Isolation of Clinical Microbes Isolated during Treatment with Orthodontic Appliances

Isolation of Clinical Microbes Isolated during Treatment with Orthodontic Appliances Nihad Nasser  Department of Medical Microbiology, College of Dentistry University of Thi-Qar, Thi-Qar, 64001, Iraq Abstract After one day of use, the microbial content of aligners will be determined. Many options for correcting bites are available in the dentistry market nowadays from orthodontists. The aligners are nearly undetectable because they are clear and translucent. Once thought to be reserved for braces, these are now worn daily to rectify the misaligned teeth. While researchers throughout the world have pondered the subject of aligners and the microbiota in the mouth on multiple occasions, recent discussions have focused more on antibiotic resistance and species composition. Furthermore, both these traits and the microbes themselves exhibit qualities that are contextand time-dependent. The issue of antibiotic resistance is relevant here. remains pertinent in the field of dentistry. Because relapses in caries and inflammatory disorders of the mouth can be caused by high contamination of orthodontic materials, it is necessary to evaluate it constantly. This is because local anti-inflammatory medication can be reduced. The virulence factors of microorganisms are provided by adhesive qualities, which are an important component of biofilm architecture. These properties are defined by an increase in optical density, which determines the duration and retrospectivity of diagnostic tests. The potential for antibiotic resistance among clinical microbiological isolates obtained after aligner therapy is the primary topic of this article. We hope to develop a safe and effective biofilm-destroying chemical using the microbes we collected in the future. Introduction Bacteria and other microbes are ever-present in the mouth [1]. Plaque biofilm is an intricate microbial community that shields harmful microbes from external medicinal agents and evades host defense mechanisms; it is the principal culprit in dental disorders such as caries and periodontitis [2]. Many studies have attempted to address this issue by creating antimicrobial drugs, but thus far, the majority of these compounds have shown to be both ineffective and dangerous [3,4]. It was initially investigated by R.G. Gibbons et al. [5,6] how members of the oral microbiota interact with and adhere to the surface of filling materials. Saliva plays a significant role in the development of caries by encouraging the adherence of bacteria to the surfaces of filling material and enamel [7]. Saliva glycoprotein content is positively correlated with S. mutans adhesion on dental filling and enamel surfaces; that is, a larger concentration of saliva glycoproteins increases the possibility of S. mutans adhesion. Research by R.G. Gibbons [8,9] led him to conclude that S. sanguis, as opposed to S. mutans, attaches more effectively to composite fillings. Nonetheless, this goes against the predominant idea, which states that S. mutans is the most dangerous (cariogenic) species because it produces insoluble glycans that help the microbes stick to the surface of the seal better [10,11]. Since the introduction of clear aligners in 1998, a tool for mild to moderate orthodontic movement of teeth, the issue of biofilm production has gained significance. Orthodontists have been using transparent aligner therapy for a long time, and it's just growing in popularity. Hundreds of articles are currently focused on the issue of biofilm production during orthodontic treatment, according to Internet searches. Since this is the pilot study of a series, we choose not to highlight biofilm compositional differences in this first piece. In More Information How to cite this article: Nasser N. Isolation of Clinical Microbes Isolated during Treatment with Orthodontic Appliances. Eur J Med Health Res, 2025;3(6):80-6. DOI: 10.59324/ejmhr.2025.3(6).13 Keywords: Clear aligners, Oral microbiota, Biofilm formation, Antibiotic resistance, Orthodontic materials, Dental caries, Virulence factors, Optical density, Microbial contamination, Oral health