Streptococcus mutans

Streptococcus mutans
Stain of S. mutans in thioglycollate broth culture.
Scientific classification
Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Streptococcaceae
Genus: Streptococcus
Species: S. mutans
Binomial name
Streptococcus mutans
Clarke 1924

Streptococcus mutans is facultatively anaerobic, Gram-positive coccus-shaped bacterium commonly found in the human oral cavity and is a significant contributor to tooth decay.[1][2] The microbe was first described by J Kilian Clarke in 1924.[3]

Two closely related species, Streptococcus mutans and Streptococcus sobrinus, can cohabit the mouth, both contributing to oral disease, and the expense of differentiating them in laboratory testing is often not clinically necessary. Therefore, for clinical purposes they are sometimes considered together as a group, called the mutans streptococci.[4] Whereas Streptococcus mutans (italic name, singular) is a single species, the mutans streptococci (nonitalic name, plural) are a group of species; the word "streptococci" [lowercase, nonitalic, plural] is the general name for all species in the genus Streptococcus [capitalized, italic]). Compare also the viridans streptococci, another group of Streptococcus species considered collectively for clinical purposes.

Introduction

Streptococcus mutans is a Gram-positive bacterium that is the primary causative agent in the formation of dental cavities in humans. Gram-positive bacteria are those that are stained dark-blue or violet by Gram staining. This is based on the physical properties of their cell walls, as opposed to gram-negative bacteria, which cannot retain the crystal violet stain. Streptococcus is a genus of spherical Gram-positive bacteria belonging to the phylum Firmicutes and the lactic acid bacteria group. S. mutans, a member of the human oral flora, is widely recognized as the main causative agent of dental cavities.

Conditions in the oral cavity are diverse and complex, frequently changing from one extreme to another. Thus, to survive in the oral cavity, S. mutans must tolerate rapidly harsh environmental fluctuations and exposure to various antimicrobial agents to survive.[5] However, the mechanisms under which this cariogenic pathogen can survive and proliferate under such extreme environmental conditions are largely unknown, as little research has been done on this matter.

Ecology

Twenty-five species of oral streptococci live in the oral cavity. Each species has developed specific specialized properties for colonizing different oral sites and constantly changing conditions to fight competing bacteria and to withstand external challenges. Imbalances in the microbial biota can initiate oral diseases. Under special conditions, commensal streptococci can switch to opportunistic pathogens, initiating disease and damaging the host. Oral streptococci have both harmless and harmful bacteria. Mutans streptococci are the most important bacteria associated with tooth decay. S. mutans, the microbial species most strongly associated with carious lesions, is naturally present in the human oral microbiota. The taxonomy of these complex bacteria remains tentative.[6] A 1970’s study found that S. mutans was more prevalent on the pits and fissures, constituting 39% of the total streptococci in the oral cavity. Fewer S. mutans bacteria were found on the buccal surface (2–9%).[7]

Role in tooth decay

Early colonizers of the tooth surface are mainly Neisseria spp. and streptococci, including S. mutans. The growth and metabolism of these pioneer species changes local environmental conditions (e.g., Eh, pH, coaggregation, and substrate availability), thereby enabling more fastidious organisms to further colonize after them, forming dental plaque.[8] Along with S. sobrinus, S. mutans plays a major role in tooth decay, metabolizing sucrose to lactic acid[2] using the enzyme glucansucrase.[9] The acidic environment created in the mouth by this process is what causes the highly mineralized tooth enamel to be vulnerable to decay. S. mutans is one of a few specialized organisms equipped with receptors that improve adhesion to the surface of teeth. Sucrose is used by S. mutans to produce a sticky, extracellular, dextran-based polysaccharide that allows them to cohere, forming plaque. S. mutans produces dextran via the enzyme dextransucrase (a hexosyltransferase) using sucrose as a substrate in the following reaction:

n sucrose → (glucose)n + n fructose

Sucrose is the only sugar that S. mutans can use to form this sticky polysaccharide.[1]

However, many other sugars—glucose, fructose, lactose—can be digested by S. mutans, but they produce lactic acid as an end product. The combination of plaque and acid leads to dental decay.[10] Due to the role S. mutans plays in tooth decay, many attempts have been made to create a vaccine for the organism. So far, such vaccines have not been successful in humans.[11] Recently, proteins involved in the colonization of teeth by S. mutans have been shown to produce antibodies that inhibit the cariogenic process.[12] A molecule recently synthesized at Yale University and the University of Chile, called Keep 32, is supposed to be able to kill S. mutans.

