Clinical efficacy of a specifically targeted antimicrobial peptide mouth rinse: targeted elimination of Streptococcus mutans and prevention of demineralization

Abstract

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.

Fig. 1

Intra-oral retainer. Sites for loading enamel chips can be seen on the left and right sides (circular punches in square mounts).

Fig. 2

Clinical study design.

Fig. 3

Activity of C16G2 against S. mutans biofilms. UA140 biofilms were grown 24 h under anaerobic conditions with 1% (w/v) sucrose. Biofilm viability was then assessed by a substrate-conversion redox assay after treatment with C16G2, G2, or Melittin B at the concentrations indicated. Biofilms treated with buffer alone were considered as having 100% viability.

Fig. 4

In vitro tissue irritation. EpiOral (a) or EpiGingival (b) tissue was exposed to 25 or 100 μM C16G2, or 1% Triton X-100, for the durations indicated. Tissue viability was recorded by a substrate conversion assay (MTT) and expressed as percentage of viability from 1× PBS-treated tissue. ET-50 (time to 50% viability) was estimated for Triton X-100-treated samples.

Fig. 5

Stability in 1× PBS and saliva. C16G2 (100 μM) was mixed with 1× PBS and held at 4 or 25°C for 20 h to assess stability (a). TIC spectra were overlayed and the C16G2 peak at ∼8.4 min shown with peak area. The y-axis for each spectrum was percent TIC (0 to 100). C16G2 (100 μM) was mixed with prepared saliva (b) and monitored for the durations indicated. Remaining C16G2 peak areas (TIC spectra) were plotted to determine STAMP half-life.

Fig. 6

S. mutans in plaque and saliva after STAMP and placebo treatment. Plaque (a) and salivary (b) cfu/ml were monitored daily from panelists treated with placebo (▴) or C16G2 (▴) rinse. Placebo treatment was associated with an increase in S. mutans cfu/ml over baseline, while STAMP treatment resulted in a reduction in S. mutans from baseline.

Fig. 7

Resting plaque pH and lactic acid production. The resting pH (a) of plaque samples was determined at baseline and daily after treatment. STAMP treatment was associated with higher pH when compared to placebo samples. Concurrently, the level of lactic acid production (b) was significantly lower in STAMP-treated samples than in placebo-treated samples after 4 days.