Results and discussion
Large numbers of bacteria were cultivable on media without triclosan from supragingival dental plaque samples taken from subjects using 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice and from supragingival dental plaque samples from subjects using dentifrice without triclosan. Subjects using the triclosan dentifrice demonstrated a mean of 1.36 ×10
8 CFU/ml while subjects using dentifrice without triclosan demonstrated a mean of 1.6 × 10
8 CFU/ml. There were fewer bacteria cultivable on media containing triclosan (p < 0.05) than on media without triclosan indicating triclosan inhibition of bacterial growth. Culture of plaque samples collected over nineteen years on media containing 7.5 μg/ml or 25 μg/ml triclosan showed reductions in the mean number of colony forming units of 99.451% (range 91.209 - 99.830%) and 99.989% (range 99.670 -100%), respectively, compared to culture on media without triclosan (Table
2). That is, plaque samples cultured on media containing 7.5 μg/ml triclosan showed an average of 8.8 × 10
5 CFU/ml while plaque samples cultured on media containing 25 μg/ml triclosan showed an average of 3.2 × 10
4 CFU/ml. Characterization of dental plaque bacteria cultivable on media containing triclosan[
10,
11] as well as the triclosan susceptibility of oral and non-oral bacteria[
15] has been previously described.
Table 2
Susceptibility of supragingival dental plaque bacteria sampled over 19 years
Mean | 99.451% | 99.989% |
Standard error | 0.092 | 0.004 |
Standard deviation | 1.145 | 0.049 |
Minimum | 91.209% | 99.670% |
Maximum | 99.830% | 100% |
Median | 99.701% | 100% |
Range | 8.791% | 0.330% |
The susceptibility of supragingival dental plaque collected from 21 subjects who used a 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice for at least five years and as long as 13 years was compared to supragingival dental plaque collected from 20 subjects who used a dentifrice without triclosan. Supragingival plaque samples from both groups demonstrated susceptibility to both concentrations of triclosan. There was no statistically significant difference in triclosan susceptibility between those who regularly used triclosan dentifrice and those who didn’t (p < 0.05).
Linear regression analysis and the runs test were used to examine antimicrobial susceptibility to triclosan in the supragingival dental plaque samples collected over 19 years. At both 7.5 μg/ml and 25 μg/ml triclosan concentrations, linear regression analyses showed that the slope of a line fitted to the results was not significantly different from zero. This indicates that there were no statistically significant changes in antimicrobial susceptibility discernible over 19 years (p = 0.159 for the 7.5 μg/ml concentration and p = 0.299 for the 25 μg/ml concentration, Table
3). Analyses using the runs test also demonstrated no trends in susceptibility patterns over the course of the study for either triclosan concentration evaluated (p > 0.05).
Table 3
Regression analysis for triclosan susceptibility in supragingival plaque samples obtained over 19 years
Model variation. Year to year variation | 2.61 | 0.0003 |
Residual variation. Unexplained variation ( σ2) | 1.30 | 0.379 |
Total Variation | 3.40 | 0.38 |
Regression analysis (p value) | 0.159 | 0.299 |
The 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice reduces dental plaque and plaque-associated gingival bleeding around teeth and also around dental implants[
16]. Gingival bleeding is a key sign of periodontal disease, especially plaque-associated gingivitis, but gingival bleeding can also be a sign of more serious forms of periodontal disease such as periodontitis. Periodontitis affects over 47% of adults 30 years of age or older in the United States and 64% of adults 65 years and older[
17]. Periodontal infections causing periodontitis affect not only the oral cavity but also increases the risk for systemic diseases and conditions such as preterm low birth weight[
18], cardiovascular disease[
19], diabetes mellitus[
20] and chronic obstructive pulmonary disease[
21]. By reducing dental plaque and gingivitis, the 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice may also reduce periodontal infection-related systemic diseases and conditions.
