Orthodontic patients are at an increased risk for developing caries. Dental caries is a biofilm-mediated disease, with mutans streptococci (MS) as the primary etiologic bacterial group. It has been suggested that persister cells (PCs), a subset of cells within the biofilm, contribute to the chronic infectious nature of dental caries. PC formation can be induced by environmental stressors such as orthodontic treatment. The aim of this study was to quantify MS, aerobic and facultative anaerobe bacterial PC proportions from plaque samples during the initial stage of orthodontic treatment. This study is the first to analyze the role of PCs in a population of patients highly susceptible to caries, that is, patients undergoing orthodontic treatment.
Plaque samples were collected from 17 participants (11 males and 6 females; age range: 11-18 years) before and 1 month after insertion of fixed orthodontic appliances. Percentages of MS and PCs were determined with selective media and a classical persister microbial assay, respectively.
There was a statistically significant decrease in %MS ( P = 0.039) but no statistically significant difference in %PCs ( P = 0.939) after 1 month of orthodontic appliance placement.
Our study illustrated the technical feasibility of analysis of PCs in plaque samples of patients during orthodontic treatment and revealed that PC formation during orthodontic treatment is highly variable across individuals.
Mutans streptococci is the primary etiologic bacterial group of caries.
Persister cells (PCs) are a dormant subpopulation that confers chronicity of diseases.
Changes in PC levels may increase caries risk in orthodontic patients.
After 1 month of orthodontic treatment, %MS decreased but %PC did not.
Studying PCs in plaque samples of patients during orthodontic treatment was technically feasible.
A population group susceptible to an increased risk of developing caries are those undergoing fixed orthodontic therapy. Orthodontic appliances provide additional new retention sites for the oral microbiota and dental plaque , that can cause a drop of plaque resting pH. The pH drop leads to subsequent enamel demineralization and caries progression resulting in white spot lesions (WSLs), , the “first sign of a caries lesion on enamel that can be detected with the naked eye.” , The relatively long duration of orthodontic treatment further compounds orthodontic patients’ risk. Oftentimes, the diagnosis of WSLs is also impaired and not made until appliance removal. Indeed, the incidence of developing WSLs during orthodontic treatment can be as high as 73%, , and clinically visible WSLs can develop in as few as 4 weeks after the initiation of fixed orthodontic therapy.
The etiology of dental caries, though multifactorial, has largely been attributed to a microbiological origin within dental plaque biofilm. Dental plaque is one of the best studied forms of human bacterial biofilms or attached microbial communities. Biofilms consist of hundreds of coexisting bacterial species encased within an extracellular matrix of bacterial origin that confer on the component bacteria protection from stressful changes in the environment. , In dental caries, one of the major bacterial groups, mutans streptococci (MS), , has been shown to play an important role. Plaque overlying WSLs may contain as much as 10% MS compared with approximately only 2% or less in healthy sites. MS have been attributed to the early stages of caries lesions, with their levels reportedly constituting about 30% of total microflora in cavitated, dentinal lesions.
Within a given population of bacteria exists a subset of cells (approximately 1% in biofilms) called persister cells (PCs). PCs, first described by Bigger, exist in a state of metabolic dormancy. , As antibiotics typically target active metabolic processes, the “growth-arrested physiological state” of PCs permits them to be tolerant to antibiotics without expressing a resistance mechanism. Because PCs are genetically identical to antibiotic-susceptible cells, and thus are phenotypic variants rather than mutants, the antibiotic tolerance mechanism of PCs is not heritable and, more importantly, is reversible. Once antibiotic concentration drops, repopulation of the entire biofilm is initiated with just a few reactivated dormant cells, leading to relapses of infection. The presence of PCs, therefore, may confer on biofilm-mediated diseases their resilience and ability to cause relapses in infection after a course of antibiotics. Recent studies have shown that PC formation can also be governed by deterministic mechanisms, including environmental and external stimuli. PC formation can be triggered by nutrient limitations, shifts in metabolism, extreme pH fluctuations, or DNA damage. Oral bacteria are constantly exposed to a wide range of environmental changes (eg, constant cycles of famine and feast, fluctuations in pH, temperature shock, and oxidative stress) that may trigger PC formation.
