ISOLATION OF ACTINOBACILLUS ACTINOMYCETUMCOMITANS (AGGREGOBACTER) ANDPORPHYROMONAS GINGIVALIS FROM SUDANESE AGGRESSIVE PERIODONTITIS PATIENTS AND TESTING INHIBITORY EFFECT OF GARLIC EXTRACT COMPARED TO CHLORHEXIDINE DIGLUCONATE 0.2%

Received: 24 June 2017 Final Accepted: 26 July 2017 Published: August 2017 Aggressive periodontitis is a destructive inflammatory condition affecting the investing and supporting tissues of the teeth. Detectable levelsof Porphyromonasgingivalis,T .forthia, Prevotellainetrmedia, Campylobacter and ActinobacillusActinomycetnsemcomita are associated with disease progression ,Adjunct use of antimicrobial agents was implemented for the treatment of the diseaseand also someantiseptic mouth wash like chlorhexidinedigluconate 0.2%. Now a day’s ,medicinal plants are usedas antimicrobial against bacteria due to increase of bacterial resistant to antibiotics, so Garlic (Allium sativum) used as antimicrobial, antifungal and antiviral against oral pathogens. So the aim of the present study was to isolateActinobacillusactinomycetumcomitansandPorphyromonasgingiv alis,from Subgingival fluid of Sudanese aggressive periodontitis patients and tostudy the effect of water garlic extract against the isolate compared to chlorhexidinedigluconate 0.2%.

Aggressive periodontitis is a destructive inflammatory condition affecting the investing and supporting tissues of the teeth. Detectable levelsof Porphyromonasgingivalis,T .forthia, Prevotellainetrmedia, Campylobacter and ActinobacillusActinomycetnsemcomita are associated with disease progression ,Adjunct use of antimicrobial agents was implemented for the treatment of the diseaseand also someantiseptic mouth wash like chlorhexidinedigluconate 0.2%. Now a day's ,medicinal plants are usedas antimicrobial against bacteria due to increase of bacterial resistant to antibiotics, so Garlic (Allium sativum) used as antimicrobial, antifungal and antiviral against oral pathogens. So the aim of the present study was to isolateActinobacillusactinomycetumcomitansandPorphyromonasgingiv alis,from Subgingival fluid of Sudanese aggressive periodontitis patients and tostudy the effect of water garlic extract against the isolate compared to chlorhexidinedigluconate 0.2%. Microbiologically: Sub-gingival plaque samples were obtained from two deepest sites per quadrant for each patient for identification of the major causative micro-organismsActinobacillusactinomycetumcomitans, andProphronomonasgingivalis. Identification was done by culture . Garlic extract was subjected to preliminary antimicrobial screening using the cup-plate diffusion method against the detected bacteria. The Minimum inhibitory concentration (MIC) of the garlic extract was determined, Chlorhexidinedigluconate 0. 2% used as positive control and distilled water as negative control (invitro study). Zones of inhibitions were measured and averaged .the mean diameter of the zone of inhibition in mm was compared to the zone of inhibition of chlorhexidine using Pearson correlation test to identify if there is a statically difference between garlic extract and chlorhexidinedigluconate 0.2%. Correlation is significant at the 0.01 and 0.05 level (2 tailed) Results: There was an increase in plaque index, gingival index, pocket depth and loss of attachment among aggressive periodontitis patients. Actinobacillusactinomycetumcomitanswas not detected (negative) but ISSN: 2320-5407 Int. J. Adv. Res. 5 (8), 1996-2003 1998 the form of mouth wash which have inhibitory effect against Gram-positive ,Gram-negative organisms and fungi by reducing bacterial counts. (26) In some studies tea tree oil, garlic, might be an alternative to chlorhexidine solutions against oral microorganisms. (27,28) Filter sterilized, aqueous extract of garlic has the ability to inhibit the growth of gram positive and gram negative micro-organisms and it has action against the arg and lys-gingipains activity of P.gingivalis and it has activity against multidrug-resistant (MDR) strains of Streptococcus mutans isolated from human carious teeth and inhibit P. gingivalis protease activity.( (29,30,31,32) .Garlic extracts in concentrations of 12.5% is cytotoxic to human gingival fibroblasts but it is safe at 6.25, 3.12, and 1.5% , the high concentration of garlic is less cytotoxic than chlorhexidinedigluconate. (33) In Sudan there are many studies on the prevalence of aggressive periodontitis among different age groups, the A. actinomycetemcomitan were detected in the subgingival plaque of 12 (70.6%) patients with aggressive periodontitis and from only one (5.9%) control subject, showing a significantly higher frequency of detection in cases than in controls. The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of high school subjects in Sudan. The detection of non-JP2 types of A. actinomycetemcomitans may be a useful marker of increased risk for development of aggressive periodontitis in young subjects. (34) so the present study was designed to investigate the periodontal health of the aggressive periodontitis patients including determination of major causative micro-organisms, Actinobacillusactinomycetumcomitansand,porphyromonasgingivalis and to test the effect of a garlic extract on their inhibition compared to chlorhexidine 0.2 % ( in invitro study).

