Abstract
Purpose
Genotypic strains of mutans streptococci (MS) may vary in important virulence properties, and may be differentially affected by specific components of full-mouth caries restorative therapy. The purpose of this pilot study was to identify MS strains that predominate following caries restorative therapy.
Methods
Plaque from seven children with severe early childhood caries was collected before and following therapy. MS isolates (N=828) were subjected to polymerase chain reaction (PCR), and arbitrarily primed-PCR (AP-PCR) for assignment within MS strains. Determining the longitudinal changes in MS strain distribution over time within each patient required the isolation of larger numbers of isolates per patient, but from fewer patients.
Results
Up to 39 genotypic strains of S. mutans and S. sobrinus, and seven genotypic strains of non-MS streptococci were identified by AP-PCR and 16S ribosomal rRNA gene sequencing. The number of MS strains isolated from each patient were 3–7 prior to treatment, diminishing to 1–2 dominant MS strains in most patients 6 months post-therapy.
Conclusions
Caries restorative therapy resulted in shifts of specific MS and non-MS streptococci strains. The implications are that caries restorative therapy affects the distribution of MS strains, and that well-accepted practices for caries prevention should be more closely examined for efficacy.
Keywords: mutans streptococci, selection and distribution of genotypic MS strains, Streptococcus mutans, oral streptococci, severe early childhood caries, caries restorative therapy
INTRODUCTION
Dental caries or tooth decay is one of the most common chronic diseases affecting young children.1–3 Mutans streptococci (MS) represent the most predominant microorganisms associated with dental caries, and consist of seven species, with S. mutans and S. sobrinus, the most frequently isolated from human dental plaque and associated with caries. S. mutans has been further classified into four serotypes (c, e, f, and k), based on the chemical composition of the rhamnose-glucose polymers expressed on the cell surface.4 Most S. mutans isolates are serotype c (70–80%), with serotypes e, f and k comprising progressively smaller numbers (20%, 5%, 2–5%, respectively).
While serotype analyses have been widely used to distinguish MS strains, this method has limited utility in the identification of genetic differences within the same serotype. Recent molecular approaches have been utilized to identify MS strain differences, including multilocus enzyme electrophoresis, ribotyping, pulsed-field gel electrophoresis, multi-locus sequence typing, arbitrarily-primed polymerase chain reaction (AP-PCR; also known as random amplification of polymorphic DNA or RAPD), and 16S ribosomal RNA gene sequencing.4–14,15 AP-PCR has been used extensively in multiple laboratories to characterize the genetic diversity of oral microorganisms.4,9–13 Unlike conventional PCR, which uses high stringency annealing of primers to template DNA, AP-PCR uses a single primer and lower annealing temperatures to allow recognition of the primer to both precise as well as partially mismatched sequences. This results in the generation of a series of fragments or “genetic fingerprint” for identification of specific unique strains.
An increasing number of reports have begun to demonstrate the importance of MS strain variation among patients and dental caries. Genetic approaches including AP-PCR have currently identified 51 genotypes of S. mutans and 8 genotypes of S. sobrinus.4,5,9–11 Individuals with differences in caries prevalence have distinct MS strains exhibiting variable virulence and caries-promoting activities.16 Genotypic determinations also indicate that there are differences in the distribution of S. mutans genotypes according to location in the oral cavity, and that genetic populations of S. mutans differ in acid tolerance properties and ability to form biofilms.11 When examining pre-school children, AP-PCR has identified greater numbers of MS genotypic strains in caries-active children than caries-free children,17 and has implicated horizontal transmission between some children.18,19 In cases of severe early childhood caries, evidence of maternal transmission was detected in 41% of mother-child pairs.20 The coexistence and concurrent virulence of distinct genotypes of S. mutans in caries-active individuals has been proposed to serve as important determinants for increased caries incidence.4,5,9–11
The colonization of cariogenic MS strains begins with the accumulation of salivary proteins and adhesive glucans on the enamel surface to form the enamel pellicle, thereby allowing for the adherence of cariogenic MS to the tooth surface.16 Lactic acid production by cariogenic MS near the surface of the tooth causes demineralization of the tooth enamel and leads to decalcification, dissolved tooth structure and potential tooth loss. Accumulation of S. mutans can alter the pH of plaque biofilm, which can subsequently select for and increase the proportion of acidogenic microorganisms. Healthy plaque flora generates higher pH observed during the fermentation of carbohydrates and can arrest the initiation of dental caries.16,21,22 Fitzgerald et al.23 have shown that S. mutans strains deficient in lactate dehydrogenase have reduced cariogenicity.
In this pilot study, we examined the profiles of MS genotypic strains and non-MS oral streptococci strains from seven pediatric patients that exhibited severe early childhood caries (S-ECC). Isolates were collected both prior to and following full-mouth caries restorative therapy, which included the removal and/or repair of carious lesions and application of antimicrobial rinse and fluoride varnish. This pilot study determines the MS genotypic identification and acidogenesis potential for every isolate, unlike future work that will utilize larger numbers of patients with S-ECC, but will only examine representative members of specific MS genotypic strains. This pilot study may help provide the beginning framework for evaluating the efficacy of the current regimen for caries preventive treatment. The significance of this research is the potential impact on the standard-of-care practices for caries preventive therapy in individual children, including implications in defining the use of antimicrobial rinse and fluoride in oral health care.
