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Annali di Stomatologia | 2026; 17(3): 698-703 ISSN 1971-1441 | DOI: 10.59987/ads/2026.3.698-703 Articles |
Ultraviolet light versus chlorhexidine plus cetylpyridinium chloride: in vitro efficacy on Streptococcus Mutans–contaminated toothbrushes
Article History
Received: May 29, 2026
Accepted: July 20, 2026
Published: July 30, 2026
Abstract
Background
Toothbrushing is the most widely used method of oral hygiene; however, toothbrushes may become contaminated during routine use and act as reservoirs for microorganisms.
Objective
To compare, in vitro, the disinfectant efficacy of ultraviolet light (UVL) with a solution containing 0.12% chlorhexidine (CHX) and 0.05% cetylpyridinium chloride (CPC) on toothbrushes contaminated with Streptococcus mutans.
Methods
Forty manual toothbrushes were inoculated with Streptococcus mutans ATCC 25175 and allocated in a 1:1 ratio to UVL exposure or immersion in CHX+CPC (n = 20 per group). Bacterial load was quantified as colony-forming units (CFU) and transformed as log10(CFU/mL + 1). Intragroup changes were assessed with the Wilcoxon signed-rank test, and logarithmic reductions were compared with the Mann-Whitney U test; p < 0.05 was considered statistically significant.
Results
Both interventions significantly reduced bacterial counts (p < 0.001). Median bacterial load decreased from 8.26 to 7.83 log10(CFU/mL + 1) in the UVL group and from 8.28 to 0.00 log10(CFU/mL + 1) in the CHX+CPC group. Median logarithmic reductions were 0.43 and 8.23, respectively, with a significant intergroup difference (p < 0.001).
Conclusion
Under the reported in vitro conditions, CHX+CPC produced a substantially greater apparent reduction in recoverable Streptococcus mutans than UVL. Because an antimicrobial neutralization procedure and the assay detection limit were not reported, the 0.00 post-treatment value should be interpreted as no recoverable colonies under the assay conditions rather than proof of sterilization. UVL produced a modest reduction and requires further protocol optimization and clinical evaluation.
Keywords: Chlorhexidine; Cetylpyridinium chloride; Disinfection; Streptococcus mutans; Toothbrushes; Ultraviolet light.
Introduction
Oral health is a fundamental component of overall health and contributes to general well-being. Because the oral cavity is colonized by diverse microbial communities, adequate oral hygiene is important for preventing oral disease [1]. Toothbrushing is the most commonly practiced method of oral care; however, repeated use may contaminate toothbrushes and allow them to serve as microbial reservoirs [2–3].
During brushing, plaque, saliva, and microorganisms are transferred to the bristles, where persistent microbial communities may develop [4]. Studies have recovered Streptococcus mutans, Escherichia coli, Micrococcus spp., Streptococcus mitis, and other microorganisms from toothbrushes [5–6]. Fungi and viruses may also be present, while environmental conditions — including proximity to a toilet, inadequate ventilation, and improper storage — may increase contamination [2,7].
Oral microbial dysbiosis has been associated with systemic conditions, including diabetes mellitus, respiratory disease, and cardiovascular disease [8]. Although the direct clinical consequences of toothbrush contamination remain incompletely defined, reducing avoidable microbial reservoirs is a relevant hygiene objective.
The densely packed bristle tufts and retained moisture of a toothbrush can hinder complete decontamination by rinsing with tap water alone [5]. Proposed methods include chemical agents such as CPC, sodium hypochlorite, triclosan, and CHX, as well as physical approaches such as ultraviolet irradiation [9–10].
A recent systematic review and meta-analysis found that chemical, household, and physical disinfection methods can reduce toothbrush contamination; CHX was among the most effective agents, although heterogeneity was substantial and the overall certainty of evidence was very low [11]. In vivo studies have also reported reductions with several synthetic and herbal solutions [12]. UV-based devices are attractive because they are compact and do not require repeated handling of chemical solutions, but their effectiveness varies across devices and protocols [13].
There is therefore no standardized protocol that is simultaneously effective, safe, cost-efficient, and practical for domestic use. This study aimed to compare the effect of ultraviolet light with a solution containing 0.12% CHX and 0.05% CPC on toothbrushes experimentally contaminated with Streptococcus mutans.
Materials and Methods
Study design and sample
This in vitro experimental study used 40 manual toothbrushes with medium bristles (Kolynos®, Colgate-Palmolive Company, New York, NY, USA), each inoculated with Streptococcus mutans ATCC 25175. The sample size was based on a previous study [14], resulting in 20 samples per group. Toothbrushes in unopened original packaging were included; brushes with damaged or deformed bristles were excluded.