This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if they made any changes. EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 81 the upcoming publication, we will examine variations in biofilms, strains, and results using participants of varying ages. Two methods exist via which orthodontic appliances, whether fixed or removable, might impact the composition of the oral microbiota: (1) the buildup of plaque and (2) the neglect of proper oral hygiene. Numerous scientific articles [12-15] indicate the qualitative and quantitative changes in the oral cavity's microbiota that are linked to orthodontic treatments. This is because these procedures include covering a large surface area of teeth. The primary causes of dental caries and gingivitis/periodontitis, or their worsening, are the buildup of food debris and bacterial plaque, as well as the growing difficulty of patients to practice appropriate oral hygiene [16-18]. Researching morphometric and densitometric markers of biofilm persistence on aligners was our primary objective. Materials and Methods Microbial Strains Ten people, six female and four males, who had aligners taken from adolescents (13–19 years old) were microbiologically tested during the course of the study. The significant prevalence of this orthodontic operation compared to other groups is the reason this age group was chosen. The next step is to conduct the same studies with different age ranges, such as those between 20 and 29 and 30 and 39. Wearing braces or other orthodontic appliances is less common among adults. No one under the age of thirteen should undergo the operation unless necessary. Invisa® aligners were utilized by patients. Their construction is based on the translucent, tough, and lightweight polyethylene terephthalate glycol (PETG). After patients wore their aligners for one day, we asked that they not wash them. A new set of aligners was given to patients at the clinic, while their old ones were kept for the trial. We used sterile wide test tubes to hold 10 mL of a 0.85% NaCl solution after slicing these aligners that had microorganism biofilms. Following a three-hour incubation period in the solution, the test tubes were vortexed for ten minutes at three thousand revolutions per minute to ensure thorough microbial removal. The entire sample, 1 milliliter in volume, was taken from a test tube. After that, we plated the diluted suspensions, counted the number of colony-forming units, and made repeated dilutions. We utilized Sabouraud dextrose agar (BioMerieux, France) for yeast-like fungus (YLF). Blaurock medium (HEM, Moscow, Russia) was utilized for Bifidobacterium spp. We employed MRS medium (HiMedia, India) for Lactobacillus spp. The Staphylococcus spp. was cultured in peptone-salt medium and yolk-salt agar, both sourced from HiMedia in India. Streptococcus spp. was cultured in mitissalivarius agar (HiMedia, India). We utilized Endo's medium, Ploskirev's medium, and bismuth-sulfite agar (HEM, Moscow, Russia) for the Enterobacteria family. We used clostridial agar from HiMedia in India for Clostridium spp. and meat-peptone agar (MPA) from HEM in Moscow, Russia, for all other bacteria. There was an incubation period of 24-72 hours at 37–38 °C for the plates. For 72 hours at 37 °C, we created anaerobic conditions using the AnaeroJar anaerostat and the Anaerocult gas-generating kits (Merk, Germany). A MALDI Biotyper (Bruker Daltonik Inc., Billerica, MA, USA) was used to identify the pure cultures by matrixactivated laser desorption/ionization technology. The X score might take on values between 0 and 3, with 2 and 3 indicating success. A score greater than 2.3 was deemed extremely dependable [19,20]. The contamination index, as measured in microorganisms per cubic centimeter of sample, was determined using the following formula and given as a logarithm with base 10 (lg CFU/1 mL), where CFU stands for colony forming unit: 𝐎 =(𝑁/𝑉)×𝐾. (1) N is the average colony count per bacteriological cup, V is the volume of suspension used to seed the agar surface, and K is the multiplicity of dilution [21,22]. When the MALDI Biotyper showed distinct results, it meant that the microbial culture belonged to a different strain. We relied on the