Targeted antimicrobial therapy against Streptococcus mutans establishes protective non-cariogenic oral biofilms and reduces subsequent infection

Int J Oral Sci. Author manuscript; available in PMC 2010 October 13.

Published in final edited form as:

Int J Oral Sci. 2010 June; 2(2): 66–73.

PMCID: PMC2953616

NIHMSID: NIHMS238840

Targeted antimicrobial therapy against Streptococcus mutans establishes protective non-cariogenic oral biofilms and reduces subsequent infection

Lina Li,¹ Lihong Guo,³ Renate Lux,¹ Randal Eckert,⁴ Daniel Yarbrough,^1,⁴ Jian He,¹ Maxwell Anderson,⁴ and Wenyuan Shi^1,²^*

¹UCLA School of Dentistry, University of California, Los Angeles, CA, 90095

²UCLA Molecular Biology Institute, University of California, Los Angeles, CA, 90095

³Department of Oral Biology, Peking University Dental School, Beijing, China

⁴C3 Jian Inc, Inglewood, CA 90301

^*Corresponding author: Wenyuan Shi UCLA School of Dentistry 10833 LeConte Avenue Los Angeles CA 90095-1668 Tel: + 1 310 825 8356 FAX: + 1 310 794 7109 ; Email: wenyuan/at/ucla.edu

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Abstract

Aim

Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus (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 very significant protection against further S. mutans colonization (5min treatment: 38 ± 13 fold reduction p=0.01; 16 hr treatment: 96 ± 28 fold reduction p=0.07).

Conclusions

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.

Keywords: Targeted antimicrobial therapy, antimicrobial peptide, biofilm, Streptococcus mutans, protective colonization, caries

Introduction

Dental caries (tooth decay) is one of the most prevalent and costly diseases in the United States and throughout the world [Evans and Kleinman, 2000]. Although the ultimate manifestation of the disease is the dissolution of tooth structure, the biological nature of the disease is a microbial infection caused primarily by the cariogenic bacterium Streptococcus mutans (S. Mutans) (Clarke, 1924; Loesche, 1986; Loesche et al., 1975; Tanzer et al., 2001). Most current dental therapies are focused on eradicating the entire dental plaque via mechanical removal or broad-spectrum antimicrobial treatments. However, the improved understanding of oral microbial ecology, especially the importance of the balance between oral pathogens and commensal residents, has prompted interest in novel approaches focused on selective pathogen inhibition and modulation of the microbial composition of dental plaque to control pathogenesis.

Most of these new approaches aim to achieve the eradication of S. mutans by targeting its virulence factors, such as the colonization of the tooth surface via both sucrose dependent and independent adhesion mechanisms [Koga et al., 2002; Smith, 2002], cell-cell signaling [Lonn-Stensrud et al., 2007], or acid production. Numerous approaches include the development of anti-caries vaccines against either cell surface antigens [Abiko, 2000] or a glucosyltransferase enzyme that is responsible for glucan production [Xu et al., 2007]. In an effort to reduce acid production of naturally occurring S. mutans, replacement therapy using a genetically engineered S. mutans that is able to outcompete indigenous strains while producing significantly less acid has been attempted [Hillman et al., 2000]. Several recent studies have also focused on understanding the interactions between S. mutans and other neighboring streptococci in the dental plaque and their impact on S. mutans pathogenesis. For example, S. sanguinis and S. oligofermentans have been observed to inhibit S. mutans growth through hydrogen peroxide production [Kreth et al., 2005; Tong et al., 2007; Kreth et al., 2009].

Our research group has initiated a targeted approach to control oral microbial pathogenesis via a new class of antimicrobials, called Specifically-Targeted Antimicrobial Peptides (STAMPs) [Eckert et al., 2006]. A typical STAMP molecule consists of two functionally independent moieties conjoined in a linear peptide sequence: a nonspecific antimicrobial peptide serves as the killing moiety while a species-specific binding peptide comprises the targeting moiety that provides specific binding to a selected pathogen and facilitates the targeted delivery of the attached antimicrobial peptide. STAMPs have shown effective elimination of S. mutans from a mixed-species environment without affecting closely related non-cariogenic oral streptococci [Eckert et al., 2006]. Based on these features STAMPs have a strong potential for development into novel therapeutics that may selectively eliminate pathogens while preserving the benefits associated with a healthy normal flora.

In the present study, we used a controlled in vitro approach to examine the effect of normal flora on S. mutans colonization. In particular, we tested the hypothesis that S. mutans-specific STAMPs can protect against S. mutans infection by maintaining biofilms composed of benign residential species.