METHODS
Patient Selection and Treatment
Pediatric patients from the OHSU Pediatric Dentistry clinic were the source of the participants for this research study. The use of human participants in this study was approved by the Institutional Review Board (IRB) of the Oregon Health & Science University, and written informed consent was obtained from parents or guardians of the children prior to specimen collection. The inclusion parameters for recruitment were children 3–12 years old and in general good health. Children who had been subjected to antibiotic treatment, topical fluoride application, and/or antiseptic mouth rinses within the previous three months were excluded, in addition to individuals undergoing any orthodontic therapies. From the age range of 3–12 years, for this pilot study, we purposefully selected participants with ages 3–5, who underwent full-mouth caries restorative therapy conducted under general anesthesia at Doernbecher’s Children Hospital of the Oregon Health & Science University; the selection of children within the age range of 3–5 years permitted full-mouth caries restorative therapy to be completed during a single patient visit.
Pediatric dentistry residents reviewed the patients’ medical history, performed comprehensive oral exams, and determined the number of decayed, missing and filled teeth (dmft score) and decayed, missing and filled surfaces (dmfs score). Each patient participant was diagnosed with severe early childhood caries (S-ECC) and was subjected to caries restorative therapy under general anesthesia. Caries restorative therapy included application of 0.12% chlorhexidine gluconate to the gingiva and dentition using a sterile gauze to prepare the surgical area prior to beginning the procedure, followed by amalgam (Valiant® PH.D®), composite (Pulpdent ® Etch-Rite 38% Phosphoric Acid Etching Gel, Optibond™, Z100™ and Filtek™ Supreme), and stainless steel crown (3M ESPE, Unitek) restorations, formocresol (Patterson Dental), pulpotomies, extractions, sealants (Patterson Dental), dental prophylaxis (NUPRO® prophylaxis paste [1.23% fluoride]), and sodium fluoride varnish [Cavity Shield™] application with a brush.
Sampling Procedure and Processing of Specimens
Plaque samples were taken from each participant at three time points: 1) prior to the initiation of caries restorative therapy, 2) at the 2–4 weeks post-treatment visit, and 3) at the 6-month recall visit. Plaque samples from each patient were individually collected by one of two clinicians, whose collection methods were previously calibrated. Two plaque samples were taken from each patient at each collection time point. Each plaque sample was obtained using a disposable sterile swab that was brushed along the buccal and lingual surfaces of the entire dentition. The swabs were then placed in a sterile test tube and immediately transported to the nearby laboratory for processing. Samples were then directly plated from the swab onto mitis salivarius agar, supplemented with sodium tellurite and bacitracin.
Control Streptococci Strains and Selection of Mutans Streptococci (MS) Isolates
Control streptococci strains include S. mutans ATCC strains 700610 (also known as UA159), 25175, and 35668, S. sobrinus, and non-MS oral streptococci strain S. salivarius. All streptococcal strains were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Plaque specimens were plated on mitis salivarius agar (MSA; product number 229810, Difco, Becton, Dickinson and Company, Sparks, MD), supplemented with 1% sodium tellurite and the antibiotic bacitracin (0.2 Units/ml), to isolate MS, as described in Fazilat et al. (2010). MSA contains high sucrose and inhibitory dyes (e.g.: crystal violet and bromphenol blue) that in combination with sodium tellurite and bacitracin will select for MS. Colonies were allowed to grow on MSA plates for 48–72 hours, and then propagated in brain heart infusion (BHI) broth for 24–48 hours at 37°C in 5% CO2. Cultures were then tested by Gram stain for verification of isolates as Gram-positive cocci.
Genomic DNA Isolation, Conventional Polymerase Chain Reaction (PCR) and Arbitrarily-Primed PCR (AP-PCR)
Genomic DNA was extracted from overnight liquid cultures using the PureLink Genomic DNA Kit (Invitrogen). S. mutans and S. sobrinus species were independently identified using conventional PCR. Conventional PCR was conducted using initial denaturation at 94°C for 5 minutes, then 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. The complete S. mutans UA159 genome has been sequenced24 and highly specific primers have been developed for both S. mutans and S. sobrinus.25,26 Serotype-specific primers for PCR-based identification of serotype c, e, f and k strains of S. mutans have also been developed 27,28, and were used in this study to analyze strains from Patient G. OPA2 and OPA3 are random primers that have been used to distinguish genotypic strains of S. mutans and have been described.12,13, Primers used for identification of S. mutans and S. mutans serotypes and S. sobrinus, as well as those used for AP-PCR (OPA2/OPA3) are described in Table 1. The amplification parameters for AP-PCR were similar to conventional PCR, with the exception of annealing at reduced temperatures (35°C for 30 seconds). PCR products were subjected to agarose gel electrophoresis, and fragments were stained with ethidium bromide for visualization using UV transillumination. Images were quantitated using Quantity One software (BioRad).
Table 1.
