed strains of S. mutans and S. sanguis. SEM analysis showed biofilm formation on the surface of a tooth shaken only in saline solution, whereas almost no biofilm was observed on a tooth incubated in TPP solution. This result suggests that TPP is effective against adherent cells of S. mutans and S. sanguis. Thus, TPP would be useful for development as an antimicrobial agent against oral microorganisms, and has great potential for use in mouthwash solutions for the prevention and treatment of dental caries. Yun-Seok Cho, Kye-Heon Oh* Department of Biotechnology, Soonchunhyang University, Chung-Nam 336-600, Korea Tel: +82-41-530-1353; Fax: +82-41-530-1493 E-mail: [email protected] Jay Jooyoung Oh Program of Microbiology, Indiana University, Bloomington, IN 47402, USA

oral microorganism, dental care

1. Introduction The oral cavity is perhaps the most complex and heterogeneous microbial habitat in the human body. Thus, it is no surprise that oral health is being increasingly linked with other conditions such as heart disease, pregnancy, and stroke [1,2]. The oral cavity (e.g. teeth, gums, inner cheek linings, and tongue) also contains some of the more common species of bacteria (e.g. streptococci, lactobacilli, staphylococci, and micrococci). Most of these oral microorganisms are either facultative or obligate anaerobes, and bad breath, which is caused by various oral bacteria, is primarily produced by the sulfur-based odorous waste products of these microorganisms [3-7]. Recently, oral microbiologists have identified the specific types of bacteria that are most damaging to the cardiovascular system, and reported a relationship between gum disease and the risk of heart attack [8-10]. The production of acids by oral bacteria is significant in the formation of dental caries. There is a direct correlation between the number of acid producing bacteria found in the oral cavity and the incidence of dental caries [11]. Two representative organisms that have been implicated in dental caries are S. sobrinus and S. mutans. S. sobrinus is able to colonize the smooth surfaces of teeth, while S. mutans is found predominantly in crevices and small fissures [12]. Until now, these oral microorganisms have been controlled by synthetic chemicals, which accompany possible side-effects. Thus, it is of great interest to develop natural and non-toxic mouth wash solutions, which can effectively act against oral microorganisms that cause bad breath, plaque, and the gum disease gingivitis. Consumption of Green tea (Camellia sinensis L.) can

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Biotechnology and Bioprocess Engineering 15: 359-364 (2010)

help relieve stress and allow one to appreciate unique flavors due to its comparative lack of caffeine. Green tea contains tea polyphenols (TPP), which are natural substances and beneficial for human heath [13]. Green tea has been reported to potentially have anti-cancer, anti-oxidant, anti-aging, anti-microbial, and anti-dental caries properties [11,14-16]. The antibacterial activity of green tea extracts against cariogenic streptococci and other harmful mouth flora has been reported [17-19]. In this study, we evaluated the antimicrobial activity of TPP against 12 oral microorganisms. The survival rates of the microbes were determined by exposure to various concentrations of TPP. Further, scanning electron microscopy (SEM) analysis was performed under lethal concentrations of TPP to examine the morphological changes that occur in the cell envelope following TPP exposure. Finally, the inhibition of biofilm formation on human teeth treated with TPP solution was investigated and examined by SEM. 2. Materials and Methods

2.1. Microorganisms and culture media

The microorganisms and complex, selective, and differential culture media used in this study are shown in Table 1. All strains were maintained routinely on LB agar. Incubation before a series of tests was performed aerobically on proper media at 35oC for 24~48 h.

2.2. Green tea polyphenols

Tea polyphenols (TPP, purity >97%) extracted from the leaf of Korean green tea (Camella sinensis L.) were obtained from COSIS Co. (Cheonan, Korea). High-performance liquid chromatography (HPLC) analysis of catechins contained in List of microorganisms and media Microorganisms L. acidophilus ATCC 4356 L. plantarum ATCC 8014 S. mutans ATCC 25175 S. sanguis ATCC 10556 S. sobrinus ATCC 27607 S. mitis ATCC 9188 S. salivarius ATCC 13419 S. aureus ATCC 12600 N. meningitidis ATCC 13077 E. coli ATCC 9637 E. cloacae ATCC 13047 E. faecalis ATCC 29212 C. albicans ATCC 90028

TPP was performed. Stock dilutions were prepared in 0.15 mM H3PO4 to avoid oxidation.