Triclosan employs several mechanisms to kill bacteria and reduce dental plaque. It kills some bacteria such as
Escherichia coli[
22],
Staphylococcus aureus[
23] and
Bacillus subtilis[
24] by inhibiting NADH- or NADPH-dependent enoyl-acyl carrier protein reductase (
fab I)[
25] during fatty acid synthesis. It also kills bacteria by means of other membranotropic effects[
26], by uncoupling oxidative phosphorylation and by inhibiting glycolysis[
27].
However, dental plaque infection and bacterial virulence are not the only mechanisms in the pathogenesis of periodontitis. Inflammation plays a significant role. Plaque bacteria initiate an inflammatory cascade leading to chronic inflammation[
28]. In addition to its antimicrobial activity, triclosan also has anti-inflammatory properties. Triclosan can suppress microbial pathogen recognition pathway molecules as well as acute and chronic inflammatory mediators[
29]. Wallet et al.[
30], for example, showed triclosan to be a potent inhibitor of oral epithelial cell LPS-induced pro-inflammatory responses by regulating the TLR-signaling pathway. Further, Mustafa et al.[
31] found that triclosan reduced biosynthesis of PGE
2 by inhibiting mPGES-1 in gingival fibroblasts.
Some bacteria are naturally resistant to triclosan[
32] and some laboratory strains can be made resistant. Pseudomonads, for example, exhibit natural resistance to triclosan by means of multidrug efflux pumps[
33].
E. coli is not naturally resistant but can be made resistant in the laboratory by long term exposure to sub lethal concentrations of triclosan. Induced resistance in the laboratory occurs by increased production of the enoyl-acyl carrier protein reductase or by overexpression of drug efflux pumps[
32] or by development of an alternative fatty acid synthesis pathway[
34].
In the present study, there was no change in ex vivo triclosan susceptibility among supragingival plaque bacteria collected over 19 years and there was no change in ex vivo triclosan susceptibility among supragingival plaque bacteria collected from subjects using a triclosan dentifrice for at least five years and as long as 13 years. This study differs from previous reports in that those samples were taken from subjects participating in clinical trials of triclosan-containing dentifrice. In the present study, subjects were neither included nor excluded based on the type of dentifrice, amount of dental plaque, degree of gingival inflammation or oral health status. The subjects in this study used different mouth rinses, chewing gum, and oral hygiene aids and were possibly exposed to other sources of triclosan as in hand soaps and cosmetics. They had different access and utilization of professional oral health care services and exposure to antibiotics or other antimicrobial agents.
Findings in the present community-level assessment are consistent with previous clinical trials (Table
4). Zambon et al
.[
9] examined supragingival dental plaque in 40 adults who used a 0.3% triclosan, 2% copolymer and 0.243% sodium fluoride dentifrice compared to 41 adults using the same dentifrice without triclosan/copolymer. After 28 weeks, there was no difference between groups except for statistically significant reductions in fusiforms, spirochetes and staphylococci and significant increases in
Streptococcus sanguis in the test group compared to the control group. Jones et al
.[
8] examined interproximal plaque bacteria and salivary
S. mutans and candida in 13 female adults who used a 0.2% triclosan, 0.5% zinc citrate dentifrice for seven months compared to 13 female adults who used the same dentifrice without 0.20% triclosan, 0.50% zinc citrate. They found no difference between groups in the number of salivary
S. mutans or candida, no evidence for a change in the number of predominant plaque bacteria i.e. population shifts, nor evidence for an increase in bacterial resistance to triclosan. Walker et al
.[
