J Bagh College Dentistry Vol. 29(2), June 2017 Antimicrobial Effect Oral and maxillofacial Surgery and Periodontics 78 Anti-Microbial Effect Of Different Time’sexposureofozonized Gas And Ozonized Water Onperiodontalpathogens (In Vitro Study) Ban Zuhair Ahmmad, B.D.S.(1) Leqaa Mahmood Ibraheem. B.D.S., M.Sc.(2) ABSTRACT Background: the oral cavity is consider to be an open ecosystem, with the balance between the microorganism’s entrance and the defenses of the host. The initiation of periodontitis has been associated with restricted kinds of anaerobic bacteria, such as Aggregatibacter actinomycetemcomitans (A.a) and Porphyromonas gingivalis (P.g) in plaque subgingivally. Ozone has a biological effects on bacteria due to oxidation of bio-molecules and its toxins. The aim is to determine and compare the antimicrobial effect of gaseous ozone and ozonized water on the growth of isolated anaerobic bacteria (A.a and P.g) when exposed to different time intervals. Materials and methods:This experiment is done byozone generator OLYMPIC- III(600mg/hr) to generator the gaseous ozone (218ppm/W-air)which bypassed around the agar plates containing on of the isolated bacteria with different time intervals (1-10 minutes).And with special aeration stone for generation of ozonized water (0.6 ppm) with different time intervals (1-15 minutes). Results: Gaseousozone have a significant reduction in the bacterial growth on the agar plates for (A.a) was 7 minutes and (P.g) was 4 minutes. While ozonated water have also a significant reduction in the bacterial growth on the agar plates for (A.a) was 5 minutes and (P.g) was 4 minutes. Conclusion: Bothgaseousozone and ozonized water are a powerful antimicrobialeffects on anaerobic microorganism isolated from chronic periodontitis patients. Keywords: gaseousozone, ozonized water, Aggregatibacter actinomycetemcomitans (A.a) , Porphyromonas gingivalis (P.g) (J Bagh Coll Dentistry 2017; 29(2): 82-78 ) INTRODUCTION Periodontitis is a destructive and inflammatory disease of the connective tissues that supportingthe teeth and is caused either by one specific type of microorganism or by a group of specific microorganisms, leading to progressive destruction of periodontal ligament and alveolar bone with the formation of periodontal pocket, gingival recession, or both (1). Bacteria are the prime etiological agents in periodontal disease, and it is estimated that more than 500 different bacterial species are capable of colonizing the adult mouth (2). Aggregatibacter actinomycetemcomitans is considered a primary pathogen in localized and generalized chronic periodontitis (3). While, Prohormones gingivalis is implicated in chronic and aggressive periodontitis (4). It is considered one of the main etiologic agents of destructive periodontal disease (5,6). Ozone is a potent oxidant and an important disinfectant, acting on microorganisms by means of oxidation of their biological material (7). It has been reported that ozone can be employed as a bactericidal agent under various forms, such as ozonizedwater (8), ozonized oil, (9), ozone associated with other substances (10), and more frequently the gaseous O3/O2 mixture (11). (1) Master Student. (2) Assistant Professor, Depart ment of Periodontics, College of Dentistry, University of Baghdad. Gaseous ozone has a high oxidation capacity and is greater than chloride for about1.5 times when is use as an antimicrobial agent against several bacteria, viruses, fungi, and protozoa. It has also the ability to stimulate blood circulation and the immune response. Such characteristic features can be applied in medicine and dentistry and have been indicated for the treatment of 260 different pathologies(12).Ozonized water have A high level of biocompatibility on human oral epithelial cells, gingival fibroblast cells, and periodontal cells(13). Ozonized water strongly inhibited the accumulation of dental plaque and is effective in killing grampositive, gram- negative bacteria and oral Candida albicans causing periodontal disease MATERIALS AND METHODS 1-Isolation and identification of the pathogens: The subgingival plaque samples were collected from 10 systemically healthy patients with chronic periodontitis attending the clinic at the Department of periodontics in the teaching hospital of the College of Dentistry / University of Baghdad. The age range was (35-55) years old; the subgingival plaque samples were collected from the periodontal pocket of more J Bagh College Dentistry Vol. 29(2), June 2017 Antimicrobial Effect Oral and maxillofacial Surgery and Periodontics 79 than˃6 