Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on sociodemographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supragingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, ex- traction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cario- genic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.
Severe early childhood caries (S-ECC) is a particularly aggressive form of dental caries associated with a history of maternal malnutrition and illness, tooth developmental defect [
Studies have also demonstrated that the presence of lactobacilli alone in caries lesions or saliva is insufficient to make inferences about their specific contribution in caries development. Although more than 150 validly described species of Lactobacillus have been isolated from various human body sites, plants, foods and the environment [
The lack of knowledge about the genetic characteristics and diversity of lactobacilli is, in part, hindered by the taxonomic challenges from the initial step of clinical sample collection to the final step of species identification. Traditionally, studies of caries-associated lactobacilli are based on laboratory cultivation, but results vary due to the lack of reliable protocols for screening and characterizing clinical isolates that enable the identification of lactobacilli. Recently, advancements in molecular genetics and comparative genomics have been applied to microbiology, largely facilitated by 16S rRNA sequencing and sophisticated analytical methods [
Previously, we reported a pilot study to demonstrate methods for determining the diversity of lactobacilli in the oral cavity [
This exploratory study consists of four components: 1) subject recruitment, clinical and bacterial sample collection; 2) assessment of Lactobacillus abundance relative to other microbes; 3) determination of lactobacilli diversity at the species level in an aggressive S-ECC group and a caries-free (CF) group; 4) comparison of whole genome sequences from the clinical Lactobacillus species/strains. The overall study design is illustrated in
early childhood caries. In order to accomplish the goal, both culture-based and culture-independent methods will be used to evaluate the level of lactobacilli in saliva and dental plaque, carious lesions and sound fissures and, consequently, to obtain individual Lactobacillus isolates. The final Lactobacillus species identification will be based on 16S rRNA gene sequence analysis. Furthermore, whole genome intra-species and inter-species comparisons will be conducted to identify niche-specific genetic elements (
The study is conducted at two sites: New York University College of Dentistry and the Pediatric Dental Clinic of Bellevue Hospital Center. Applications for this study have been approved by the Institutional Review Boards of the New York University School of Medicine, New York University College of Dentistry (Research Proposal Oversight Committee), and the New York City Health and Hospital Corporation (for Bellevue Hospital Center) for human subjects participating in research activities.
The study recruitment began in February 2012 and ended in May 2014. A total of 80 children, 3 to 6 years of age, were recruited for this study from the Bellevue Hospital Pediatric Dental Clinic. Forty of them were diagnosed with S-ECC according to the criteria set forth by the American Academy for Pediatric Dentistry [
S-ECC is defined as caries in children younger than 3 years of age with one or more non-cavitated or cavitated lesions, filled or missing (due to caries) smooth surfaces in primary maxillary anterior teeth (dmfs), or a dmfs score ³ 4 at age 4, ³ 5 at age 5, or ³ 6 at age 6 [
To be eligible for this study, children must be 3 to 6 years old, healthy without chronic diseases other than dental caries, and not having taken antibiotics within 3 months prior to bacterial sample collection. Children with chronic disease other than dental caries or a history of antibiotic use within 3 months prior to the dental treatment or missing more than 6 primary teeth are excluded.
The study is designed to recruit 80 children divided into an S-ECC group (n = 40) and a CF group (n = 40). The sample size of estimation is based on data previously reported that the composition of oral microbiota of S-ECC children exhibits less diversity than that of CF children [
Before undergoing dental examination, all primary caregivers who agreed to participate in this study were interviewed by a trained project coordinator to obtain the child’s current and past medical history, nursing practices, dietary habits, oral hygiene habits, and prescribed medications. Both English and Spanish versions of the questionnaire survey were available. The entire interview took no more than 20 minutes. Minimum or no risks result from participation in this study, and all information related to the project will be kept confidential following state laws and regulations governing confidentiality of medical records.
A standardized clinical examiner performed the dental examination for each child in the Bellevue Pediatric Den- tal Clinic using standard dental mirrors and established criteria and scoring system. Using a sterilized soft toothbrush (Soft P-20 Oral-B Cat. 33259737, Henry Schein, Melville, NY), saliva and plaque samples were collected from all S-ECC children in the operating room by the standardized attending dentist. The same samples were collected from the CF children by a trained dental hygienist. A second bacterial sample was also obtained from caries lesions of the S-ECC children by the same dentist. More specific procedures of bacterial sample collection are described in the following sections.
