molecules
Article
Evaluation of the Antimicrobial Effect of Thymoquinone Against Different Dental Pathogens : An In Vitro Study
Khalifa S. Al-Khalifa1 Rasha AlSheikh2, Moahmmed T. Al-Hariri 3, Hosam El-Sayyad \ Mäher S. Alqurashi \ Saqib Ali6 and Amr S. Bugshan6
Citation: Al-Khalifa KS, AlSheikh R, Al-Hariri MT, El-Sayyad H, Alqurashi MS, Saqib Ali S, Bugshan AS .   Evaluation of the antimicrobial effect of Thymoquinone against different dental pathogens: An in vitro Study. Molecules 2021, 26, x. https://doi.org/10.3390/xxxxx
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1 Department of Preventive Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, 6 Dammam, Saudi Arabia; kalkhalifa@iau.edu.sa 7
2 Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, 8 Dammam, Saudi Arabia; ralsheikh@iau.edu.sa 9
3 Department of Physiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, 10 Saudi Arabia; mtalhariri@iau.edu.sa 11
4 Middle Eastern Regional Radioisotope Centre for the Arab Countries, Dokki, Giza, Egypt; hosealsay- 12 yad@gmail.com 13
5 Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, 14 Saudi Arabia; msalqurashi@iau.edu.sa 15
6 Department of Biomedical Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, 16 Dammam, Saudi Arabia; samali@iau.edu.sa; abugshan@iau.edu.sa 17
* Correspondence: kalkhalifa@iau.edu.sa 18
Abstract: This study aimed to evaluate the antimicrobial effect of Thymoquinone (TQ) on four dif- 19
ferent oral microorganisms. Minimum Bactericidal Concentration (MBC), Minimum Inhibition 20
Concentration (MIC), Broth microdilution, and Well diffusion tests were used to determine the op- 21
timum antimicrobial concentrations of TQ against Streptococcus Salivarius, Oralis, Mutans, and Staph- 22
ylococcus aureus over 1, 3, 6, 12, and 24 h. Chlorhexidine (CHX) 0.12% was selected as a positive 23
control. The inhibitory effect of TQ on bacterial growth was most noticeable with Streptococcus Sal- 24
ivarius while the least affected was Staphylococcus Aureus. TQ's MBC and MIC for Streptococcus Oralis 25
and Staphylococcus Aureus were comparable 2 mg/ml and 3 mg/ml, respectively. Streptococcus Soli- 26
varius was most resistant to TQ and displayed a value of 5 mg/ml and 4 mg/ml for MIC and MBC, 27
respectively. The viable count of different strains after exposure to TQ's MBC values was most no- 28
ticeable with Staphylococcus aureus followed by Streptococcus Oralis and Streptococcus Mutans, while 29
Streptococcus Salivarius was least affected. This study emphasized the promising antimicrobial effect 30
of TQ against the four main oral microorganisms. It has a potential preventive effect against dental 31
caries as well as other oral diseases. 32
Keywords: Black seeds; Thymoquinone; Dental caries; anti-bacterial agents; herbs 33
34
1. Introduction 35
The oral cavity is inhabited by hundreds of microorganisms of different species 36
mainly bacteria forming the oral biofilm. The formation of the biofilm and the type of 37
microorganisms are influenced by numerous host and environmental factors. Biofilm for- 38
mation in the oral cavity can cause multiple different diseases depending on the promi- 39
nent microorganisms such as caries, root canal infections, periodontal diseases, and peri- 40
implant diseases, and other gastric intestinal track-related diseases [(1-3)]. 41
Dental caries is an infectious disease that starts with a bacteria-colonized dental 42
plaque. Dental plaque is a sticky, highly hydrated extracellular biofilm that forms on the 43
tooth surface then later is colonized by cariogenic bacteria that ferment dietary carbohy- 44
drate-producing acid. The acid then dissolves tooth mineral content causing cavitation, 45
known as dental caries [(4)]. It is a multifactorial disease that represents a significant 46
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public health challenge worldwide [(5)]. Despite all the advancements in dental education 47
and oral hygiene awareness, dental caries remain the leading health challenge in most 48
developed countries where 60 - 90% of children and most adults suffer from dental caries. 49
On the contrary, developing countries have caries prevalence reported to reach as high as 50