It is believed that Streptococcus mutans acquired the gene that enables it to produce biofilms through horizontal gene transfer with other lactic acid bacterial species, such as Lactobacillus.[13]

Life in the oral cavity

Surviving in the oral cavity, S. mutans is the primary causal agent and the pathogenic species responsible for dental caries (tooth decay or cavities) specifically in the initiation and development stages.

Dental plaque, typically the precursor to tooth decay, contains more than 600 different microorganisms, contributing to the oral cavity’s overall dynamic environment that frequently undergoes rapid changes in pH, nutrient availability, and oxygen tension. Dental plaque adheres to the teeth and consists of bacterial cells, while plaque is the biofilm on the surfaces of the teeth. Dental plaque and S. mutans is frequently exposed to "toxic compounds" from oral healthcare products, food additives, and tobacco.

While S. mutans grows in the biofilm, cells maintain a balance of metabolism that involves production and detoxification. Biofilm is an aggregate of microorganisms in which cells adhere to each other or a surface. Bacteria in the biofilm community can actually generate various toxic compounds that interfere with the growth of other competing bacteria.

S. mutans has over time developed strategies to successfully colonize and maintain a dominant presence in the oral cavity. The oral biofilm is continuously challenged by changes in the environmental conditions. In response to such changes, the bacterial community evolved with individual members and their specific functions to survive in the oral cavity. S. mutans has been able to evolve from nutrition-limiting conditions to protect itself in extreme conditions.[5] Streptococci represent 20% of the oral bacteria and actually determines the development of the biofilms. Although S. mutans can be antagonized by pioneer colonizers, once they become dominant in oral biofilms, dental caries can develop and thrive.[5]

Survival under stressful conditions

Transformation is a bacterial adaptation involving the transfer of DNA from one bacterium to another through the surrounding medium. Transformation is a primitive form of sexual reproduction. For a bacterium to bind, take up, and recombine exogenous DNA into its chromosome, it must enter a special physiological state termed “competence”. In S. mutans, a peptide pheromone quorum-sensing signaling system controls genetic competence.[14] This system functions optimally when the S. mutans cells are in crowded biofilms.[15] S. mutans cells growing in a biofilm are transformed at a rate 10- to 600-fold higher than single cells growing under uncrowded conditions (planktonic cells).[14] Induction of competence appears to be an adaptation for repairing DNA damages caused by crowded, stressful conditions.[16]

Cariogenic potential

The causative agent of dental caries is associated with its ability to metabolize various sugars, form a robust biofilm, produce an abundant amount of lactic acid, and thrive in the acid environment it generates.[17]

Dental caries is a dental biofilm-related oral disease associated with increased consumption of dietary sugar and fermentable carbohydrates. When dental biofilms remain on tooth surfaces, along with frequent exposure to sugars, acidogenic bacteria (members of dental biofilms) will metabolize the sugars to organic acids. Persistence of this acidic condition encourages the proliferation of acidogenic and aciduric bacteria as a result of their ability to survive at a low-pH environment. The low-pH environment in the biofilm matrix erodes the surface of the teeth and begins the "initiation" of the dental caries.[17] If the adherence of S. mutans to the surface of teeth or the physiological ability (acidogenity and aciduricity) of S. mutans in dental biofilms can be reduced or eliminated, the acidification potential of dental biofilms and later cavity formations can be decreased.[17]

Susceptibility to disease varies between individuals and immunological mechanisms have been proposed to confer protection or susceptibility to the disease. These mechanisms have yet to be fully elucidated but it seems that while antigen presenting cells are activated by S. mutans in vitro, they fail to respond in vivo. Immunological tolerance to S. mutans at the mucosal surface may make individuals more prone to colonisation with S. mutans and therefore increase susceptibility to dental caries.[18]

Evolution

There are three key traits that have evolved in S. mutans that help increase its adaptability to the oral cavity, but at the same time, these traits have increased its virulence. These characteristics are increased organic acid production, the capacity to form biofilms on the hard surfaces of teeth, and the ability to survive and thrive in a low pH environment.[19]

During its evolution, S. mutans acquired the ability to increase the amount of carbohydrates it could metabolize, and consequently more organic acid was produced as a byproduct.[20] This is significant in the formation of dental caries because increased acidity in the oral cavity amplifies the rate of demineralization of the tooth, which leads to carious lesions.[21] It is thought that the trait evolved in S. mutans via lateral gene transfer with another bacterial species present in the oral cavity. There are several genes, SMU.438 and SMU.1561, involved in carbohydrate metabolism that are up-regulated in S. mutans. These genes possibly originated from Lactococcus lactis and S. gallolyticus, respectively.[20]

Another instance of lateral gene transfer is responsible for S. mutans’ acquisition of the glucosyltransferase (GTF) gene, which allows the bacteria to produce polysaccharides from sucrose. These sticky polysaccharides are responsible for the bacteria’s ability to aggregate with one another and adhere to tooth enamel, i.e. to form biofilms. The GTF genes found in S. mutans most likely are derived from other anaerobic bacteria found in the oral cavity, such as Lactobacillus or Leuconostoc. Additionally, the GTF genes in S. mutans display homology with similar genes found in Lactobacillus and Leuconostoc. The common ancestral gene is believed to have been used for hydrolysis and linkage of carbohydrates.[22]

The third trait that evolved in S. mutans is its ability to not only survive, but also thrive in acidic conditions. This trait gives S. mutans a selective advantage over other members of the oral microbiota. As a result, S. mutans could outcompete other species, and occupy additional regions of the mouth, such as advanced dental plaques, which can be as acidic as pH 4.0.[23] Natural selection is most likely the primary evolutionary mechanisms responsible for this trait.

In discussing the evolution of S. mutans, it is imperative to include the role humans have played and the co-evolution that has occurred between the two species. As humans evolved anthropologically, the bacteria evolved biologically. It is widely accepted that the advent of agriculture in early human populations provided the conditions S. mutans needed to evolve into the virulent bacteria it is today. Agriculture introduced fermented foods, as well as more carbohydrate rich foods, into the diets of historic human populations. These new foods would have introduced new bacteria into the oral cavity, as well as created new environmental conditions. For example, Lactobacillus or Leuconostoc are typically found in foods such as yogurt and wine. Also, consuming more carbohydrates would have increase the amount of sugars available to S. mutans for metabolism as well as lowered the pH of the oral cavity. This newly acidic habitat would select for those bacteria that could survive and reproduce at a lower pH.[20]

Another significant change to the oral environment occurred during the Industrial Revolution. More efficient manufacturing of foodstuffs increased the availability and amount of sucrose consumed by humans. This provided S. mutans with more energy resources, and thus exacerbated an already rising rate of dental caries.[22]

Children

In general, S. mutans is acquired in the oral cavity at the moment of tooth eruption. But S. mutans has been detected in the oral cavity of predentate children. This suggests that the eruption of teeth is not a necessary prerequisite. Thus, this species may not be confined to dental plaque. Additionally, acquisition of the virulent bacteria is most often due to vertical transmission from mother to child, most likely shortly after birth.[24] The adhesion, invasion, and persistence within the oral cells are considered the virulence mechanism of S. mutans to colonize and survive in the oral cavity in the absence of a tooth surface.[25]

Cardiovascular disease

S. mutans is implicated in the pathogenesis of certain cardiovascular diseases, and is the most prevalent bacterial species detected in extirpated heart valve tissues, as well as in atheromatous plaques, with an incidence of 68.6% and 74.1%, respectively.[26]

Prevention and treatment

Practice of good oral hygiene including daily brushing, flossing and the use of appropriate mouthwash can significantly reduce the number of oral bacteria and inhibit their proliferation. Oral bacteria often live in plaque, a kind of biofilm, hence mechanical removal of plaque is the most effective way of getting rid of harmful oral bacteria, as bacterial biofilms are notoriously resistant to antibiotics and antimicrobial rinses.[27] However, there are some remedies used in the treatment of oral bacterial infection, in conjunction with mechanical cleaning.

Antimicrobial agents used in dentistry

Fluoride has a direct inhibitory effect on the enolase enzyme.

Chlorhexidine reduces populations of S. mutans, presumably by interfering with bacterial adherence.

