Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide
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- 作者：Ai Yasunaga (7)
Akihiro Yoshida (7)
Kazumasa Morikawa (8)
Kenshi Maki (8)
Suguru Nakamura (7)
Inho Soh (7)
Shuji Awano (7)
Toshihiro Ansai (7) - 关键词：Dental caries ; Dental plaque ; Biofilm ; Propidium monoazide (PMA) ; Quantification ; qPCR (Real ; time PCR) ; Streptococcus mutans ; Streptococcus sobrinus ; Viability
- 刊名：BMC Microbiology
- 出版年：2013
- 出版时间：December 2013
- 年：2013
- 卷：13
- 期：1
- 全文大小：928KB
- 参考文献：1. Loesche WJ: Role of Streptococcus mutans in human dental decay. / Microbiol Rev 1986, 50:353鈥?80.
2. de-Soet JJ, Toors FA, de-Graaff J: Acidogenesis by oral streptococci at different pH values. / Caries Res 1989, 23:14鈥?7. CrossRef
3. Fujiwara T, Sasada E, Mima N, Ooshima T: Caries prevalence and salivary mutans streptococci in 0鈥?-year-old children of Japan. / Community Dent Oral Epidemiol 1991, 19:151鈥?54. CrossRef
4. Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T: Development of a 5' nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus . / J Clin Microbiol 2003, 41:4438鈥?441. CrossRef
5. Nagashima S, Yoshida A, Ansai T, Watari H, Notomi T, Maki K, Takehara T: Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification. / Oral Microbiol Immunol 2007, 22:361鈥?68. CrossRef
6. Rudi K, Moen B, Dr酶mtorp SM, Holck AL: Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. / Appl Environ Microbiol 2005, 71:1018鈥?024. CrossRef
7. Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I: Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR. / Res Microbiol 2007, 158:405鈥?12. CrossRef
8. Nocker A, Cheung CY, Camper AK: Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs dead bacteria by selective removal of DNA from dead cells. / J Microbiol Methods 2006, 67:310鈥?20. CrossRef
9. Nocker A, Sossa-Fernandez P, Burr MD, Camper AK: Use of propidium monoazide for live/dead distinction in microbial ecology. / Appl Environ Microbiol 2007, 73:5111鈥?117. CrossRef
10. Pan Y, Breidt F Jr: Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. / Appl Environ Microbiol 2007, 73:8028鈥?031. CrossRef
11. Loozen G, Boon N, Pauwels M, Quirynen M, Teughels W: Live/dead real-time polymerase chain reaction to assess new therapies against dental plaque-related pathologies. / Mol Oral Microbiol 2011, 26:253鈥?61. CrossRef
12. Hamada S, Slade HD: Biology, immunology, and cariogenicity of Streptococcus mutans . / Microbiol Rev 1980, 44:331鈥?84.
13. Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, Kozai K: Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in pre-school children. / J Med Microbiol 2005, 54:661鈥?65. CrossRef
14. Klein MI, Scott-Anne KM, Gregoire S, Rosalen PL, Koo H: Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries. / Mol Oral Microbiol 2012, 27:350鈥?1. CrossRef
15. Ammann TW, Bostanci N, Belibasakis GN, Thurnheer T: Validation of a quantitative real-time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species-specific quantification of an in vitro subgingival biofilm model. / J Periodontal Res 2013, 48:517鈥?6. CrossRef
16. Lindquist B, Emilson CG, Wennerholm K: Relationship between mutans streptococci in saliva and their colonization of the tooth surfaces. / Oral Microbiol Immunol 1989, 4:71鈥?6. CrossRef
17. Li Y, Ge Y, Saxena D, Caufield PW: Genetic profiling of the oral microbiota associated with severe early-childhood caries. / J Clin Microbiol 2007, 45:81鈥?7. CrossRef
18. Boulos L, Pr茅vost M, Barbeau B, Coallier J, Desjardins R: LIVE/DEAD BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. / J Microbiol Methods 1999, 37:77鈥?6. CrossRef
19. Takahashi Y, Yoshida A, Nagayoshi M, Kitamura C, Nishihara T, Awano S, Ansai T: Enumeration of viable Enterococcus faecalis , a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time polymerase chain reaction. / Microbiol Immunol 2011, 55:889鈥?92. CrossRef
20. Sambrook J, Fritsch EF, Maniatis T: / Molecular Cloning: a Laboratory Manual. 2nd edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 1989.
21. Nadkarni MA, Martin FE, Jacques NA, Hunter N: Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. / Microbiology 2002, 148:257鈥?66.
22. Yoshida A, Kuramitsu HK: Streptococcus mutans biofilm formation: utilization of a gtfB promoter-green fluorescent protein ( PgtfB :: gfp ) construct to monitor development. / Microbiology 2002, 148:3385鈥?394. - 作者单位：Ai Yasunaga (7)
Akihiro Yoshida (7)
Kazumasa Morikawa (8)
Kenshi Maki (8)
Suguru Nakamura (7)
Inho Soh (7)
Shuji Awano (7)
Toshihiro Ansai (7)
7. Division of Community Oral Health Development, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku Kitakyushu, 803-8580, Japan
8. Division of Developmental Stomatognatic Function Science, Kyushu Dental University, Kitakyushu, 803-8580, Japan - ISSN：1471-2180
文摘
Background Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). Results We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. Conclusions PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status.