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1.  Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection 
Aim
Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
Methodology
Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization.
Results
S. mutans colonization was considerably reduced (9 ± 0.4 fold reduction, P=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with significant protection against further S. mutans colonization (5 minutes treatment: 38 ± 13 fold reduction P=0.01; 16 hours treatment: 96 ± 28 fold reduction P=0.07).
Conclusion
S. mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or “normal” biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.
doi:10.4248/IJOS10024
PMCID: PMC3733586  PMID: 20737932
targeted antimicrobial therapy; antimicrobial peptide; biofilm; Streptococcus mutans; protective colonization; caries
2.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide 
Antimicrobial Agents and Chemotherapy  2006;50(11):3651-3657.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
doi:10.1128/AAC.00622-06
PMCID: PMC1635210  PMID: 17060534
3.  A Lactotransferrin Single Nucleotide Polymorphism Demonstrates Biological Activity That Can Reduce Susceptibility to Caries 
Infection and Immunity  2013;81(5):1596-1605.
Streptococcus mutans is prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutans activity, characterize its genotype, and determine genotypic variants associated with S. mutans activity and reduced caries. An S. mutans affinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killed S. mutans in vitro. Saliva from a subject with moderate caries and with the LTF “wild-type” R form at position 47 had no such activity. A pilot genetic study (n = 30) showed that KK subjects were more likely to have anti-S. mutans activity than RR subjects (P = 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reduced S. mutans killing in a dose-dependent manner (P = 0.02). KK subjects were less likely to have caries (P = 0.02). A synthetic 11-mer LTF/K peptide killed S. mutans and other caries-related bacteria, while the LTF/R peptide had no effect (P = 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutans activity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.
doi:10.1128/IAI.01063-12
PMCID: PMC3647982  PMID: 23460521
4.  Biofilm formation and virulence expression by Streptococcus mutans are altered when grown in dual-species model 
BMC Microbiology  2010;10:111.
Background
Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.
Results
As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.
Conclusions
These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.
doi:10.1186/1471-2180-10-111
PMCID: PMC2867949  PMID: 20398271
5.  Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces 
Background
10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces.
Methods
Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically.
Results
500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells.
Conclusion
Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely.
doi:10.1186/1476-0711-11-21
PMCID: PMC3495742  PMID: 22839724
Biofilm; Caries; Glucosyltransferase; Streptococcus
6.  Probiotic Lactobacillus paracasei effect on cariogenic bacterial flora 
Clinical Oral Investigations  2010;15(4):471-476.
Lactobacillus paracasei has been demonstrated to inhibit the growth of many pathogenic microbes such as Streptococcus mutans, in vitro. However, its clinical application remains unclear. Here, we examined whether a novel probiotic L. paracasei GMNL-33 may reduce the caries-associated salivary microbial counts in healthy adults. Seventy-eight subjects (aged 20 to 26) had completed this double-blinded, randomized, placebo-controlled study. A probiotic/test (n = 42) and a control group (n = 36) took a L. paracasei GMNL-33 and a placebo oral tablet three times per day for 2 weeks, respectively. Bacterial counts of salivary S. mutans, lactobacilli, and salivary buffer capacity were measured with chair-side kits at the beginning (T1), the completion (T2) of medication, and 2 weeks after medication (T3). The results did not show differences in the counts of S. mutans and lactobacilli between probiotic and control groups at T1, T2, and T3. Nevertheless, within the probiotic group, an interesting probiotic effect was noticed. Between T1 and T2, no inhibitory effect against S. mutans was observed. However, a significant count reduction in the salivary S. mutans was detected between T2 and T3 (p = 0.016). Thus, a 2-week period of medication via oral administration route may be needed for L. paracasei GMNL-33 to be effective in the probiotic action.
doi:10.1007/s00784-010-0423-9
PMCID: PMC3133768  PMID: 20502929
Probiotic; Lactobacillus paracasei; Streptococcus mutans; Lactobacilli; Salivary buffer capacity
7.  Clinical Efficacy of a Specifically Targeted Antimicrobial Peptide Mouth Rinse: Targeted Elimination of Streptococcus mutans and Prevention of Demineralization 
Caries Research  2011;45(5):415-428.
