Anti-Biofilm Effect of Oral Rinses on an Engineered Tooth Model

OBJECTIVES: We had previously reported on the development of an Oral Biofilm-Minimal Elimination Concentration (OBF-MEC) Assay for biofilms using the CBD (Calgary Biofilms Device) as a substratum to which was engineered a tooth surface using calcium hydroxyapatate. Here we further evaluated the biofilm-tooth model comparing the efficacy of two standard, oral rinses. METHODS: MEC methods were previously described; the tooth surface was engineered using a microtitre lid immersed in a 2:1 mixture of rubber cement in thinner to glue calcium hydroxyapatite beads (BioRad) to 96 microtitre pins. Dental rinses (1x) were: 1) Listerine with alcohol (21.6%) and 2) Crest Pro-Health with cetylpyridinmine chloride (0.07%). 3 dilutions for each rinse (1x) were made at 0.1x, 0.01x, and 0.001x. Clinical isolates were recovered from the WVUH Oral Facial-Dental Microbiology Laboratory where the Etest (AB BioDisk) had established the planktonic Minimal Inhibitory Concentration (MIC): Staph aureus (SA)/Clindamycin (4μg/ml) and Pseudomonas aeruginosa (PA)/Ciprofloxicin (6μg/ml). 48hr-biofilm pins were immersed for 30min or 48hrs using a rocker to establish shear, 8 rocks/min after preconditioning with artificial saliva. Anti-biofilm impact was assayed by crystal violet (CV) staining of individually removed pins at 10/40x microscope and SEM (NIOSH) using a JOEL 6400 electron microscope. RESULTS: 4+ biofilms (>90% confluency/3-D architecture) developed on the 96 pins 18-36hrs. Quantitative changes between Listerine and Crest Pro-Health were minimal with greatest activity at undiluted (1x) concentrations for either SA or PA. There was no inhibition at 30min. SEM highlighted biofilm growth on the beads with minimal alteration in 3-D architecture and confluency comparing the two rinses. Listerine showed the greater loss of susceptibility (change in MEC/MIC). CONCLUSIONS: The 96-pin replicate microtitre tooth model (OBF-MEC) was a viable biofilm susceptibility measurement replicating a clinical environment. The ratio of OBF-MEC/MIC may help eliminate ineffective biofilm anti-infectives.