2.3. HPLC analysis of TPP

TPP (purity >97%) extracted from the leaf of Korean green tea was analyzed by HPLC. For HPLC analysis, a Zorbax ODS reverse-phase column (5 µm, 250 mm × 4.6 mm, and particle size 5 µm) was eluted with mobile phases, which were mixed with solution A (2% (v/v) acetic acid) and solution B (15% (v/v) acetonitrile) [25]. The flow rate of the mobile phase was 1.0 mL/min and the injection volume was 10 µL. TPP solution at a concentration of 2,000 µg/mL was prepared for further analyses.

2.4.

In vitro

antimicrobial activity of TPP

Cells grown on LB broth were harvested by centrifugation at 2,000 g for 10 min. These cells were washed 3 times with 0.85% physiological saline. After centrifugation, the cells were formed into pellets and then the killing effect against the target microorganisms (initial cell density of approximately 104 cells/mL) was determined in 2,000 µg/ mL TPP solution based on the >90% reduction in viable cell count within 5 min. After exposure for an adequate period, the vital cells were counted by enumeration of colony-forming units (CFU) on complex, selective, and differential agar media.

2.5.

In vivo


The killing effects of 2,000 µg/mL TPP solution against oral microorganisms in the human mouth were examined with the participation of 25 individuals, who gargled 20 mL of TPP solution for 3 min after meals. The gargled and rinsed samples were collected, spread onto different selective and differential solid media, incubated for 24~48

Table 1.

Culture Media* deMan, Rogosa, and Sharpe (MRS) agar deMan, Rogosa, and Sharpe (MRS) agar Brain heart infusion (BHI) agar Brain heart infusion (BHI) agar Brain heart infusion (BHI) agar Mitis-salivarius Mitis-salivarius Mannitol salts agar Brain heart infusion (BHI) agar Eosin-methylene blue agar LB agar LB agar BiGGY agar

*All media were purchased from Difco Co. (Sparks, MD, USA).

References [20] [20] [21] [21] [21] [22] [22] [22] [21] [23] [24] [24] [22]

Antimicrobial Activity and Biofilm Formation Inhibition of Green Tea Polyphenols on Human Teeth

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h, and CFU were enumerated on the media.

2.6. Biofilm formation on human teeth

Human tooth samples were placed in BHI broth inoculated with mixed cultures of S. mutans and S. sobrinus. For 3 min, 3 times a day, the 2 teeth samples were removed from their cultures and one was shaken in TPP solution while the other one was shaken in physiological saline. This was repeated for 4 days, after which biofilm formation on the surface of the teeth was examined with SEM.

2.7. SEM analysis

Colonies of each microorganism grown on LB agar plates for 24 h were excised on small agar blocks of 0.5 cm3. The blocks were then exposed to TPP solution for 5 min at 37oC. The colonies treated with TPP were fixed, dehydrated, coated with gold, and examined with a Hitachi S-2500C scanning electron microscope (Hitachi Co., Japan), as described previously [26,27]. Samples of human teeth treated periodically with physiological saline or TPP solutions were also prepared for SEM analysis according to methods described above. 3. Results

3.1. HPLC analysis of TPP

HPLC analysis of catechins contained in TPP was performed. Based on the retention times of the peaks from the chromatograms, each peaks of 2 samples, TPP and authentic standards (mixtures of EGC, EC, EGCG, GCG, and ECG), were compared. We found that the catechins contained in TPP belong to 5 main compounds: ~50% EGCG, ~24% ECG, ~16% GCG, ~2% EC, and ~1% EGC

HPLC chromatogram of green tea polyphenols. The peak numbers indicate: 1, EGC; 2, EC; 3, EGCG; 4, GCG; and 5, ECG. Fig. 1.

(Fig. 1).

3.2.