Background/Aims
Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact.
Methods
In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model.
Results and Conclusions
C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic.
doi:10.1159/000330510
PMCID: PMC3169368  PMID: 21860239
Antimicrobial; Antimicrobial peptide; Caries; Demineralization; Dental plaque; Lactic acid; Mouth rinse; Oral therapeutic; Selective antibiotic; Selective therapeutic; Specifically targeted antimicrobial peptide; Streptococcus mutans; Targeted antimicrobial
8.  Dental plaque as a biofilm and a microbial community – implications for health and disease 
BMC Oral Health  2006;6(Suppl 1):S14.
Dental plaque is a structurally- and functionally-organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that, in health, remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift toward community dominance by acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other species with relevant traits may be involved. Strategies to control caries could include inhibition of biofilm development (e.g. prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.), or enhancement of the host defenses. Additionally, these more conventional approaches could be augmented by interference with the factors that enable the cariogenic bacteria to escape from the normal homeostatic mechanisms that restrict their growth in plaque and out compete the organisms associated with health. Evidence suggests that regular conditions of low pH in plaque select for mutans streptococci and lactobacilli. Therefore, the suppression of sugar catabolism and acid production by the use of metabolic inhibitors and non-fermentable artificial sweeteners in snacks, or the stimulation of saliva flow, could assist in the maintenance of homeostasis in plaque. Arguments will be presented that an appreciation of ecological principles will enable a more holistic approach to be taken in caries control.
doi:10.1186/1472-6831-6-S1-S14
PMCID: PMC2147593  PMID: 16934115
9.  Antimicrobial Activity of Antrodia camphorata Extracts against Oral Bacteria 
PLoS ONE  2014;9(8):e105286.
Antrodia camphorata (A. camphorata) is a unique, endemic and extremely rare mushroom species native to Taiwan, and both crude extracts of and purified chemical compounds from A. camphorata have been reported to have a variety of significant beneficial effects, such as anti-tumor and anti-inflammatory activity. However, reports on the effects of A. camphorata against dental pathogens have been limited. Oral health is now recognized as important for overall general health, including conditions such as dental caries, periodontal disease and rheumatoid arthritis. Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) are the most common bacteria associated with dental plaque and periodontopathic diseases, respectively. Thus, our study examined the ability of five various crude extracts of A. camphorata to inhibit the growth of dental bacteria and anti-adherence in vitro. Among the extracts, the ethanol, ethyl acetate and chloroform extracts exhibited the lowest MICs against P. gingivalis and S. mutans (MIC = 4∼16 µg/mL). The MIC of the aqueous extract was greater than 2048 µg/mL against both P. gingivalis and S. mutans. In vitro adherence of S. mutans was significantly inhibited by the addition of either the ethyl acetate extract or chloroform extract (MIC = 16∼24 µg/mL), while the ethanol extract (MIC = 32∼64 µg/mL) exhibited moderate inhibitory activity. Based on the result of this study, the ethyl acetate and chloroform extracts of A. camphorata may be good candidates for oral hygiene agents to control dental caries and periodontopathic conditions.
doi:10.1371/journal.pone.0105286
PMCID: PMC4140745  PMID: 25144619
10.  Natural History of Streptococcus sanguinis in the Oral Cavity of Infants: Evidence for a Discrete Window of Infectivity 
Infection and Immunity  2000;68(7):4018-4023.
