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Topical Effect of a Medically Prescribed Pediatric Antibiotic on Dental Biofilm: A Cross-Over, In Situ Study

  • Viviane Santos da Silva Pierro ,

    vivipierro@gmail.com

    Affiliation Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Dennis de Carvalho Ferreira,

    Affiliation Institute of Microbiology Paulo de Goés, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Hugo Emiliano de Jesus,

    Affiliation Institute of Microbiology Paulo de Goés, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Alexandre Soares Rosado,

    Affiliation Institute of Microbiology Paulo de Goés, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Ronir Raggio Luiz,

    Affiliation Institute of Studies of Public Health (IESC), Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Kátia Regina Netto dos Santos,

    Affiliation Institute of Microbiology Paulo de Goés, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

  • Lucianne Cople Maia

    Affiliation Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, Brazil

Topical Effect of a Medically Prescribed Pediatric Antibiotic on Dental Biofilm: A Cross-Over, In Situ Study

  • Viviane Santos da Silva Pierro, 
  • Dennis de Carvalho Ferreira, 
  • Hugo Emiliano de Jesus, 
  • Alexandre Soares Rosado, 
  • Ronir Raggio Luiz, 
  • Kátia Regina Netto dos Santos, 
  • Lucianne Cople Maia
PLOS
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Abstract

Objective

This study aimed to investigate the possible topical effect of a broad-spectrum antibiotic on dental biofilm formed in situ in the absence or presence of sucrose.

Methods

A crossover study was conducted in three phases of 14 days each, during which 11 volunteers wore palatal devices containing 6 enamel blocks covered with meshes to allow biofilm formation. Dental blocks were extraorally submitted to a 20% sucrose solution at three different frequencies of exposure (0, 3 and 8 times/day), and to a suspension of amoxicillin/clavulanate potassium (A/CP) or a placebo (P) suspension at an 8-hour time interval application regimen. On the 14th day of each phase, biofilms were collected for microbiological (conventional culture) and molecular (Denaturing Gradient Gel Electrophoresis – DGGE) analyses.

Results

In the absence of sucrose exposure (SE) and at the 3-time daily frequency, dental biofilms treated with A/CP showed lower total biofilm weight and lower counts of total microbiota than the ones treated with P (p>0.05). A/CP presented higher counts of Candida spp. when compared with P in the presence of SE, especially at the 8-time daily frequency (p<0.05). Considering the DGGE analysis, the mean number of bands was higher for P (p>0.05), regardless of SE. However, DGGE profiles demonstrated large interindividual variability.

Conclusion

Both conventional culture and DGGE have demonstrated some differences on total microbiota of dental biofilms when exposed to the A/CP or P suspensions, mainly in the absence of sucrose, which suggests a possible topical effect of the sugar-free A/CP suspension on dental biofilm.

Introduction

Dental caries is an infectious disease caused by oral acidophilic bacteria which metabolize fermentable sugars. It is viewed as a consequence of an imbalance in the resident microflora due to frequent conditions of low pH in plaque biofilms, for example, as a result of a sugared diet or a reduction in saliva flow [1]. Therefore, considering the role of oral bacteria in dental caries, frequent oral intakes of medically prescribed antibiotic suspensions could influence caries establishment because of their possible additional topical effect on dental biofilms.

Some clinical studies tried to elucidate the role of antibiotic suspensions on children's oral health status [2][11]. Although most of them have been conducted with sick children taking long-term antimicrobial medication compared with their healthy siblings or students from near communities [2][7], conflicting results pointed out either a protective effect [2][6], [10][11] or a contributing effect [7], [9] of antibiotics on dental caries.

Many liquid pharmaceutical preparations (including antibiotics) for children are made palatable by the addition of sucrose, glucose, or fructose as sweeteners. These fermentable carbohydrates in thick liquid preparations may contribute significantly to the dental caries potential in young patients [12]. Furthermore, preoccupation with the main medical problem often results in neglect of oral health, and sugars are frequently offered to patients as a demonstration of comfort or as a necessary source of energy [13][14]. With the increasing formulation of sugar-free medicines [15][16], the frequency of dietary sugar consumption can be considered of further importance for sick children receiving recurrent and/or prolonged oral medication.