MALDI Biotyper database (Bruker Daltonics GmbH, Bremen, Germany) and the CLSI Microbiology Standards for MALDI-TOF MS (https://clsi.org/standards/products/microbiology/doc uments/m58/, accessed on 6 December 2022) [23,24]. Absolute numbers (A.n.) represent the number of distinct strains within a given species. The table also includes the percentage of strains. The cultures of microorganisms were kept at 4 ± 1 °C in a semi-liquid, freeze-dried form of 0.5% meat-peptone agar. Densitometric Indicators of Microbial Biofilms At 490 nm, the amount of crystal violet binding (HiMedia, India) was used to identify microbial biofilms. The samples that were subjected to testing were placed into the wells of a 96-well plate (made by Medpolymer Company, Russia) and allowed to grow in a controlled aerobic environment at 37 °C for a duration of 48 hours. We poured out the liquid and used 200 μL of phosphate-buffered solution (PBS) to wash the wells three times until they reached a pH of 7.3. Every washing step included shaking the plates for 5 minutes. Following a 15-minute fixation with 150 μL of 96% ethanol, the samples were dried at 37 °C for 20 minutes. Before cultivating the microbial biofilms at 37 °C for 5 minutes, a 0.5% dye solution was added to each well. After discarding the contents of the wells, the EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 82 plates were washed three times with 200 μL of PBS (pH 7.3) and allowed to dry. After 30 minutes, 200 μL of 96% ethanol was used to elute the bound dye from the connected cells [25,26]. Biofilms' optical densities (ODs) were found by measuring how strongly they bound crystal violet [27]. Confocal Laser Scanning Microscopy Using the CSLM LSM510/ConfoCor2 system (Carl Zeiss, Oberkochen, Germany), we examined the aligners to see if biofilms remained after washing. Concanavalin A (50 mg/L) from Molecular Probes in Eugene, OR, USA was used to stain the aligners for 1 hour at 37 °C (green fluorescence). From the surfaces that were investigated, about one hundred slices were made. The lack of fluorescent signal indicated that the aligners were free of biofilms. Statistics Using SPSS 20.0 (IBM Corp., Armonk, NY, USA), we conducted the analysis of the results. When p < 0.05, the results were considered significant according to Student's t-test. Results and Discussions Analysis of the oral microbiota in the study group led to the isolation and identification of microorganisms from 28 different species (Table 1). By analyzing the contamination index, we were able to determine which microbial groups were most crucial to the development of aligners' microecosystems by comparing their degrees of dominance. According to Table 1, the oral microflora of teenagers who wear aligners is dominated by bifidobacteria, yeasts of the genus Candida (Candida albicans), E. coli, Peptostreptococcus anaerobius, Porphyromonas gingivalis, Staphylococcus aureus, and Streptococcus mitis, with a concentration of at least 5 lg CFU/1 mL. Thirteen Gram-positive and thirteen Gram-negative bacteria were identified, suggesting that both types of bacteria contribute equally to the formation of plaque microbiota by interacting with the cell wall structure. Other microflora representatives' digital expression of ecological relevance is much lower than the values set for the prominent species (<5 lg CFU/1 mL), suggesting that they do not significantly contribute to the structure of biofilm biocenoses. Table 1. Groups of Microorganisms According to the Degree of Dominance on Aligners Species Gram-Negative (−) or Positive (+) Number of Isolates lg CFU/1 mL Absolute Number % Actinomyces israelii + 2.45 ± 1.01 2 2.94 Actinomyces naeslundii + 4.54 ± 1.38 3 4.41 Candida albicans YLF 6.38 ± 1.86 4 5.88 Candida parapsilosis YLF 2.45 ± 0.60 3 4.41 Clostridium aminobutyricum + 1.19 ± 0.29 2 2.94 Escherichia coli − 5.67 ± 2.05 5 7.35 Lactobacillus rhamnosus + 4.38 ± 1.27 2 2.94 Porphyromonas gingivalis − 6.40 ± 2.01 3 4.41 Staphylococcus aureus + 6.05 ± 1.44 4 5.88 Streptococcus gordonii + 3.88 ± 1.20 3 4.41 Streptococcus mitis + 7.51 ± 1.64 3 4.41 Streptococcus salivarius + 0.31 ± 0.20 1 1.47 Total: 28 13 Gr+ and 13 Gr− 68 100% Note: CFU—colony forming