Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol

Yihong Li, Silvia Argimón, Catherine N Schön, Prakaimuk Saraithong, Page W Caufield, Yihong Li, Silvia Argimón, Catherine N Schön, Prakaimuk Saraithong, Page W Caufield

Abstract

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 socio-demographic 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 supra-gingival 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, extraction 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 cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.

Keywords: 16S rRNA; AP-PCR; Bacterial Diversity; Dental Plaque; Early Childhood Caries; Lactobacilli; Saliva.

Conflict of interest statement

Authors’ Conflict of Interest Statement

The authors declare to have no financial competing interests.

Figures

Figure 1
Figure 1
Schematic illustration of the overall study design. S-ECC = severe early childhood caries; CF = caries free; LB = lactobacilli; AP-PCR = arbitrarily primed polymerase chain reaction.
Figure 2
Figure 2
Flowchart of the culture-based component for Lactobacillus isolation, genotyping and species identification from clinical samples.

References

    1. Li Y, Navia JM, Bian JY. Caries Experience in Deciduous Dentition of Rural Chinese Children 3 – 5 years old in Relation to the Presence or Absence of Enamel Hypoplasia. Caries Research. 1996;30:8–15. .
    1. Caufield PW, Li Y, Bromage TG. Hypoplasia-Associated Severe Early Childhood Caries—A Proposed Definition. Journal of Dental Research. 2012;91:544–550. .
    1. Navia JM. Carbohydrates and Dental Health. The American Journal of Clinical Nutrition. 1994;59:719S–727S. .
    1. Carlsson J, Grahnen H, Jonsson G. Lactobacilli and Streptococci in the Mouth of Children. Caries Research. 1975;9:333–339. .
    1. Granath L, Cleaton-Jones P, Fatti LP, Grossman ES. Salivary Lactobacilli Explain Dental Caries Better than Salivary Mutants Streptococci in 4- to 5-Year-Old Children. Scandinavian Journal of Dental Research. 1994;102:319–323. .
    1. Roeters FJ, van der Hoeven JS, Burgersdijk RC, Schaeken MJ. Lactobacilli, Mutants Streptococci and Dental Caries: A Longitudinal Study in 2-Year-Old Children Up to the Age of 5 Years. Caries Research. 1995;29:272–279. .
    1. van Houte J, Gibbs G, Butera C. Oral Flora of Children with “Nursing Bottle Caries”. Journal of Dental Research. 1982;61:382–385. .
    1. Loesche WJ, Eklund S, Earnest R, Burt B. Longitudinal Investigation of Bacteriology of Human Fissure Decay: Epidemiological Studies in Molars Shortly after Eruption. Infection and Immunity. 1984;46:765–772. .
    1. Leverett DH, Proskin HM, Featherstone JD, Adair SM, Eisenberg AD, Mundorff-Shrestha SA, Shields CP, Shaffer CL, Billings RJ. Caries Risk Assessment in a Longitudinal Discrimination Study. Journal of Dental Research. 1993;72:538–543. .
    1. Badet C, Thebaud NB. Ecology of Lactobacilli in the Oral Cavity: A Review of Literature. The Open Microbiology Journal. 2008;2:38–48. .
    1. Beighton D. The Complex Oral Microflora of High-Risk Individuals and Groups and Its Role in the Caries Process. Community Dentistry and Oral Epidemiology. 2005;33:248–255. .
    1. Li Y, Ge Y, Saxena D, Caufield PW. Genetic Profiling of the Oral Microbiota Associated with Severe Early-Childhood Caries. Journal of Clinical Microbiology. 2007;45:81–87. .
    1. Tanner AC, Mathney JM, Kent RL, Chalmers NI, Hughes CV, Loo CY, Pradhan N, Kanasi E, Hwang J, Dahlan MA, Papadopolou E, Dewhirst FE. Cultivable Anaerobic Microbiota of Severe Early Childhood Caries. Journal of Clinical Microbiology. 2011;49:1464–1474. .
    1. Callaway A, Kostrzewa M, Willershausen B, Schmidt F, Thiede B, Kupper H, Kneist S. Identification of Lactobacilli from Deep Carious Lesions by Means of Species-Specific PCR and MALDI-TOF Mass Spectrometry. Clinical Laboratory. 2013;59:1373–1379. .
    1. Teanpaisan R, Thitasomakul S, Piwat S, Thearmontree A, Pithpornchaiyakul W, Chankanka O. Longitudinal Study of the Presence of Mutans Streptococci and Lactobacilli in Relation to Dental Caries Development in 3 – 24 Months Old Thai Children. International Dental Journal. 2007;57:445–451. .
    1. Leverett DH, Featherstone JD, Proskin HM, Adair SM, Eisenberg AD, Mundorff-Shrestha SA, Shields CP, Shaffer CL, Billings RJ. Caries Risk Assessment by a Cross-Sectional Discrimination Model. Journal of Dental Research. 1993;72:529–537. .
    1. Marsh P, Martin MV. Oral Microbiology. 4. Reed Educational and Professional Publishing Ltd; Woburn: 1999. The Resident Oral Microflora; pp. 17–33.
    1. Salvetti E, Fondi M, Fani R, Torriani S, Felis GE. Evolution of Lactic Acid Bacteria in the Order Lactobacillales as Depicted by Analysis of Glycolysis and Pentose Phosphate Pathways. Systematic and Applied Microbiology. 2013;36:291–305. .
