Journal of Current Biomedical Reports jcbior.com Volume 3, Number 4, 2022 eISSN: 2717-1906 1 Original research Evaluation of the antibacterial effect of nickel oxide nanoparticles against bacteria involved in dental caries Chanour Tavakol1, Sara Nasiri2, Elahe Keshavarz3, Hossein Ghafori4, Hirad Rokni5, Meysam Hasannejad6, Milad Jafari Sayadi7, Fereshte Naser Alavi7, Hasan Pourmoshtagh8,* 1Student Research Committee, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2Heshmat Hospital, Guilan University of Medical Sciences, Rasht, Iran 3Department of Sciences, Farhangian University, Rasht, Iran 4Department of Microbiology, Faculty of Biological Sciences, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran 5School of Dentistry, Islamic Azad University of Medical Sciences, Tehran, Iran 6Department of Microbiology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran 7Razi Clinical Research Development Unit, Razi Hospital, Guilan University of Medical Sciences, Rasht, Iran 8Department of Pediatrics, Loghman-Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran Abstract Tooth decay is one of the most common diseases in the oral cavity and is one of the most widespread diseases in the human population. This study aimed to determine the antibacterial effect of nickel oxide nanoparticles against bacteria involved in tooth decay. In this study, the disk diffusion method was used to determine the antibiotic susceptibility and the microdilution broth method was used to determine the minimum inhibitory concentration (MIC). Nanoparticles were also synthesized in two molecular size (A: 8.1 and B: 12 nm) by the sol-gel method. The MIC of the first nanoparticle for Streptococcus sanguinis and Streptococcus mutans was 31.25 and 125 μg/ml, respectively. The MIC of the second nanoparticle for S. sanguinis was 125 μg/ml. In the case of S. mutans up to a concentration of 500 μg/ml, no growth inhibition was observed. The results showed that nickel oxide nanoparticles have acceptable antibacterial properties against S. mutans and S. sanguinis, which can be used in dental materials to prevent dental caries. However, this requires the determination of cellular toxicity and its side effects in future studies. Keywords: Nanoparticles, Nickel oxide, Streptococcus sanguinis, Streptococcus mutans 1. Introduction Tooth decay is one of the most common diseases in the oral cavity and is one of the most widespread diseases in the human population. About half of people 6 to 19 years old who are economically disadvantaged have decayed teeth [1]. Tooth decay is also a problem for adults, with more than 90% of people over the age of 40 having it. A quarter of people over the age of 60 have lost all their teeth permanently *Corresponding author: Hasan Pourmoshtagh, MD Department of Pediatrics, Loghman-Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran Tel/Fax: +98 912 6402128 Email: hpourmoshtagh@gmail.com http://orcid.org/0000-0001-8478-8226 Received: April, 30, 2022 Accepted: July, 30, 2022 due to the impact of caries on their self-esteem and involvement in nutritional problems [2]. Bacteria are the main cause of tooth decay. The main mechanism of bacteria in causing tooth decay is the formation of plaque on tooth enamel. Plaque is actually a kind of biofilm that is made up of one or more types of microorganisms that can grow at different levels. The formation of dental biofilm is not necessarily a sign of © The Author(s) 2022 https://jcbior.com/ Tavakol et al. 2 disease, but the formation of biofilm is an important mechanism of disease for bacteria [3]. Streptococcus mutans is a Gram-positive and aerobic cocci in the oral cavity flora in humans. This bacterium is the most important cause of tooth decay. S. mutans damages tooth enamel by fermenting sucrose and producing lactic acid. The bacterium also uses sucrose to make dental plaque. S. mutans is known to produce lactic acid as part of its metabolism [4, 5]. Unlike most edible microorganisms, S. mutans grows in low acid conditions and becomes the dominant bacterium in cultures with a permanent decrease in pH. In addition, contrary to the characteristics of plaque, whose metabolism is significantly slowed down at such a pH, the metabolism of S. mutans is actually improved [4, 6]. Streptococcus sanguinis is a Gram-positive, aerobic cocci and normal oral flora and is found in dental plaque. S. sanguinis is widely present in the oral cavity [7-9]. Studies show that S. sanguinis has two genes which produces glucan. Glucan is also formed in the presence of glucose and various forms of sucrose. Glucan is the major biofilm polymer in S. sanguinis and increases the adhesion of bacteria to hydroxyapatite, promoting biofilm development [10, 11]. When particles are prepared in nanoscale (less than 100 nm) they can react with organic and inorganic molecules more easily due to the increase in surface area [12]. The main advantages of hydroxyapatite (HA) nanoparticles are their similarity to tooth mineral structure, biological activity and biocompatibility [13, 14]. In one design, the effect of hydroxyapatite magnesium carbonate nanopowder was used to prevent and repair primary caries lesions and the results showed that MgHa nanoparticles are adsorbed on the enamel surface and by changing the mechanical properties of the tooth, a new layer is formed in Enamel surface and filling all cavities improves