The heterogeneous group of oral bacteria within the sanguinis (sanguis) streptococci comprise members of the indigenous biota of the human oral cavity. While the association of Streptococcus sanguinis with bacterial endocarditis is well described in the literature, S. sanguinis is thought to play a benign, if not a beneficial, role in the oral cavity. Little is known, however, about the natural history of S. sanguinis and its specific relationship with other oral bacteria. As part of a longitudinal study concerning the transmission and acquisition of oral bacteria within mother-infant pairs, we examined the initial acquisition of S. sanguinis and described its colonization relative to tooth emergence and its proportions in plaque and saliva as a function of other biological events, including subsequent colonization with mutans streptococci. A second cohort of infants was recruited to define the taxonomic affiliation of S. sanguinis. We found that the colonization of the S. sanguinis occurs during a discrete “window of infectivity” at a median age of 9 months in the infants. Its colonization is tooth dependent and correlated to the time of tooth emergence; its proportions in saliva increase as new teeth emerge. In addition, early colonization of S. sanguinis and its elevated levels in the oral cavity were correlated to a significant delay in the colonization of mutans streptococci. Underpinning this apparent antagonism between S. sanguinis and mutans streptococci is the observation that after mutans streptococci colonize the infant, the levels of S. sanguinis decrease. Children who do not harbor detectable levels of mutans streptococci have significantly higher levels of S. sanguinis in their saliva than do children colonized with mutans streptococci. Collectively, these findings suggest that the colonization of S. sanguinis may influence the subsequent colonization of mutans streptococci, and this in turn may suggest several ecological approaches toward controlling dental caries.
PMCID: PMC101685  PMID: 10858217
11.  Passive Transfer of Immunoglobulin Y Antibody to Streptococcus mutans Glucan Binding Protein B Can Confer Protection against Experimental Dental Caries 
Infection and Immunity  2001;69(5):3135-3142.
Active immunization with Streptococcus mutans glucan binding protein B (GBP-B) has been shown to induce protection against experimental dental caries. This protection presumably results from continuous secretion of salivary antibody to GBP-B, which inhibits accumulation of S. mutans within the oral biofilm. The purpose of this study was to explore the influence of short-term (9- or 24-day) passive oral administration of antibody to S. mutans GBP-B on the longer-term accumulation and cariogenicity of S. mutans in a rat model of dental caries. Preimmune chicken egg yolk immunoglobulin Y (IgY) or IgY antibody to S. mutans GBP-B was supplied in lower (experiment 1) and higher (experiment 2) concentrations in the diet and drinking water of rats for 9 (experiment 1) or 24 (experiment 2) days. During the first 3 days of IgY feeding, all animals were challenged with 5 × 106 streptomycin-resistant S. mutans strain SJ-r organisms. Rats remained infected with S. mutans for 78 days, during which rat molars were sampled for the accumulation of S. mutans SJ-r bacteria and total streptococci. Geometric mean levels of S. mutans SJ-r accumulation on molar surfaces were significantly lower in antibody-treated rats on days 16 and 78 of experiment 2 and were lower on all but the initial (day 5) swabbing occasions in both experiments. Relative to controls, the extent of molar dental caries measured on day 78 was also significantly decreased. The decrease in molar caries correlated with the amount and duration of antibody administration. This is the first demonstration that passive antibody to S. mutans GBP-B can have a protective effect against cariogenic S. mutans infection and disease. Furthermore, this decrease in infection and disease did not require continuous antibody administration for the duration of the infection period. This study also indicates that antibody to components putatively involved only in cellular aggregation can have a significant effect on the incorporation of mutans streptococci in dental biofilm.
doi:10.1128/IAI.69.5.3135-3142.2001
PMCID: PMC98269  PMID: 11292733
12.  The Bifidobacterium dentium Bd1 Genome Sequence Reflects Its Genetic Adaptation to the Human Oral Cavity 
PLoS Genetics  2009;5(12):e1000785.
Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.
