Rat model of dental caries

The animal experiment was reviewed and approved by the Ethical Committee on Animal Research at the University of Campinas (CEUA – Ethics Committee on Animal Use/UNICAMP, Campinas, SP, Brazil, process number 4463–1/2017) and was performed according to methods previously described [29,30]. An overview of the experimental design is shown in Figure 1. A total of 56 SPF (Specific Pathogen Free) female Wistar rats, aged 19 days were provided by CEMIB/UNICAMP (Multidisciplinary Center for Biological Research, Campinas, SP, Brazil). From the arrival of the animals until the end of the experiment, the animals were kept in a room of the vivarium for research using Genetically Modified Organisms (GMOs), authorized and approved by the GMO Biosafety Committee from FOP/UNICAMP (A001-2017).

Experimental design for the rat model of dental caries.

On the day after the arrival of the animals, they were randomized to form four groups composed of 14 animals, using ear tags. The four groups were color-coded, so the investigators handling the animals were blinded to the strain that would be used for infection. The black group was infected with the parental strain, UA159 (positive control strain). The red group was infected with ΔgtfB (a reduced-caries-causing control to which the caries-causing potential of the tested strains was compared). The blue and green groups were infected with ΔlytS and ΔdltD, respectively.

Each animal was weighed (COMTEC 426–1070) and the data recorded. Initial screening was performed to verify that the animals were not colonized with mutans streptococci. A sterile cotton bud (Labor Import), wetted in saline solution (0.89% NaCl), was used to swab the rat mouths. Each oral swab was struck on a Mitis Salivarius agar plate without antibiotic (MSA) and on a Mitis Salivarius agar plate containing 0.2 U of bacitracin per ml (MSB). The plates were incubated (37°C, 95% (v/v) air/5% (v/v) CO₂, 48 h) and the morphologies of colonies were visually evaluated to ensure that the animals were free of mutans streptococci colonization.

The four strains (parental strain UA159 and deletion strains ΔgtfB, ΔlytS, and ΔdltD) were grown in biofilm cultures on glass rods in the presence of sucrose. It has been shown that using biofilm cultures as the inoculum in the rat mouth enhances their ability to colonize tooth surfaces [31,32]. On day one of biofilm growth, 50 µL of each strain was transferred to a tube containing TY (2.5% tryptone plus 1.5% yeast extract, Difco) and 2% sucrose (w/v) (Dinamica®/1894–1), antibiotic was added to media for cultures of the deletion strains. After incubation at 37°C in a 95% (v/v) air/5% (v/v) CO₂ atmosphere for 24 h (day two of biofilm growth), each rod was transferred to new tubes with fresh media containing 2% sucrose. These cultures were incubated under the same conditions described above, and the rods transferred to fresh media again on day three of biofilm growth. On day four of biofilm growth, biofilms were scraped off the rods and transferred to a new tube containing fresh media, then incubated as above. After 18 h, these cultures were plated on MSB agar plates and incubated for 48 h, after which time the plates were stored at 4°C to be utilized as inocula to infect the animals.

On the day before the arrival of the animals, starter cultures were prepared by inoculating 10 mL of TY liquid medium containing 1% glucose (w/v; Synth) with colonies from the plates described above. Two tubes were prepared for each strain and incubated at 37°C in a 95% air/5% CO₂ atmosphere. After 18 h, the starter cultures were diluted in TY liquid medium containing 1% glucose (1:20 dilution). The diluted cultures were allowed to grow to an O.D._540nm ~ 0.5. Each culture was then transferred to a 50 mL tube containing 14 swabs. These tubes were maintained on ice until shortly before inoculating the animals' mouths, at which time the cultures were warmed to room temperature.

Rats were infected with the cultures described above by oral swabbing four times, on four consecutive days (as outlined in Figure 1). After these four infection procedures, a confirmatory screening was performed to verify the presence of the S. mutans strains in the mouth of each rat, similar to the procedure used in the initial screening. Immediately after the confirmatory screening, the animals were transferred to suspended cages (two animals per cage), where they remained for five weeks until sacrificed.

After the first infection, the rats’ diet was replaced with Diet 2000 (containing 56% sucrose [Table S1; 33]). The animals were given sterile, distilled, deionized, water containing 5% sucrose, ad libitum. The ad libitum cariogenic diet used during the five-week experimental period is an established method for allowing the development of carious lesions on the dental surfaces [9]. The animals were weighed weekly (Figure S1). In the second and fourth weeks, the vitamin Vitagold Potenciado (Fabiani Saúde Animal, São Paulo, Brazil) was added to the drinking water (20 mL per 10 L of water) to prevent hair loss.

The animals were euthanized by anesthesia using Ketamine (Dopalen 1,000mg/10 mL – Ceva ®) (300 mg/kg) and Xylazine (Anasedan 2,000 mg/10 mL – Ceva ®) (30 mg/kg), followed by decapitation.

The lower left jaw of each animal was aseptically dissected and was suspended in 5 mL of sterile 0.89% NaCl solution. The jaws were sonicated twice, with a 10-s interval between probe sonication for 30 s at 7 W (Vibracell, Sonics and Material Inc., Newtown, CT). Aliquots of each biofilm suspension were used for 10-fold serial dilutions. The dilutions 1:10, 1:100 and 1:1,000 and undiluted suspension were seeded on blood agar medium (for total cultured microbiota) and MSB agar medium (for S. mutans), and the plates were incubated for 48 h at 37°C in a 95% (v/v) air/5% (v/v) CO₂ atmosphere. The blood agar plates were incubated for another 24 h at 37°C. Colonies were counted and used to calculate CFU in 5 mL of biofilm suspension (CFU/biofilm). All jaws were stored at −20°C until manual dissection for caries scoring.

The teeth were prepared for caries scoring according to Larson’s modification of Keyes system [34]. One calibrated examiner performed the caries score of the codified jaws. The smooth surface caries scoring (buccal, morsal, proximal, lingual and palatinal) was done using a stereoscopic Zeiss magnifying lens (Stemi SV 6).

Before performing caries scoring on sulcal surfaces, the samples were prepared as follows: The jaws were painted with transparent nail polish to strengthen the structure and left to dry for 24 h. Murexide (SIGMA M-2628) dye solution (0.24 mg/mL) was used to stain the jaw and left to dry for 18 h. A sagittal cut was made in the jaws with a carbide disk coupled to a micromotor (mc52 – dent) to allow for better visualization of the sulcal surface. Caries scoring was performed on the sulcal surfaces using a stereoscopic Zeiss magnifying lens (Stemi SV 6).