Xylitol is a noncariogenic sugar alcohol found in gum and oral health care products which is not able to be metabolized into the cariogenic acids that commonly cause tooth demineralization and decay. Xylitol-containing gum can reduce dental caries by decreasing levels of S. mutans in plaque and saliva.[28][29]

It has been suggested that deglycyrrhizinated licorice root extract may have use as it demonstrates antimicrobial effects in cultures of S. mutans as well.[30]

Green tea extract

Green tea extract is rich in catechin, a class of antioxidants. Topically applied green tea extract inhibits S. mutans growth, kills oral bacteria, combats oral plaque, and inhibits collagenase activity.

Tea tree oil

Tea tree oil, when used in the form of a mouthwash, has been shown to be effective in killing several bacteria, including S. mutans, and fighting gingivitis.[31]

Macelignan from nutmeg

Macelignan, a compound found in nutmeg, is shown to decrease the biofilm level of S. mutans.[32]

Curcuminoids

Curcuminoids, the main components of turmeric, have a wide range of pharmacological uses. Many studies of turmeric have revealed antimicrobial properties. Researchers discovered that a fraction could be separated from turmeric and showed that it had anti-biofilm activity. Researchers based this on a comparison of curcuminoid content and antiacidogenic activity against S. mutans. The data showed that the separated turmeric fraction and curcuminoids may be effective in controlling both dental biofilms and dental cavity formations, as they are related.[33]

Barley tea

In addition, a compound in roasted barley tea, a popular drink in East Asia, has been demonstrated to inhibit S. mutans biofilms.[34]

Lollipop

In January 2011, scientists developed safe and effective sugar-free herbal lollipops that kill cavity-causing bacteria. As already established, tooth decay is caused by cariogenic bacteria like S. mutans. S. mutans converts sugars into acids that dissolve minerals in the tooth enamel. A previous study found that novel compound from the extraction of licorice roots, glycyrrhizol A, has strong antimicrobial activity against cariogenic bacteria. Killing such bacteria would control or prevent tooth decay. Researchers produced specific herbal extracts to develop a sugar-free lollipop to kill bacteria such as S. mutans. Subsequent studies on humans showed a reduction of cariogenic bacteria in the oral cavity after eating these lollipops.[35][36]