    1. Claesson MJ, van Sinderen D, O’Toole PW. Lactobacillus Phylogenomics—Towards a Reclassification of the Genus. International Journal of Systematic and Evolutionary Microbiology. 2008;58:2945–2954. .
    1. Piwat S, Teanpaisan R, Dahlen G, Thitasomakul S, Douglas CW. Acid Production and Growth by Oral Lactobacillus Species in Vitro. Journal of Investigative and Clinical Dentistry. 2012;3:56–61. .
    1. Broadbent JR, Neeno-Eckwall EC, Stahl B, Tandee K, Cai H, Morovic W, Horvath P, Heidenreich J, Perna NT, Barrangou R, Steele JL. Analysis of the Lactobacillus casei Supragenome and Its Influence in Species Evolution and Lifestyle Adaptation. BMC Genomics. 2012;13:533. .
    1. O’Sullivan O, O’Callaghan J, Sangrador-Vegas A, McAuliffe O, Slattery L, Kaleta P, Callanan M, Fitzgerald GF, Ross RP, Beresford T. Comparative Genomics of Lactic Acid Bacteria Reveals a Niche-Specific Gene Set. BMC Microbiology. 2009;9:50. .
    1. Gross EL, Leys EJ, Gasparovich SR, Firestone ND, Schwartzbaum JA, Janies DA, Asnani K, Griffen AL. Bacterial 16S Sequence Analysis of Severe Caries in Young Permanent Teeth. Journal of Clinical Microbiology. 2010;48:4121–4128. .
    1. Woese CR. Bacterial Evolution. Microbiological Reviews. 1987;51:221–271. .
    1. Furrie E. A Molecular Revolution in the Study of Intestinal Microflora. Gut. 2006;55:141–143. .
    1. Yang R, Argimon S, Li Y, Zhou X, Caufield PW. Determining the Genetic Diversity of Lactobacilli from the Oral Cavity. Journal of Microbiological Methods. 2010;82:163–169. .
    1. American Academy of Pediatric Dentistry. Definition of Early Childhood Caries (ECC) American Academy of Pediatric Dentistry; 2007. .
    1. Dye BA, Tan S, Smith V, Lewis BG, Barker LK, Thornton-Evans G, Eke PI, Beltran-Aguilar ED, Horo-witz AM, Li CH. Vital and Health Statistics Series. Vol. 11. National Center for Health Statistics; 2007. Trends in Oral Health Status: United States, 1988–1994 and 1999–2004. .
    1. Caufield PW, Li Y, Dasanayake A, Saxena D. Diversity of Lactobacilli in the Oral Cavities of Young Women with Dental Caries. Caries Research. 2007;41:2–8. .
    1. Dal Bello F, Hertel C. Oral Cavity as Natural Reservoir for Intestinal Lactobacilli. Systematic and Applied Microbiology. 2006;29:69–76. .
    1. Mahenthiralingam E, Marchbank A, Drevinek P, Garaiova I, Plummer S. Use of Colony-Based Bacterial Strain Typing for Tracking the Fate of Lactobacillus Strains during Human Consumption. BMC Microbiology. 2009;9:251. .
    1. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and Direct Complete Nucleotide Determination of Entire Genes. Characterization of a Gene Coding for 16S Ribosomal RNA. Nucleic Acids Research. 1989;17:7843–7853. .
    1. Heilig HG, Zoetendal EG, Vaughan EE, Marteau P, Akkermans AD, de Vos WM. Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA. Applied and Environmental Microbiology. 2002;68:114–123. .
    1. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tiedje JM. The Ribosomal Database Project (RDP-II): Introducing myRDP Space and Quality Controlled Public Data. Nucleic Acids Research. 2007;35:D169–D172.
    1. Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, Lakshmanan A, Wade WG. The Human Oral Microbiome. Journal of Bacteriology. 2010;192:5002–5017. .
    1. Lane DJ. 16S/23S rRNA Sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons Ltd; West Sussex: 1991. pp. 115–147.
    1. Li Y, Ku CY, Xu J, Saxena D, Caufield PW. Survey of Oral Microbial Diversity Using PCR-Based Denaturing Gradient Gel Electrophoresis. Journal of Dental Research. 2005;84:559–564. .
    1. Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative Analysis of Diverse Lactobacillus Species Present in Advanced Dental Caries. Journal of Clinical Microbiology. 2004;42:3128–3136. .
    1. Gotell NJ, Chao A. Measuring and Estimating Species Richness, Species Diversity, and Biotic Similarity from Sampling Data. In: Levin SA, editor. Encyclopedia of Biodiversity. 2. Vol. 5. Academic Press; Waltham, MA: 2013. pp. 195–211. .
    1. Loesche WJ. Role of Streptococcus mutans in Human Dental Decay. Microbiological Reviews. 1986;50:353–380. .
    1. van Houte J, Sansone C, Joshipura K, Kent R. Mutans Streptococci and Non-Mutans Streptococci Aci-dogenic at Low pH, and in Vitro Acidogenic Potential of Dental Plaque in Two Different Areas of the Human Dentition. Journal of Dental Research. 1991;70:1503–1507. .
    1. Jay P. Lactobacillus acidophilus and Dental Caries. American Journal of Public Health and the Nations Health. 1938;28:759–761. .
    1. Owen OW. A Study of Bacterial Counts (Lactobacilli) in Saliva Related to Orthodontic Appliances: A Preliminary Report. American Journal of Orthodontics. 1949;35:672–678. .

Source: PubMed

스폰서 및 공동 작업자

건강 상태

약물 개입

3
구독하다