Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT05372835 |
| Other study ID # |
KMUHIRB-F(I)-20200042 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
March 20, 2020 |
| Est. completion date |
February 22, 2022 |
Study information
| Verified date |
May 2022 |
| Source |
Kaohsiung Medical University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Mouthwash is one method of maintain oral health and to reduce the dental plaque and gum
inflammation. However, the effects of mouthwash on oral bacteria were not consistent among
different brands of them. The aims of present study were to determine the effects of a new
designed mouthwash with hypochlorous acid on oral bacteria and Staphylococcus aureus. All
participants were invited as a volunteer to attend this study from a private dental clinic
and diagnosed by the same dentist. Patients with periodontal diseases were randomized
selected as mouthwash group and mouthwash plus dental flossing device (La Chlogen, Taiwan)
group. Patients for regular dental visit and without periodontal disease were invited as a
control group having mouth rinse with water. After the inform consent was signed,
participants completed the intervention study, saliva collection, and a simple survey under
the guide of a dental assistant. Saliva samples were collected before and after the
intervention for bacterial DNA extraction. A real-time polymerase chain reaction and S.
aureus with serial dilutions were applied for the estimation of total oral bacterial count
(TOBC) in saliva. An in vitro assay with CCK-8 reagent was apply to test the antibacterial
ability of mouthwash.
Description:
Finally there were 83 person recruited into the present study. A total of 53 patients with
periodontal disease diagnosed by a periodontist, who were randomly assigned to the
mouthwash-only group (26 patients) or the mouthwash plus periodontal flosser group (27
patients). There were 30 controls recruited into the present study. For the determination of
oral bacterial counts, a real-time polymerase chain reaction and a pure strain of
Staphylococcus aureus with a serial dilutions were applied to create a standardized growth
curve for the estimation of oral bacterial counts in saliva. Before the intervention,
participants were asked to rinse 5 ml of water to deplete the food debris and the other
residues in mouth. The intervention was asked participants to rinse the 15 ml mouthwash or
water for 5 minutes in oral cavity.
Participants were asked their health history and use of dental cleaning tools. The
participants underwent relevant oral health examinations, and their oral health indices were
also recorded. The mouthwash used for intervention in the present study was the 15 mL of
commercially available La Chlogen mouthwash (Republic of China Patent No. M616466) for 5
minites. The main ingredient in the mouthwash is low-concentration high-purity HOCl (100
ppm). The HOCl solutions can be also used in conjunction with the La Chlogen periodontal
flosser (Republic of China Patent No. M590033). The controls were instructed to gargle with
pure water (15 ml) for 5 minutes. Each participant first underwent a pretest, in which after
the participants' saliva samples were collected, their total oral bacterial count (TOBC) were
measured. Thereafter, each participant rinsed their mouth with their assigned liquid
(mouthwash, mouthwash plus periodontal flosser or water) without spitting. After 5 minutes,
the participants spit out the liquid, and these saliva samples were collected and tested to
determine the TOBC.
Each participant underwent two saliva sample collection sessions: pretest and posttest.
Before the saliva collection, participant rinsed with 5 ml of water. After this, participants
were asked to expectorate over a maximum period of 3 minutes into a 50 ml sterile
centrifugation tube (Creative Biotechnology Co., Ltd., Taiwan) for the collection of
unstimulated whole saliva. The collected specimen was then immediately placed in a portable
ice box for storage, returned to the laboratory for the same-day bacterial DNA extraction.
The saliva volume and weight of each sample was also recorded before the bacterial genomic
DNA extraction process.
TOBC analysis: After the extraction of the bacterial genomic DNA, The real-time polymerase
chain reaction (RT-PCR) techniques were used to quantify the total counts of oral bacteria in
the saliva samples. Saliva sample was centrifuged at 2500 rpm for 5 minutes. The pellet was
suspended with 300µl lysozyme solution (2.5 mg/mL). After being mixed evenly, the mixture was
transferred into a 1.5mL eppendorf tube and placed on ice for 1 hour. Thereafter, 10% SDS
(20µL), 0.5M EDTA (80µL), and 20mg/mL proteinase K (10µL) were added to each tube in
sequence; the contents of each tube were mixed evenly and placed in a 55°C oven for
overnight. Next, 10M NH4OAC was added to the mixture in equal volume. The mixture was placed
on ice for 5 minutes and centrifuged at 13,000 rpm for 10 minutes. The resulting supernatant
was placed into a 1.5-mL centrifuge tube. Isopropanol was added to the resulting mixture in
equal volume, and the mixture was stored at -20°C for overnight or -80°C for 1 hour. After
centrifugation for 10 minutes at 13,000 rpm, the supernatants were removed from the
precipitates, 75% alcohol (500µL) was added and left to stand for 5 minutes, and the mixture
was centrifuged for 10 minutes at 13,000 rpm. The alcohol was then removed. Next, 100%
alcohol (500µL) was added to the pellet. The mixture was left to stand for 5 minutes and
subsequently centrifuged for 10 minutes at 13,000 rpm. Finally, the alcohol in the tube was
poured out, and the centrifuge tube was placed upside down to allow any remaining alcohol to
evaporate. Finally, the DNA precipitates were dissolved using an appropriate amount of
sterilized water, placed into an oven at 55°C for 5 minutes, and stored at -20°C. Generally,
the DNA specimens with an OD260/ OD280 ratio of 1.4 or higher. The concentration of recovered
DNA was calculated on the basis of a concentration of 50 µg/mL at OD260 = 1.00, which was
determined to be suitable for estimating the TOBC of each sample after the DNA
quantification. Staphylococcus aureus (ATCC 29213) was used as the reference strain in
bacterial growth curve estimation. After overnight culturing, the bacterial solution was
serially diluted five times to generate specimens with different concentrations of S. aureus.
