A Drug Repositioning Approach Reveals that Streptococcus mutans Is Susceptible to a Diverse Range of Established Antimicrobials and Nonantibiotics

Abstract

Streptococcus mutans is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. An adenylate kinase-based assay was optimized for S. mutans to detect cell lysis when exposed to the Selleck library (Selleck Chemical, Houston, TX) of 853 FDA-approved drugs in, to our knowledge, the first high-throughput drug screen in S. mutans We found 126 drugs with activity against S. mutans planktonic cultures, and they were classified into six categories: antibacterials (61), antineoplastics (23), ion channel effectors (9), other antimicrobials (7), antifungals (6), and other (20). These drugs were also tested for activity against S. mutans biofilm cultures, and 24 compounds were found to inhibit biofilm formation, 6 killed preexisting biofilms, 84 exhibited biofilm inhibition and killing activity, and 12 had no activity against biofilms. The activities of 9 selected compounds that exhibited antimicrobial activity were further characterized for their activity against S. mutans planktonic and biofilm cultures. Together, our results suggest that S. mutans exhibits a susceptibility profile to a diverse array of established and novel antibacterials.

Keywords: Streptococcus mutans; antibiotics; dental caries; high-throughput screen.

FIG 1

(A) Optimization of the AK assay for detection of AK release from S. mutans UA159. Shown is AK activity versus CFU of S. mutans UA159. The values for live cells and heat-killed cells were statistically significant for each pairwise comparison using Student's t test (two tailed). *, P < 0.001. (B) Z′ score determination for detection of S. mutans AK release. The Z′ score was calculated according to the following equation: Z′ = 1 − 3[(σ⁺ + σ⁻)/(∣μ⁺ − μ⁻∣)], where σ and μ are the standard deviation and average, respectively, for the positive (+; 20 μg/ml ciprofloxacin) and negative (−; 0.5% DMSO) controls. The error bars indicate standard deviations.

FIG 2

AK assay data from a representative plate. Shown is a graphical representation of relative luminescence units (RLU) (y axis) versus plate position. The dashed line shows the cutoff value of 2-fold above background (DMSO). The drugs contained in these wells were used for further analysis.

FIG 3

(A) Drugs with activity against biofilm cultures of S. mutans. Of the 126 hits with activity against planktonic cells, 24 exclusively inhibited in vitro biofilm formation (inhibits), 6 exclusively induced AK release in preformed biofilms (kills), and 84 hits displayed both biofilm inhibition and activity against preformed biofilms; 12 compounds had no effect on biofilms. (B) Classes of hit compounds from the Selleck library with activity against planktonic cultures of S. mutans UA159.

FIG 4

Biofilm inhibition in the presence of select hit drugs from the AK assay. Select hit drugs from each class identified from the Selleck screen were assayed at 0.25×, 0.5×, and 1× MIC for their ability to prevent biofilm formation in vitro. The data were normalized to the DMSO control. *, P < 0.05 (Student's t test; two tailed). The error bars indicate standard deviations.

FIG 5

(A) Select statin class drugs lysed S. mutans in an AK assay screen of the Selleck library. Statins were identified in the screening of the Selleck library against planktonic cultures of S. mutans UA159. The data are representative of 2 independent screens. (B) Simvastatin inhibits growth of S. mutans. Growth of log-phase S. mutans UA159 was measured via OD₆₀₀ in the presence of various amounts of simvastatin (n = 5). (C) Simvastatin inhibits biofilm formation by S. mutans. Simvastatin was tested at 0.5× and 1× MIC for the ability to prevent in vitro biofilm formation by cells grown in TY plus 1% (wt/vol) glucose or TY plus 1% (wt/vol) sucrose, as detected by crystal violet staining (OD₅₇₅). The data were normalized to the DMSO control. *, P < 0.05 (Student's t test; two tailed). The error bars indicate standard deviations.