Material and method:-
Cross-sectional analytical clinical and microbiological hospital based study at Department of Periodontology Faculty of Dentistry, University of Khartoum. Thirty aggressive periodontitis patients both males and females Patients were within the age group 18-35 years(,mean 25.4±5.5 years) newly diagnosed according to clinical and radiographic examination as aggressive periodontits patients For all participants the clinical examination was done using a mouth mirror and periodontal probe (Universityof Michigan-0 probe) for 10 teeth recorded in special form ,The patients were examined for plaque accumulation according to PLI (Sillness and Loe)(,35) examination of gingivitis according to GI (Loe and Sillness)(36), in addition to measuring pocket depth and loss of attachment in mm for the deepest site of the examined teeth. Panoramic X-rays or priapical were done for all patients. Patients excluded if they had systemic diseases, those who were immunocompromised, pregnant ladies, patients used antibiotics 3 months before the examination and patients who had periodontal treatment 3 months before the start of the study.
Ethical approval:-Ethical clearance was given by the Research Committee (Faculty of Medicine/ University of Al-Neelain 2007) and approval from the director of faculty of dentistry , head department of periodontology for sample collection . Since the patient are adult, seeking periodontal treatment and capable of giving informed consent, voluntary and freely they agrees verbally to participate in the study after explanation of the aim of the study .

Bacterial isolate:-Isolation of Actinobacillusactinomycetumocomitans:
Bacterial suspension was vortex and homogenized and incubated anaeropically with cooked meat media. To obtain pure culture Samples were subcultured in blood agar with vancomycin 5Mg/ml and incubated anaerobically in anaerobic gar at 37Cº using gas generating kits for carbon dioxide (Micromaster LaboratoriesPVT. LTD38/39, KalpataruInd Estate, Thane (W) MAH. INDIA.

Isolation of prophyromonasegingivalis:
Bacteria incubated anaeropically in blood agar plates for 48 hours and 7-14 days in anaerobic jar at 37C using gas generating kits for carbon dioxide (Micromaster laboratories PVT. LTD 38/39, KalpataruIndEstate,Thane(W) MAH.INDIA .To obtain pure culture the isolate was sub-cultured in Kanamycin blood agar (by dissolving 1.5 g Kanamycin in 100ml of sterile water and then mixed with blood agar.
Bacterial identification:-Colony feature:-Characterization of bacteria was done based on colony features, size, color, shape and consistency.

1999
Gram stain:-Bacterial film fixed and stained with Triphenylmethane dye, Crystal violet, in conjunction with iodine solution and subsequently treated with an organic solvent as alcohol and counter stained with safranin. Bacteria which retain the dye are designated Gram positive and the gram negative bacteria lose the dye.

Simplfied routine biochemical tests for identification of bacterial isolates:-
Biochemical tests will be done under anaerobic condition (in anaerobic jar)The biochemical activities of the purified isolates were then studied for identification and confirmation of these organisms. The biochemical tests carried out include Fermentation tests: (Harris,1964). (37) Methyl red tests,Voges-Proskauer test,Indole production test: Hydrogen sulphide production test). Catalase test, Oxidase test: (Cruickshank et al ., 1975, Salle,1961 Urease test ( Cheesbrough, 1996). (40) Biochemical tests for P.gingivalis, important characteristics leading to proper identification include , negative oxidase and positive catalase results, negative spot indole results, and the ability to ferment glucose but not lactose.
Culture charcteristics:-It grows as white, translucent smooth non-hemolytic colonies in blood agar colonies sticky and adherent M notfement g/lucose, catalase, heamolysis and urease positive ,oxidase,indole and vogesproskauer negative.

Microscopical examination showed gram -ve rods . Biochemical tests:
None of the isolates fermented glucose, All isolates were urease positive, vogesproskaur negative. All isolates were indole -ve, catalase positive and oxidase negative. Some of isolates make gelatin hydrolysis.

Result of Isolation ofActinobacillusactinomycetumocomitans:
Actinobacillusactinomycetumocomitansnot detected in the samples (Negative result) Result of Isolation of prophryomonasegingivalis Microscopical examination: Gram negative, short rod Culture characteristics On blood agar, black hemolytic colonies were seen after 48 hours incubation under anaerobic condition at 37°C.Sample sub-cultured in blood agar with Kanamycin for 7-14 days anaerobically, colonies changed to brown.

Biochemical tests:-
Most isolates ferment glucose do not ferment sucrose and lactose,oxidase negative, catalase positive, β hemolytic and indole negative Water garlic extract:-Fresh garlic cloves (100g) were blended in 20 ml distilled water filtered using cotton wool and ultrafilterd under reduced pressure by using a Buchner funnel and a side armed flask. By subtracting the weight of insoluble material from the weight of the original cloves, the final concentration of garlic extract in solution was determined to be 16.5% (w/v). The garlic extract was stored at -20°C and used for antibacterial testing.