mm depthwith attachment loss of one to two mm.The subgingival plaque was put on a swap that was inserted immediately in transfer media to preserve the sample which is then was spread on selective agar media (tryptic soy agar) for both A.a and P.gand incubated anaerobically using anaerobic jar and anaerobic gas packs in the incubator for 72 hours at 37ºC(15,16). The procedure must be done within a period of less than 30 minutes from collecting the sample from the patient. The identification of both A.a and P.g was done byMorphological characteristic(17), Gram’sstain(18) and by using Analytical profile index (API) test for the biochemical testes (19) 2. General description of theexperiments: The gaseous ozone was generated form ozone generator OLYMPIC- III (600mg/hr).In this experiment a small plastic jar was used. The plastic cover has one ozone gas inlet port to inject the ozone gas and distribute it evenly throughout the jar, and one gas outlet for the release of the ozone gas. The ozone generator was fed with 1 LPM of dry compressed air as a feed gas. Ozonized air was bypassed around the agar plates to supply a total air flow of ozone of 218 ppm/w-air. The ozone gas/dry air mixture flowed into the jar for different times (1-10 minutes) according to the experimental design the ozone level inside the plastic jar was kept consistent during the time periods by adjusting the outlet port (Fig.1). Figure 1: General description of the devise While ozonized water was generated by using special aeration stone, with concentration (0.6 ppm) measured by special CHEMets-Kit.As shown in (Fig 2). Figure 2: shows the: 1) Ozone Generator 2) plastic container 3) CHE Mets Kit 3. Procedure: Five well-isolated colonies of the same morphological type which was incubated anaerobically at 37ºC for 24hrs, were selected from tryptic soy agar plate culture. The top of each colony is touched with a loop, and the growth is transferred into a tube containing 5 ml of tryptic soy broth medium. This results in a suspension containing approximately 1 x 108 CFU/ml (equivalent to a 0.5 tube McFarland standard)(20). Using a sterilized Loop, a loopful of bacterial growth was streaked on agar petri plate (A.A. /P.g. agar). Each plate was exposed to the ozonatedgas for a specific time, and then incubated anaerobically at 37ºC for 24hrs. The bacterial growth corresponding to each exposure time on each plate was performed visually, and recorded (21). While the ozonized water experiment was done with the same suspension by adding 1.0ml of bacterial broth in a test tube and then 1.0ml of ozonized water (0.6ppm) was added to the first tube and mixed well then bystreaking on agar petri plate (A.A. / P.g. agar) for 1,2,3,4,5,10, and 15 minutes respectively. The plates were sealed and incubated in an anaerobic jar at 37ºC overnight .The bacterial growth corresponding to each contact time on each plate was performed visually, and recorded (21). Plates showing no bacterial growth means highly efficient exposure time of both ozonized water and the gaseous ozone. RESULTS The results for A.a for colony morphology were white radiating star shaped with no black pigmentation with Gram negative, coccobacilli and give appositive reaction to API NH test (According to API, A.a is listed as (Haemophilus actinomycetemcomitans). While the results for P.g for colony morphology were appeared as 2 1 3 J Bagh College Dentistry Vol. 29(2), June 2017 Antimicrobial Effect Oral and maxillofacial Surgery and Periodontics 80 round spherical in shape with raised or convex surface, black-pigmented colonies with Gram negative, coccobacilli and give appositive reaction to API 20A(According to API, P.g is listed as (p.saccharolytica) with index number (10000004). The results obtained for the qualitative evaluation of ozonated gas (218 ppm/W-Air), is presented in Table 1. The inactivation effect of ozonated gas was observed on both A. a, and P.g colonies. After ozonated gas exposure, the numbers of bacterial colonies on the agar surface decreased in a time-dependent manner and the colony's growth was no longer detected in 7, and 4 minutes of treatment against A.a and P.g. Respectively. The analysis of the results of Table 1 verified that the P.g bacteria were much more sensitive toward ozonated gas compared to A.a. Table 1: Efficiency of ozonated gas at different exposure times onA.a and P.g. colonies growth. ExposurTimes(minutes) Antimicrobial agent Ozonated gas (218 ppm/W-air) A.a P.g 1 +ve +ve 2 +ve +ve 3 +ve +ve 4 +ve -ve 5 +ve -ve 6 +ve -ve 7 -ve -ve 8 -ve -ve 9 -ve -ve 10 -ve -ve +ve =Bacterial growth, -ve = no bacterial grow. While the results obtained for the ozonized water is presented in Table 2. The inactivation effect of ozonized water was observed on both A. a, and P.g colonies. After ozone exposure, the numbers of bacterial colonies on the agar surface decreased in a time-dependent manner and the colony's growth was no longer detected in 5 and 4 minutes of treatment against A.a and P.g. respectively. Table 2: Efficiency of ozonated water at different exposure times on A.a and P.g. colonies growth. Contact Times (minutes) Antimicrobial agents Ozonated Water(0.6 ppm) A.a P.g 1 +ve +ve 2 +ve +ve 3 +ve +ve 4 +ve - ve 5 -ve -ve 10 -ve -ve 15 -ve -ve +ve = Bacterial growth (Uncountable colonies). -ve= No bacterial growth (no colonies). DISCUSSIONS Ozone has been proposed in clinical practice of dentistry and medicine due to its several actions such as antimicrobial, anti-inflammatory, immunostimulating, etc. (22). Ozone has been used for treatment of early carious lesions, periodontal pockets, wound healing such as ulceration, bleaching and in treatment of peri- implantitis(23). The antimicrobialaction of ozone is by damaging the cytoplasmic membrane of the bacteria and cell lysis (24).The results of gaseous ozone experiment was showed that ozonated gas was highly effective in eliminating of both A.a and P.g, (7 and 4 minutes respectively).These results could be explained by the conclusions reached by Hauser et al.,in 2011(25)who investigated the use of gaseous ozone on bacteria adhering to implant surfaces and showed a selective reduction in bacteria, concluding that gaseous ozone may have a role in treatment of peri-implantitis. Huth et al., in 2011(26) similarly showed significant results with gaseous and aqueous ozone and concluded that they merit further investigation. Pereira et al., in 2005(27)reported that application of a gaseous O3/O2 mixture (0.4%/99.6%) for 1 h, at constant pressure and flow (11 mm Hg and 2 L/min, respectively) and controlled temperature, in plates containing 104 CFU/mL of E. coli, S. aureus, and P. aeruginosa lead to total inhibition of growth of these bacteria. And Fontes et al.,in 2012(28), concluded that the application of a low dose of gaseous ozone (dose of 20 μg of O3/mL in a gaseous O3/O2 mixture) for 5 minutes completely prevented the in vitro growth of gram-positive and negative pathogenic bacteria commonly present in patients with J Bagh College Dentistry Vol. 29(2), June 2017 Antimicrobial Effect Oral and maxillofacial Surgery and Periodontics 81 severe nosocomial infections, with known resistance to antibiotics. While, the result of ozonized water (0.6 ppm) was highly effective in eliminating of both A.a and P.g (5 and 4 minutes respectively ).This results were in agreement with kshitish and vandana in 2010 (29).It was reported that ozone at low concentration of 0.1 ppm, is sufficient to inactivate bacterial cells including their spores (8).This reacts could explained by the parcens of various chemical compounds in two different and coexisting modes, one involving direct reactions of molecular ozone and the other a free radical-mediated reaction ,both these mechanisms may be involved in the destruction of bacteria by ozone(30).Ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite and also the metabolic activity of fibroblasts was high when the cells were treated with ozonated water. 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Indian J Dent Res 2010;21:341-348. المستخلص الثھ مع انواع المقدمھ: یعتبر التجویف الفمي كنظام بیئي مفتوح مع التوازن بین البكتریا الداخلھ والنظام الدفاعي للجسم. فلھذا یعتبر تطور التھاب في الصفیحھ الجرثومیھ في (البورفوریموناساللثویھ واالجریجاتیباكتر اكتینومایسیتیمكومیتانس)الموجوده محدده من البكتریا الالھوائیھ وخاصھ داخل الجیوب اللثویھ .االوزون لھ تاثیر بایولوجي على البكتربا من خالل اكسده الماده البایولوجیھ والتوكسین للبكتریا. المعزولھ عند تعرضھا الوقات على البكتریا الالھوائیھ و ماء االوزن تاثیر المضاد البكتیري لغاز االوزوندقیقھ) ومقارنھ ھدف الدراسھ : لتحدید مختلفھ دقیقھ) ومع 10- 1) لتولید غاز االوزون بوقت مابین (3المواد والطریقھ: في دراسھ مختبریھ ,اجریت التجربھ من خالل جھاز االوزون (اولمبك بالبكتریا المعزولھ. دقیقھ) والذي سوف یمر على االطباق المزروعھ15- 1استعمال حجر خاص لتولید ماء االوزون بوقت مابین ( النتیجھ: وجد بأن غاز االوزون و ماء االوزون قد قلل بشكل ملحوظ على نمو البكتریا المتواجده باالطبق لكال النوعین. ثھاالستنتاج: غاز االوزون وماء االوزون یعتبر مضاد بكتیري فعال جدا ضد البكتریا االھوائیھ المعزولھ من المرضى المصابین بالتھاب الل المفتاح: غاز االوزون,ماء االوزون, البورفوریموناساللثویھ , االجریجاتیباكتر اكتینومایسیتیمكومیتانس.