A total of 10 µL of each of the specimens were taken and spread on the agar medium to attain
concentrations of 2.5 × 103 to 3.9 × 107 CFU/mL. A volume of 1 mL of each different bacterial
concentration was taken for bacterial DNA extraction. The extracted bacterial genomic DNA was
stored frozen at -80°C until the RT-PCR test was conducted. Before the RT-PCR test, the
bacterial genomic DNA concentration was measured. RT-PCR tests mainly detect 16S rRNA, and a
StepOnePlus system (Applied Biosystems, Foster City, CA, USA) was used to establish a
standard curve of S. aureus concentration. The sequence of the forward primer and reverse
primer used were 5'-CCT ACG GGA GGC AGC AG-3' and 5'-CCG TCA ATT CMT TTR AGTT T-3',
respectively.16 The coverage rates of selected forward and reverse primer pair for common
bacterial species are 94.9% and 92.8%, respectively.16 The total PCR volume was 25 uL,
comprising 5.5 µL of ddH2O, 1.0 µL of 5-µM preprimer, 1.0 µL of 5-µM reverse primer, 12.5 µL
of SYBR solution, and 5.0 µL (0.25 µg) of DNA. The RT-PCR reaction conditions were as
follows: 94°C for 10 minutes; followed by 35 cycles of 95°C for 45 seconds, 58°C for 40
seconds, and 72°C for 60 seconds; and followed by one cycle of 72°C for 7 minutes. A
regression equation derived from the standard curve and the RT-PCR results were used to
calculate the bacterial concentration in each saliva sample. The RT-PCR assay was conducted
two duplicates for each sample, and the coefficient of variation of the threshold was varied
between 2% and 12%. Human blood DNA was used as a negative control group.
Antibacterial activity of the mouthwash solution: The pure-cultured S. aureus used as the
standard strain was plated on a culture plate (10 cm in diameter) containing 1.5% (g/L) luria
broth agar and cultured overnight. A single colony was selected and placed in a conical flask
containing LB nutrient liquid, shaken, and recultured overnight, and the resulting solution
was serially diluted and coated on the plate to calculate the number of bacteria per unit
volume. Finally, a Cell Counting Kit 8 (CCK-8, Engreen Biosystem) commercial reagent was used
to evaluate the antibacterial effect of the mouthwash. Each experiment was performed in
duplicate. Solutions containing different colony forming units (0, 10, 100, 1000, 10000,
100000, and 1000000) of S. aureus were centrifuged at 2000 rpm for 5 minutes to concentrate
the bacteria; after the liquid was removed, 180 µL of fresh culture medium was added to
suspend the bacteria in the culture medium. The bacteria solution was transferred to a
96-well plate. To evaluate the antibacterial effect of the mouthwash, 20 µL of the mouthwash
was added to the mixture of the bacterial solution. The mixture was incubated at 37°C for 2
hours. Finally, 10 µL of CCK-8 reagent was added and mixed well. The resulting mixture was
incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured using an ELISA
reader. The standard growth curve could be established for the estimation of the number of
bacteria in the mixture and their survival rate of the bacteria, which were then compared
with the standard curve.
Statistical analysis: After the data collection and checking were complete, the finalized
debugged files were transferred to a computer with statistical software for statistical
analysis. The descriptive statistics used included frequency distribution tables,
percentages, means, and standard deviations. In the TOBC analysis, in addition to descriptive
statistics, a t test, chi-square test, and linear regression analysis were conducted to
assess and compare the changes in the bacterial counts of the participants' saliva after the
intervention period. Considering the small sample size and the distribution of TOBC,
nonparametric statistical methods were used for analysis. The Wilcoxon rank-sum test was used
to identify differences in the numerical data of the intervention group and the control
group, the Wilcoxon signed-rank test was used to identify changes in numerical data between
the baseline and post-intervention, and the Kruskal-Wallis test and a post hoc Tukey test
were used to identify differences in numerical data of three or more groups.