Material and Methods

Strains and growth conditions

S. mutans strains UA140 [Qi et al., 2001] and JM11 (UA140::pldh-luc, Spc^r) [Merritt et al., 2005] were grown anaerobically (N₂ 85%, H₂ 10%, and CO₂ 5%) in Todd–Hewitt (TH) medium (Difco) or on TH agar plates at 37 °C. JM11 was grown in TH supplemented with 800 μg/ml spectinomycin (Sigma, St Louis, MO). For growth of saliva-derived biofilms, TH medium was supplemented with 1% mannose, 1% sucrose, and 1% dextrose. All biofilms were grown anaerobically at 37°C.

Peptide synthesis and purification

Two STAMPs were used in this study: C16G2 (Eckert et al., 2006) and 2_1G2, a derivative in which the S. mutans-specific CSPC16 targeting domain was replaced with the newly identified S. mutans-specific targeting peptide 2_1 (FIKHFIHRF). Peptides were synthesized using Fmoc (9-fluorenylmethoxy carbonyl) solid-phase chemistry (431A peptide synthesizer; Applied Biosciences, Foster City, USA) as previously described (Eckert et al., 2006) and purified to >95% by high-performance liquid chromatography (Waters; Milford, USA), as described previously [He et al., 2009]. Peptide mass was determined by electrospray ionization (ESI) mass spectrometry (data not shown, 3100 Mass Detector, Waters, Milford, USA).

Screening of saliva samples using confocal laser scanning fluorescence microscopy (CLSM)

An anti-S. mutans monoclonal antibody was employed for detecting S. mutans colonization within saliva-derived biofilms, as described previously [Gu et al., 2002; Gu et al., 2005]. Since this approach cannot distinguish endogenous and exogenously added S. mutans, individuals were screened for saliva that was naturally free of S. mutans to enable better controlled experiments. For screening, biofilms were grown from unstimulated saliva samples collected from 12 subjects 6 hr post-cleaning. Individual saliva samples were diluted 1:4 into TH supplemented with 1% glucose, 1% mannose and 1% sucrose. This suspension was subjected to a low speed centrifugation for 10 min at 600 × g to remove eukaryotic cells and large debris and 400 μL of the supernatant was inoculated into each well of a sterile 8-well Lab-Tek™ Chambered Coverglass (Nalge Nunc International; Naperville, USA) that had been coated with 50% (filtered microorganism-free) saliva in phosphate buffered saline (PBS pH 7.0) for pellicle formation. To enhance attachment of salivary bacteria, 2 cycles of “starter” biofilms were allowed to deposit matrix material on the glass surface for 24 hr followed by removal and chamber re-sterilization, prior to seeding of experimental biofilms. Biofilms were incubated for 16 hr prior to staining and microscopic evaluation.

For visualization, biofilms were stained with the cell permeant general nucleic acid stain SYTO59 (Invitrogen, Carlsbad, USA) according to manufacturer's instructions, and S. mutans was specifically identified utilizing SWLA1–IgG2a as described previously (Gu et al., 2002; Gu et al., 2005) with the following modifications: the primary antibody was diluted in PBS to 50 μg/mL⁻¹ ; 100 μL of this diluted antibody solution was used for each sample and incubated for 30 min. Biofilms were then washed three times with PBS and further incubated with fluorescein isothiogyanate (FITC) conjugated goat anti-mouse IgG diluted 1:50 in PBS for an additional 30 min according to manufacturer's instructions (Sigma, St Louis, USA). Biofilms were examined with a PASCAL 5 confocal laser scanning microscope using LSM 5 PASCAL software (Zeiss, Jena, Germany). The scanning module of the system was mounted onto an inverted microscope (Axiovert 200M) and samples were viewed through a 40× oil-immersion objective (Plan-Neofluar/1.3). Excitation at 488 nm with an argon laser in combination with a 505–560 nm bandpass emission filter was used for FITC fluorescence imaging. An excitation at 633 nm with a helium-neon laser and a 650 nm longpass emission filter was utilized to reveal SYTO59-stained cells within the samples.

Saliva samples that appeared free of S. mutans were further tested for their ability to integrate exogenously added S. mutans UA140 upon co-inoculation. S. mutans at 1.25×10⁶ cells per well was added directly to the processed saliva and biofilms were allowed to form for 16 hr prior to visualization as described above. Samples without detectable S. mutans were eliminated due to their potential ability to suppress S. mutans growth. Saliva from subjects containing no detectable S. mutans that did not inhibit growth of exogenously added S. mutans were utilized for further studies.

Confirmation of protective colonization effects

To test protective biofilm effects, processed saliva suspensions were allowed to form biofilms for 16 hours and then replenished with fresh medium containing 1.25×10⁶S. mutans cells. After an additional 24 hours growth period, samples were stained and subjected to CLSM analysis as described above or cfu analysis described below.