Bacterial PC have been extensively studied in other bacteria but very few in MS. In this study, we analyzed PCs in the orthodontic population because of this population group’s increased susceptibility and risk of developing caries. Although the role of PCs in the etiology of chronic bacterial diseases is well recognized and studied in many fields, the dearth of studies in dentistry is surprising in light of the fact that caries is a major chronic health problem worldwide. This study is among the first to analyze the role of PCs in orthodontics; information generated from the study is expected to provide information useful in understanding the role of PCs in the early stages of the caries process and will provide guidance for future studies of this nature.
We hypothesized that the introduction of fixed orthodontic appliances represents a local environmental stress that would induce PC formation. We aimed to analyze short-term changes in MS and aerobic and facultative anaerobic bacterial PC levels from plaque samples of patients commencing orthodontic treatment.
Material and methods
Study participants were recruited from the Graduate Orthodontic Clinic at the University of Toronto, Faculty of Dentistry. All participants and their guardians signed informed consent and assent forms before taking part in the research study. The study was approved by the Research Ethics Board at the University of Toronto (ethics approval #32462) and conducted in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki.
Participant screening was completed by 1 assessor (C.K.A.). Participants were recruited if they were healthy, required fixed orthodontic treatment, possessed good-to-fair oral hygiene as indicated by healthy noninflamed gingiva at the time of recruitment, and a full complement of permanent dentition (except third molars). Exclusion criteria included a history of antibiotic use within the past 6 months, professional cleaning within the past 3 days (to allow time for the biofilm to develop and mature), presence of major dental or medical conditions—such as untreated carious lesions at baseline or loss of periodontal support requiring intervention—and fixed ceramic or self-ligating appliances or Invisalign treatment.
Intraoral photographs and panoramic radiographs of 30 eligible participants were identified; 3 declined to participate, and 10 others were not included either because of a lack of follow-up or a late start in orthodontic treatment, resulting in a recruitment of 17 participants (11 males and 6 females; age range: 10.9-18.1 years). All recruited participants completed the study.
At the end of the initial bonding appointment, an oral hygiene kit (toothbrush, floss, floss threaders, and proxybrush) was given to all participants. Standardized oral hygiene instructions were given—participants were shown how to use the items in the kit followed by participants practicing their use.
Plaque samples were collected at 2 time points: T0 = at the bonding appointment (baseline, before bonding the brackets) and T1 = 4 weeks into fixed orthodontic therapy. Supragingival plaque from the buccal surface of maxillary right canine and mandibular left canine was collected using sterile wooden toothpicks and placed into prelabeled sterile microcentrifuge tubes for microbial analysis. The samples were immediately stored in a secured −80°C freezer until microbiological analyses were conducted.
Plaque control was assessed using a modified set of Ramfjord teeth, wherein the canines rather than first premolars were included because the premolars were often extracted as part of the orthodontic treatment plan. The modified Ramfjord teeth, maxillary right first molar, maxillary left central incisor, maxillary left canine, mandibular left first molar, mandibular right central incisor, and mandibular right canine were disclosed using HurriView Snap-n-Go swabs (Beutlich Pharmaceuticals LLC, Bunnell, Fla) according to the manufacturer’s instructions ( Fig 1 ).
Using the orthodontic plaque index outlined by Attin and later applied by Bock, a plaque index (PI) for each patient at each time point was calculated ( Figs 2 and 3 ). The formula used for calculating PI was PI = (sum of plaque scores × 100)/(number of teeth evaluated × 3). Teeth that could not be disclosed owing to orthodontic bands were not included in the determination of the PI.
Plaque samples from the −80°C freezer were resuspended in 300 μL of phosphate buffered saline (pH 7) by pipetting up and down and vortexing for a few seconds. Serial dilutions (by a factor of 10) were plated using an automated spiral plater, dispensing 50 μL-aliquots on brain-heart-infusion medium (BHI) and incubated at 37°C in air containing 5% carbon dioxide (CO 2 ). After 48 hours, the colony-forming units(CFUs) were counted using the segment pair counting grids. Serial dilutions were also spiral-plated on mitis salivarius–bacitracin agar (Thermo Fisher Scientific Difco, Waltham, MA) supplemented with 20% (weight/volume) sucrose, a selective medium for MS, and incubated for 72 hours under the same conditions (37°C in 5% CO 2 ). The MS CFUs were then counted, and the percentage of MS from each sample was determined.