Author Summary
The accessibility of complete bacterial genome sequences has provided important changes to the field of microbiology by significantly enhancing our understanding of the physiology, genetics, and evolutionary development of bacteria. Bifidobacteria are among such microorganisms, being mammalian commensals of biotechnological significance due to their perceived role in maintaining a balanced gastrointestinal (GIT) microflora. Bifidobacteria are therefore often applied as health-promoting or probiotic components in functional food products and represent a growing area of scientific interest. However, within the genus Bifidobacterium not all species provide beneficial effects on the host's health. In fact, the Bifidobacterium dentium species is considered an opportunistic pathogen since it has been associated with the development of dental caries. In this manuscript, we describe the complete genetic make-up of the B. dentium Bd1 genome and discuss functions that explain how this microorganism has adapted to the oral human cavity and imparts a cariogeneous phenotype. Moreover, we performed comparative genomic analyses of B. dentium genome with other bifidobacterial genomes in order to trace genetic differences/similarities between the opportunistic oral pathogen B. dentium Bd1 and closely related intestinal bifidobacteria.
doi:10.1371/journal.pgen.1000785
PMCID: PMC2788695  PMID: 20041198
13.  Inhibition of ecological emergence of mutans streptococci naturally transmitted between rats and consequent caries inhibition by Streptococcus salivarius TOVE-R infection. 
Infection and Immunity  1985;49(1):76-83.
The ability of Streptococcus salivarius strain TOVE-R to inhibit the ecological emergence of virulent representatives of the most prevalent human mutans streptococci on the teeth of specific pathogen-free Osborne-Mendel rats was studied. Rats which were infected by TOVE-R, or either S. mutans 10449S or S. sobrinus 6715-13WT, or uninfected were transiently co-caged so as to allow natural fecal transfer of organisms due to coprophagy. The infectants were differentially recovered from swabs of the teeth over the time course of the experiments and from sonified teeth at termination. Data were expressed on both relative (percentage) and absolute (CFU) bases. Initial oral colonization of rats by TOVE-R inhibited the ecological emergence of fecally transmitted S. mutans 10449S and S. sobrinus 6715-13WT. There was a generally inverse relationship between the percentages and absolute numbers of TOVE-R and the mutans streptococci on the teeth, which strongly suggested their competition for tooth sites. Absolute numbers of total recoverable flora from the teeth upon sonification were correlated with caries scores, thus suggesting that total recoverable flora counts substantially reflect cavitation status. TOVE-R itself induced no apparent caries activity and its transmission to rats already infected by 10449S or its colonization of rats before 10449S infection inhibition caries induction by this S. mutans strain; similar anticaries effects were not statistically significant for TOVE-R against 6715-13WT in these experiments. These data on the inhibition of the ecological emergence of the mutans streptococci supplement the already reported ability of TOVE-R to preempt initial colonization of teeth and partially displace the colonization of teeth by the mutans streptococci.
PMCID: PMC262061  PMID: 4008050
14.  The virulence of Streptococcus mutans and the ability to form biofilms 
In some diseases, a very important role is played by the ability of bacteria to form multi-dimensional complex structure known as biofilm. The most common disease of the oral cavity, known as dental caries, is a top leader. Streptococcus mutans, one of the many etiological factors of dental caries, is a microorganism which is able to acquire new properties allowing for the expression of pathogenicity determinants determining its virulence in specific environmental conditions. Through the mechanism of adhesion to a solid surface, S. mutans is capable of colonizing the oral cavity and also of forming bacterial biofilm. Additional properties enabling S. mutans to colonize the oral cavity include the ability to survive in an acidic environment and specific interaction with other microorganisms colonizing this ecosystem. This review is an attempt to establish which characteristics associated with biofilm formation—virulence determinants of S. mutans—are responsible for the development of dental caries. In order to extend the knowledge of the nature of Streptococcus infections, an attempt to face the following problems will be made: Biofilm formation as a complex process of protein–bacterium interaction. To what extent do microorganisms of the cariogenic flora exemplified by S. mutans differ in virulence determinants “expression” from microorganisms of physiological flora? How does the environment of the oral cavity and its microorganisms affect the biofilm formation of dominant species? How do selected inhibitors affect the biofilm formation of cariogenic microorganisms?
doi:10.1007/s10096-013-1993-7
PMCID: PMC3953549  PMID: 24154653
15.  Oral ecology and virulence of Lactobacillus casei and Streptococcus mutans in gnotobiotic rats. 
Infection and Immunity  1981;33(3):690-696.