The relationship between medically prescribed antibiotics and dental caries is mainly based on the possible antibacterial effect of these medications on dental biofilm, which could be considered an additional effect of this therapeutic class on oral health status. Considering that amoxicillin and its combined formulation with clavulanate potassium have been pointed out as the most commonly prescribed antibiotics for hospitalized children [17] and for preschool children [18] to treat a variety of infections usually through oral administration, the present study aimed at evaluating, in situ, the possible topical effect of a sugar-free antibiotic suspension containing amoxicillin/clavulanate potassium on dental biofilm.

Methods

Ethics Statement

This in situ study was approved by the Research and Ethics Committee of IESC/UFRJ (protocol No. 03/2010), and was performed with undergraduate and graduate dental students who signed an informed consent for participation. All procedures were conducted in accordance with the Declaration of Helsinki.

Experimental Design

The study had a crossover and “split-mouth” design, performed in three experimental phases of 14 days each; with a washout period of 7 days between each phase (Figure 1).

Eleven volunteers (3 males and 8 females, aged 19–31 years) met the inclusion criteria proposed by Zero [19] for in situ studies and did not violate the main exclusion criterion, which was the use of antibiotics or any other antimicrobial substances 60 days prior to the study and throughout all experimental phases. Each volunteer wore acrylic intraoral palatal devices containing 2 sets of 3 blocks (3×3×2 mm) of polished sound human primary dental enamel covered by a plastic mesh [20]. Both sets of dental enamel blocks were extraorally submitted to 20% sucrose solution (two drops onto each set) at three different daily frequencies of exposure (no exposure – 0 times/day; exposure – 3 times/day and 8 times/day), which were randomly assigned to each experimental phase undertaken by each volunteer. Additionally, during all the phases, volunteers were instructed to remove the palatal devices and drip two drops of an sugar-free antibiotic suspension containing 250 mg of amoxicillin and 62.50 mg of clavulanate potassium/5 mL onto one set of enamel blocks, and two drops of a placebo suspension onto the other set of blocks, following an 8-hour time interval application regimen. All volunteers were blind to this treatment regimen [21][25]. Furthermore, excess fluid was removed with gauze and the device was replaced in the mouth after 5 minutes [24][25]. The use of two treatments (split-mouth) in the same intraoral device was supported by the absence of a crossover effect in previous studies [21][26]. All intraoral devices were sterilized with ethylene oxide before the beginning of each experimental phase.

The volunteers received instructions to wear the devices all the time, including at night, but to remove them during meals, drinking and oral hygiene. All volunteers were advised to keep their usual dietary pattern during all the experimental phases.

Dental Biofilm Collection

On the 14th day of each experimental phase, plastic meshes were cut with sterile surgical blades (No. 11) and dental biofilms formed on the 2 opposite sets of 3 enamel blocks were separately collected with sterilized plastic curettes for microbiological analyses.

The collected biofilms were weighed in pre-weighed sterile microcentrifuge tubes and approximately 1.0–3.0 mg (wet weigh) were transferred to other sterile microcentrifuge tubes containing 5 glass beads (2 mm in diameter) to be suspended in 1 mL of 0.85% NaCl solution. The biofilms were vortexed for 30 s and ten-fold serial dilutions in sterile saline were inoculated in duplicate by the drop-countig technique [27] on the following culture media: blood agar (Plast Labor, Brazil), for total microbiota; and CHROMagar Candida (CHROMagar, France), for Candida spp. CHROMagar plates were incubated for 48 h at 37°C in aerobiosis, while blood agar plates were incubated in microaerophilic condition (candle jar) at 37°C for 24 h. The colony-forming units (CFU) were counted and the results expressed in number of CFU/mg dental biofilm (wet weight) and converted to log10 for the statistical analyses.

To proceed with bacterial molecular analysis, an aliquot of 300 µL from the above mentioned original suspensions was transferred to microcentrifuge tubes containing 1.0 mL of TE buffer (10 mM Tris- and 1 mM EDTA [pH 7.8]), which were agitated and transported on ice to a microbiology laboratory at the Dept. of Microbiology (UFRJ).

DNA extraction

The 1.3 mL samples were vortexed for 30 sec. Then, the microbial suspension was washed three times with 100 µL of sterilized Milli-Q water and bacteria were pelleted from the suspension by centrifugation at 2,500×g. The pellets were resuspended in 100 µL of Milli-Q water and bacterial DNA was extracted using QIAamp DNA Mini Kit (Qiagen, USA) accord