10] examined 70 subjects using 0.3% triclosan, 2% copolymer and 0.243% sodium fluoride dentifrice twice a day for six months with surveillance for an additional six months. They found no evidence for unfavorable shifts in supragingival plaque bacteria and no correlation between use of the triclosan dentifrice and the number of triclosan-resistant bacteria, the resistant bacterial taxa, or the number of subjects harboring triclosan-resistant bacteria. Zambon et al
.[
11] examined 73 subjects using 0.3% triclosan, 2% copolymer and 0.243% sodium fluoride dentifrice twice a day for six months with surveillance for an additional six months. Both test and control groups (71 subjects) harbored triclosan resistant supragingival plaque bacteria, mainly
Veillonella dispar. There was no evidence for unfavorable shifts in supragingival plaque bacteria from use of the triclosan dentifrice and no evidence for the development of triclosan resistance. Fine et al
.[
12] examined 71 subjects using a 0.3% triclosan, 2% copolymer and 0.243% sodium fluoride dentifrice for six months and for an additional six months post-treatment. There were no detrimental shifts in supragingival plaque bacteria and no overgrowth in opportunists, periodontal pathogens, or cariogenic bacteria in either test or control. There was no increase in the proportion of supragingival plaque bacteria resistant to triclosan and there was no increase in the minimum inhibitory concentrations for triclosan against
Actinomyces viscosus or
Veillonella parvula in test or control groups. Sullivan et al
.[
13] examined the
in vitro sensitivity of triclosan against viridans streptococci and normal oral flora in saliva samples obtained from 9 adults who used a 0.3% triclosan, 2% copolymer and 0.243% sodium fluoride dentifrice twice a day for two weeks. No major changes were detected in the normal oral flora and there were no differences in susceptibility to triclosan, benzyl penicillin, gentamicin, erythromycin, tetracycline or fusidic acid between streptococci obtained pre- or post-treatment.
Table 4
Studies of the effect of triclosan dentifrice on oral microorganisms
| 0.2% triclosan | • Test = 13 adult females | • 4 mon. washout | • Interproximal plaque and saliva samples | • No detectable shifts in plaque oral ecology |
0.5% zinc citrate | • Control = 13 adult females | • 7 mon. use | • Bacterial culture | • No difference between groups in numbers of S. mutans or candida |
| • 20–50 years of age | | • Salivary S. mutans and candida enumerated | • No evidence of increased bacterial resistance to triclosan |
| | | • MIC’s by agar dilution | |
| 0.3% triclosan | • Test = 40 adults | • 6 mon. use | • Supragingival plaque samples | • No unfavorable shifts in plaque bacteria |
2% copolymer 0.243% Na fluoride | • Control = 41 adults | • 6 mon. post-use | • Bacterial culture | • Significant reductions in fusiforms, spirochetes and staphylococci and increases in S. sanguis in triclosan group |
| | | • Phase contrast microscopy | |
| | | • Immunofluorescence microscopy | |
| | | • MIC’s by agar dilution | |
| 0.3% triclosan | • Test = 70 adults | • 6 mon. use | • Supragingival plaque samples | • No detrimental shifts in plaque bacteria |
2% copolymer 0.243% Na fluoride | • Control = 74 adults | • 6 mon. post-use | • Bacterial culture | • No correlation between triclosan dentifrice and number of triclosan-resistant bacteria, resistant bacterial taxa, or number of subjects harboring tricosan-resistant bacteria |
| | | • Phase contrast microscopy | |
• Immunofluorescence microscopy |
| | | • MIC’s by agar dilution | |
| 0.3% triclosan | • Test = 73 adults | • 6 mon. use | • Supragingival plaque samples | • No unfavorable shifts in plaque bacteria |
2% copolymer 0.243% Na fluoride | • Controls = 71 adults | • 6 mon. post-use | • Bacterial culture | • Higher levels of fusiforms in test group |
| | | • Phase contrast microscopy | • Higher levels of neisseria and P. gingivalis-infected |
| | | • Immunofluorescence microscopy | subjects in controls |
| | | • MIC’s by agar dilution | • Both test and controls subjects exhibited triclosan resistant bacteria |
| | | | • No evidence for development of triclosan resistance |
| 0.3% triclosan | • Test = 20 adults | • 36 mon. | • Subgingival plaque samples | • No detrimental shifts in plaque bacteria |