Intervention groups
The toothbrushes were allocated in a 1:1 ratio to two intervention groups.
UVL group: Toothbrushes were exposed for 3 minutes in the Toothbrush Sterilizer TS-02 (Ultrawave, ULTRAWAVE®, BNSoft Inc., Seoul, South Korea), equipped with UV LEDs emitting at 265–285 nm. The device contains a 120 mAh rechargeable lithium-polymer battery and measures 45 × 45 × 20 mm.
CHX+CPC group: Toothbrushes were immersed for 10 minutes in 10 mL of Perio·Aid Intensive Care® (Dentaid S.L., Cerdanyola del Vallès, Barcelona, Spain), containing 0.12% chlorhexidine digluconate and 0.05% cetylpyridinium chloride.
Microbiological preparation
A total of 5 L of nutrient agar was prepared according to the manufacturer’s instructions. The medium was weighed on an analytical balance, hydrated with distilled water, autoclaved for 15 minutes at 121 °C, and cooled to 45 °C in a thermostatic bath.
The reference strain Streptococcus mutans ATCC 25175 was reactivated in Brain Heart Infusion (BHI) broth and incubated at 37°C for 24 hours under anaerobic conditions. All procedures were performed under sterile conditions.
After incubation, the microorganism was inoculated onto nutrient agar plates using streaking and serial-dilution techniques. The plates were incubated for 24 hours at 37°C under anaerobic conditions to obtain young colonies of Streptococcus mutans.
Under sterile conditions near a Bunsen burner, a portion of an isolated colony was transferred into 800 mL of sterile BHI broth and incubated at 37°C for 2 hours.
For inoculation, each group of toothbrushes was immersed for 1 hour in a beaker containing 400 mL of BHI broth with an active Streptococcus mutans culture.
UVL treatment
Twenty toothbrushes were positioned with their heads inside the TS-02 device on a flat surface. After the device was closed, the brushes were exposed to UVL for 3 minutes.
CHX+CPC treatment
The remaining 20 toothbrushes were immersed for 10 minutes in test tubes containing 10 mL of the CHX+CPC solution.
Microbiological analysis
Immediately after treatment, each toothbrush was placed in a test tube containing 10 mL of 0.9% saline solution. Seven serial dilutions were prepared, and 1 mL of each dilution was inoculated into Petri dishes. Nutrient agar was poured into the dishes and gently mixed to promote uniform colony distribution.
After solidification, plates were incubated anaerobically at 37 °C for. Streptococcus mutans colonies were counted with a dark-field colony counter equipped with a 4× magnifying lens and white light. For each sample, plates containing no more than 300 colonies were selected, and counts were multiplied by the corresponding dilution factor to obtain CFU values.
Statistical analysis
Data were recorded on a standardized collection form and analyzed with IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA). Bacterial counts were transformed using log10(CFU/mL + 1). The Shapiro-Wilk test was used to assess normality. Because the data were non-normally distributed, the Wilcoxon signed-rank test was used for paired pretreatment-post-treatment comparisons within each group, and the Mann-Whitney U test was used to compare logarithmic reductions (Δlog10) between groups. Statistical significance was set at p < 0.05.
Results
Table 1 summarizes bacterial counts as median [interquartile range (IQR)] log10(CFU/mL + 1). Pretreatment medians were numerically close between groups; no formal baseline intergroup comparison was reported. In the UVL group, the median bacterial load decreased from 8.26 to 7.83. In the CHX+CPC group, it decreased from 8.28 to 0.00, indicating that no colonies were recovered under the reported assay conditions. The Wilcoxon signed-rank test showed significant intragroup reductions in both groups (p < 0.001).
| Group | n | Before, median [IQR] | After, median [IQR] | p value* | r |
|---|---|---|---|---|---|
| UVL | 20 | 8.26 [8.20–8.31] | 7.83 [7.77–7.88] | <0.001 | 0.88 |
| CHX+CPC | 20 | 8.28 [8.23–8.32] | 0.00 [0.00–0.00] | <0.001 | 0.88 |
*Wilcoxon signed-rank test. IQR, interquartile range; r, effect size.
Table 2 compares the logarithmic reductions between groups. The median reduction was 0.43 log10 in the UVL group and 8.23 log10 in the CHX+CPC group. The Mann-Whitney U test showed a significant intergroup difference favoring CHX+CPC (p < 0.001).
| Group | n | Δlog10, median [IQR] | p value* | r |
|---|---|---|---|---|
| UVL | 20 | 0.43 [0.42–0.43] | <0.001 | 0.86 |
| CHX+CPC | 20 | 8.23 [8.19–8.28] |
*Mann-Whitney U test. IQR, interquartile range; r, effect size.