In vitro


The antimicrobial activity of TPP solution against 12 representative oral microorganisms [i.e. Lactobacillus spp. (L. acidophilus and L. plantarum), Streptococcus spp. (S. mutans, S. sanguis, S. mitis, and S. salivarius), S. aureus, N. meningitidis, E. coli, E. cloacae, E. faecalis, and C. albicans] was evaluated. As a representative oral microorganism, S. sanguis was the most sensitive bacterium, and was killed immediately when treated with TPP, while L. acidophilus and N. meningitides were killed within 3 min. Complete elimination of the following oral microorganisms was achieved with 5 min after treatment with TPP to S. salivarius, S. sobrinus, S. mitis, S. mutans, and L. plantarum. During this time, only a few colonies of S. aureus, E. coli, E. faecalis, E. cloacae, and C. albicans could grow on solid plates. Based on these results (summarized in Table 2), TPP demonstrated powerful antimicrobial activity on all

Number of CFUs of different oral microorganisms treated with 2,000 µg/mL TPP solution at different exposure times Number of CFU/mL Microorganisms Control 1 min 2 min 3 min 4 min 5 min 0 S. sanguis ~104 1.1 × 10 0 L. acidophilus ~104 N. meningitidis ~104 1.7 × 102 1.3 × 10 0 4 3 2 S. salivarius ~10 1.5 × 10 3.1 × 10 8.6 × 10 5.3 × 10 0 S. sobrinus ~104 1.7 × 103 7.4 × 102 2.5 × 102 4.8 × 10 0 S. mitis ~104 9.1 × 102 7.8 × 102 8.3 × 10 5.2 × 10 0 4.2 × 103 1.2 × 103 7.6 × 102 2.1 × 102 0 S. mutans ~104 4 3 3 2 L. plantarum ~10 4.1 × 10 1.2 × 10 8.2 × 10 2.7 × 10 0 S. aureus ~104 4.6 × 103 2.5 × 103 3.7 × 102 1.3 × 102 7 E. coli ~104 8.9 × 102 7.7 × 102 5.4 × 102 7.5 × 10 6 4 3 3 E. faecalis ~10 5.4 × 10 2.5 × 10 4.7 × 102 6.3 × 10 7 E. cloacae ~104 3.4 × 103 2.2 × 103 8.7 × 102 4.6 × 10 4 6.8 × 103 2.9 × 103 8.8 × 102 2.6 × 102 2 C. albicans ~104

Table 2.

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oral microorganisms tested in this work.

3.3. SEM analysis of oral microorganisms treated with TPP solution

Based on the results in Table 1, morphological changes of several oral bacteria treated with TPP solution were examined by SEM. In scanning electron micrographs of Streptococcus spp. (e.g. S. mutans and S. sobrinus) and S. aureus, cells grown in complex medium in the absence of TPP exhibited typical coccus shapes with smooth surfaces (micrographs not shown). However, cells treated with TPP for 5 min (Figs. 2A~2C) showed major structural cell surface changes, as well as, a preponderance of irregular forms with aggregates among cells. Compared to scanning electron micrographs of Streptococcus spp. and S. aureus treated with TPP solution, both L. plantarum and N. meningitidis demonstrated different morphological patterns; we observed a leakage of cellular material from these bacterial cells (Figs. 2D~2E). No leakage of cytoplasmic material was observed from C. albicans treated with TPP, but irregular protrusions did occur on the surface of cells (Fig. 2F).

3.4.

In vivo

Number of CFUs of oral microorganisms on different solid media after gargling with TPP solution Number of CFU/mL percentage of survival (%) Media Control 1 min 2 min 3 min 4 min 5 min MSa 2.7 × 107 1.8 × 107 1.3 × 107 9.1 × 106 2.3 × 105 0 (100) (66.7) (48.2) (33.7) (0.9) (0.0) BHI 2.6 × 107 2.0 × 107 1.1 × 107 4.8 × 106 8.0 × 105 0 (100) (76.9) (42.2) (18.4) (0.3%) (0.0) LB 1.9 × 107 1.3 × 107 8.7 × 106 4.4 × 106 6.1 × 105 0 (100) (63.1) (45.7) (23.1) (3.2) (0.0) MRS 1.6 × 107 9.7 × 106 5.7 × 106 1.0 × 106 0 (100) (60.6) (35.0) (6.3) (0.0) TJ 8.6 × 106 1.3 × 106 9.3 × 105 3.3 × 105 0 (100) (15.1) (10.9) (3.8) (0.0) EMB 7.3 × 106 5.1 × 106 3.4 × 106 6.7 × 105 0 (100) (70.8) (46.5) (9.3) (0.0) MSb 9.5 × 102 2 0 (100) (0.2) (0.0) Table 3.