1) Subject-specific
a) For S-ECC children scheduled for treatment under general anesthesia in the operating room: before comprehensive restorative treatment, the dentist brushed the child’s teeth for 1 min using a sterile soft toothbrush (Soft P-20 Oral-B).
b) For CF children in the dental clinic: the hygienist brushed a child’s teeth for 1 min using a sterile soft toothbrush without swallowing saliva.
2) Procedure
a) The dentist/hygienist slowly brushed the surfaces of the teeth, and then mixed with saliva.
b) The toothbrush was immediately placed into a pre-labeled glass tube containing 9 mL of a pre-reduced liquid dental transport (LDT) medium (custom order; Anaerobe Systems, Morgan Hill, CA).
c) The bacterial sample was removed from the toothbrush by shaking the toothbrush vigorously for 30 sec (approximately 30 strokes) in the LDT medium; the toothbrush was then discarded.
d) The bacterial sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.
1) Caries lesion site-specific sample for S-ECC group
a) Bacterial sample was collected from 1 caries lesion at the most severe site.
b) A sterile regular No. 6 dental round bur (Dentsply International, Des Plaines, IL) was used with a slow- speed handpiece to remove caries lesion for 5 sec.
c) Bacterial sample along with the bur was immediately placed into a pre-labeled glass tube containing 1 mL of LDT medium (AS-916, Anaerobe Systems, Morgan Hill, CA), and the tube was capped.
d) The sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.
2) Fissure site-specific sample for CF group
a) Bacterial sample was collected from 4 sound occlusal fissures (molar or premolar; 1 per quadrant).
b) A sterile 30-gauge extra-short dental needle (Henry Schein, Melville, NY) was held in a hemostat and gently passed across the 4 occlusal fissures to collect as much bacterial sample as possible.
c) The needle was immediately placed into a pre-labeled glass tube containing 1 mL of the LDT medium, and the tube was capped.
d) The sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.
Previously, we observed that the saliva of caries-active mothers could harbor between 2 to 8 distinct genotypes of lactobacilli [
Upon arrival of the bacterial sample, sterile glass beads (3 mm diameter) (Pyrex, Corning Incorporated, Corning, NY) will be added to the samples and mixed by vortexing for 30 s. Three 10-fold serial dilutions (10−1 to 10−3) of the sample will be used in order to obtain accurate colony forming unit (CFU) reads. The diluted samples (50 ml) will be plated on LBS medium, (Dickinson and Company, Sparks, MD) for recovery of lactobacilli using an AutoplateÒ Spiral Plating System (Advanced Instruments, Inc., Norwood, MA). The samples will be incubated anaerobically (10% CO2, 10% H2, and 80% N2) at 37˚C (standard conditions) for 4 days, as well as in microaerophilic conditions (6% - 16% O2 and 2% - 10% CO2) at 30˚C (alternative conditions) for 4 days to capture as many Lactobacillus species as possible [
After cultivation, 50 individual colonies per sample will be randomly selected with sterile toothpicks from the LBS plates and pure streaked on MRS agar plates (MRS, Becton Dickinson and Company, Sparks, MD). The MRS plates will be cultivated overnight anaerobically at 37˚C. DNA will be extracted from the freshly grown culture by using a modified heating technique. Briefly, each of the bacterial samples will be transferred from the MRS agar plates, suspended in a 20 μl lysis solution (10 mM Tris-HCl buffer, 1 mM EDTA, 1% Triton X-100, pH 8.0) in a 96-well PCR plate, and incubated in a thermal cycler at 95˚C for 10 min. The lysed samples will be immediately placed on ice for 3 - 5 min, and 1 µl of the supernatant will be used for PCR.