Primers Used for Conventional and Arbitrarily-Primed PCR
| Primer Description | Amplicon Size (bp) | Primer Sequence | Reference |
|---|---|---|---|
| S. mutans specific | |||
| Forward (NC 004350)1 | 479 | 5′ TCG CGA AAA AGA TAA ACA AAC A 3′2 | Chen et al. (2007) |
| Reverse (NC 004350) | 5′ GCC CCT TCA CAG TTG GTT AG 3′3 | ||
| S. sobrinus specific | |||
| Forward (M96978) | 1610 | 5′ TGC TAT CTT TCC CTA GCA TG 3′4 | Igarashi et al. (2000) |
| Reverse (M96978) | 5′ GGT ATT CGG TTT GAC TGC 3′4 | ||
| S. mutans serotype | |||
| Serotype c | |||
| Forward (SC-F)5 | 727 | 5′ CGG AGT GCT TTT TAC AAG TGC TGG 3′ | Shibata et al. (2003) |
| Reverse (SC-R) | 5′ AAC CAC GGC CAG CAA ACC CTT TAT 3′ | ||
| Serotype e | |||
| Forward (SE-F) | 517 | 5′ CCT GCT TTT CAA GTA CCT TTC GCC 3′ | Shibata et al. (2003) |
| Reverse (SE-R) | 5′ CTG CTT GCC AAG CCC TAC TAG AAA 3′ | ||
| Serotype f | |||
| Forward (SF-F) | 316 | 5′ CCC ACA ATT GGC TTC AAG AGG AGA 3′ | Shibata et al. (2003) |
| Reverse (SF-R) | 5′ TGC GAA ACC ATA AGC ATA GCG AGG 3′ | ||
| Serotype k | |||
| Forward (CEFK-F) | 296 | 5′ ATT CCC GCC GTT GGA CCA TTC C 3′ | Nakaro et al. (2009) |
| Reverse (K-R) | 5′ CCA ATG TGA TTC ATC CCA TCA C 3′ | ||
| OPA 2 | 5′ TGC CGA GCT G 3′ | Baca et al. (2008) | |
| OPA 3 | 5′ AGT CAG CAC 3′ | Tabchoury et al. (2008) | |
Accession Number
Forward primer sequence was derived from htrA gene; position 2029599–2029620 in S. mutans genome
Reverse primer sequence was derived from downstream noncoding region of htrA gene; position 2030077–2030058 in S. mutans genome
Forward and reverse primer sequences were derived from the dex gene; position 134–153 (forward) and 1743–1726 (reverse) in M96978 sequence
Primer names for serotype c, e, f, and k were originally defined in Shibata et al. (2003).
16S Ribosomal RNA Gene Sequencing, Acidogenesis Assays and Statistical Analyses
PCR of 16S ribosomal RNA gene targets15, using isolates determined not to be S. mutans or S. sobrinus, were conducted by the Human Microbe Identification Microarray Core Laboratory (Forsyth Institute, Cambridge, MA). Sequencing was conducted by GENEWIZ (South Plainfield, NJ). Acidogenesis determinations were based on final pH values obtained at 72 hours of growth in phenol red dextrose, supplemented with 1% glucose, following procedures described in Lembo et al.11 Cultures for acidogenesis determinations were conducted with replicates (n = 3 or 4), and calculations for mean, variance, standard deviation and standard error were determined. The Mann-Whitney test was used to determine differences in the distribution of pH values between the MS and non-MS oral streptococci.
RESULTS
Description of Study Participants
Nine patients were originally enrolled in this study, but seven (patients G, H, I, J, K, L and M) were available for their 2–4 week recall visits and five patients (patients G, J, K, L and M) were available for their 6 month recall visits. The American Academy of Pediatric Dentistry (AAPD) defines S-ECC in children ages 3–5 years old as: one or more cavitated, missing (due to caries) or smooth filled surfaces in primary maxillary anterior teeth, or dmfs score of >4 (age 3), >5 (age 4), or >6 (age 5)29. At the time of caries restorative therapy, every subject had only primary dentition present, and only patient H was missing any teeth (tooth #K, as defined by the Primary Universal Numbering System). The patients were diagnosed with severe early childhood caries (S-ECC) with dmft (decayed, missing or filled teeth) and dmfs (decayed, missing or filled smooth surfaces) scores ranging from 11–20 and 25–87, respectively (Table 2).
Table 2.
Patient Demographics and dmfs and dmft Scores of Patient Cohort
| G | H | I | J | K | L | M | |
|---|---|---|---|---|---|---|---|
| Treatment age1 | 5yo | 5yo | 4yo | 5yo | 3yo | 3yo | 5yo |
| Sex | Female | Female | Female | Male | Male | Male | Male |
| ASA Classification2 | ASAI | ASAII | ASAI | ASAI | ASAI | ASAI | ASAI |
| dmft score3 | 11 | 20 | 11 | 13 | 18 | 13 | 12 |
| dmfs score4 | 25 | 87 | 56 | 38 | 61 | 48 | 41 |
| Teeth present5 | A-T | A-J, L-T | A-T | A-T | A-T | A-T | A-T |
Treatment age: Age of patient on day of full-mouth dental rehabilitation
ASA Classification: Classification according to the American Society of Anesthesiologists physical status classification system
dmft: The sum of the primary teeth that are decayed (d), missing (m) or filled (f) due to dental caries
dmfs: The sum of the primary tooth surfaces that are decayed (d), missing (m) or filled (f) due to dental caries
Teeth present: Teeth present on day of full-mouth dental rehabilitation. Letters denote primary teeth present following the Primary Universal Numbering System. Patient H had tooth #K extracted ~3 months prior to treatment.
Identification of Mutans Streptococci Strains
Based on colony morphology on mitis salivarius agar (MSA) plates and Gram stain analysis, we selected up to 50 isolates using plaque specimens from each patient at each collection period, with each isolate being confirmed as bacitracin-resistant, Gram-positive oral streptococci. This cohort of Gram-positive cocci was found to include a considerable proportion of MS, including S. mutans. Using primers specific for S. mutans or S. sobrinus, and testing genomic DNA from all 828 isolates obtained from the seven pediatric patients (patients G, H, I, J, K, L and M), we identified 37 genotypic strains of S. mutans and two genotypic strains (K2 and K3 strains) of S. sobrinus (Table 3). Using primers designed by Nakano et al.,4 specific for the serotype stains c, e, f, and k of S. mutans, almost all isolates identified by PCR as S. mutans in patient G were serotype c, with the exception of a single isolate as serotype e (E. A. Palmer, T. Nielsen and C. A. Machida; unpublished observations).