MSa, mitis-salivarius; BHI, brain heart infusion; LB, Luria-Bertani; MRS, de Man-Rogosa-Sharpe; TJ, tomato juice; EMB, eosin-methylene blue; and MSb, mannitol salt.


In order to evaluate the in vivo killing effects of TPP on oral microorganisms in the human mouth, samples (gargled with 20 mL TPP for 1~5 min) from 25 individuals were inoculated on different selective solid media, after which CFUs were enumerated and the kill percentage calculated. We found that the number of colonies grown on different media decreased with increasing TPP treatment time (Table 3).

3.5. Biofilm formation on human teeth

Scanning electron micrographs of biofilms formed by S. mutans and S. sanguis colonized (A) on the surface of a human

Fig. 3.

tooth and (B) no biofilm formation after rinsing with TPP solution.

In order to examine whether TPP inhibits biofilm formation on the surface of human teeth, Streptococcus spp. were inoculated into 2 BHI broth samples and a single tooth was placed in each. For 3 min, 3 times a day, the tooth samples were taken out of their cultures and one was shaken in TPP solution while the other one was shaken in physiological saline. Four days later, SEM micrographs showed biofilm formation on the surface of the tooth shaken only in physiological saline, but no biofilm was observe on the tooth shaken in TPP solution (Fig. 3). 4. Discussion

Scanning electron micrographs of morphologically changed (A) S. mutans, (B) S. salivarius, (C) Staphylococcus aureus, (D) N. meningitidis, (E) L. plantarum, and (F) C. albicans treated with TPP solution for 5 min. The upper left in each micrograph includes control strains. Fig. 2.

TPP extracted from green tea mainly consists of 5 kinds of catechins: (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechin gallate, (−)-gallocatechin gallate, and (−)-epigallocatechin gallate, as shown in Fig. 1. Recent studies have presented data on a number of biological activities of TPP or cate-

Antimicrobial Activity and Biofilm Formation Inhibition of Green Tea Polyphenols on Human Teeth

chins and have been reported to exhibit anti-caries activity against many oral bacteria [11,28]. The present work focuses on the killing of oral bacteria and the inhibition of biofilm formation on human teeth by TPP. Mortality tests revealed that the CFUs of several oral bacteria dramatically decrease as a function of treatment time and killing rates are proportional to the exposure time to TPP. Interestingly, complete removal of all of the bacteria tested in this work was achieved within 5 min after exposure to TPP. Several studies have shown that the polyphenolic components in green tea and oolong tea effectively inhibit the growth of oral streptococci including S. mutans and S. sobrinus [12,29]. Based on this work, we report that TPP exerts effective toxicity to oral bacteria cells. SEM analysis demonstrated that all oral bacterial cells treated with TPP exhibit considerable morphological changes. All Streptococcus spp. treated with TPP morphologically changed, developing cellular aggregates with irregularly shaped clusters (Figs. 2A~2B). Matsumoto et al. [28] have reported that oolong tea extract induces remarkable cellular aggregation of S. mutans, S. oralis, S. sanguis, and S. gordonii. In that respect, data obtained from SEM analysis for Streptococcus spp. in this work are consistent with morphological changes of Streptococcus spp. against TPP found in other reports. As with expected patterns of morphological changes, some disruptive opening and leakage of cellular material was observed in the cells of L. plantarum and N. meningitidis after exposure to TPP. Morphological changes have been previously reported in several bacterial cells by toxic compounds [18,26,30]. For instance, Stenotrophomonas sp. OK-5 treated with lethal concentrations of the explosive TNT displayed morphologically altered cells, which developed rippled cell surfaces with irregularly shaped and significant alterations [26]. Recently, Cho et al. [18] reported that green tea extracts, at high concentrations, are toxic to E. coli due to the disruption of membrane components, which ultimately leads to cell death. As such, data obtained from SEM analysis for oral bacteria used in this work are consistent with cellular responses against toxic compounds observed with other bacteria. Based on the findings of this study, it is evident that cells of oral microorganisms react to TPP as a toxic substance. The inhibition of biofilm formation and the attachment of streptococci on human teeth by TPP were also examined. SEM analysis demonstrated that teeth treated with TPP had no biofilm formation, whereas cell clusters of S. mutans and S. sobrinus produced biofilms on tooth surfaces not treated with TPP (Fig. 3). Otake et al. [29] reported that TPP inhibits the attachment of S. mutans to saliva-coated hydroxyapatite discs; TPP also inhibits glucosyltransferase