tooth remineralization [15]. Nickel oxide is of interest due to the chemical and magnetic properties of an oxide. Nickel oxide nanoparticles are widely used. These nanoparticles are also used as an effective catalyst for the oxidation of many organic compounds. Nickel oxide nanoparticles as an antibacterial agent have also been effective on some bacteria. Numerous methods for the preparation of nickel oxide nanoparticles are known. The size distribution and morphology of nanoparticles depend on the way they are synthesized. Among the nanoparticle synthesis methods, we can mention the most common methods called sol-gel method [16]. Removing plaque from teeth reduces the risk of problems such as tooth decay, gum disease and bad breath. Since aerobic and anaerobic bacteria are an important part of dental plaque, new substances with antibacterial properties are essential to prevent their growth. Metal nanoparticles show significant antibacterial activity. This property is due to the very small size and surface to volume ratio of these particles. In this regard and the lack of similar studies, we aimed to investigate the antibacterial effect of nickel oxide nanoparticles against bacteria involved in tooth decay. 2. Materials and Methods 2.1 Bacterial strains The studied strains were standard ATCC of S. mutans, and S. sanguinis which was lyophilized from the Center for Genetic and Biological Resources of Iran. To prepare the strain for the experiment, the stored strain was first inoculated in 5 ml of tryptone soy broth (Merck, Germany) and incubated for 24 hours at 37 °C. Then, a fresh culture of a bacterial suspension equal to half McFarland standard was prepared in Müller-Hinton broth medium and used for further experiments. 2.2 Synthesis of nickel oxide nanoparticles At first, 100 ml of 2 M ammonium hydroxide solution dropwise to 50 ml of 0.5 M solution of Ni (NO3) 2.6H2O stirred by magnetic magnet for 4 h at 100 °C. C was added. The solution was kept at room temperature for 24 hours. The prepared light green suspension was centrifuged and then the dry precipitate was calcined at 300 and 700 °C (17) [17]. The structure and morphology of the synthesized nanoparticles were identified by FTIR and TEM techniques. The size of synthetic nanoparticles was estimated to be about 5 to 15 nanometers with this technique. The synthesized nanoparticle powder was first weighed by a sensitive scale on sterile aluminum foil. Then pour one gram of nanoparticles in a sterile tube 16 x 20 mm screw cap, and add 10 ml of sterile distilled water. After vortexing, we mixed it well in the sonicator and at room temperature for 10 minutes. Then the desired serial dilutions were prepared. All Tavakol et al. 3 steps were performed in full compliance with sterile conditions. 2.3 Determination of antibiotic susceptibility In order to determine the antibacterial susceptibility, well diffusion method on Muller- Hinton agar (MHA; Merck, Germany) described by Nanda et al. were applied. In each well 25 μl of nanoparticles in desired concentrations (500, 250, 125, and 62.5 µg/ml) was added [18]. Finally, the plates were transferred to an incubator at 35-37 °C and the results were evaluated after 16-18 hours. Finally, the results were reported based on the measurement of the diameter of the growth inhibition zone by a millimeter ruler. Determination of minimum inhibitory concentration (MIC) was performed by standard broth microdilution based on the clinical and laboratory standards institute (CLSI) recommendation [19]. First, 96-well microplates different concentrations of nanoparticles (500-15.625 µg/mL) were provided using Müller-Hinton broth (Merck, Germeny) medium containing 5% Sheep blood agar. After providing the required concentrations, 10 μl of the diluted microbial suspension was added to each well (according to CLSI method), the optimum concentration of bacteria for inoculation into the wells is 106 CFU/ ml. Following 24 h incubation at 37 °C, the wells were inspected for microbial growth, and the MIC results were recorded based on the lowest concentration that did not produce visual growth. 2.4 Statistical analysis Analysis was performed by using SPSSTM software, version 22.0 (IBM Corp., USA). The results are presented as descriptive statistics in terms of relative frequency. 3. Results Morphological studies determined the size of calcifying nanoparticles at 300 °C (NP1) and 400 °C (NP2) were 8.1 and 12 nm, respectively. As the calcination temperature decreases, the size of the nanoparticles decreases, and as the size decreases, the surface-to-volume ratio increases. According to the Table 1, the growth inhibition zone for the concentration of 500 to 62.5 µg/ml of nanoparticles for S. sanguinis varied from 12 mm to 17 mm. For S. mutans, the minimum zone diameter was 10 mm at the 250 µg/ml of nanoparticles and the maximum zone diameter was 14 mm at both 125 and 62.5 µg/ml of nanoparticles. Based on Table 2, the amount of the first MIC nanoparticle for S. sanguinis and S. mutans was 31.25 and 125 μg/ml, respectively. 