Table 3.
Identification of MS and Oral Streptococci Genotypes and the Number of Isolates Identified at Each Visit
| Patient G
| |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes1 | Other Genotypes2 | Number of Genotypes | Dominant Strain | |||||
|
| |||||||||
| n3 | G1 | G2 | G4 | G4a | n | G3 | |||
| Pre-Treatment | 30 | 6 | 18 | 5 | 1 | 2 | 2 | 5 | G2 (56%) |
| Post-Treatment (4 weeks) | 45 | 0 | 45 | 0 | 0 | 1 | 1 | 2 | G2 (98%) |
| Post-Treatment (6 months) | 50 | 0 | 50 | 0 | 0 | 0 | 0 | 1 | G2 (100%) |
| Patient H
| |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | |||||||
|
| |||||||||||
| n | H1a | H1c | H2 | H3 | H4 | n | H1 | H1b | |||
| Pre-Treatment | 12 | 1 | 2 | 8 | 1 | 0 | 38 | 37 | 1 | 6 | H1 (74%) |
| Post-Treatment (2 weeks) | 3 | 0 | 0 | 2 | 0 | 1 | 15 | 11 | 4 | 4 | H1 (61%) |
| Post-Treatment (6 months) | - | - | - | - | - | - | - | - | - | - | - |
| Patient I
| ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | ||||||||
|
| ||||||||||||
| n | I1 | I1a4 | I1b | I2 | I3 | I4 | I5 | I6 | n | |||
| Pre-Treatment | 50 | 43 | 1 | 0 | 3 | 1 | 1 | 0 | 1 | 0 | 6 | I1 (86%) |
| Post-Treatment (2 weeks) | 50 | 46 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 5 | I1 (92%) |
| Post-Treatment (6 months) | - | - | - | - | - | - | - | - | - | 0 | - | - |
| Patient J
| ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | ||||||||
|
| ||||||||||||
| n | J1 | J2 | J2a | J2b | J3 | J3a | J4 | J5 | n | |||
| Pre-Treatment | 45 | 6 | 25 | 3 | 1 | 7 | 2 | 1 | 0 | 0 | 7 | J2 (56%) |
| Post-Treatment (2 weeks) | 50 | 0 | 0 | 0 | 0 | 49 | 0 | 0 | 1 | 0 | 2 | J3 (98%) |
| Post-Treatment (6 months) | 50 | 0 | 0 | 0 | 0 | 50 | 0 | 0 | 0 | 0 | 1 | J3 (100%) |
| Patient K
| ||||||||
|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | ||||
|
| ||||||||
| n | K15 | K1a | n | K2 | K3 | |||
| Pre-Treatment | 29 | 27 | 2 | 1 | 1 | 0 | 3 | K1 (90%) |
| Post-Treatment (2 weeks) | 4 | 4 | 0 | 3 | 0 | 3 | 2 | K1 (57%) |
| Post-Treatment (6 months) | 50 | 50 | 0 | 0 | 0 | 0 | 1 | K1 (100%) |
| Patient L
| |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | |||||
|
| |||||||||
| n | L1 | L1a | L1b | L2 | L3 | n | |||
| Pre-Treatment | 50 | 48 | 0 | 0 | 1 | 1 | 0 | 3 | L1 (96%) |
| Post-Treatment (4 weeks) | 50 | 48 | 1 | 1 | 0 | 0 | 0 | 3 | L1 (96%) |
| Post-Treatment (6 months) | 50 | 50 | 0 | 0 | 0 | 0 | 0 | 1 | L1 (100%) |
| Patient M
| |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient Visit | S. mutans Genotypes | Other Genotypes | Number of Genotypes | Dominant Strain | |||||||||
|
| |||||||||||||
| n | M2 | M3 | M3a | M5 | M7 | n | M1 | M1a | M46 | M66 | |||
| Pre-Treatment | 30 | 7 | 22 | 0 | 1 | 0 | 20 | 5 | 1 | 10 | 4 | 7 | M3 (44%) |
| Post-Treatment (4 weeks) | 49 | 1 | 44 | 2 | 0 | 0 | 1 | 0 | 0 | 2 | 1 | 5 | M3 (88%) |
| Post-Treatment (6 months) | 26 | 0 | 20 | 0 | 0 | 6 | 24 | 24 | 0 | 0 | 0 | 3 | M3 (40%), M1 (48%) |
Genotypes confirmed as S. mutans by conventional PCR with S. mutans specific primers. Note that genotypes containing an “a”, “b”, or “c” suffix as in G4a or J2a and J2b differ from the its matched comparison strains (in this case: G4 and J2) with the addition of one or more AP-PCR fragments (“a” suffix implies one additional band, “b” suffix implies two additional bands and “c” suffix implies three additional bands, all when compared to the AP-PCR profile of its matched genotypic strain). Note that individual MS genotypes were determined for each patient alone, and thus, there was no attempt to compare MS genotypes between patients.
Other genotypes are classified as non-MS oral streptococci, except genotypes K2 and K3 that were confirmed as S. sobrinus by conventional PCR with S. sobrinus specific primers. Note that individual genotypes were determined for each patient alone, and thus, there was no attempt to compare genotypes between patients. Using 16S ribosomal RNA gene sequencing, we have made the following bacterial species identifications for genotypes classified in the non-MS oral streptococci group: G3 = S. gordonii, H1 and H1b = both S. anginosus, M1 = S. anginosus, M1b = G. adiacens, M4 and M6 = both S. gordonii.
n = Number of isolates
To further characterize selected isolates that were difficult to identify by conventional PCR, acidification reactions (D-ribose, L-arabinose, D-mannitol, D-sorbitol, D-lactose, D-trehalose, inulin, D-raffinose, amidon (or starch), and glycogen; derived from API-20 Strep kit, Biomurieux SA) were also conducted and were compared against S. mutans strains ATCC 700610, ATCC 25175 and ATCC 35668, and S. salivarius. Genotype I1a (single isolate) did not yield robust PCR products using S. mutans-specific primers and conventional PCR; however, I1a was counted as a member of the S. mutans group because its metabolic fermentation profile appeared identical to other control S. mutans strains.
Genotype K1 isolates obtained at the 2 week post-treatment collection did not yield robust PCR products using S. mutans-specific primers and conventional PCR; however, these isolates were counted in the MS group because they retained identical AP-PCR fingerprints when compared to other K1 isolates.
M4 and M6 isolates obtained at the pre-treatment collection did not yield robust PCR products using S. mutans-specific primers and conventional PCR. And small numbers of isolates from genotypes M4 (2 isolates) and M6 (1 isolate) obtained at the 2 week post-treatment collection yielded robust PCR products using S. mutans-specific primers and conventional PCR. We counted these three isolates from M4 and M6 obtained from the 2 weeks post-treatment collection as members of the non-MS group, because they retained metabolic fermentation profiles inconsistent with those obtained for several S. mutans strains.
Identification of Non-MS Oral Streptococci Strains
Several genotypic strains of Gram-positive oral streptococci, including G3, when tested with primers specific for S. mutans, yielded weak amplification bands. In total, seven genotypic strains of bacitracin-resistant Gram-positive oral streptococci, with either weak band intensity, or no bands generated when using the S. mutans primers, were identified from the seven-patient cohort (Table 3). Representative isolates from the seven genotypic strains classified as non-MS oral streptococci strains were subjected to 16S ribosomal RNA gene sequencing, and identified as three strains of S. gordonii, three strains of S. anginosus, and one strain of Granulicatella adiacens. G. adiacens was previously known as Streptococcus adjacens.30
Distribution Shifts of Genotypic Strains Between Pre-Treatment and Post-Caries Restorative Therapy Collections Within Each Patient
The numbers of genotypic strains identified from any one patient over the entire collection period ranged from 4–9, or from any one visit from 3–7 (Table 3 and Figure 1). In all seven patients, the highest numbers of strains were observed at the pre-treatment collection, and dominant strains representing >44% of the population examined were prevalent at all pre-treatment collections. At 2–4 weeks post-caries restorative therapy, the numbers of strains isolated decreased in six of the seven patients, and patients seen at 6 months post-treatment contained only 1–2 dominant strains (Table 3 and Figure 1). Four out of five patients, who were present at the 6-month recall visit, had only one genotypic strain at the 6 months post-treatment collection (Patients G, J, K and L; Table 3 and Figure 1). Two of the seven patients, specifically patients H and I, did not arrive at their scheduled 6 month recall visits, and data was not subsequently tracked for the 6 month collection for these individuals. It is also very interesting to note that in almost all cases and collection times (with the exception of Patient M), there was only one dominant strain, each representing >56% of the strain population examined (Table 3 and Figure 1). The genotypic distribution patterns for the patient cohort were distinctive and unique for each patient, and it should be noted that all comparisons of genotypes were conducted with strains collected within each individual patient, and not between patients.
Figure 1. Clonal diversity of MS and non-MS oral streptococci populations in pediatric dentistry patients at pre-treatment and post-caries restorative therapy (2–4 weeks and 6 months).
Each line represents distinct genotypes identified by AP-PCR (n = the number of isolates identified at each collection; E, O and 6 Mo. represents pre-treatment, post-treatment (2–4 weeks) and post-treatment (6 month) collections. The dominant genotypes are marked in bold at each collection point (greater than 40% of the isolates). Dotted lines indicate the periods when genotypes detected at pre-treatment were not found at 2–4 weeks post-treatment, but were re-detected at 6 month post-treatment. Isolates were genotyped (1–7) within each patient (G, H, I, J, K, L and M). Genotypes described in Figure 1 are abbreviated with numbers only (e.g.: genotype G1 is abbreviated as genotype 1 in the figure illustration for patient G).
Patient G Exhibits Five Genotypes During Pre-Treatment with One Dominant Strain Persisting At 6 Months Post-Caries Restorative Therapy
Figure 2 displays a typical experiment showing the AP-PCR profiles of several isolates obtained at the pre-treatment and post-treatment collections (4 weeks and 6 months) for Patient G. Up to 50 isolates, which were confirmed as Gram-positive streptococci grown on MSA containing bacitracin, were isolated from each collection for each patient, propagated in BHI medium, and analyzed by conventional PCR using MS-specific primers and also by AP-PCR to identify specific genotypic strains. For Patient G, we tested the discriminatory potential of two random primers, OPA2 and OPA3, in the identification of distinct genotypes. Consistent with the findings of Baca et al.,12 Napimoga et al.9 and Pieralisi et al.,17 who have identified OPA2 as having high discriminatory potential, we also found OPA2 to generate the greatest number of bands following AP-PCR (E. A. Palmer, T. Nielsen and C. A. Machida, unpublished observations). However, as with Tabchoury et al.,13 we have determined that the use of OPA3 in AP-PCR, with its slight reduction in numbers of bands, was more reproducible in our laboratory and allowed satisfactory discrimination into distinct genotypes (E. A. Palmer, T. Nielsen and C. A. Machida, unpublished observations). Because of this reason, we chose to focus on the use of OPA3 for all AP-PCR experiments examining this seven patient cohort. Representative AP-PCR profiles using the OPA3 primer for each collection from Patient G are displayed in the electrophoretic analyses (Figure 2). Patient G exhibited five genotypic strains, with genotype G2 (S. mutans strain) increasing from 56% to 98% of the population immediately (4 weeks) following treatment and to 100% of the population at the 6 month visit (Table 3). DNA from genotypes G1, G2, G4 and G4a all yielded robust PCR products when using S. mutans-specific primers and were classified as members of the S. mutans group, while G3 yielded weak intensity bands of the same size and were classified in the non-MS oral streptococci group. Using 16S ribosomal RNA sequencing, G3 has been identified as S. gordonii. Genotype G2 was the dominant S. mutans strain (100% of the population surveyed) at 6 months post-caries restorative therapy (Table 3). Caries restorative therapy effectively reduced all other strains, including other genotypes of S. mutans and S. gordonii (G3) observed at pre-treatment.
