Abstract
The presence of Helicobacter pylori in the oral cavity has been associated to the failure of antimicrobial therapy in patients with gastrointestinal infection and the development of oral diseases. However, it has been reported that the maintenance of good oral hygiene can improve the therapeutic success rates, where the use of mouthwashes with anti-Helicobacter activity would help to achieve it. The aim was to evaluate the antimicrobial activity of OxOral® mouthwash against H. pylori and its effect on biofilm formation. The minimum inhibitory concentration (MIC) of OxOral® (pH = 6.4–7.5, ORP = 650–900 mV) against H. pylori was calculated testing serial dilutions 0.117–15 ppm against 1 × 108 CFU/mL of H. pylori (ATCC® 700824™) by broth microdilution method using 96‐well plates. The H. pylori biofilm formation was determined by the optical density measurement at 600 nm from coverslips stained with 0.1% crystal violet. The gene expression of ureA, luxS, flaA, omp18, and lpxD were analyzed by RT‐qPCR. OxOral® cytotoxicity was evaluated in a human gingival fibroblast cell line by MTT assay. MIC was of 3.75 ppm, with 99.7 ± 7.7% bacterial growth inhibition. In the negative control, the biofilm formation was observed, whereas when bacteria were treated with OxOral® at 0.234, 0.469, and 0.938 ppm, an inhibition of 35.5 ± 0.9%, 89.1 ± 1.2%, and 99.9 ± 5.5% were obtained, respectively. The gene expression analysis showed that flaA, omp18, and lpxD genes were down‐regulated with OxOral® compared with control (p < 0.05). Low cytotoxicity of 16.5 ± 7.6% was observed at the highest dose (15 ppm); no significant differences were observed from 15 to 0.469 ppm compared to the control of untreated cells (p > 0.05). Our results reveal an important anti-Helicobacter activity of OxOral® and open the possibility of its therapeutic use new studies, which would increase the success rate of conventional therapies against H. pylori.
Highlights
Introduction eHelicobacter pylori gastric infection has been associated with the development of chronic gastritis, peptic ulcer, and gastric cancer [1, 2]. us the Agency for Research on Cancer (IARC) recognized H. pylori as a group 1 carcinogen to humans [3, 4]
Antibacterial Activity Assay. e minimal inhibitory concentration (MIC) was performed in 96-well at-bottom plates containing 100 μL of each OxOral® dilution in trypticase soy broth (TSB) supplemented with 10% fetal bovine serum (FBS) and 100 μL of 1.0 × 108 CFU/mL of H. pylori, up to a nal volume of 200 μL per well; 5 μg/mL tetracycline was used as a positive control for inhibition of bacterial growth and saline solution (SS) as a negative control
An important antimicrobial activity of OxOral® was observed from 15 to 3.75 ppm, in those doses no di erence was observed compared to 5 μg/mL tetracycline ( > 0.05)
Summary
E minimal inhibitory concentration (MIC) was performed in 96-well at-bottom plates containing 100 μL of each OxOral® dilution in TSB supplemented with 10% FBS and 100 μL of 1.0 × 108 CFU/mL of H. pylori, up to a nal volume of 200 μL per well; 5 μg/mL tetracycline was used as a positive control for inhibition of bacterial growth and saline solution (SS) as a negative control. The MIC value was de ned as the lowest concentration of OxOral® that inhibited 99% of H. pylori growth. Each plate was lled with 12 mL of MH broth supplemented with 0.3% glucose and 10% FBS and 2 mL of OxOral® until reaching a nal concentration from 0.938 ppm to 0.117 ppm; 5 μg/mL tetracycline and SS were used as positive and negative controls, respectively. CAG TAT AGA TGG TCG TGG GAT TG CTA AAT TCT GTG CGC CCT CTA A GGA GTA CGG TCG CAA GAT TAA A
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