unit; YLF—yeast-like fungi. Source: The table is created based on the research of [46] When Candida albicans colonizes mucosal membranes, it may acquire and maintain a stable Candida population, which can stop clinical illness from developing. The pathogenicity factors of Candida albicans, which can be conditionally grouped into five groups, explain the greater YLF concentration. But when illness strikes, all of these things happen at once in the body: 1. The capacity to attach to host tissues is the initial stage in interacting with a microbe; 2. The secretory aspartyl proteases (SAP) are proteolytic enzymes that aid Candida in penetrating and invading host tissues. 3. Changes in morphology leading to the "yeast-hyphal form," which aids in YLF's ability to penetrate tissues and evade host defensive mechanisms; 4. Candida's phenotypic flipping in response to environmental changes; 5. Different immunomodulatory processes of some C. albicans compounds that may lower the efficacy of antifungal immunity. Morphometric and Densitometric Indicators of Biofilms Biofilm formation symbolizes the species’ ability to occupy a dominant position in a community and exert a predominant impact on biocenotic processes, which EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 83 determine the kind of biocenosis according to the main ecological groupings [28-31,46]. Here, it was fascinating to look at the bacterial and fungal community structure and see how much biofilm each isolate within the species produced (Table 2). The strongest producers of biofilms were (ODS ≥ 0.3): Actinomyces israelii, Actinomyces naeslundii isolate 3, Campylobacter concisus, Candida albicans, Candida parapsilosis, Escherichia coli, Lactobacillus rhamnosus, Peptostreptococcus anaerobius, and Staphylococcus aureus [46]. The biofilm production rates were significantly lower for all other community types on the liners. For future usage, all isolates that could form biofilms effectively were freeze-dried and stored at 4 ± 1 °C. Figure 1. Isolation of Microorganisms Figure 2.Antibiotic Figure 3. Bacteria Culture Medium Table 2. Determination of Bacterial Biofilm Formation Intensity by Optic Density Optic Density ODS ODA Average Error 1 2 3 4 5 Actinomyces israelii 0.312 0.331 np np np 0.3215 0.0095 Actinomyces naeslundii 0.244 0.293 0.302 np np 0.2797 0.0238 Candida albicans 0.420 0.502 0.431 0.384 np 0.4343 0.0338 Candida parapsilosis 0.390 0.347 0.333 np np 0.3567 0.0222 Clostridium aminobutyricum 0.279 0.193 Np np np 0.236 0.043 Escherichia coli 0.462 0.411 0.380 0.376 0.382 0.4022 0.0274 Lactobacillus rhamnosus 0.325 0.303 Np np np 0.314 0.011 Porphyromonas gingivalis 0.132 0.176 0.173 np np 0.1603 0.0189 EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 84 Staphylococcus aureus 0.421 0.394 0.385 0.400 np 0.4 0.0105 Streptococcus gordonii 0.189 0.195 0.215 np np 0.1997 0.0102 Streptococcus mitis 0.244 0.230 0.261 np np 0.245 0.0107 Streptococcus salivarius 0.253 np Np np np np np Note: ODS—tested sample; ODA—average value; np—not possible. Source: The table is created based on the research of [46] Clear aligners are susceptible to bacterial biofilm formation, even though aligner orthodontic therapy has demonstrated promising outcomes in comparison to traditional fixed orthodontic treatment in terms of plaque index and gum condition control [32-34]. Enamel, metal orthodontic braces, and transparent aligners all share similarities in the early stages of microbial adhesion and biofilm development in aerobic and anaerobic oral cavity types, according to research by Tektas et al. [35-37]. The shape of the aligner, which is not straight and has grooves and protrusions [46], also improves the likelihood of bacterial adherence. Also, even brand-new aligners have surface imperfections including micro-scratches, microcracks, and small elevations, as demonstrated by Low et al. [38,39]. These imperfections serve as attachment points for microbial and fungal biofilms. Furthermore, when the aligners were worn, Gracco et al. [40] showed that they underwent physical modifications. They developed microcracks, worn and delaminated spots, and a loss of transparency after 14 days, which encouraged adhesion