See also

References

  1. ↑ 1.0 1.1 Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0-8385-8529-9.
  2. ↑ 2.0 2.1 Loesche WJ (1996). "Ch. 99: Microbiology of Dental Decay and Periodontal Disease". In Baron S et al. Baron's Medical Microbiology (4th ed.). University of Texas Medical Branch. ISBN 0-9631172-1-1. PMID 21413316.
  3. ↑ Clarke, J. Kilian (1924). "On the Bacterial Factor in the Ætiology of Dental Caries". British Journal of Experimental Pathology 5 (3): 141–7. PMC 2047899.
  4. ↑ Newcastle University Dental School. "Streptococcus mutans and the mutans streptococci. In: The Oral Environment, online tutorial". Retrieved 2013-11-04.
  5. ↑ 5.0 5.1 5.2 Biswas, S; Biswas, I (2011). "Role of VltAB, an ABC transporter complex, in viologen tolerance in Streptococcus mutans". Antimicrobial agents and chemotherapy 55 (4): 1460–9. doi:10.1128/AAC.01094-10. PMC 3067168. PMID 21282456.
  6. ↑ Nicolas, Guillaume G.; Lavoie, Marc C. (2011). "Streptococcus mutans et les streptocoques buccaux dans la plaque dentaire". Canadian Journal of Microbiology 57 (1): 1–20. doi:10.1139/W10-095. PMID 21217792.
  7. ↑ Ikeda, T.; Sandham, H.J. (1971). "Prevalence of Streptococcus mutans on various tooth surfaces in negro children". Archives of Oral Biology 16 (10): 1237–40. doi:10.1016/0003-9969(71)90053-7. PMID 5289682.
  8. ↑ Vinogradov AM, Winston M, Rupp CJ, Stoodley P; Winston; Rupp; Stoodley (2004). "Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans". Biofilms 1: 49–56. doi:10.1017/S1479050503001078.
  9. ↑ http://blogs.discovermagazine.com/discoblog/2010/12/08/dental-researchers-to-mouth-bacteria-dont-get-too-attached/
  10. ↑ Madigan M, Martinko J (editors). (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1.
  11. ↑ Klein, J.P.; Scholler, M. (December 1998). "Recent Advances in the Development of a Streptococcus mutans Vaccine". European Journal of Epidemiology 4 (4): 419–25. doi:10.1007/BF00146392. JSTOR 3521322. PMID 3060368.
  12. ↑ Hajishengallis G, Russell MW (2008). "Molecular Approaches to Vaccination against Oral Infections". Molecular Oral Microbiology. Caister Academic Press. ISBN 978-1-904455-24-0.
  13. ↑ Hoshino, T., T. Fujiwara, and S. Kawabata. 2012. Evolution of Cariogenic Character in Streptococcus mutans: Horizontal Transmission of Glycosyl Hydrolase Family 70 Genes. Scientific Reports 2:518-525
  14. ↑ 14.0 14.1 Li YH, Lau PC, Lee JH, Ellen RP, Cvitkovitch DG; Lau; Lee; Ellen; Cvitkovitch (February 2001). "Natural genetic transformation of Streptococcus mutans growing in biofilms". J. Bacteriol. 183 (3): 897–908. doi:10.1128/JB.183.3.897-908.2001. PMC 94956. PMID 11208787.
  15. ↑ Aspiras MB, Ellen RP, Cvitkovitch DG; Ellen; Cvitkovitch (September 2004). "ComX activity of Streptococcus mutans growing in biofilms". FEMS Microbiol. Lett. 238 (1): 167–74. doi:10.1016/j.femsle.2004.07.032. PMID 15336418.
  16. ↑ Michod RE, Bernstein H, Nedelcu AM; Bernstein; Nedelcu (May 2008). "Adaptive value of sex in microbial pathogens". Infect. Genet. Evol. 8 (3): 267–85. doi:10.1016/j.meegid.2008.01.002. PMID 18295550. as PDF
  17. ↑ 17.0 17.1 17.2 Argimón, S; Caufield, PW (2011). "Distribution of putative virulence genes in Streptococcus mutans strains does not correlate with caries experience". Journal of clinical microbiology 49 (3): 984–92. doi:10.1128/JCM.01993-10. PMC 3067729. PMID 21209168.
  18. ↑ Butcher, JP; Malcolm, J; Benson, RA; Deng, DM; Brewer, JM; Garside, P; Culshaw, S (2011). "Effects of Streptococcus mutans on dendritic cell activation and function". Journal of dental research 90 (10): 1221–7. doi:10.1177/0022034511412970. PMID 21690565.
  19. ↑ Banas, J. A.; Miller, J. D.; Fuschino, M. E.; Hazlett, K. R. O.; Toyofuku, W.; Porter, K. A.; Florczyk, M. A.; McDonough, K. A.; Michalek, S. M. (2007). "Evidence that accumulation of mutants in a biofilm reflects natural selection rather than stress-induced adaptive mutation". Applied and Environmental Microbiology 73 (1): 357–361. doi:10.1128/aem.02014-06.
  20. ↑ 20.0 20.1 20.2 Cornejo, O. E.; Lefébure, T.; Bitar, P.D. Pavinski; Lang, P.; Richards, V. P.; Eilertson, K.; Do, T.; Beighton, D.; Zeng, L.; Ahn, S. J.; Burne, R. A.; Siepel, A.; Bustamante, C. D.; Stanhope, M. J. (2012). "Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans". Mol. Biol. Evol 30 (4): 881–893. doi:10.1093/molbev/mss278.