68% [(6)]. Latest global reports noted that untreated dental caries is still a common disease 51
worldwide [(6)]. Recent studies in different countries around the world indicated an in- 52
crease in caries prevalence [(7, 8)]. Caries lesions can be asymptomatic in their initial 53
stages. In contrast, advanced caries lesions can cause toothache, which adversely affects 54
life quality by affecting the mastication efficiency, patient smile, and self-consciousness. 55
Children suffering from caries suffer relatively from poor nutritional health, poor child 56
growth, and low weight gain [(9)]. 57
Although several different bacteria have been associated with dental caries' patho- 58
genesis, Streptococcus Mutans (SM) is the most relevant acidogenic-aciduric bacterial spe- 59
cies. It plays the primary role in initiating dental caries lesions. [(10,11)] Other cariogenic 60
bacteria are responsible for the sustainability and progression of the caries lesion. Studies 61
investigating the prevalence of cariogenic bacteria and other bacterial microorganisms in 62
caries lesions revealed a high prevalence of SM, Streptococcus Salivarius (SS), and Strepto- 63
coccus Oralis (SO) in samples collected from active supra gingival caries lesions [(2, 3)]. 64
Staphylococcus aureus (SA) is associated with dental implant infection and has established 65
a high tolerance to common antimicrobial treatments [(1)]. 66
The use of herbs and plant extract by humans as medicine is an ancient practice that 67
goes back thousands of years. Lately, medicinal plants have been experiencing a growing 68
popularity and interest due to the public concern with the adverse effect of synthetic drugs 69
[(12,13)]. Plant extract medicine is being widely investigated and proven to have exceeded 70
expectations in preventing and treating a wide array of diseases and medical conditions 71
[(14,15)]. 72
Black cumin (Nigella sativa) belonging to the Ranunculaceae family has been used 73
historically by various cultural traditional medicinal treatments. Traditionally has been 74
used to treat various diseases such as headaches, influenza, dyspepsia, diabetes, and 75
asthma [(16)]. Black seeds extract has been confirmed to improve oral health and reduce 76
dental caries, [(17) Jperiodontitis, gingivitis, and pulp diseases [(18) ]. 77
Black seeds extract includes essentials oils, fixed oils, alkaloids, saponins, and pro- 78
teins. Thymoquinone (TQ) is the core ingredient of the black seed oil extract with proven 79
medical benefits [(19)]. Literature suggested a significant decrease in the count of Strepto- 80
coccus Mutans when exposed to TQ [(17)]. 81
Most published studies focused on the antibacterial effect of TQ against a single 82
strain of bacteria or by using one method of evaluation. There have not been any extensive 83
studies exploring the antibacterial effect of TQ on several bacterial strains. Hence, this 84
study was aiming to evaluate the antimicrobial effect of the TQ (concentration and expo- 85
sure duration) on four different oral microorganisms. The tested hypothesis states that 86
there is no difference in bacterial growth when using the TQ solution's different concen- 87
trations with the control groups (negative and positive). 88
2. Materials and Methods 89
Thymoquinone (TQ) preparation 90
Dry powder extract of TQ (Sigma-Aldrich) and TQ oil was obtained and used in the 91
experiments. Five hundred mg of TQ was dissolved in 1 ml of methanol. The tested com- 92
pounds were diluted in brain heart infusion broth (BHIB) to prepare seven different con- 93
centrations (0.2, 0.5, 1.0,1.5, 2.0, 3.0, and 4.0 mg/ml) and two control test tubes, one nega- 94
five control (1ml brain-heart fusion broth without TQ) and one positive -control (1 ml 95
(0.12%) Chlorohexidine; Avohex, Middle East Pharmaceutical Industries Ltd, Riyadh, 96
Saudi Arabia).(20, 21) 97
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Microorganism 98
Four Gram-positive strains were used in this research Streptococcus Mutans (SM) 99
(ATCC 25175), Staphylococcus aureus (SA) (ATCC 25923), Streptococcus Oralis (SO) (ATCC loo
6249), and Streptococcus Salivarius (SS) (ATCC 13419), obtained from Microbiologics, St. 101
Cloud, Minnesota, USA and cultivated on Mueller-Hinton Agar with 5% Sheep Blood 102
(HiMedia Laboratories Pvt. Ltd., India) for 48 h at 37 °C, with 5% C02. After this period, 103
bacteria colonies were picked up from the new culture and suspended in 2 ml of sterile 104
distilled water, then serial diluted (fivefold) from 1:10 to 1:105. 105
Antibacterial activity assay of Thymoquinone (TQ) against tested organisms 106
This study used seven different concentrations of TQ where each test tube containing 107
1 ml of sterile BHIB with different concentrations of TQ separately (0.2 mg/ml, 0.5 mg/ml, 108
1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, and 4.0 mg/ml) and 0.1 ml of tested organ- 109
isms. Furthermore, 0.1 ml of tested organisms were mixed with 0.9 ml sterile BHIB as a 110
negative control. The positive control was 0.1 ml of tested organisms added to 0.9 ml chlo- ill
rohexidine. Then 250 |jl from each concentration was cultured on Mueller-Hinton Agar 112
with 5% Sheep Blood and then incubated for 48 hours at 37°C in a microaerophilic condi- 113
tion (10% CO2) candle gar method for SM, SO, and SS and with aerophilic conditions for 114
SA Triplicate of the TQ as well as the control solution were performed in this study. 115
Minimum Inhibitory Concentration (MIC) test 116
After 24 hours of incubation, the number of colonies forming units per millimeter 117
was determined for each concentration to detect the minimum inhibitory concentration 118
(MIC) by the lowest TQ concentration that inhibits bacteria growth [(22)]. 119
Minimum Bactericidal Concentration (MBC) test 120
From all MIC test tubes that showed no growth, 250 |jl of culture was inoculated into 121
fresh Mueller-Hinton Agar with 5% Sheep Blood plates. After incubation for 48 h under 122
suitable conditions according to the tested microorganism, as previously described. The 123
minimum bactericidal concentration (MBC) was determined by observing bacterial 124
growth. The concentration of antimicrobial agents that eliminate more than 99.9% after 24 125
h of incubation was recorded as MBC. 126
The following equation calculated the reduction of bacterial count percentage for 127
each concentration: 128
C control - C sample
Reduction of bacterial count % = _ * 100
C control
After 48 hours of incubation, the colonies were counted. 129
The bacterial count of MBC values during different periods 130
The MBC value of each tested organism for TQ was inculcated with bacterial inocu- 131
lums (1.7* 105 CFU/ ml) in sterile Eppendorf containing 1 ml of BHIB. Also, tested organ- 132
isms were mixed with one ml of positive control (Chlorhexidine). All tested groups and 133
control were incubated for 24 h at 37 °C. Then, 0.1 ml of each group was assessed periodi- 134
cally after 1,3,6,12 and 24 hours from inoculum by preparing serial dilutions in sterile sa- 135
line solution (0.9%). After that, 250 |jl from these dilutions was added and spread on 136
Mueller-Hinton Agar with 5% Sheep Blood in duplicate and incubated under suitable 137
conditions according to the tested microorganism, as previously described. 138
139
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Well diffusion test 140
The well diffusion method was performed by using blood Muller- Hinton agar 141
(BMHA) [(23)]. The BMHA plate surface was inoculated for a defined volume of all test 142
isolates using a sterile cotton swab over the entire plate surface. A hole (6 mm in diameter) 143
was cut in the agar gel, and then 100 |jL of five various concentrations of TQ (12.5, 25, 50, 144
75, and 100 mg / mL) were added to each well, separately. Chlorohexidine was used as a 145
positive control.  Then all plates were incubated under suitable conditions according to 146
the tested microorganism for 24 h. All experiments were performed in duplicates for each 147
antimicrobial agent. After the incubation period, bacterial growth inhibition (the endpoint 148
of the clear zone diameter) was measured (mm). 149
Statistical analysis 150
The obtained CFU data were pooled for the different time points and processed with 151
LoglO transformation. The comparisons between test (TQ) and control [CHX] were per- 152
formed, for each bacterial strain, using an independent t-test. The statistical analysis was 153
performed in IBM SPSS® Statistic software (Version 25). The CFU results are provided in 154
dot-plots, showing the individual measurements and the mean and the standard error of 155
the mean.   The microbial inhibition zone results and the minimum inhibitory and bacte- 156
ricidal concentrations are provided descriptively in tables. 157
3. Results 158
The antimicrobial potential of TQ at different concentrations compared to compli- 159
mentary control CHX mouth wash is shown in Table 1. The findings indicated that the 160
microbial activity of TQ was concentration-dependent. Interestingly, there was no bacte- 161
rial growth for TQ concentrations of 75 and 100 mg/ml.   The inhibitory effect of TQ on 162
bacterial growth was most noticeable with Streptococcus Salivarius followed by Streptococ- 163
cus Mutans and Streptococcus Oralis, as the least affected was Staphylococcus Aureus. 164
165
Figure 1. Bacterial growth; MIC (Minimal Inhibitory Concentrate) & MBC (Minimal Bacteriocidal Concentrate) of TQ with 166
the four bacterial strains. 167
To ensure TQ's antibacterial effect, colonies of the four bacterial strains were counted 168
after applying the same TQ doses using cell culture counts to estimate the MIC and MBC 169
(Figure 1).    TQ's minimum concentration required for inhibition (MIC) or killing (MBC) 170
of Streptococcus Oralis and Staphylococcus Aureus was comparable 2 mg/ml and 3 mg/ml, 171
respectively, as they were the most sensitive to TQ. On the contrary, Streptococcus Salivar- 172
ius showed the highest resistance to TQ with the values of 5 mg/ml and 4 mg/ml for MBC 173
and MIC, respectively. TQ's minimum concentration required for inhibition (MIC) and 174
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killing (MBC) of Streptococcus Mutans was 3 mg/ml and 4 mg/ml, respectively. On the 175
other hand, CHX exhibiting better antibacterial activity when compared to TQ; presented 176
MBC > 0.0009 mg/mL (>0.00009%), and MIC > 0.00002 mg/mL (>0.000002%) for the micro- 177
organisms tested (Table 2). 178
The viable count of different strains after exposure to TQ's MBC values was most 179
noticeable with Staphylococcus Aureus followed by Streptococcus Oralis and Streptococcus 180
Mutans least affected was Streptococcus Salivarius. Significant drop overtime in CFU counts 181
was noted, except at 24 h, where no difference was detected (Table 3). The LoglO transfer- 182
mation results showed only a statistically significant difference between TQ and CHX 183
with the Staphylococcus Aureus bacterial strain. The colony-forming units (CFU) of the dif- 184
ferent bacterial strains after exposure to the minimal bactericidal doses of TQ and the cor- 185
responding doses of the control Chlorhexidine (CHX) for each bacterial strain. The dot- 186
plot shows the distribution of the individual measurements and the mean (horizontal line) 187
and the standard error of the mean (capped vertical line). A statistically significant differ- 188
ence (p<0.05) is indicated with bars and asterisk. (Figure 2) 189
Table 1. Microbial inhibition zone in millimeter provided by TQ at different concentrations compared to a positive control (CHX).
Inhibition Zone (mm)				
_            . Streptococcus Concentration ,. fma'mn SallVOriUS	Streptococcus Mutans		Streptococcus Staphylococcus Oralis Aureus	
5 Mean&StD		Mean&StD	Mean&StD Mean&StD	
12.5 37	29.5		41.5	36.5
25 38.5	37.5		44	44
50 38.5	44		44	44
75 44	44		44	44
100 44	44		44	44
CHX (+ve control) 11.5	17.5		21.5	12
Note: The plate diameter is 88 mm (half diameter =44), which designates no bacteria growth.				
Table 2. Minimum Inhibitory and Bactericidal Concentration (mg/ml) of TQ and positive control CHX against the four bacterial strains.				
Microorganisms		TQ		CHX
		MIC MBC		MIC MBC
Streptococcus Salivarius		4.0	5.0	0.00857 0.0171
Streptococcus Mutans		3.0	4.0	<0.00002 0.0009
Streptococcus Oralis		2.0	3.0	0.00095 0.0038
Staphylococcus Aureus		2.0	3.0	0.00857 0.0171
Table 3. The viable count of different strains after exposure to the MBC values of TQ and positive control (CHX) during different observation periods.				
190
191
192
193
194
Time
Streptococcus Salivarius
Streptococcus Mutans
Streptococcus Oralis Staphylococcus
(hours)	TQ 5 mg/ml	CHX	TQ 4 mg/ml	CHX	TQ 3 mg/ml	CHX	TQ 3 mg/ml	CHX
lh	0.38 xlO3	0.54 xlO3	1.8 xlO4	1.0 xlO2	0.71 xlO3	0.45 xlO3	0.31xl03	0.21 xlO4
3h	0.11 xlO3	0.40 xlO3	1.2 xlO4	NG	0.34 xlO2	0.28 xlO3	0.28 xlO2	0.80 xlO3
6h	NG	0.20 xlO3	4.9 xlO3	NG	0.11 xlO2	0.14 xlO3	0.22 xlO2	0.48 xlO3
12 h	NG	0.48 xlO2	NG	NG	NG	0.80 xlO2	0.14 xlO2	0.13 xlO3
24 h	NG	0.28 xlO2	NG	NG	NG	NG	NG	0.74 102
195
196
*NG; negative.
197
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m 2-
Streptococcus mutans
o o
-e-e*e-e-
o o
CHX TQ Streptococcus ora//s
o
o
o °
O
o
o
o
4n
LL
o
§ 2-o
Streptococcus sativarius
o o
c
o o
-0-Q-&-
CHX TQ Staphylococcus aureus •k
O o
CHX
O O TQ
CHX
TQ
Figure 2. Viable bacterial count using LoglO transformation CHX (chlorohexidine) TQ (Thimoqui-non).
198
199
200
4. Discussion 201
Dental caries persist to be a worldwide health problem despite clinical and medical 202
advancement. The cariogenic bacteria colonize the dental plaque to induce the disease; 203
different strategies to inhibit the bacterial growth and prevent colonization have been in- 204
vestigated and later implemented to control dental caries. Cariogenic bacteria have been 205
linked to several systemic diseases, such as infective endocarditis, ulcerative colitis, peri- 206
tonitis, and atherosclerosis. (24, 25) Recently, natural products have been experimentally 207
investigated to inhibit the growth of dental plaque microorganisms.(12) in addition to the 208
public interest in organic natural products the such as ease of extraction and large-scale 209
production render such products more alluring to manufacturers.(26) 210
The medicinal uses of the Nigella sativa have been well documented over the years. 211
Its active ingredient available literature has proven the effectiveness of TQ, has against 212
various pathologies such as cancer, inflammation, allergies, and microbial.(27-30) 213
Minimal inhibitory concentrate (MIC) and the minimal bactericidal concentration 214
(MBC) are considered the golden standards to evaluate the antimicrobial effect of an agent 215
against microorganisms and such is used to judge other methods to evaluate antimicrobi- 216
als.(31) 217
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In the current study, TQ displayed promising results in inhibiting the growth of oral 218
bacterial strains. The null hypothesis was rejected as the TQ was most potent against SO, 219
SM showed MIC of 3mg/ml and MBC of 4mg/ml whereas, SS showed most resistance with 220
MIC of 4 mg/ml and MBC 5 mg/ml of TQ. All four species showed the same resistance 221
pattern when using the control chlorohexidine CHX, where both SA & SO showed the 222
least resistance and SS the most resistance.  SM is one of the main bacteria that initiate 223
dental caries. In previous studies, Streptococcus Salivarius (SS) presence in a high number 224
in active carious lesions helps sustain and the progression of carious lesions. (12-14, 25) 225
Our results are in line with other studies showing bactericidal as well as growth inhibition 226
against SM. (32) SO is also one of the bacteria found in the active carious lesion. They are 227
also considered to take part in the progression and the sustainability of the carious pro- 228
cess. (15,16, 27). These results were consistent with the study on different bacteria strains 229
showing Streptococcus Salivarius, Mutans, and Aureus sensitivity to TQ, with MIB and 230
MIC values being significant. (33, 34) 231
Another interesting finding of this study was that there was no microbial activ- 232
ity/growth when TQ concentrations were 75 and 100 mg/ml. previous studies have stated 233
that TQ can be toxic at a certain concentrate that starts from 92%-106.6%; the concentrate 234
at which showed no microbial activity is still below the toxic concentrate. (34)It can be 235
reported that the microbial activity of TQ is concentration-dependent. SA & SO showed 236
maximum inhibition at TQ concentration of 25 mg/ml, followed by SM at 50 mg/ml, then 237
SS at 75 mg/ml. A previous study has shown dose-dependent antibacterial activity, which 238
is in line with our results. It also reported it better against Gram-positive in comparison 239
with gram-negative bacteria. Staphylococcus Aureus exhibit the highest sensitivity to TQ 240
amongst the different gram-positive and gram-negative species tested. (35) Staphylococcus 241
Aureus is one of the commonest pathogens which is faced in clinical settings. Besides this, 242
Staphylococcus Aureus is found in infections related to dental implants, and it is of utmost 243
importance as infection can decide the faith of success of the dental implant. Our result is 244