Figure 2. AP-PCR fingerprints of independent isolates collected from patient G at pre-treatment (Panel A) and post-caries restorative therapy (2–4 weeks and 6 months (Panels B and C, respectively).
OPA3 primer was used. Note distribution change in MS isolates based on genotypic profiles that occurred between pre- and post-treatment collections. Isolates 1 and 9 in Panel A are representative of genotype G1. Isolates 4, 10 and 13 in Panel A are representative of genotype G2. Isolates 2 and 24 in Panel A are representative of genotype G3. Isolates 6 and 7 in Panel A are representative of genotype G4. Genotype 4a, present as only a single isolate (not shown in Figure 2), differs from genotype G4 with the addition of another PCR fragment. Representative bacitracin-sensitive oral streptococci (isolated from MSA) from Patient G were also isolated and identified, and are displayed here (representative isolates 3, 8, 11 and 12 in Panel A). Densitometric enhancement of band intensities for the 6-month collection resulted in the appearance of two additional bands, above and below the major visible band (as observed in Panel C), that were consistent in size with the bands found in G2 during the pre-treatment and the 2–4 week post-treatment collections (Panels A and B). Thus, the dominant strain found in both the 2–4 week and the 6-month collection is G2.
Other Patients Exhibited Diverse MS Genotypes with Unique Distribution Patterns
The total number of genotypic strains isolated from each patient ranged from 4–9 (composite for all visits), or 3–7 genotypic strains for any one visit, and diminished to one dominant S. mutans strain in four patients (patients G, J, K, L) at 6 months post-caries restorative therapy (Figure 1 and Table 3).
Caries Restorative Therapy Diminishes the Non-MS Oral Streptococci Group and Allows Specific, More Acidogenic MS Strains to Persist or Become Dominant Strains
In almost all patients observed in this pilot study, caries restorative therapy diminishes the strains within the non-MS oral streptococci group, which includes three strains of S. gordonii, three strains of S. anginosus, and one strain of G. adiacens, and one of the S. mutans strains persists or becomes the dominant strain during the post-treatment period, either at 2–4 weeks or 6 months post-treatment (Figure 1).
Acid generation from sugar fermentation is a major feature of cariogenic bacteria, and we conducted experiments to determine acidogenic potential of every isolate from these patients. Acidogenesis determinations were based on final pH values obtained after 72 hours of growth in phenol red dextrose, supplemented with 1% glucose. In Table 4, we compared the mean pH values of all isolates classified as S. mutans by conventional PCR to all isolates within the non-MS oral streptococci group, collectively examined across all patients but calculated by patient visit (pre-treatment, post-treatment [2–4 weeks] and post-treatment [6 months]). As expected, isolates within the S. mutans group were highly acidogenic, with mean pH values of 4.41–4.64, at all collection periods. In marked contrast, mean pH values for the non-MS oral streptococci group, collectively assessed using data from all patients, was much less acidic with values at 4.81–5.3 and were statistically different from the mean pH values of the S. mutans group (p values ≪ 0.01). Mean pH values were also examined for the S. mutans group for each patient and at each visit, and from this analysis, we clearly observe that the S. mutans group, when categorized by patient and by patient visit, yielded highly acidic pH values ranging from 4.17–4.7 (unpublished observations). In general, caries restorative therapy eliminated the less acidogenic non-MS oral strepococci group and allowed highly acidogenic S. mutans to persist or become the dominant strains.
Table 4.
Average pH for S. mutans Group and Non-MS Oral Streptococci Group at Each Visit
| Patient Visit | Group | Mean pH | Var1 | SD2 | SE3 | p-value5 |
|---|---|---|---|---|---|---|
| Pre-Treatment | S. mutans | 4.41 | 0.1144 | 0.338 | NA4 | 2.20E-16 |
| Non-MS Oral Streptococci | 5.16 | 0.8201 | 0.906 | NA | ||
| Post-Treatment (2–4 weeks) | S. mutans | 4.64 | 0.3564 | 0.597 | 0.038 | 2.20E-16 |
| Non-MS Oral Streptococci | 5.21 | 0.5327 | 0.730 | 0.0757 | ||
| Post-Treatment (6 months) | S. mutans | 4.41 | 0.0858 | 0.293 | 0.0195 | 2.80E-14 |
| Non-MS Oral Streptococci | 5.30 | 0.0306 | 0.175 | 0.0357 |
Var = The variance of the distribution is the expectation or mean of the squared deviation of that variable from its expected value or mean.