4. Discussion According to our results, it was illustrated that S. sanguinis represented a higher antibacterial effects than S. mutans. Numerous studies have been performed on the effects of nanocomposites on bacteria that cause tooth decay. Similar to our study, a study by Khodaee et al., the antibacterial and anti- biofilm effects of silver nanoparticles against dental plaque microorganisms, S. mutans and Agrigatebacter Actinomycetemcomitans represented that the active ingredient in silver nanoparticles showed a greater antibacterial effect than nickel oxide nanoparticles [20]. In the study of Emrani et al., the results showed that the MIC for biosynthesized nanoparticles of licorice extract against S. mutans, Actinomyces viscosus and Lactobacillus rhamnosus bacteria were 1.56, 6.25, and 50 µg/ml, respectively (P ≤0.05), and the MIC for nanoparticles biosynthesized with peppermint extract was 12.5, 12.5, and 200 µg/ml (P ≤0.05), respectively [21]. Both expression and mint showed greater antibacterial effect than nickel oxide nanoparticles. In another study conducted by Vahid Dastjerdi et al., it was found that the largest diameter of the growth inhibition disk in the disk diffusion method for S. mutans, S. sanguinis, Streptococcus salivarius, Streptococcus sobrinus and Enterococcus faecalis at a concentration of 100 micrograms per milliliter and equal to 14, 18, 15, 18, and 10.5 µg/ml, respectively. The MIC values for S. mutans, S. sanguinis, S. salivarius, S. sobrinus and E. faecalis were 50, 25, 6.25, 25, 25, and 50 µg/ml, respectively [22]. Similar to the previous study on nanoparticles biosynthesized with licorice and peppermint, in this study the results showed that the aqueous extract of pomegranate flower showed slightly more antibacterial properties than nickel oxide nanoparticles, and perhaps this hypothesis suggests that the use of biological compounds along with nanoparticles increases their effectiveness, which of course requires further studies to prove this. Tavakol et al. 4 In another study conducted by Sadeghi et al., the MIC of silver and chlorhexidine nanoparticles in relation to S. sanguinis was 25 and 16 µg/ml, respectively, and for Actinomyces viscosus was 4 and 64 µg/ml, respectively. Comparing the results of this study with the present study, both types of nickel oxide nanoparticles showed more antibacterial effect than chlorhexidine and less antibacterial effect than silver nanoparticles [23]. An investigation by Lu et al., in which the MIC of 5 nanometer silver nanoparticles for anaerobic bacteria such as Actinomycetemcomitans, Fusobacterium nucleotum, Streptococcus mitis, S. mutans, and S. sanguinis were 50, 50, 25, 25, 25 and 25 µg/ml, respectively [24]. Comparing the results of this study with our study, showed that the first type of nickel oxide nanoparticles showed less antibacterial effect against S. mutans and more antibacterial effect against S. sanguinis than silver nanoparticles. In the study of Aflatoonian et al., the minimum growth inhibitory concentration and the minimum bactericidal concentration of nanoparticles on Pseudomonas aeruginosa and E. faecalis strains were determined using broth microdilution method. Visible-ultraviolet spectroscopy showed an absorption peak in the range of 370 nm. Transmission electron microscopy images showed the synthesis of more spherical zinc oxide nanoparticles with a size of less than 50 nm. The lowest inhibitory concentrations of ZnO nanoparticles against P. aeruginosa and E. faecalis were determined to be 6.25 and 12.5 µg/ml, respectively [25]. A study by Davari et al. represented that copper and zinc oxide nanoparticles at concentrations of 0.1 and 0.5% at 15 and 30 days compared to the control group significantly reduced the number of bacteria. Zinc oxide nanoparticles at 0.5% in the composite composition showed the highest and both oxidized nanoparticles and zinc at the concentration of 0.05% showed the lowest amount of antimicrobial properties [26]. Despite the promising effects of nickel oxide nanoparticles, further study to investigate the toxicity on different human cell lines is suggested as the most important limitation of the recent study. The results showed that nickel oxide nanoparticles have acceptable antibacterial properties against S. mutans and S. sanguinis, which can be considered as preventative agents for tooth decay in materials and compounds used in dentistry. Authors' contributions Concept and Study design: EK, MH, FN, HP; Methods, data collection and experimental work: EK, MJ, MH; Results analysis and drafting: CT, SN, HG, HR, HP; Critical revisions: CT, MH, HR, FN, HP. All authors read and approved the final version. Conflict of interests None to be declared. Ethical declarations The study deign was approved by the ethical committee at the Guilan University of Medical Science [IR.GUMS.REC.1399.569]. Table 1. Diameter of zone inhibition by well-diffusion Nanoparticle Streptococcus sanguinis Streptococcus mutans 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml NP (1) 12mm 12 16 17 11mm 10 14 14 NP (2) 11 12 14 14 0 0 12 12 Table 2. The Minimum inhibitory concentration (MIC) of nanoparticles Nanoparticle MIC Streptococcus sanguinis Streptococcus mutans NP (1) 31.25 µg/ml 125 µg/ml NP (2) 125 µg/ml >500 µg/ml Tavakol et al. 5 Financial support Self-funded. References 1. Allaker RP, Memarzadeh K. Nanoparticles and the control of oral infections. Int J Antimicrob Agents. 2014;43(2):95-104. 2. Song W, Ge S. Application of antimicrobial nanoparticles in dentistry. Molecules. 2019;24(6):1033. 3. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, et al. Bacterial biofilms in nature and disease. 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