and bacterial development and caused localized deposits of calcined biofilm. Removing protrusions and adhering buccal epithelial cells mixed with microbial biofilms were discovered when used aligners were evaluated by both our study and Schuster et al. [41]. Lastly, the morphology and ultrastructure of biofilms on aligners were investigated by Low et al. [42]. Researchers identified streptococci and staphylococci as the primary bacterial species present in the early biofilm. Bacteria and, eventually, fungi that are Gram-negative were present in this biofilm. The authors of a related study compared contamination markers in 25 patients before and after six months of aligner treatment to learn about microbial alterations in the mouth cavity during this time [43]. The presence of periodontal pathogens and cariogenic bacteria, such as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus sobrinus, was uncovered [46]. They concluded that to determine if the oral and periodontal microbiological alterations caused by orthodontic appliance insertion go back to their pretreatment levels, more high-quality, long-term studies are required. An intriguing study conducted by Sfondrini M.F. et al. [44,45] examined the effects of clear aligner treatment of periodontal condition and the microbiological composition of the oral microbiota. Twenty orthodontic patients first had expert teeth cleanings and then got aligners. During the initial two months of treatment, they demonstrated that aligner therapy had no discernible effect on periodontal and microbiological parameters compared to those who did not get treatment. Conclusions Antibacterial medications for orthodontic patients could be developed using morphometric and densitometric markers of heterogeneous biofilms, according to our findings. Two yeast-like fungus and thirteen Gram-negative bacteria were discovered. The capacity of isolated microbes to form biofilms was evaluated, and the most robust producers were identified. Currently, no method exists to guarantee the thorough and direct elimination of biofilms. But we learned how to make and build new medications when we exposed the pathogenetic mechanisms underlying the microbial relationships in biofilms. A small sample size of 10 patients and an age restriction of 13–19 was two of the drawbacks of our study. Other age groups and those with more than 10 participants should be considered in future research. Furthermore, we intend to develop a safe and efficient biofilm-degrading agent using the acquired microbial strains. References [1] Fernandes GL, Delbem ACB. Nanosynthesis of silver-calcium glycerophosphate: promising association against oral pathogens. Antibiotics (Basel). 2018;7:52. doi:10.3390/antibiotics7030052. [2] Saafan A, Zaazou MH, Sallam MK, Mosallam O, El Danaf HA. Assessment of photodynamic therapy and nanoparticles effects on caries models. Open Access Maced J Med Sci. 2018;6:1289–1295. doi:10.3889/OAMJMS.2018.241. [3] Li F, Weir MD, Xu HHK, et al. Effect of salivary pellicle on antibacterial activity of novel antibacterial dental adhesives using a dental plaque microcosm biofilm model. Dent Mater. 2014;30:182–191. doi:10.1016/j.dental.2013.11.004. [4] Wiedmer D, Petersen FC. Antibacterial effect of hydrogen peroxide–titanium dioxide suspensions in the decontamination of rough titanium surfaces. Biofouling. 2017;33:451–459. doi:10.1080/08927014.2017.1322585. EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 85 [5] Gibbons RJ, van Houte J. On the formation of dental plaques. J Periodontol. 1973;44:347–360. doi:10.1902/jop.1973.44.6.347. [6] Gibbons RJ, Nyggard M. Interbacterial aggregation of plaque bacteria. Arch Oral Biol. 1970;15:1397–1400. doi:10.1016/00039969(70)90031-2. [7] Leonhardt Å, Renvert S, Dahlen G. Microbial findings at failing implants. Clin Oral Implants Res. 1999;10(5):339–345. doi:10.1034/j.16000501.1999.100501.x. [8] Kobayashi H. Saliva-promoted adhesion of Streptococcus mutans MT8148 associated with dental plaque and caries experience. Caries Res. 2007;41:217. doi:10.1159/000099321. [9] Kolenbrander PE, London J. Adhere today, here tomorrow: oral bacteria adherens. J Bacteriol. 