AP-PCR fingerprints will be obtained from each individual isolate. All PCR procedures will be performed using a Gene Amp PCR 9700 thermocycler (PE Applied Biosystems, Foster City, CA). The total volume of 50 μl consists of PCR buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4), 200 μM of each dNTP (Invitrogen, Carlsbad, CA), 7 mM MgCl2, 2.5 U of Taq DNA polymerase (Invitrogen), 100 pmol of primer of 272 (5’-AGCGGGCCAA-3’) [
Isolates displaying unique AP-PCR fingerprints and Lactobacillus phenotype by Gram staining are subjected to 16S rRNA gene sequencing analysis for species identification. The DNA templates isolated as described above will also be used for sequence analysis. A partial 16S rRNA gene will be amplified by PCR with universal bacterial primers 8F (5'-AGA GTT TGA TCC TGG CTC AG-3') [
Taxonomic identification based on the 660 bp amplicon will be used to confirm membership in the Lactobacillus genus, and will, in most cases, provide a species-level assignment. Taxonomic identification to known species will be performed by RDF Sequence Match with an S_ab score greater than or equal to 0.95, or on the best blastn match to HOMD sequences with sequence identity greater than or equal to 98.5%. After initial screening, high-quality DNA of each representative Lactobacillus isolate will be obtained using Qiagen Genomic-tips 20/G (Qiagen Inc., Valencia, CA, USA). Lactobacillus species identification will be confirmed by sequencing the full-length 16S rRNA gene. A fragment of approximately 1.4 Kb will be amplified with universal primers 8F and 1492R [
In addition to culture-based analysis, the study plans to conduct a second analysis using a culture-independent approach to measure the overall abundance, diversity, and species affiliation of lactobacilli in the plaque samples. This method estimates both cultivable and uncultivable populations of lactobacilli, thus serving as a benchmark estimation of true diversity comparing the findings of the culture-based approach. To examine cultivation independent approach, multiple plaque samples will be collected from various sites: 1) pooled supragingival plaque samples; 2) pooled plaque samples within caries lesions, one from each of the four quadrants; 3) pooled plaque samples from non-caries fissures, one from each of the four quadrants. Genomic DNA will be obtained directly from each sample by methods that are well established in our lab [
The 232-bp amplicon will be cloned into pCR4-TOPO vector and transformed into competent E. coli cells (Invitrogen). Fifty clones for each sample will be randomly selected and sequenced using vector primer T7 at the Genewiz sequencing facility (South Plainfield, NJ). Species assignment will be conducted as described above. The Lactobacillus phylotypes present in each sample will be tabulated, creating a taxonomic fingerprint for each sample. The final parameters for Lactobacillus diversity analysis will include the numbers and distribution of Lactobacillus species/phylotypes (distinct 16S rRNA gene sequences), the abundance and richness of Lactobacillus species, as well as phylotypes for different groups of samples.
The primary outcome of the culture-based method will be the level of Lactobacillus colonization measured by colony forming units (CFU) for each sample. The quantitative results will be analyzed against other independent variables including caries score, children’s medical history, oral hygiene behavior, and family socio-demo- graphic factors.
The second outcome of the study will be the number of unique Lactobacillus genotypes per individual as determined by AP-PCR. All of the AP-PCR fingerprint profiles will be examined; pair-wise similarity (inter- and intra-lactobacilli isolates) will be calculated based on the Dice coefficient. Hierarchical cluster analyses will be performed using BioNumerics 6.0 program (Applied Maths, Austin, TX). The results will be compared between S-ECC and CF children, and among different types of clinical samples, such as total plaque versus carious lesion and sound fissure.
The third outcome of the study will be the abundance and distribution of Lactobacillus species between S-ECC and CF groups. For Lactobacillus sequence analysis, the Chi-square test will be used to compare phylogenetic differences between the two groups. Species diversity and richness will be evaluated using Chao1, abundance-based coverage estimator (ACE), rarefaction curves, rank abundance and diversity indices, Shannon (Hʹ), as well as Good’s percent coverage [
The fourth outcome of the study will involve carrying out the delineation of caries-associated genetic elements Lactobacillus strains obtained from S-ECC and CF children. Intra- and inter-species comparisons with advanced classification algorithms will be performed to identify the loci specific to all S-ECC Lactobacillus species and to all CF Lactobacillus species. The analysis will focus on oral Lactobacillus genomes and genetic loci associated with niche specialization. Further genomic comparisons with environmental and food-associated lactobacilli will allow us to deduce species-specific genetic signatures for the oral niche.
Finally, all of the data, socio-demographics, medical history, questionnaires, dental examine, Lactobacillus isolates, specie identifications, as well as documents and images will be entered, stored, and managed by a custom-designed program for this project using FileMaker Pro 12 program (FileMaker, Inc. Santa Clara, CA) for this study. In addition, lab materials will also be labeled with 2D barcodes to protect subject identification, prevent data entry errors, and track sample process. Information in the dataset can be exported into Microsoft Excel files for statistical analysis.