Discussion
This study compared two methods for disinfecting toothbrushes contaminated with Streptococcus mutans. Both interventions reduced recoverable bacterial counts, but CHX+CPC produced a markedly larger logarithmic reduction than UVL. The CHX+CPC result should nevertheless be interpreted as an absence of recoverable colonies under the assay conditions, not as confirmed sterilization, because the detection limit and an antimicrobial neutralization step were not reported.
The 0.43-log reduction observed with UVL corresponds to an approximate 63% reduction when calculated from the median log values. Boylan et al. [15] reported an 86% reduction in total CFU with a commercial Violight device, whereas Gujjari et al. [16] reported a 42% reduction. Differences in device geometry, wavelength, irradiance, exposure time, initial bacterial load, bristle arrangement, and recovery methods may account for the variable findings.
The bactericidal effect of UV radiation is primarily mediated by absorption of photons by microbial nucleic acids, producing photochemical lesions that inhibit replication and transcription [17]. Sensitivity varies across bacterial species and wavelengths; peak germicidal effectiveness has been reported near 263–270 nm [18]. The device used in this study emitted at 265–285 nm. Because Streptococcus mutans is Gram-positive and organisms embedded between bristle tufts may be shielded by geometry or organic material, incomplete inactivation is biologically plausible.
CHX is a cationic bisbiguanide that adsorbs to negatively charged bacterial surfaces, disrupts membrane integrity, and causes leakage of intracellular components [19–20]. CPC is also a cationic antimicrobial agent used in mouthrinses, with evidence supporting antiplaque and antigingivitis activity [21]; however, the present design tested a fixed CHX+CPC formulation and therefore cannot determine the independent contribution or interaction of the two active ingredients. Evidence from mouthrinse trials supports the antimicrobial effectiveness of the combination [22].
A randomized controlled trial found that immersion in a mouthrinse containing 0.05% CHX and 0.05% CPC reduced viable microorganisms on toothbrush bristles [22]. Although the CHX concentration in that study was lower than the 0.12% concentration used here, both findings support the activity of CHX+CPC formulations. Direct comparison remains limited by differences in exposure time, microorganisms, sampling, and recovery procedures.
A comparative study of several antimicrobial agents similarly found UV sanitation less effective than CHX- or CPC-based approaches [23]. CHX use in the oral cavity has been associated with extrinsic staining, taste alteration, mucosal irritation, and xerostomia, particularly with prolonged or frequent exposure [20,24]. These clinical adverse effects cannot be directly extrapolated to toothbrush immersion, but any home protocol should specify concentration, contact time, rinsing, solution replacement, and safe handling.
Adherence is an important implementation issue. Surveys have found limited knowledge and inconsistent toothbrush-decontamination practices among health-science students and dental professionals [25–26]. Education and simple instructions may therefore be as important as microbiological efficacy when translating a protocol into routine use.
UV-LED devices have practical advantages, including compact size, low energy consumption, and absence of chemical residues [27]. However, convenience was not measured in this study, and home-use adherence cannot be inferred from the in vitro findings.
The study has several limitations. It used a single reference strain under in vitro conditions and did not include an untreated control group. The UV irradiance and delivered dose were not reported, limiting reproducibility. The method used to obtain individual pretreatment counts was unclear. Most importantly, no validated neutralization step for residual CHX/CPC was described; antimicrobial carryover during dilution and plating could overestimate the apparent reduction. The post-treatment incubation duration, technical replicates, and assay detection limit were also not reported. These limitations are particularly relevant to interpreting the 0.00 median in the CHX+CPC group.
Conclusions
Under the reported in vitro conditions, both UVL and CHX+CPC reduced recoverable Streptococcus mutans from contaminated toothbrushes, with a substantially greater apparent reduction after immersion in 0.12% CHX plus 0.05% CPC. The zero-growth result should be interpreted cautiously because neutralization and detection-limit information were not provided. UVL achieved only a modest reduction with the tested 3-minute protocol. Future studies should use untreated controls, validated antimicrobial neutralization, reported UV fluence, predefined detection limits, multiple microorganisms, and clinically realistic use conditions before recommending a domestic protocol.
Author Contributions
Brenda Luz Rojas Chuquimbalqui: Conceptualization, formal analysis, data acquisition, writing—original draft, and final approval of the manuscript.
Hernán Vásquez Rodrigo: Conceptualization, methodology, data acquisition, writing—original draft, and final approval of the manuscript.
Rosa Josefina Roncal Espinoza: Data acquisition, data analysis, writing—review and editing, supervision, and final approval of the manuscript.
Funding
No external funding was received for this study.
Conflicts of Interest
The authors declare no conflicts of interest.
Ethics Approval
Not applicable. The study used commercially available toothbrushes and a reference bacterial strain and involved no human participants or animals.
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