Antibacterial Activity of Freshly Prepared Ozonated Water and Chlorhexidine on Mutans Streptococcus When Used as an Oral Rinse – A Randomised Clinical Study

Background

Dental caries remains one of the most common chronic conditions worldwide, and Mutans Streptococcus (MS) is the primary bacterial species responsible for initiating tooth decay. Chlorhexidine mouth rinse has long been considered the benchmark antimicrobial agent for reducing MS levels in the mouth, but it comes with drawbacks over long-term use, including tooth staining, altered taste and limited suitability for daily, ongoing use. This has prompted researchers to look for alternative rinses that could offer similar antibacterial benefits without these downsides, particularly for patients identified as being at high risk of caries. Ozonated water - water infused with ozone gas via a tabletop generator - has been proposed as one such alternative, and this study set out to test how its antibacterial performance compared with chlorhexidine when used as a daily oral rinse.

Study design

The trial recruited 46 healthy adults who were randomly allocated, via block randomisation, into two equal groups of 23. Group A rinsed daily with a 0.2% chlorhexidine solution, while Group B rinsed with freshly prepared ozonated water generated by a tabletop ozone generator at a concentration of 2.4 mg/l. Saliva samples were collected at baseline, then again at seven and 14 days. MS counts were measured by serially diluting each sample in phosphate buffer and culturing it on a selective agar (MSB agar), incubated for 48 hours at 37 degrees Celsius, with colony counts taken as the measure of bacterial load.

Findings

Both rinses produced a statistically significant reduction in MS counts from baseline to day 14 (p<0.001). Starting counts were similar between groups (chlorhexidine 196.52 plus/minus 48.86; ozonated water 202.17 plus/minus 59.23). By day 14, the ozonated water group had fallen further, to 68.70 plus/minus 32.24, compared with 109.13 plus/minus 37.04 in the chlorhexidine group. Looked at another way, all 23 participants in the ozonated water group achieved at least a 50% reduction in MS counts by day 14, compared with only seven of the 23 participants in the chlorhexidine group. The difference in efficacy between the two rinses was also statistically significant (p<0.001), favouring ozonated water.

What the authors concluded

The authors concluded that ozonated water, when used consecutively as a daily mouth rinse, produced a significant reduction in MS counts, and that on this evidence it could serve as an alternative to chlorhexidine. They noted that tabletop ozone generators are relatively inexpensive and easy to access, and suggested ozonated water could be recommended as a daily rinse for patients at high risk of caries, as well as a general preventive measure for other patients.

Limitations of this study

The authors themselves flagged the small sample size (46 participants at a single dental college) as a limitation of the trial, along with the short 14-day follow-up period, which does not establish whether the antibacterial effect is sustained over months or years of regular use. The study also did not assess other clinically relevant outcomes, such as actual caries incidence, gingival health or patient tolerability over time. The authors recommended further research into ozonated water's efficacy against the smear layer in tooth preparation and root canal procedures, indicating this specific trial's findings should not be extended beyond the mouth-rinse context it tested.