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activity derived from the same strain. In this study, the data obtained from SEM analysis of biofilm formation on human teeth by cariogenic bacteria (e.g. S. mutans and S. sobrinus) are consistent with the inhibition of biofilm formation on other solid matrices such as hydroxyapatite discs. 5. Conclusion

The antimicrobial effects and biofilm formation inhibition of TPP extracted from Korean green tea (Camellia sinensis L) were evaluated against 12 oral microorganisms and effective antimicrobial activity against all microbes tested in this work was shown at 2,000 µg/mL TPP within 5 min of incubation. Depending on the bacteria, SEM analysis revealed various morphological changes. In the experiments of biofilm formation inhibition on human teeth using 2 mixed strains of S. mutans and S. sanguis, biofilm formation was inhibited on teeth shaken in TPP solution. This result suggests that TPP is effective against adherent cells of S. mutans and S. sanguis. In consequence, TPP was found to have antimicrobial effects and inhibit the formation of biofilms on human teeth. It could thus be a useful agent for the development of oral health products, such as mouth washes, to prevent and treat dental caries. References

1. Lowe, G. D. O. (2004) Dental disease, coronary heart disease and stroke, and inflammatory markers what are the associations and what do they mean? Circulation 109: 1076-1078. 2. Meurman, J. H., M. Sanz, and S. J. Janket (2004) Oral health, atherosclerosis, and cardiovascular disease. Crit. Rev. Oral Biol. Med. 15: 403-413. 3. Awano, S., K. Gohara, E. Kurihara, T. Ansai, and T. Takehara (2002) The relationship between the presence of periodontopathogenic bacteria in saliva and halitosis. Int. Dent. J. 52: 212-216. 4. Burton, J. P., C. N. Chilcott, and J. R. Tagg (2005) The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics. Oral Dis. 11: 29-31. 5. Kazor, C. E., P. M. Mitchell, A. M. Lee, L. N. Stokes, W. J. Loesche, F. E. Dewhirst, and B. J. Paster (2003) Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J. Clin. Microbiol. 41: 558-563. 6. Krespi, Y. P., M. G. Shrime, and A. Kacker (2006) The relationship between oral malodor and volatile sulfur compoundproducing bacteria. Otolaryngol. Head Neck Surg. 135: 671-676. 7. Loesche, W. J. and C. Kazor (2002) Microbiology and treatment of halitosis. Periodontol. 28: 256-279. 8. Nakano, K., H. Nemoto, R. Nomura, H. Inaba, H. Yoshioka, K. Taniguchi, A. Amano, and T. Ooshima (2009) Detection of oral bacteria in cardiovascular specimens. Oral Microbiol. Immunol. 24: 64-68. 9. Nemoto, H., K. Nakano, R. Nomura, and T. Ooshima (2008) Molecular characterization of Streptococcus mutans strains isolated from the heart valve of an infective endocarditis patient.