Evaluation of STAMP ability to inhibit S. mutans biofilm incorporation at innoculation

To test the ability of S. mutans-specific STAMPs to remove S. mutans at inoculation, 100 μmol/L⁻¹ of the respective peptide was directly added to the mixture of salivary bacteria and JM11 (1.25×10⁶ cfu/mL⁻¹). The STAMPs would then either be left in the inoculum or be removed via an immediate high speed spin (16,000 × g) for 5 minutes. The latter samples were washed once and resuspended in an equal volume of fresh medium prior to inoculation. 400 μl of the mixtures were inoculated into each well of a pellicle-coated and UV sterilized 48-well cell culture plate. As bacterial cells attach significantly better to plastic than to glass surfaces, the “conditioning” process was omitted for these experiments. Biofilms were grown anaerobically for 16 hours, and were then washed with PBS and subject to viability evaluation via quantitation of viable colony forming units (cfu) as follows: biofilms were mechanically disrupted by vigorous pipetting in 1 ml TH and a series of 1:10 dilutions was plated onto both TH plates and TH plates supplemented with 800 μg·ml⁻¹ spectinomycin to assess total cell viability and JM11 viability, respectively.

Evaluation of STAMP-treated S. mutans-free biofilms to inhibit S. mutans incorporation

An S. mutans JM11 and saliva mixture was prepared and treated with the respective STAMPs as described above prior to biofilm formation for 16 hours before and a subsequent challenge with 1.25×10⁶ JM11 cells in fresh medium. After an additional 24 hours of anaerobic incubation, S. mutans incorporation was determined by biofilm disruption and plating to quantitate cfu (described above).

Results

Establishment of a saliva-derived biofilm system and observation of protective colonization

Biofilms were grown with saliva collected from subjects who are not colonized by detectable levels of S. mutans (selection criteria described in Methods) (Fig. 1A). To evaluate if we could control exogenous S. mutans addition, UA140 was inoculated together with the salivary bacteria to form biofilms which were then examined for S. mutans incorporation. S. mutans became an integral part of the biofilm community (Fig. 1B), comprising 2.7 % ± 1.0 % of the total population after 16 hr of biofilm growth (Fig. 1D, 0 hr). This finding confirmed that the in vitro saliva-derived biofilm model employed in this study enables the controlled addition of S. mutans to address the ability of this bacterium to invade preexisting biofilms.

Figure 1

Detection of S. mutans in the saliva derived biofilms. Saliva containing no detectable S. mutans was chosen for this study. (A)–(C): CLSM imaging. S. mutans cells within the biofilm were labeled with anti-S. mutans monoclonal antibody and FITC-conjugated (more ...)

Interestingly, the proportion of S. mutans incorporated into the biofilm was significantly reduced (0.3 % ± 0.1 % of the total biofilm population, representing 9 ± 0.4 fold reduction p=0.01) when S. mutans was added after saliva biofilms were allowed to form for 16 hr. Although biofilms were grown for an extra 24 hr after addition of S. mutans, this further growth period did not significantly increase the number of total biofilm cells (p=0.14, Figs. 1C and 1D). These results indicate that the observed difference reflects a protective colonization effect when a biofilm is already formed prior to addition of S. mutans.

Generation of protective S. mutans-free biofilms via STAMP treatment

The results in Fig. 1 suggested that the ability of S. mutans to colonize a surface was reduced in the presence of a preexisting biofilm derived from natural occurring S. mutans-free saliva. Based on this finding, we hypothesized that targeted elimination of S. mutans could yield a biofilm with similar protection capability. Previous studies have demonstrated that the STAMP C16G2 possessed specific antimicrobial activity against S. mutans cells in both planktonic and early stages of mixed species biofilm formation [Eckert et al., 2006]. Here, we evaluated if removal of S. mutans from the saliva inoculum with C16G2 or the functionally analogous STAMP 2_1G2 leads to the generation of protective biofilms with the ability to prevent secondary surface colonization by this cariogenic species.

Biofilm inocula containing salivary bacteria and the spectinomycin-resistant S. mutans derivative JM11 were incubated with the STAMPs for 16 hours. No S. mutans cells were detected in the resulting biofilms for both peptide-treated samples, while the untreated control contained ~10⁷S. mutans cells in the biofilm. More importantly, these in vitro generated S. mutans-free saliva biofilms were able to prevent subsequent infection from exogenous S. mutans (Figure. 2). A striking difference in the degree of colonization prevention was apparent in 2_1G2 and C16G2-treated samples: a 96 ± 28 fold (p=0.01) and 92 ± 14 fold (p=0.01) reduction, respectively, compared to S. mutans levels detected in the untreated control (Figure. 2).