Lactobacilli comprise a small percentage of the normal oral microbial flora of humans and are isolated commonly from saliva and frequently from an active caries lesion. We have compared the pathogenesis and colonization pattern of Lactobacillus casei with that of Streptococcus mutans strain 6715 in gnotobiotic rats. Of the two L. casei strains tested, L. casei strain ATCC 4646 caused slightly more caries than L. casei strain ATCC 11578. However, the level of caries induced by either L. casei strain was significantly lower (P less than 0.01) than that observed in similar-aged rats monoassociated with S. mutans strain 6715. When groups of rats were infected with mixtures of L. casei strain ATCC 4646 and S. mutans strain 6715, or with L. casei followed by S. mutans, higher numbers of L. casei than S. mutans were found associated with the tongue and in saliva; S. mutans always predominated in plaque. The level of caries observed in these groups of rats was similar to that seen with rats monoassociated with S. mutans except when L. casei comprised greater than 1% of the plaque microflora. In this latter situation, the level of caries was significantly lower (P less than or equal to 0.05) than that obtained in S. mutans-monoassociated rats. The results of this study suggest that L. casei colonizes sites in the oral cavity (including the tongue and saliva) other than the tooth surface in rats. The effect of L. casei in plaque toward reduction of S. mutans-induced dental caries in rats is discussed.
PMCID: PMC350763  PMID: 6793515
16.  Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model. 
Molecular Medicine  2002;8(6):313-317.
BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.
PMCID: PMC2039998  PMID: 12428062
17.  Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin. 
Infection and Immunity  1993;61(5):1964-1971.
The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.
PMCID: PMC280790  PMID: 8478086
18.  Interactions between Oral Bacteria: Inhibition of Streptococcus mutans Bacteriocin Production by Streptococcus gordonii 
Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.
doi:10.1128/AEM.71.1.354-362.2005
PMCID: PMC544254  PMID: 15640209
19.  A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria 
PLoS Pathogens  2012;8(7):e1002815.
The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.
Author Summary
The beneficial contribution of commensal bacteria to host health led to the concept that exogenous and non-pathogenic bacteria (probiotics) could be used to prevent infectious disease. However, the absence of relevant experimentally tractable in vivo models severely limits our understanding of the molecular processes behind probiotic effects, therefore hampering prophylactic and therapeutic use of probiotics against infections. Here we developed a protocol to raise microbe-free zebrafish larvae fed on microbe-free live food. We placed this microbiologically controlled model in contact with known pathogens and potential probiotics to investigate molecular events underlying pathogen exclusion by probiotic bacteria. We showed that Edwardsiella ictaluri, the causative agent of catfish enteric septicemia, causes rapid death of infected larvae following exposure via the natural immersion route. We used this mortality to screen potential probiotic bacteria able to extend zebrafish survival to E. ictaluri infection and thereby identified 3 protective strains. While host immune response modulation did not contribute to protection against E. ictaluri infection, comparison of protective and non-protective strains demonstrated a key role for their adhesion factors. Our in vivo approach constitutes a relevant new model of vertebrate oro-intestinal infection and provides new insight into molecular events underlying probiotic effects against incoming pathogens.
doi:10.1371/journal.ppat.1002815
PMCID: PMC3406073  PMID: 22911651
20.  Targeted Antimicrobial Treatment to Re-establish a Healthy Microbial Flora for Long-term Protection 
Advances in Dental Research  2012;24(2):94-97.
