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, 2019, 6154867
eCollection

Antimicrobial and Anti-Biofilm Effect of an Electrolyzed Superoxidized Solution at Neutral-pH Against Helicobacter pylori

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Antimicrobial and Anti-Biofilm Effect of an Electrolyzed Superoxidized Solution at Neutral-pH Against Helicobacter pylori

Daniela Guadalupe Lucio-Sauceda et al. Biomed Res Int.

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.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of OxOral® on H. pylori growth. 1 × 108 CFU/mL of H. pylori (ATCC® 700824™) suspension was treated with 15–0.117 ppm OxOral® in 96-well microtiter plates. Plates were then incubated for 72 h in a microaerobic atmosphere at 37°C. After incubation, the effect of OxOral® on H. pylori growth was determined by measuring optical densities at 600 nm and the percentage of growth inhibition was calculated, as explained in the text. The data represent the percentage mean ± the percentage deviation. TET, 5 μg/mL tetracycline; SS, saline solution; ∗∗, p < 0.001; ns, not significant.
Figure 2
Figure 2
Evaluation of the H. pylori biofilm formation in the presence of OxOral®. The H. pylori biofilm formation was carried using sterile 22 × 22 mm glass coverslips that were placed in Petri dishes. Each plate was filled with MH broth supplemented and OxOral® at different doses. Biofilm formation was started by inoculating H. pylori at an initial concentration of 1 × 106 CFU/mL. The dishes were incubated under microaerobic conditions at 37°C for 168 h. After incubation, coverslips were washed and stained with 0.1% violet crystal. After staining, the dye associated to biofilms was dissolved using 33% glacial acetic acid, then the optical density was measured at 600 nm and the percentage of biofilm inhibition was calculated. The data represent the percentage mean ± the percentage deviation. TET, 5 μg/mL tetracycline; SS, saline solution; ∗∗, p < 0.001; ns, not significant.
Figure 3
Figure 3
The H. pylori biofilm treated with OxOral®. The H. pylori biofilm formation was carried using sterile glass coverslips that were placed in Petri dishes. Each plate was filled with MH broth supplemented and OxOral® at different doses: (a) saline solution, (b) 0.938 ppm OxOral®, and (c) 0.469 ppm OxOral®. Biofilm formation was started by inoculating H. pylori at an initial concentration of 1 × 106 CFU/mL. The dishes were incubated under microaerobic conditions at 37°C for 168 h. After incubation, coverslips were washed and stained with 0.1% violet crystal. The coverslips were observed by optical microscopy at a magnification of 100x.
Figure 4
Figure 4
Effect of OxOral® on mRNA relative expression levels of H. pylori biofilm genes. The relative expression was determined by the ΔΔCT method and the relative expression ratio (R) was calculated. Data represent means ± SE of triplicate determinations from three independent experiments; 16s rRNA gene was used as reference. ∗∗, p < 0.001.
Figure 5
Figure 5
Evaluation of the OxOral® cytotoxicity on ATCC®PCS-201-018™ cell line. Human gingival fibroblast cells (5 × 104 cells/mL) were incubated in the presence of OxOral® at various concentrations for 24 h. After incubation, the cytotoxicity of OxOral® on ATCC®PCS-201-018™ cell line was determined by the MTT reduction assay. The optical densities was measured at 570 nm and the percentage of cytotoxicity was calculated. The data represent the percentage mean ± the percentage deviation. ∗∗, P < 0.001; ns, not significant.

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