364

J. Med. Microbiol. 57: 891-895. 10. Poveda-Roda, R., Y. Jiménez, E. Carbonell, C. Gavaldá , M. M. Margaix-Muñoz, and G. Sarrión-Pérez (2008) Bacteremia originating in the oral cavity. Med. Oral Patol. Oral. Cir. Bucal. 13: 355-362. 11. Hirasawa, M., K. Takada, and S. Otake (2006) Inhibition of acid production on dental plaque bacteria by green tea catechins. Caries Res. 40: 265-270. 12. Sasaki, H., M. Matsumoto, T. Tanaka, M. Maeda, M. Nakai, S. Hamada, and T. Oshima (2003) Antibacterial activity of polyphenol compounds in oolong tea extract against Streptococcus mutans. Caries Res. 38: 2-8. 13. Yang, C. S. and J. M. Landau (2000) Effects of tea consumption on nutrition and health. J. Nutr. 130: 2409-2412. 14. Cowan, M. M. (1999) Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564-582. 15. Hamilton-Miller, J. M. T. (1995) Antimicrobial properties of tea (Camella sinensis L.). Antimicrob. Agents Chemother. 39: 23752377. 16. Yang, C. S., J. Hong, Z. Hou, and S. Sang (2004) Green tea polyphenols: antioxidative and prooxidative effects. J. Nutr. 134: 3181S. 17. Blanco, A. R., A. Sudano-Roccaro, G. C. Spoto, A. Nostro, and D. Rusciano (2005) Epigallocatechin gallate inhibits biofilm formation by ocular staphylococcal isolates. Antimicrob. Agents Chemother. 49: 4339-4343. 18. Cho, Y. S., N. L. Schiller, H. Y. Kahng, and K. H. Oh (2007) Cellular responses and proteomic analysis of Escherichia coli exposed to green tea polyphenols. Curr. Microbiol. 55: 501-506. 19. Cho, Y. S., N. L. Schiller, and K. H. Oh (2008) Antibacterial effects of green tea polyphenols on clinical isolates of methicillin-resistant Staphylococcus aureus. Curr. Microbiol. 57: 542546. 20. De Man, J. C., M. Rogosa, and M. E. Sharpe (1960) A medium used for the cultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135.


21. Creitz, J. R. and T. F. Puckett (1954) A method for cultural identification of Coccidioides immitis. Am. J. Clin. Pathol. 24: 1318-1323. 22. Murray P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (1999) Manual of Clinical Microbiology. 7th ed. American Society for Microbiology, Washington, DC, USA. 23. Holt-Harris, J. E. and O. Teague (1916) A new culture medium for the isolation of Bacillus typhosa from stools. J. Infect. Dis. 18: 596. 24. Hoffmann, H., S. Stindl, W. Ludwig, A. Stumpf, A. Mehlen, J. Heesemann, D. Monget, K. H. Schleifer, and A. Roggenkamp (2005) Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E. cloacae subspecies dissolvens comb. nov. and emended description of Enterobacter asburiae and Enterobacter kobei. Syst. Appl. Microbiol. 28: 196-205. 25. Xu, J. Z., S. Y. Yeung, Q. Chang, Y. Huang, and Z. Y. Chen (2004) Comparison of antioxidant activity and bioavailability of tea epicatechins with their epimers. Br. J. Nutr. 91: 873-881. 26. Ho, E. M., H. W. Chang, S. I. Kim, H. Y. Kahng, and K. H. Oh (2004) Analysis of TNT (2,4,6-trinitrotoluene)-inducible cellular responses and stress shock proteome in Stenotrophomonas sp. OK-5. Curr. Microbiol. 49: 346-352. 27. Ng, L. K., R. Sherburne, D. E. Taylor, and M. E. Stiles (1985) Morphological forms and viability of Campylobacter species studied by electron microscopy. J. Bacteriol. 164: 338-343. 28. Matsumoto, M., T. Minami, H. Sasaki, S. Sobue, and S. Hamada (1999) Inhibitory effects of oolong tea extract on caries-including properties of mutans streptococci. Caries Res. 33: 441-445. 29. Otake, S., M. Makimura, T. Kuroki, Y. Nishihara, and M. Hirasawa (1991) Anticaries effects of phenolic compounds from Japanese green tea. Caries Res. 25: 438-443. 30. Chang, H. W., H. Y. Kahng, S. I. Kim, J. W. Chun, and K. H. Oh (2004) Characterization of Pseudomonas sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5triazine). Appl. Microbiol. Biotechnol. 65: 323-329.