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Ozone is strong oxidizing agent that is applied in aqueous form for sanitation. However, ozonated water is unstable and has a short half-life. Ultrafine bubble technology is promising to overcome these issues. Ultrafine bubble is nanoscale bubble and can exist in water for a considerable duration of time. This study aims to investigate the application of ozone ultrafine bubble water (OUFBW) as a disinfectant. We produced an OUFBW generator which generates OUFBW containing 4-6 ppm of ozone. Thereafter, we examined the bactericidal activity of the OUFBW against various pathogenic bacteria in oral cavity and upper airway, including antibiotic-susceptible and antibiotic-resistant Streptococcus pneumoniae, Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis. Exposure of planktonic culture of these bacterial species to OUFBW reduced viable bacteria by > 99% within 30s. Additionally, OUFBW exerted bactericidal activity against S. pneumoniae and P. aeruginosa adhered to toothbrush and gauze, respectively. We also observed disruption of bacterial cell wall of S. pneumoniae exposed to OUFBW by transmission electron microscope. Additionally, OUFB did not show any significant cytotoxicity toward the human gingival epithelial cell line Ca9-22. These results suggest that OUFBW exhibits bactericidal activity against broad spectrum of bacteria and has low toxicity towards human cells.

Streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. Antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. Hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against S. pneumoniae in vitro regardless of antimicrobial resistance. In this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. Male 8-week-old BALB/c mice were intratracheally infected with S. pneumoniae strains D39 (antimicrobial susceptible) and NU4471 (macrolide resistant). After 1 h, hinokitiol was injected via the tracheal route. Hinokitiol significantly decreased the number of S. pneumoniae in the bronchoalveolar lavage fluid (BALF) and the concentration of pneumococcal DNA in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. In addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the BALF and serum. Repeated hinokitiol injection at 18 h intervals showed downward trend in the number of S. pneumoniae in the BALF and the concentration of S. pneumoniae DNA in the serum with the number of hinokitiol administrations. These findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. Accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia.