Antibacterial Efficacy of 940 nm Diode Laser against Cariogenic Bacteria (Tooth Decay-causing Bacteria)

Dental caries is an extremely prevalent infectious disease caused by gram-positive bacteria mainly streptococcus mutans which is considered the major etiological factor causing dental caries. In recent years, there has been an enormous increase in the use of laser technology in medicine and dentistry and it has been demonstrated to have a considerable antibacterial action with no harm to the oral tissues. The study goal was to assess a diode laser's effectiveness as an antibacterial agent against the bacterium species S. mutans at various doses. The study was performed on streptococcus mutans microorganism at 106 CFU/ml concentration which received irradiation from a diode laser with 940 nm a central wavelength to investigate three output powers (1 watt, 2 watts, and 3 watts) for 30 s exposure time, and chlorohexidine (CHX) was used as a positive control group. Bacterial growth (CFU/ml) was calculated 24 hours after laser exposure. A significant diminish in CFUs/ml of S. mutans bacteria was observed 24 hours following irradiation by the three different powers. The result showed a statistical difference (p-value < 0.01) as compared to the negative control group without treatment. The current study demonstrated that the 940 nm diode laser was successful and efficient in the reduction of S. mutans growth at different doses.


Introduction
Streptococcus mutans, a gram-positive, facultative anaerobic cocci that is typically found in the mouth is also non-motile, non-spore-forming, and catalasenegative 1 .It is the main etiological factor of dental caries, which affects 95% of people of all ages worldwide 2 .There are many factors that enable S.mutans to cause dental caries, including adhesion to enamel surfaces, formation of acidic metabolites, the capability to accumulate glycogen reserves, and synthesis of extracellular polysaccharides 3 .Mutans streptococci create a lot of acids, which makes the environment acidic and increases the risk of cavities also it has the ability to survive in an acidic medium (greater tolerance to low pH) 1 .This acidic state results from sugar fermentation by cariogenic microorganisms that contribute to dental plaque buildup and the development of biofilms 4 .Dental caries is a multifactorial, chronic dynamic disease, that is mediated by biofilms and sugar and involves the presence of cariogenic bacteria 5 .Bacterial plaque, fermentable substrate (sugar), vulnerable tooth surfaces, and time, into which dental hard tissues undergo phasic demineralization by acid produced from food fermentation by bacteria 6 .It is

Abstract
Dental caries is an extremely prevalent infectious disease caused by gram-positive bacteria mainly streptococcus mutans which is considered the major etiological factor causing dental caries.In recent years, there has been an enormous increase in the use of laser technology in medicine and dentistry and it has been demonstrated to have a considerable antibacterial action with no harm to the oral tissues.The study goal was to assess a diode laser's effectiveness as an antibacterial agent against the bacterium species S. mutans at various doses.The study was performed on streptococcus mutans microorganism at 106 CFU/ml concentration which received irradiation from a diode laser with 940 nm a central wavelength to investigate three output powers (1 watt, 2 watts, and 3 watts) for 30 s exposure time, and chlorohexidine (CHX) was used as a positive control group.Bacterial growth (CFU/ml) was calculated 24 hours after laser exposure.A significant diminish in CFUs/ml of S. mutans bacteria was observed 24 hours following irradiation by the three different powers.The result showed a statistical difference (p-value < 0.01) as compared to the negative control group without treatment.The current study demonstrated that the 940 nm diode laser was successful and efficient in the reduction of S. mutans growth at different doses.becoming common practice among dentists all over the world to use antibacterial agents which can aid in lowering the prevalence of pathogenic bacteria 7 .
The majority of studies presented findings related to the use of cavity disinfectants like chlorhexidine, laser systems, and sodium hypochlorite (NaOCl).In some of the studies, additional disinfectant solutions were also assessed 8 .There are numerous techniques to lower the prevalence of dental caries, such as fluoride and chemical antibacterial agents, although they are not always highly efficient and may have negative side effects 9,10 or the bacteria may develop a resistance to this antibacterial agent, there has been an obvious increase in the percentage of bacterial pathogens that are resistant to numerous antibacterial agents 11 due to inappropriate utilization and widespread consumption of antibiotics 12 .As a result, various antibacterial methods have a good bactericidal effect and do not have any negative side effects are required such as lasers.Rapid advancements in laser technology (including wavelengths, methods, and delivery systems) have allowed for its usage in a wide range of fields, including medicine, physics, biology, biotechnology, biochemistry, and dentistry 13 .It has been demonstrated that a variety of lasers, including diode lasers, Er-YAG, and Nd-YAG, exhibit bactericidal properties 14 .Because of its low cost, portable, effective bactericidal action through its thermal effect, and temperature rise that is within a safe range for permanent teeth, the diode laser has gained popularity recently and is now commonplace in dental offices 15 .In addition, diode laser irradiation can reach into the dentinal tubules up to 1000 μm from the surface while the chemical disinfectants have a penetrating depth of only 100 μm, so this makes it potent for disinfection 16 .Also, the effectiveness of Er,Cr:YSGG lasers in terms of bacterial elimination has attracted a lot of study interest, as the bacteria's water molecules make them an ideal target for this laser, which causes bacterial destruction when energy absorption happens 17 .
Laser treatment is recognized as an effective method thanks to many benefits, including a reduction in the bacterial count.Diode lasers, in particular, have been demonstrated in studies to be successful at reducing bacterial counts and eliminating microorganisms through their photothermal effect, hence they are utilized for disinfection 18 .Also, the effectiveness of Er,Cr:YSGG lasers in terms of bacterial elimination has attracted a lot of study interest, as the bacteria's water molecules make them an ideal target for the Er,Cr,YSGG laser, which causes destruction when the laser energy is absorbed 19 .
Photothermal interaction is a reaction that occurs when a laser beam strikes a tissue and then the light energy is converted into heat through a thermal interaction with the cellular molecules.It develops several impacts on tissue like hyperthermia, coagulation, vaporization, carbonization, and finally melting 20 .
Theoretically, laser energy might kill bacteria directly by disrupting their cell membranes and killing them as a result of the production of reactive oxygen species brought on by the strong absorption of laser light 21 .It has been hypothesized that the inhibition of bacterial growth after exposure to laser light has resulted from the effect of free radicals on membrane lipids and DNA 22 .Free radicals are extremely reactive chemical substances that can interact with bacterial cell molecules including membrane lipids, proteins, and nucleic acids.This is going to damage the bacterial plasma membrane, massive vacuole development, leak cytoplasmic material, and complete cell distraction 23 .
The aim of this study is to evaluate the antibacterial effectiveness of a diode laser with 940 nm wavelength on the viability of S. mutans bacteria and compare this antibacterial effect (if ever found) with that of chlorohexidine.

Selection of Bacterial Species
As previously mentioned, dental caries is thought to be mainly caused by Streptococcus mutans (S. mutans) according to numerous epidemiological, experimental, and animal research 24 .This pathogenic bacterium promotes rapid carbohydrate digestion and produces an acidic environment in the oral cavity which results in tooth demineralization and caries 25 .This bacterium is a member of a group of acidogenic and aciduric Gram-positive bacterial species and is most prevalent in humans, comprising the specific microorganisms that have long been the subject of research into how caries begins and progresses 26   Group C: Irradiation of bacterial suspension by diode laser 940 nm 1 W output power, CW, 30 s exposure time.
Group D: Irradiation of bacterial suspension by diode laser 940 nm 2 W output power, CW, 30 s exposure time.
Group E: Irradiation of bacterial suspension by diode laser 940 nm 3 W output power, CW, 30 s exposure time.

Antibacterial Activity Determination
The study's objective is to assess the antibacterial properties, so the reduction in bacterial number https://doi.org/10.21123/bsj.2024.9041P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal following exposure to different antibacterial treatments was the main focus of this study and to check how effective the treatment is.After treating all the samples of bacteria with different treatment modalities, bacterial counting using CFU/ml was made by taking a part of the bacterial suspension and streaking it on a bacterial growth selective media (MSBA), cultured for 24 hours after being serially diluted by 3-dilution fold 10 -1 , 10 -2 , and 10 -

3
. By using the following equation, the number of CFU that were counted on the MSA plates was determined per milliliter of the initial sample: The number of CFU/ml = Number of CFU x Dilution factor 28,29 .

Results and discussion
For each group, the mean and standard deviation values were calculated, and the data were processed by a one-way analysis of variance test (ANOVA) to compare the means of different groups, Table 1.
Outcomes were presented as mean and SD, p values less than or equal to 0.05 denoting statistical insignificance while when it is greater or equal to 0.05, 0.01, and 0.001 denoting statistically significant differences.The statistical analysis was finished by using SPSS (v 20).The letters A, B, C, D, and E represent the various degrees of significance.The significant differences between the tested means were ascertained using the LSD test.Highly significant starting with the letter (A) and ending with the letter (E).The maximum reduction in the number of colonies forming unit of bacteria was achieved by group B, while the minimum reduction was obtained by group D as represented by Fig. 3.One of the primary goals of restorative treatment is to restore function and aesthetics while minimizing or completely eliminating bacteria in and around the filling to assure the treatment's success and decrease the risk of developing secondary caries and treatment failure.
The objective of the current investigation was to assess the effect of the 940 nm diode laser antibacterial property on the survival of the S. mutans microorganisms.The study's findings indicated that there was a decrease in the number of bacterial colonies and a statistical difference between the CFU of the bacteria before and after irradiation (high significance P value 0.001).It is worth mentioning that this type of laser therapy is considered a HLLT as the powers used are 1 watt, 2 watts, and 3 watts.
Diode lasers have been shown in numerous studies to be able to remove layers generated by bacteria and enhance the efficacy of tooth restoration 4 .The photo-disruptive and thermal effects of the diode laser's irradiation, which caused sub-lethal damage to the bacteria, can be linked to the antimicrobial action proposed when it was utilized 30 .If done properly, laser irradiation is known to cause bacterial cell wall disintegration, bacterial integrity disturbance, a buildup of denatured proteins, cell lysis, and ultimately microbial death.Additionally, the diode laser's near-infrared light can directly destroy bacteria that are pigmented and contain protoporphyrin IX, strengthening disinfection 31 .
Hendi et al study on the antibacterial effects of 940 nm diode laser on E. faecalis bacteria employed the laser parameters of 1 W output power and (45 s) exposure period of 15 s for three separate exposures.The results showed a reduction in bacterial colonies between the time they were exposed to 940 nm diode laser light and before exposure (P value 0.001), which agreed with the findings of the current investigation 32 .
Numerous studies were conducted to determine the effects of lasers on the growth of Streptococcus mutans.As an example, in a study by Robati et al 4 ., Streptococcus mutans and Lactobacillus bacteria at 10 8 CFU/ml concentration were irradiated by a diode laser source with a central wavelength of 980 nm in order to assess the effectiveness of the laser using different doses and times.The results revealed that the 980 nm diode laser is particularly successful at inhibiting the growth of the two types of bacteria at various times and doses 24 hours after the irradiation 4 and this is significantly related to the findings of the current study because the laser action was examined 24 hours after exposure.
In Castelo et al study 33 , E. faecalis was eliminated using a 940-nm diode laser, they reported a 70% rate of bacterial elimination by applying laser output was 3.5 watts, and in pulsed mode for 1 minute exposure time.While the current study, laser radiation of bacterial suspension was done in a continuous mode for 30 s irradiation time, so more bacterial colonies were eradicated than Castelo et al investigation, and this agreed with the results of our study as the outpower 1 watt has strongest bacterial killing than the powers 2 watts and 3 watts.
Chemical disinfection using chlorhexidine is one of the most often used disinfecting techniques in dentistry for preventing and reducing the growth of microbes, particularly S. mutans 34 .Given that it is a disinfectant that is readily available on the market, the standard 2% CHX was used in this study as a positive control.The study's findings showed that 2% CHX was effective against the S. mutans bacterium and had good antibacterial characteristics, as evidenced by a reduction in CFUs/ml that gave a statistically significant difference (P value 0.001) when compared to the control negative group.
The efficiency of the disinfectant is determined by the process of CHX's adherence to microbe cell walls, which causes intracellular component leakage.
At low concentrations, CHX's bacteriostatic activity results in the release of the microorganism's tiny molecular weight https://doi.org/10.21123/bsj.2024.9041P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal components; However, at higher concentrations, CHX causes cytoplasmic precipitation and/or coagulation, which is most likely caused by protein cross-linkage and exhibits the bactericidal effect 34 .
According to the Hassaballah et al study, which compared the effectiveness of grape seed extract, CHX, and laser diode as primary disinfectants for tooth cavities, the diode laser system is a more potent disinfectant for caries lesions than CHX and Grape Seed Extract 35 .In light of the fact that the diode laser group's results are better than those of the CHX group with a static difference (P value 0.001), there is ample agreement with the findings of our current investigation.

Conclusion
From this work results, it is concluded that the application of the diode laser is a successful and effective technology for the elimination of S. mutans bacterial colonies at different exposure powers and it can be used as a potent disinfectant in dental clinics by all dentists to help in reducing the incidence of secondary tooth caries by reducing the number of tooth decay bacteria.
. https://doi.org/10.21123/bsj.2024.9041P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal Samples Collection, Isolation & Identification of Mutans Streptococci Bacteria Plaque and saliva samples were collected from 30 patients with dental caries between the ages of 15 and 40 years of both sexes, non-smokers and without any systemic disease, who visited the dental clinics of the College of Dentistry/University of Baghdad.The samples were collected by swabs using sterile wet transport media and then it is transported by ice box to the Ministry of Science and Technology/Food Contamination Research Center Laboratories.100 microliters from the collected samples were cultured on the mitis salivarius bacitracin agar MSBA selective medium, which prevents the growth of all bacterium types except S. mutans, S. salivarius, and S. oralis as shown in Fig. 1.The isolated colonies from the sample culture on the MSBA plate were cultured in an anaerobic jar for 24 hours at 37-celsius degrees with 5-10% CO2.The isolated bacteria were identified by the conventional method which involved microscopic examination and biochemical tests and the final identification was done by polymerase chain reaction (PCR).

Figure 1 .Figure 2 .
Figure 1. S. mutans growth on MSBA after 24 hours at 37°C.Bacterial Samples PreparationStreptococcus mutans was the bacteria used in this study.It was cultivated in brain heart infusion broth (HIMEDIA, India) at 37°C for 24 hours with turbidity adjusted to 0.5 scale McFarland (1 ml of 0.5 McFarland usually contains 10 8 bacteria), and 10-fold dilution to 0.5 McFarland suspension was decided to make to achieve a concentration of 5x 10 6 bacteria in 1 ml for easy counting them.
Figure 3. Graphical representation of antibacterial activity among the tested groups.