SD = Standard deviation
SE = Standard error
NA = Not applicable
P-values indicate statistically significant differences between mean pH values of the S. mutans group versus the non-MS oral streptococci group at all patient visits.
DISCUSSION
Streptococcus mutans is considered as an important cariogenic microorganism because of its acidogenic and acid-tolerant properties, and its ability to form biofilms and utilize glucose in the production of insoluble extracellular glycans. Distinct genotypes of S. mutans have been found to produce differential levels of glucosyltransferases31 and caries-active individuals have been identified to contain higher numbers of MS genotypes with increased abilities to synthesize water-insoluble glycans.10 Thus, the genetic diversity of S. mutans, as opposed solely to oral microbial numbers, is now assuming consideration as an important virulence factor of dental caries.
This pilot study was undertaken to develop the beginning framework for understanding the genetic diversity of S. mutans in individuals with severe early childhood caries, and to define changes in the genotypic population within each of these patients following caries restorative therapy, including restorations and the use of antimicrobial rinse and fluoride varnish. Many publications describing the genotypic diversity of S. mutans in children examine smaller numbers of MS isolates within larger numbers of individuals, such as Mattos-Graner et al.18 who examined 76 isolates from 24 children, Napimoga et al.9 who examined 299 isolates from 16 young adults, Pieralisi et al.17 who examined 280 isolates from 28 children, and Baca et al.12 who described 367 isolates from 20 school-age children. In this pilot study, we purposely chose to examine larger numbers of isolates per patient over a longitudinal period of time (yielding 828 isolates from seven patients) in order to more accurately examine the changes in distribution of MS isolates following full-mouth dental rehabilitation.
Genetic Analysis and Identification of MS Strains
Genetic and molecular analysis has recently gained greater acceptance in identification of diverse strains of oral microorganisms due to enhanced reliability and reproducibility of analytical methods. Studies by Napimoga et al.,9 Lembo et al.,11 and Baca et al.12 have validated the use of arbitrarily primed-PCR (AP-PCR) in discriminating MS genotypes within individuals. In our laboratory, consistent with the report of Pierasali et al.,17 we find that the OPA3 primer allowed efficient and clear discrimination of MS genotypes, even though OPA2 generated a larger number of amplicons (bands). This is in contrast to the determinations by Li and Caufield,32 who found that OPA3 retained a lower discriminatory capacity than OPA2. AP-PCR can accurately discriminate genotypes on the same gel or in multiple gels within the same diagnostic laboratory, but may not have the same discriminatory capacity between laboratories. We conjecture that the differences in discriminatory capacity for the use of OPA3 between laboratories, including our laboratory, may be due to differences in the purity of the genomic DNA used as templates in AP-PCR. In the majority of published articles describing the use of AP-PCR, crude DNA lysates were prepared by heating bacteria to 100°C, followed by clarification of debris using low-speed centrifugation. In our laboratory, we utilized commercial DNA extraction kits to prepare genomic DNA, which were free from inhibitors that could affect PCR efficiency, and allowed us to obtain clear and highly reproducible AP-PCR fingerprints from each isolate.
MS Colonization and Genotypic Diversity in Children with Severe Early Childhood Caries
Severe early childhood caries (S-ECC) is found in 1–6% of children (age ≤ 5 years) in the United States, and disproportionately affects lower socioeconomic populations.20 Mitchell et al.20 suggested that the acquisition and loss of specific strains within the MS population is dynamic in patients with S-ECC, and we also observed similar MS population shifts in patients described in our pilot study. Patient M may have undergone potential re-infection with genotypic strains from external sources at some point following the pre-treatment collection, but prior to the 6 months collection date, as observed with the reappearance of genotype M1 as the dominant strain at the 6 month collection (Figure 1 and Table 3). While undetected at the 4 week collection, M1 may have been present at very low numbers below the threshold of detection with the numbers of isolates analyzed. In the case of Patient M and other individuals with S-ECC, it is plausible that MS genotypes could be acquired vertically from their mother or horizontally between family members or extended care group. The diagnoses of ECC and S-ECC are associated with several risk factors including caregivers with high levels of MS and untreated carious lesions, frequent ingestion of sucrose-rich diets, and poor oral hygiene practices. In combination, these factors can result in MS colonization at earlier ages, with higher bacterial levels and greater number of genotypes than in caries-free children.
Caries-active adults and children may carry multiple genotypes of S. mutans.9,17,33 Caries-active adults contain up to 8 distinct genotypes of S. mutans,9,33 while pre-school children appear to exhibit fewer strains. Pieralisi et al.17 have shown that out of 28 pre-school aged children, 10 children within the caries-free group exhibited only a single genotype of S. mutans, while 13 other children within the caries-active group had more than one genotype, typically within 2–5 genotypes. In this current pilot study, our enrolled children with S-ECC, ages 3–5 years, exhibited 3–7 genotypes prior to caries restorative treatment, and all exhibited only 1–3 genotypes at 6 months post-treatment. Single genotypes, or the most dominant strains, have been identified in four out of five pediatric patients at 6-months post-caries restorative therapy.
The relationship between genotypic diversity and caries activity has been controversial. Pieralisi et al.17 have demonstrated a positive relationship between caries activity and MS genetic diversity, while Kreulen et al.34 and Lembo et al.11 have demonstrated negative relationships, or no significant differences respectively, between caries activity and numbers of MS genotypes. Moreover, it has been proposed that the concurrent and cumulative effects of different MS genotypes in a single patient may accentuate the risk of dental caries.35 The results of our pilot study are most similar to those of Pieralisi et al.17 where we observe genetic diversity of MS strains in children with S-ECC, but also demonstrate that caries restorative therapy results in the appearance of single dominant MS strains.