The overnight treatment with peptides efficiently enabled growth of S. mutans-free saliva biofilms despite the presence of significant numbers of S. mutans in the inoculum. Based on these encouraging results we further explored the STAMPs' efficacy during shorter exposure times that are most relevant for potential therapeutic applications. As shown in Figure 3, treatment of an S. mutans containing saliva inoculum with either STAMP for as little as five minutes prior to biofilm growth resulted in biofilms containing approximately four- (2-1G2) and five- (C16G2) orders of magnitude less S. mutans cells (Figure 3). Upon re-infection with S. mutans, 38 ± 13 (2_1G2) to 61 ± 13 fold (C16G2) less S. mutans cells were detected in treated biofilms compared to the untreated control (p=0.07 for both peptides).

Figure 3

Evaluation of 5 minutes peptide treat- ment on S. mutans colonization 1.25•10⁶S. mutans strain JM11 was added to processed saliva suspensions and treated with 100 μmol/L⁻¹ STAMPs for 5 minutes. Samples were washed and then used (more ...)

Discussion

The data presented here support the hypothesis that the cariogenic organism S. mutans exhibits greatly reduced colonization in the presence of a preexisting saliva-derived biofilms containing no or a minimal amount of S. mutans. These findings provided evidence for the protective effect of a “normal” oral biofilm in concurrence with the proposed `window of infection' identified based on previous clinical studies [Caufield et al., 1993].

According to this report, S. mutans colonization mainly occurs between the second to the third year of life. Study participants who were infected during this crucial time period acquired persistent S. mutans colonization, while uninfected individuals remained S. mutans-free until the eruption of secondary dentition provided new colonization opportunities. It is likely that these uninfected individuals formed a protective biofilm community void of S. mutans, similar to the ones we observe in the in vitro saliva-derived biofilm model system, which significantly reduced the ability of S. mutans to become part of the biofilm community when added later on.

Though the mechanism behind the ability of biofilms to exclude S. mutans remains to be elucidated, recent studies by Kreth et al. [Kreth et al., 2005; Kreth et al., 2008] demonstrated that the sequence of inoculation determines whether cariogenic S. mutans or the health-associated S. sanguinis [Corby et al., 2005; Corby et al., 2007;] compete or co-exist with one another. Both species can persist in a biofilm when inoculated at the same time, but given the chance to establish a biofilm first, either species can preclude colonization of the other. In the context of this study, it is possible that the presence of certain species (such as S. sanguinis) in the established biofilms could mediate the protective effects against exposure to S. mutans. Interestingly, this protective effect was observed even in the presence of 1% sucrose in the growth medium which, according to the “Ecological Plaque Hypothesis” [Marsh, 2003], is likely to give the acid-tolerant S. mutans a competitive advantage through its ability to produce lactic acid from sucrose fermentation. Furthermore, the microbial homeostasis within oral biofilms is suggested to shift when oral environmental changes, such as the ingestion of fermentable sugars, create low pH environments where acid tolerant/generating organisms thrive and promote demineralization. Given consistent sucrose intake, these changes can compound and result in an oral biofilm comprised of predominantly cariogenic pathogens such as S. mutans. Our results suggest that established S. mutans-free biofilms, both naturally occurring or through STAMP treatment, are able to reduce the competitive advantage of S. mutans even in the presence of high sugar content, thus preventing the shift in the biofilm composition toward cariogenesis.

In addition to demonstrating that prior establishment of an S. mutans-free biofilm provides considerable protection against subsequent infection with this oral pathogen, we were able to significantly reduce S. mutans in the inocula with short 5 min exposure, a duration short enough for the application of most oral care products. More importantly, this reduction in S. mutans load achieved via STAMP treatment resulted in biofilms with considerable protective effects against further S. mutans colonization. Taken together, these results suggest that STAMP technology is a potentially useful method for removing S. mutans from saliva, thus establishing an oral flora that can help prevent or reduce further infection of S. mutans.

STAMP technology is by no means limited to modulating oral biofilms. It could potentially extend to other simple or complex microbial communities where a dynamic shift in microbial diversity is required to promote a healthy state for the host. Studies have shown that chronic conditions such as Crohn's disease and ulcerative colitis are associated with shifts in the homeostasis of the flora, and it is reasonable to speculate that inter-species interaction plays a role in determining the disease or non-disease state of the complex community within the gut as well. STAMP technology could be used to remove species that have high occurrence rates in patients suffering these diseases, such as Bacteroides vulgatus [Mangin et al., 2004], thus helping to reestablish a balanced flora where the specific niche of B. vulgatus is filled by other, more benign species. Further studies are underway to examine these potential applications.

Figure 2

Evaluation of overnight STAMP treatment on S. mutans colonization 100 μmol/L⁻¹ STAMPs were added to a saliva/S. mutans (1.25 •10⁶) mixture and incubated anaerobically for 16 hours, S. mutans surviving cfu were presented (dotted (more ...)

Acknowledgement

This work was supported by a grant from NIH (5R42MD001831).

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