1993;175:3247–3250. doi:10.1128/jb.175.11.32473252.1993. [10] Kolenbrander PE. Intragenic coaggregation among strains of human oral bacteria: potential role in primary colonization of the tooth surface. Appl Environ Microbiol. 1990;56:3890–3894. doi:10.1128/AEM.56.12.3890-3894.1990. [11] Noiri Y, Li L, Ebisu S. The colonization of periodontal disease-associated bacteria in human periodontal pocket. J Dent Res. 2001;80:1930–1934. doi:10.1177/00220345010800101301. [12] Sukontapatipark W, el-Agroudi MA, Selliseth NJ, Thunold K, Selvig KA. Bacterial colonization associated with fixed orthodontic appliances: a scanning electron microscopy study. Eur J Orthod. 2001;23:475–484. doi:10.1093/ejo/23.5.475. [13] Kilian M, Chapple LC, Hannig M, Marsh PD, Meuric V, Pedersen AM. The oral microbiome—an update for oral healthcare professionals. Br Dent J. 2016;221:657–666. doi:10.1038/sj.bdj.2016.865. [14] Xu X, He J, Xue J, Wang Y, Li K, Zhang K. Oral cavity contains distinct niches with dynamic microbial communities. Environ Microbiol. 2015;17:699–710. doi:10.1111/1462-2920.12502. [15] Jabur SF. Influence of removable orthodontic appliance on oral microbiological status. J Fac Med Baghdad. 2008;50:199–202. doi: not found. [16] Sun F, Ahmed A, Wang L, Dong M, Niu W. Comparison of oral microbiota in orthodontic patients and healthy individuals. Microb Pathog. 2018;123:473– 477. doi:10.1016/j.micpath.2018.08.011. [17] Rossini G, Parrini S, Castroflorio T, Deregibus A, Debernardi CL. Periodontal health during clear aligners treatment: a systematic review. Eur J Orthod. 2015;37:539–543. doi:10.1093/ejo/cju083. [18] Kim S, Choi D, Jang I, Cha B, Jost-Brinkmann P, Song J. Microbiologic changes in subgingival plaque before and during the early period of orthodontic treatment. Angle Orthod. 2012;82:254–260. doi:10.2319/030311-156.1. [19] Rudenko P, Vatnikov Y, Sachivkina N, et al. Search for promising strains of probiotic microbiota isolated from different biotopes of healthy cats for use in the control of surgical infections. Pathogens. 2021;10:667. doi:10.3390/pathogens10060667. [20] Rudenko P, Sachivkina N, Vatnikov Y, Shabunin S, Engashev S, Kontsevaya S, Karamyan A, Bokov D, Kuznetsova O, Vasilieva E. Role of microorganisms isolated from cows with mastitis in Moscow region in biofilm formation. Vet World. 2021;14:40–48. doi:10.14202/vetworld.2021.40-48. [21] Sachivkina N, Vasilieva E, Lenchenko E, Kuznetsova O, Karamyan A, Ibragimova A, Zhabo N, Molchanova M. Reduction in pathogenicity in yeast-like fungi by farnesol in quail model. Animals (Basel). 2022;12:489. doi:10.3390/ani12040489. [22] Arsene MMJ, Viktorovna PI, Sergei GV, Hajjar F, Vyacheslavovna YN, Vladimirovna ZA, Aleksandrovna VE, Nikolayevich SA, Sachivkina N. Phytochemical analysis, antibacterial and antibiofilm activities of Aloe vera aqueous extract against selected resistant Gramnegative bacteria involved in urinary tract infections. Fermentation. 2022;8:626. doi:10.3390/fermentation8110626. [23] Patel R. A moldy application of MALDI: MALDITOF mass spectrometry for fungal identification. J Fungi (Basel). 2019;5:4. doi:10.3390/jof5010004. [24] Angeletti S, Ciccozzi M. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiology: an updating review. Infect Genet Evol. 2019;76:104063. doi:10.1016/j.meegid.2019.104063. [25] Sachivkina N, Podoprigora I, Bokov D. Morphological characteristics of Candida albicans, Candida krusei, Candida guilliermondii, and Candida glabrata biofilms, and response to farnesol. Vet World. 2021;14:1608–1614. doi:10.14202/vetworld.2021.1608-1614. [26] Sachivkina NP, Lenchenko EM, Mannapova RT, Strizhakov AA, Romanova EV, Lukina DM. Candida biofilm modeling: past and present. Farmatsiya. 2019;68:18–22. doi:10.29296/25419218-2019-03-03. [27] Lenchenko EM, Blumenkrants D, Sachivkina N, Shadrova N, Ibragimova A. Morphological and adhesive properties of Klebsiella pneumoniae biofilms. Vet World. 2020;13:197–200. doi:10.14202/vetworld.2020.197-200. EUR J MED HEALTH RES Volume 3 | Number 6 | 2025 86 [28] Łysik D, Deptuła P, Chmielewska S, Bucki R, Mystkowska J. Degradation of polylactide and polycaprolactone as a result of biofilm formation assessed under experimental conditions simulating the oral cavity environment. Materials (Basel). 