Caries Restorative Therapy and Elimination of S. Mutans
In this pilot study, we examined the genotypic strains of MS and non-MS oral streptococci strains from seven pediatric patients that exhibited S-ECC, with the secondary objective in evaluating the efficacy of the current regimen for caries restorative therapy. Caries restorative therapy included restorations of various material types (amalgam, composite, and stainless steel crowns), as well as extractions, sealants, prophylaxis, and application of chlorhexidine gluconate and fluoride varnish. It is interesting that in most patients, caries restorative therapy reduced the diversity of oral streptococci from several genotypic strains to 1–2 dominant strains, which in almost all cases were strains of highly acidogenic S. mutans. It is likely that one or more components of the caries restorative therapy is responsible for the appearance of MS dominant strains, but at this time we cannot determine which component, or components, is responsible. Prolonged usage of fluoride can lead to development and selection of fluoride-resistance strains of S. mutans and S. sobrinus, which have been identified in many cases to be more cariogenic than their parent strain.6,8 However, Van Loveren et al.36 have found fluoride-resistant strains of S. mutans with reduced cariogenic potential. In vitro studies also suggest that restorative material characteristics such as surface hydrophobicity and roughness as well as electrostatic interactions play key roles in the attachment abilities of specific MS strains.37–39 In vivo studies should be conducted to determine if any of the restorative materials used in this study alter MS attachment and thus growth.
We do not doubt that the antimicrobial rinse and fluoride treatment reduced the total bacterial numbers in all strains immediately following treatment, but it appears that this reduction was time-limited, and that dominant strains persisted and/or emerged at 6 months post-treatment. In one patient (patient H), the dominant strain at both the pre-treatment and 2-week post-treatment collections was one member of the non-MS oral streptococci group (H1 genotypic strain identified as S. anginosus). Since patient H did not attend the 6-month recall visit, we are uncertain if the H1 strain remained as the dominant strain, but find it interesting that patient H was the only one of the seven patients surveyed, who contained strains not to be either S. mutans or S. sobrinus as the single dominant strain. We recognize that the dentition of individual patients, including missing teeth and spacing between missing teeth, may influence the nature of the oral microflora, and find it interesting that patient H, who has a missing tooth (tooth #K) also has the H1 genotype as the dominant strain. We understand that we have limited numbers of patients in this pilot study, and that additional numbers of patients should be examined to develop definitive conclusions. We also understand that a limitation of this study was the lack of the precise determinations of bacterial numbers at each collection period, but believe that the dental plaque would regenerate to contain bacterial levels consistent to levels observed at pre-treatment, especially when comparing the pre-treatment and 6-month post-treatment collections. This statement is substantiated in part by the visually equivalent numbers of MS colonies observed between platings of collected specimens, and studies conducted by others which demonstrate recolonization of the oral cavity by S. mutans within 3–6 months following mechanical and chemical asepsis, or by chlorohexidine treatment.40,41 In spite of the limitations of this pilot study, with the large numbers of isolates that were examined, we believe that the results may have potential impact on defining practices for caries preventive therapy in individual children, and may have implications in the use of antimicrobial rinse and fluoride in oral health care.
CONCLUSIONS
Full-mouth caries restorative therapy, including the use of antimicrobial rinse and fluoride, can result in population shifts and decrease in numbers of specific MS and non-MS oral streptococci strains, with the potential appearance of dominant strains at 6-months post-treatment.
The implications of this study are that well-accepted practices for caries restorative therapy should be more closely examined for efficacy.
Acknowledgments
Support was provided for the projects of EAP, PP and AN, by the Pediatric Dentistry Resident Fund of the OHSU Foundation. This research was also made possible with support to AV and TF from the Oregon Clinical and Translational Research Institute (OCTRI), grant numbers TL1 RR024159 and UL1 RR024140 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This research was also supported in part by NIDCR grant R25 DE018206, with support to KM. EAP, PP, and AN were current or former residents in the Graduate Pediatric Dentistry Program at the OHSU School of Dentistry. TN and RS were both OCMID Student Research Scholars in Caries Microbiology (2010 and 2008, respectively). TF was a 2009 OCTRI student research fellow, and AV was a 2009 OSLER student research fellow. ATN, AV, SJ, TF, and KM are all dental students at the OHSU School of Dentistry. JE, JP, TM, and CM are faculty supported by the OHSU School of Dentistry, and BW is an OHSU faculty member supported by the Oregon Clinical and Translational Research Institute. The authors thank and acknowledge Dan Lafferty, Marie Teasdale, David Poon, Ivona Ristovska, Vahid Khonsari, Chase Pappenfuss, Nick Sassen, PQ Nguyen, Samyia Chaudhry, Tyler Lundquist and Huu Vu for laboratory support. The authors thank Dr. Bruce Paster, Director of the Human Microbe Identification Microarray Core Laboratory, Forsyth Institute, for conducting the PCR of 16S ribosomal RNA gene targets and the BLAST analysis of the non-MS oral streptococci strains. The authors thank ATN and AV for expert help in the development of tables and illustrations used in this manuscript. The authors thank Drs. Tom Shearer, John Engle and Jack Clinton for their encouragement and support in the development of this project. The authors thank Professor Kazuhiko Nakano for providing information concerning the distribution of the MS serotypes in the Japanese population. The authors thank Dr. Kim Kutsch and Oral Biotech for the luminometer used in some aspects of this study, and the OCMID group for support of the dental students to travel and present this research in part at regional meetings.
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