In vitro testing of extract for antimicrobial activity:-Determination of minimum inhibitory concentration (MIC):-
The antibacterial activity of Allium sativum was evaluated by cup-plate agar diffusion method for aqueous extract. The garlic extract used in present study was 16.5% (w/v). One ml of the standard bacteria suspension were thoroughly mixed with 60 ml of molten sterile Muller Hinton agar media and poured into the petridishes. The agars was left to set and in each of plate cups (10mm diameter) were cut using a sterile cork borer and agar discs were removed. Garlic extract were prepared in series of increasing concentrations. The bottom of each plat was marked off into 8 segments. Serial dilution from 100%, ,50%, 25%, 12.5% to 10% is used. 50 µl of extracted garlic 2000 introduced into the wells using automatic microlitre pipette and all plates were incubated at 37°C for 24 hours. Chlorhexidine 0.2 % (Claradine ,Mfd by MEDPHARMA,United Arab Emirates ) used as a positive control, the negative control were distilled water .Sensitivity of P.gingivalisand control was determined by measuring the diameter of the zone of inhibition. The end point (MIC) is the least concentration of antimicrobial agent that completely inhibits the bacterial growth. The resultant growth inhibition zones were measured averaged and the mean values were tabulated. (Table 1). The data were carried out using a computer software program (SPSS version 16, Chicago, USA). ANOVA tests were used to identify significant differences between the means of the study groups. Critical P values of significance were set at 0.05 and a confidence of 95%. Pearson Correlation and the value expressed as mean± S.D .P value at 0.5

Result:-
The extract inhibited the growth of P.Gingivalisat concentration of100%,50%,25%, 12.5% and 10%. The end point(MIC) which is the least concentration of garlic that completely inhibits the growth of P.gingivalis was 10% .The sensitivity pattern as zone of inhibition (mm) of aqueous garlic extract concentration showed growth inhibition activities against Proyhyromonasgingivaliscompared to sensitivity pattern in (mm) of positive control chlorhexidine 0.2%. The pearson correlation between water garlic extract and chlorhexidine 0.2% is 0.596 and , p value > 0.069,so There was no significance difference between aqueous garlic extract and chlorhexidine 0.2%. No zone of inhibition around the negative controls, ( distilled water ). Table(2)   Table 2 Garlic water extract Chlorhexidine Garlic water extract Pearson correlation Sig.  Lau l (2004) in which they found that Actinobacillus actinomycetumcomitans was the least recovered micro-organisms from samples taken from patients with aggressive periodontitis. In the present study p.gingivalis was detected in 70% of 2001 aggressive periodontitis patients and the result is almost in agreement with the previous studies of Savit E.D.et al (1988) in which the detection was 91% , Ann L ,et al (1998) in which p.gingivalis was detected in 79% of the patients and in 25% of healthy subjects . The authors of those studies concluded that P.gingivalis is implicated in the pathogeneses of periodontitis and aggressive periodontitis but P.gingivalis may not be a normal inhabitant of periodontaly healthy subjects .The results are also in agreement with those of Atsuo Amano (1999) in which P.gingivalis was detected in examined sample of 73 patients (78.5%) with aggressive periodontitis ,as well as the results of the study by Lau L, et al (2004) in which p.gingivalis was low in healthy subjects and increased in patients with periodontitis . For the prevention and treatment of periodontal diseases a number of topically applied antimicrobial agents were found to inhibit the formation of bacterial plaque and hence , prevents the onset of gingivitis and periodontits. Chlorhexidine gluconate was commonly used in 0.2% mouthwash or gel. With the rise of bacterial resistance to antibiotics, there is need to search for other antimicrobial agents for treatment and control of oral infections. Garlic which is known to have antibacterial antiviral, anti-brotozoal and antifungal activity (Tariq Abdullah1987).It has an inhibitory effect against gram+ve and gram-ve bacteria in oral cavity (Elnima et al 1983), (Groppo 2002) inhibition of streptococcus mutans(, Bakri 2005) in study of the inhibitory effect on putative periodontal pathogens and (Fanni et al 2007) in study of inhibition of multidrug-resistant strain of streptococcus mutans,so in the present study ,which is the first study in Sudan, we confirmed the marked growth inhibition activity of garlic( grows in northern Sudan) against P. gingivalis which has been described before (Bakri 2005). Aqueous extract(concentration 16.5%) at serial dilution of 100%,, 50%, 25%,12.5% and 10% showed activity . there was no significant difference between Chlorhexidine gluconate 0.2%, and the aqueous extract , The MIC of the aqueous extracts of garlic in the present study was 10% .

Conclusion:-
Since garlic water extract in the present study had invitro inhibitory effect on P.gingivalis isolated from the examined group of aggressive periodontitis patients compared to chlohexidine and due to the widespread use of antibiotic and the spread of antibiotic resistance ,we present evidence for the antimicrobial activity of garlic extract against P.gingivalis and this raises the possibility that garlic may has therapeutic use for prevention and treatment of the onset of aggressive periodontitis.