2022;15:7061. doi:10.3390/ma15207061. [29] Guo M, Zhang Z, Lu J, Wang D, Yan Y, Zhang S, Yu X, Su S, Yuan L, Li Z, et al. Differences in supragingival microbiome in patients with and without full-crown prostheses. Dent J (Basel). 2022;10:152. doi:10.3390/dj10080152. [30] Campos MS, Marchini L, Bernardes LAS, Paulino LC, Nobrega FG. Biofilm microbial communities of denture stomatitis. Oral Microbiol Immunol. 2008;23:419–424. doi:10.1111/j.1399302X.2008.00445.x. [31] Benítez-Páez A, Belda-Ferre P, Simón-Soro A, Mira A. Microbiota diversity and gene expression dynamics in human oral biofilms. BMC Genomics. 2014;15:311. doi:10.1186/1471-2164-15-311. [32] Kormas I, Pedercini C, Pedercini A, Raptopoulos M, Alassy H, Wolff LF. Peri-implant diseases: diagnosis, clinical, histological, microbiological characteristics and treatment strategies. A narrative review. Antibiotics (Basel). 2020;9:835. doi:10.3390/antibiotics9110835. [33] Levrini L, Mangano A, Montanari P, Margherini S, Caprioglio A, Abbate GM. Periodontal health status in patients treated with the Invisalign® system and fixed orthodontic appliances: a 3 months clinical and microbiological evaluation. Eur J Dent. 2015;9:404– 410. doi:10.4103/1305-7456.163218. [34] Jiang Q, Li J, Mei L, Du J, Levrini L, Abbate GM, Li H. Periodontal health during orthodontic treatment with clear aligners and fixed appliances. J Am Dent Assoc. 2018;149:712–720.e12. doi:10.1016/j.adaj.2018.04.010. [35] Tektas S, Thurnheer T, Eliades T, Attin T, Karygianni L. Initial bacterial adhesion and biofilm formation on aligner materials. Antibiotics (Basel). 2020;9:908. doi:10.3390/antibiotics9120908. [36] Low B, Lee W, Seneviratne CJ, Samaranayake LP, Hagg U. Ultrastructure and morphology of biofilms on thermoplastic orthodontic appliances in “fast” and “slow” plaque formers. Eur J Orthod. 2011;33:577–583. doi:10.1093/ejo/cjq126. [37] Gracco A, Mazzoli A, Favoni O, Conti C, Ferraris P, Tosi G, Guarneri MP. Short-term chemical and physical changes in Invisalign appliances. Aust Orthod J. 2009;25:34–40. doi: not found. [38] Schuster S, Eliades G, Zinelis S, Eliades T, Bradley TG. Structural conformation and leaching from in vitro aged and retrieved Invisalign appliances. Am J Orthod Dentofacial Orthop. 2004;126:725–728. doi:10.1016/j.ajodo.2004.04.021. [39] Zhao R, Huang R, Long H, Li Y, Gao M, Lai W. The dynamics of the oral microbiome and oral health among patients receiving clear aligner orthodontic treatment. Oral Dis. 2020;26:473–483. doi:10.1111/odi.13175. [40] Sfondrini MF, Butera A, Di Michele P, Luccisano C, Ottini B, Sangalli E, Gallo S, Pascadopoli M, Gandini P, Scribante A. Microbiological changes during orthodontic aligner therapy: a prospective clinical trial. Appl Sci. 2021;11:6758. doi:10.3390/app11156758. [41] Ali MD, Barrak MM, Salman RI, Sa'doon NM. The combined effect of Artemisia absinthium methanolic extract and vinblastine chemotherapy on apoptosis and decreasing chemotherapy drug concentration. In: AIP Conference Proceedings. 2023 Sep 13; Vol. 2845, No. 1:020023. AIP Publishing LLC. doi: not found. [42] Barrak MM, Al-Aidy SR, Bonyadi M. Unraveling DREAM-PL: a case report on challenges and management. University of Thi-Qar Journal of Science. 2024 Jun 1;11(1):33–35. doi: not found. [43] Riyas NR, Kazim MK, Ubaid AQ, Barrak MM. Applications of biophysics in the understanding and treatment of cellular diseases. Innovative: International Multidisciplinary Journal of Applied Technology. 2024 Jun 15;2(5):100–104. ISSN 2995-486X. doi: not found. [44] Barrak MM, Bonyadi M, Hami HJ, Al Ali MD, Muttair RY, Abd RA. Exploring the heterogeneity of non-syndromic hearing loss: a comprehensive review of implicated genes and the role of whole exome sequencing. University of Thi-Qar Journal of Science. 2023 Dec 27;10(2):192–199. doi: not found. [45] Hami HJ, Barrak MM. The relationship between flavonoids and cancer. [conference/report/journal not specified]. doi: not found. [46] Baybekov O, Stanishevskiy Y, Sachivkina N, Bobunova A, Zhabo N, Avdonina M. Isolation of clinical microbial isolates during orthodontic aligner therapy and their ability to form biofilm. Dent J (Basel). 2023;11:13. doi:10.3390/dj11010013.