  21. ↑ Takahashi, N., and B. Nyvad. 2010. The role of bacteria in the caries process: Ecological perspectives. Journal of Dental Research 90(3): 294-303.
  22. ↑ 22.0 22.1 Hoshino, T.; Fujiwara, T.; Kawabata, S. (2012). "Evolution of cariogenic character in Streptococcus mutans: Horizontal transmission of glycosyl hydrolase family 70 genes". Scientific Reports 2: 518–524. doi:10.1038/srep00518.
  23. ↑ Takahashi, N.; Nyvad, B. (2010). "The role of bacteria in the caries process: Ecological perspectives". Journal of Dental Research 90 (3): 294–303. doi:10.1177/0022034510379602.
  24. ↑ Simon, L. 2007. The role of Streptococcus mutans and oral ecology in the formation of dental caries. Journal of Young Investigators.
  25. ↑ Berlutti, F; Catizone, A; Ricci, G; Frioni, A; Natalizi, T; Valenti, P; Polimeni, A (2010). "Streptococcus mutans and Streptococcus sobrinus are able to adhere and invade human gingival fibroblast cell line". International journal of immunopathology and pharmacology 23 (4): 1253–60. PMID 21244775.
  26. ↑ Nakano, K; Inaba, H; Nomura, R; Nemoto, H; Takeda, M; Yoshioka, H; Matsue, H; Takahashi, T et al. (2006). "Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens". Journal of clinical microbiology 44 (9): 3313–7. doi:10.1128/JCM.00377-06. PMC 1594668. PMID 16954266.
  27. ↑ Finkelstein, P; Yost, KG; Grossman, E (1990). "Mechanical devices versus antimicrobial rinses in plaque and gingivitis reduction". Clinical preventive dentistry 12 (3): 8–11. PMID 2083478.
  28. ↑ Ly, KA; Milgrom, P last3=Rothen (2006). "Xylitol, sweeteners, and dental caries". Pediatric Dentistry 28 (2): 154–63. PMID 16708791. |first3= missing |last3= in Authors list (help)
  29. ↑ Heinsohn, Torben (2013). "Welchen Einfluss haben Xylit-haltige Kaugummis auf die Mundflora? Entwicklung eines quantitativen Testes zum Nachweis von Streptococcus mutans auf Basis der "Real-time"-quantitativen Polymerase-Kettenreaktion" [Xylitol-containing chewing gum and the oral bacterial flora. Development of a Quantitative Test for Streptococcus mutans on the Basis of the Real-time Quantitative Polymerase Chain Reaction] (PDF). Junge Wissenschaft (Young Researcher) (in German) 97: 18–30. Retrieved 23 January 2015.
  30. ↑ Ahn, S; Cho, E; Kim, H; Park, S; Lim, Y; Kook, J (December 2012). "The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures". Anaerobe 18 (6): 590–596. doi:10.1016/j.anaerobe.2012.10.005. ISSN 1075-9964. PMID 23123832. Retrieved 13 March 2014.
  31. ↑ Carson, CF; Hammer, KA; Riley, TV (2006). "Melaleuca alternifolia (Tea Tree) oil: A review of antimicrobial and other medicinal properties". Clinical Microbiology Reviews 19 (1): 50–62. doi:10.1128/CMR.19.1.50-62.2006. PMC 1360273. PMID 16418522.
  32. ↑ Yanti; Rukayadi, Y; Kim, KH; Hwang, JK (2008). "In vitro anti-biofilm activity of macelignan isolated from Myristica fragrans Houtt. Against oral primary colonizer bacteria". Phytotherapy research : PTR 22 (3): 308–12. doi:10.1002/ptr.2312. PMID 17926328.
  33. ↑ Pandit, Santosh; Kim, Hye-Jin; Kim, Jeong-Eun; Jeon, Jae-Gyu (2011). "Separation of an effective fraction from turmeric against Streptococcus mutans biofilms by the comparison of curcuminoid content and anti-acidogenic activity". Food Chemistry 126 (4): 1565–70. doi:10.1016/j.foodchem.2010.12.005.
  34. ↑ Stauder, Monica; Papetti, Adele; Daglia, Maria; Vezzulli, Luigi; Gazzani, Gabriella; Varaldo, Pietro E.; Pruzzo, Carla (2010). "Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm". Current Microbiology 61 (5): 417–21. doi:10.1007/s00284-010-9630-5. PMID 20361189.
  35. ↑ Abstract
  36. ↑ Hu, Chu-hong; He, Jian; Eckert, Randal; Wu, Xiao-yang; Li, Li-na; Tian, Yan; Lux, Renate; Shuffer, Justin A; Gelman, Faina; Mentes, Janet; Spackman, Sue; Bauer, Janet; Anderson, Maxwell H; Shi, Wen‐yuan (2011). "Development and evaluation of a safe and effective sugar-free herbal lollipop that kills cavity-causing bacteria" (PDF). International journal of oral science 3 (1): 13–20. doi:10.4248/IJOS11005. PMID 21449211.

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