| 2% copolymer 0.243% Na fluoride | • Control = 20 adults | | • Bacterial culture | • Lower total viable bacterial counts in triclosan group compared to control |
| | | | • No changes in gingival health-associated bacteria |
| 0.3% triclosan | • 68 adults | • 6 mon. use | • Supragingival plaque samples | • No detrimental shifts in plaque bacteria |
2% copolymer 0.243% Na fluoride | | • 6 mon. post-use | • Bacterial culture | • Decreased spirochetes in triclosan group |
| | | • Darkfield microscopy | • No increased proportion of triclosan resistant bacteria |
| | | • Immunofluorescence microscopy | • No increase in triclosan MIC’s for Actinomyces viscosus or Veillonella parvula
|
| | | | • MIC’s by agar dilution | |
| 0.3% triclosan | • Test = 179 adults | • 5 years | • Subgingival plaque samples | • No overgrowth of A. actinomycetemcomitans, P. intermedia, or P. gingivalis
|
2% copolymer 0.243% Na fluoride | • Control = 178 adults | | • Enzyme-linked immunosorbent assay | |
| 0.3% triclosan | • 9 adults | • 2 weeks | • Saliva samples | • No major changes in normal oral flora |
2% copolymer 0.243% Na fluoride | | | • Bacterial culture | • No change in streptococcal susceptibility to triclosan, benzyl-penicillin, gentamycin, erythromycin, tetracycline or fusidic acid |
| | | • MIC’s by agar dilution | |
| 0.3% triclosan | • Test = 18 adults | • 5 years | • Supra and subgingival interproximal plaque samples | • Triclosan MIC’s similar for isolates from both groups |
2% copolymer 0.243% Na fluoride | • Control = 22 adults | | • Bacterial culture | • Triclosan dentifrice does not lead to increased MIC’s for oral bacteria |
| | | • MIC’s by agar dilution | |
| | | | • Isolates identified by 16S rDNA sequencing | |
In contrast to supragingival plaque, two studies reported the effect of a triclosan dentifrice on subgingival dental plaque. Rosling et al
.[
7] examined 20 test subjects and 20 control subjects over 36 months and found lower numbers of subgingival bacteria and lower numbers of subjects harboring periodontal pathogens in the triclosan dentifrice group compared to the control group. Cullinan et al
.[
6] used enzyme-linked immunosorbent assays for
Actinobacillus (now
Aggregatibacter)
actinomycetemcomitans,
Prevotella intermedia, and
Porphyromonas gingivalis and found no overgrowth of these species in 179 adults using 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice for five years compared to 178 control subjects.
A recent study by Cullinan et al
.[
14] examined supra and subgingival interproximal dental plaque samples from 18 subjects using a 0.3% triclosan, 2% copolymer, 0.243% sodium fluoride dentifrice for 5 years and 22 subjects using a control dentifrice without triclosan/copolymer. Like the present study, 5 years use of the triclosan dentifrice did not increase triclosan minimum inhibitory concentrations for oral bacteria and did not lead to triclosan-resistant oral bacteria. The minimum inhibitory concentration for oral bacterial isolates in their study from both their triclosan and control groups ranged from 12.50–50 μg/mL, consistent with the mean growth inhibition of 99.451% and 99.989% on media containing 7.5 μg/ml and 25 μg/ml triclosan, respectively, found in the present study. The majority of species cultivable at lower triclosan concentrations in their study were common to both groups and included
Veillonella parvula and
Campylobacter gracilis isolates which had the highest MICs. This is consistent with a previous study that also found that triclosan-resistant species were similar between triclosan and control groups and were predominantly
V. dispar[
11].
Competing interests
Prem K. Sreenivasan is an employee of the Colgate Palmolive Company that is paying article-processing charges for this paper. Violet Haraszthy and Joseph Zambon declare that they have no competing interests.
Authors’ contributions
All authors contributed to the conception and design of the study. VH collected samples and supervised the clinical laboratory assays. JZ collected samples and drafted the paper. PS performed the statistical analysis. All authors read and approved the final manuscript.