SPSS Statistics software v22.0 (IBM Corp., Somers, NY) and STATA v12.1 (Stata Corp, College Station, TX) will be used for the data analyses. ANOVA and nonparametric independent tests will be used for continuous variables (CFU counts, age, and caries score). Chi-square and Pearson Correlation tests will be used for comparisons of clinical data (gender, mode of delivery, use of antibiotics, and oral hygiene behavior) with Lactobacillus colonization between S-ECC and CF groups and between the two different study methods as for culture-based versus molecular-based approach.
S-ECC is a serious public health concern as a result of its early onset, rapidly clinical progression, high treatment cost and negative impact on oral health-related quality of life of young children. The pathology of S-ECC is highly complex. The primary etiologic agents of dental caries associated with the disease are the mutans streptococci, particularly, Streptococcus mutans [
Based on selective culture media, previous studies of caries-associated lactobacilli have been mostly quantitative. The culture-based approaches can be time- and labor-intensive, and results can vary widely based on colony morphology or counts on the particular medium or cultivation method used. On the other hand, using culture- independent methods for taxonomic differentiation of lactobacilli can be challenging as well because of a vast group of Gram-positive bacilli sharing similar morphological profiles as well as the molar percentage of guanine-cytosine content (% G + C). Furthermore, there are very few definitive Lactobacillus species-specific 16S rRNA gene primers/probes, thus creating more obstacles against the use of advanced molecular-based techniques to determine lactobacilli diversity, abundance and taxonomical affiliation.
The study proposes a well-designed methodology aimed to assure that the sampling depth will be sufficient to find most Lactobacillus species associated with dental caries. Unlike most culture-based studies of the microbiota of S-ECC, this protocol utilizes a dual approach that utilizes both culture-based and culture-independent approaches. The culture-based component relies on a Lactobacillus selective medium that eliminates the background growth of other acid-tolerant oral bacteria, and can be completed, from sample plating to lactobacilli screening and isolation, in 10 to 12 working days for each subject. The workflow can be staggered for multiple subjects, allowing a trained team of dentists and lab technicians to process 5 - 10 subjects in one day. Lactobacillus strains isolated based on the selective medium can also serve as source materials for the whole genome sequencing and comparative genomics leading to the identification of niche-specific loci. The culture-inde- pendent component can be completed in 5 to 7 days from sample collection, genomic DNA purification to retrieval of the Lactobacillus 16S rRNA sequences for each subject. Up to 8 subjects can easily be processed simultaneously. Moreover, the culture-independent component relies on a new set of Lactobacillus-specific primers which allows for the detection of lactobacilli in plaque samples, even though they are present in low numbers. That all elements of the study will incorporate a custom-designed barcoding identification and tracking system and a user friendly interface will greatly enhance the efficiency and accuracy of samples process and data management.
In summary, the novel culture-dependent and culture-independent approach will enable us to determine the complexity and the role of lactobacilli played in S-ECC. From a clinical perspective, Lactobacillus function- and niche-specific cariogenic variables can serve as antimicrobial targets for new therapeutic treatment for caries and help facilitate the development of new tools for future caries risk assessments.
The authors declare to have no financial competing interests.
YL and PWC are Multiple-Principal Investigators of this research project and responsible for the study design and authorship of this paper. SA, as a Co-Investigator, helped conceive the study design, designed the new Lactobacillus-specific primer (LB516F), and contributed to the manuscript. CS, who also contributed to the manuscript, was the Project Coordinator responsible for subject recruitment and the project management. PS was responsible for Lactobacillus screening experiments and also contributed to the manuscript. All of the authors reviewed and approved the final version of the paper.
We are grateful for the continued support of members of the Pediatric Dental Clinic at Bellevue Hospital Center, Dr. Peter Catapano, Dr. Untray Brown, Dr. Charles Larsen, Dr. Glen Shanock and Ms. Elaine Landa. The participation of Dr. Zhou Chen, Ruth N. Maldonado Rey, Elise Blanchet, Sharon Casey and Yue Qin is greatly appreciated. The project was supported by the National Institute of Dental and Craniofacial Research (R01DE019455).
S-ECC: severe early childhood caries;
CF: caries free;
dmft/dmfs: decay, filled or missing (due to caries) tooth/tooth surfaces in primary dentition;
MS: mutans streptococci;
LB: lactobacilli;
CFU: colony forming units;
LBS: lactobacilli selection medium;
LDT: liquid dental transport medium;
AP-PCR: arbitrarily primed polymerase chain reaction;
RDP: Ribosomal Database Project;
HOMD: Human Oral Microbiome Database.