in line with the previous results showing antibacterial activity, minimum inhibitory effect, 245
and being more tolerant than the rest of the three (SM, SO, SS) bacterial strains. (36, 37) 246
While the Staphylococcus Aureus has shown resistance against common antimicrobial treat- 247
ments, increase the dose of TQ has been effective in reducing the bacterial count and 248
growth; TQ could be the answer to overcoming this strand's high resistance. (17,18, 28). 249
To further validate the study findings against the standard laboratory testing, a well 250
diffusion test was carried. TQ inhibition effect is not only concentration-dependent but 251
also time-dependent, for SS, SO as well as SA the TQ proved to be faster than the CHX 252
control in stopping the growth of the bacteria. While the SS & SA were still present after 253
24h when using the CHX with the TQ SS was eliminated after 6h and SA after 12h. Other 254
studies reflected similar findings; Mouwakeh et al. stated they had more potent inhibitory 255
activity aver time against the SO.(38) 256
Such finding suggests that the use of TQ in low concentration can be effective against 257
caries progression as well as peri-implantitis; which is the severe inflammation around 258
dental implants. While higher concentrations can be beneficial to caries initiation preven- 259
tion. While the CHX can be providing the same benefits as TQ, the latter is showed a 260
promising advantage over CHX being effective in a short period. 261
Other studies have focused on minimal inhibitory concentration (MIC) or minimal 262
bactericidal concentration (MBC) (32, 33), others on bacterial inhibition zone (35, 39), or 263
limit the study to one or two bacterial strains. This study's strength is that it covered a 264
more comprehensive selection of cariogenic bacteria, caries initiation, and caries sustain- 265
ing and progression, enhancing microorganisms. Simultaneously, covering the MIC, 266
MBC, and the inhibitory zone are viable tests in studying experimental agents' antibacte- 267
rial activities. 268
Other essential oils have been investigated and proven effective in dental plaque con- 269
trol and reducing the count of cariogenic microorganisms. Filogonio et al. added mineral 270
Brazilian nut oil to the commercially available dentifrice. He stated that after 90 days, the 271
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tested oils were sufficient to improve dental biofilm control significantly. (40) Lobo et al. 272
compared the effectiveness of Lippia sidoides Cham essential oil (LSO) to Chlorhexidine 273
in reducing the salivary Streptococcus Mutans in children with caries. In contrast, CHX 274
reduces SM after using the mouthwash, but the SM count returns to baseline levels shortly 275
after treatment. While using the LSO, the SM count was reduced and remained low even 276
after treatment.(41) Charugundla et al. investigated the essential oil in reducing and den- 277
tal plaque compared to CHX and Fluoride mouth rinse; all three mouth rinses signifi- 278
cantly reduced plaque accumulation and gingivitis, particularly in caries-free subjects 279
against those with caries. (42) 280
This study evaluated TQ's antibacterial effect against four potent bacteria essential to 281
initiate and sustain caries lesions. However, dental caries start with the dental plaque, 282
which exists in the oral cavity in a biofilm. Further clinical studies to evaluate the effect 283
against other biofilm bacteria and in vitro evaluation of the TQ antibacterial potential are 284
recommended. Further studies with different TQ concentrations and intervals, combined 285
with other essential oils that can modify or boost its effect, can be of great benefit to ex- 286
plore TQ's therapeutic and preventive role. 287
288
5. Conclusions 289
This study emphasized the promising cariogenic antimicrobial effect of TQ against 290
the four main oral microorganisms, not only that the TQ exhibits antimicrobial effect com- 291
parable to the standard antimicrobial medicine it shows a longer duration effect. It has a 292
potential therapeutic and preventive effect against dental caries as well as other oral dis- 293
eases. A significant highlight is eliminating chemically synthesized drugs and the grow- 294
ing concerns of bacterial resistance and medical side effects. 295
Author Contributions: All authors contributed equally to the work and have read and agreed to 296
the published version of the manuscript. 297
Funding: This research received no external funding. 298
Institutional Review Board Statement: Not applicable. 299
Informed Consent Statement: Not applicable. 300
Acknowledgments: None. 301
Conflicts of Interest: The authors declare no conflict of interest. 302
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