Streptococcus mutans has been implicated as the major acid-producing (cariogenic) bacterium. Dietary sugars and other factors may cause an imbalance of oral microflora that enables S. mutans to become dominant in the multi-species biofilms on the tooth surface, which could lead to dental caries. The application of broad-spectrum antimicrobials often results in re-colonization and re-dominance of S. mutans within oral flora, while in contrast, therapies capable of selective elimination of S. mutans from oral microbial communities may help to re-establish the normal flora and provide long-term protection. C16G2, a novel synthetic antimicrobial peptide with specificity for S. mutans, was found to have robust killing efficacy and selectivity for S. mutans in vitro. A subsequent pilot human study found that a single application of C16G2 in the oral cavity (formulated in a mouthrinse vehicle) was associated with a reduction in plaque and salivary S. mutans, lactic acid production, and enamel demineralization during the entire 4-day testing period. C16G2 is now being developed as a new anticaries drug.
doi:10.1177/0022034512453725
PMCID: PMC3420366  PMID: 22899688
microbial ecology; microbiology; microbial genetics; caries; dental biofilm; microbiota
21.  Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants. 
Infection and Immunity  1983;40(2):577-591.
In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.
PMCID: PMC264894  PMID: 6840853
22.  Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach 
BMC Biotechnology  2005;5:4.
Background
Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.
Results
The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans.
Conclusions
Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.
doi:10.1186/1472-6750-5-4
PMCID: PMC548521  PMID: 15667665
23.  Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm 
Molecular Oral Microbiology  2011;26(6):337-352.
As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota which is often considered a health asset, studies of the oral commensal microbial flora have been largely limited to their implication in oral diseases such as dental caries and periodontal diseases; Little emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign/pathogenic bacteria. In this study, we used the salivary microbiota derived from healthy human subjects to investigate protective effects against the colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing and pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into salivary microbial community during biofilm formation. Furthermore, in the saliva medium supplemented with 0.05% (w/v) sucrose, the oral flora inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign/pathogenic bacterial species, such as P. aeruginosa.
doi:10.1111/j.2041-1014.2011.00622.x
PMCID: PMC3327514  PMID: 22053962
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm
24.  Cultivable Anaerobic Microbiota of Severe Early Childhood Caries▿¶ 
Journal of Clinical Microbiology  2011;49(4):1464-1474.
Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen.
doi:10.1128/JCM.02427-10
PMCID: PMC3122858  PMID: 21289150
25.  Current perspectives of nanoparticles in medical and dental biomaterials 
Journal of Biomedical Research  2012;26(3):143-151.
Nanotechnology is gaining tremendous impetus due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical and optical properties of metals. Nanoparticles have been introduced as materials with good potential to be extensively used in biological and medical applications. Nanoparticles are clusters of atoms in the size range of 1-100 nm. Inorganic nanoparticles and their nano-composites are applied as good antibacterial agents. Due to the outbreak of infectious diseases caused by different pathogenic bacteria and the development of antibiotic resistance, pharmaceutical companies and researchers are searching for new antibacterial agents. The metallic nanoparticles are the most promising as they show good antibacterial properties due to their large surface area to volume ratios, which draw growing interest from researchers due to increasing microbial resistance against metal ions, antibiotics and the development of resistant strains. Metallic nanoparticles can be used as effective growth inhibitors in various microorganisms and thereby are applicable to diverse medical devices. Nanotechnology discloses the use of elemental nanoparticles as active antibacterial ingredient for dental materials. In dentistry, both restorative materials and oral bacteria are believed to be responsible for restoration failure. Secondary caries is found to be the main reason to restoration failure. Secondary caries is primarily caused by invasion of plaque bacteria (acid-producing bacteria) such as Streptococcus mutans and lactobacilli in the presence of fermentable carbohydrates. To make long-lasting restorations, antibacterial materials should be made. The potential of nanoparticles to control the formation of biofilms within the oral cavity is also coming under increasing scrutiny. Possible uses of nanoparticles as topically applied agents within dental materials and the application of nanoparticles in the control of oral infections are also reviewed.
doi:10.7555/JBR.26.20120027
PMCID: PMC3596063  PMID: 23554743
nanoparticles; nano-medicine; nano-biomaterials; nanotechnology; antibacterial agent

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