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Isolation and identification of bacteria and parasite from teeth caries and periodontal.

INTRODUCTION

Oral cavity of human supports life for many different species of microorganisms, but bacterial infection happened when these microorganisms permeate into the tissues or in case of lower host immunity. The display of bacterial infection of dental descent is permanently changing and is a measurable reflexing of new evolution of oral flora.[1]

The studies of bacteriology have shown differences in acute microbiological dental infections. According to Rautemaa et al.,[2] surly this difference reflects variations in the bacteriological knock used for their isolation, identification, and gradual changes in flora due to injudicious use and not commitment to taking of antimicrobials.

About 700 or more different types of bacteria include complex microbial groups that live in the mouth, it is important to preserving healthy oral. The mouth gives an environment to growth diverse groups of microorganisms comprising lactic acid bacteria belonging to the genera Lactobacilli, Streptococcus, members of the genus Actinomyces and two kinds of parasite: Entamoeba gingivalis and Trichomonas tenax, these kinds of parasite attacked to periodontal. Trichomonas tenax anaerobic parasite found in oral cavity which responsible for chronic periodontitis parasite cans transmission by kissing and droplet spray Entamoeba gingivalis parasite found in mouth [3].

More recently, studies are suggesting that in addition to dental caries and periodontal disease, oral micro biota can have negative impacts on other aspects of our health [4]. This research was aimed to isolation and identification of microorganisms causes teeth decay and periodontal.

MATERIAL AND METHODS

A/ sample collection:

200 samples collected from 100 patient (children, teenager and adult) 70 female and 30 male with periodontal disease, 100 of these samples taken from teeth decay and the other 100 samples taken from periodontal to detect microorganism.

B/Isolation procedures

a/ examine under microscope:

To detect parasite used wet mount, normal saline and eosin stain then prepared slid and finely examined directly under microscope. [5].

Direct smears were prepared and stained with gram stain to identification of bacteria isolated after inculated in culture. All morphological arrangement and its reaction with the stain were studied by using light microscope.

b/ used culture:

Each sample was incubated into three blood agar plates; one of them was incubated aerobically. While the others were incubated anaerobically by using two methods of incubations; anaerobic jar with candle jar incubation. MaConkeys agar was also used and incubated aerobically. Sabouraud glucose agar was used for culture Candida spp. All plates were incubated at 37[degrees]C for 24 hours, with further 24 hours incubation if there was no growth. Cultures for anaerobes were incubated for 48-72 hours [6].

Biochemical reactions, sugar fermentation tests, bile solubility, starch hydrolysis, gelatin digestion, catalase, lecithinase, indole production and nitrate reduction test, which are the most characteristics tests to differentiate between them [7].

C/ Statistical analysis:

Statistical analysis was performed using SPSS statistical computer software.

Result:

The result show that 80% from the sample was parasite infection and 76% with bacterial infection and present 87.1% female infected with parasite and 74.2% with bacteria from Table(1) Statistical analysis show there was p < 0.05 significant difference .

Table (2, 3) show the ratio of infection with parasite and bacteria increased when the age of patient increased with p < 0.05 significant difference in parasite only.

The type of bacteria isolated belong to in gram positive, gram negative, aerobic and anaerobic bacteria as shown in table (4) the most bacteria which found were the aerobic bacteria (10 strains) including Bacillus sp. , Staphylococcus sp. , Streptococcus mutans , Micrococcus spp. , Proteus vulgaris, Klebsiella spp. , Echerichia coli and Pseudomonas aeroginosae compared to anaerobic bacteria (4 strains) which include Lactobacillus fermentum, Actinomyces naeslundii , Peptostreptococcus spp. and Bacteroides ovatus . and 6 type of parasite was Entamoeba gingivalis, Trichomonas tenax , Entamoeba coli , Iodamoeba butschlii, Chilomastix mesnili and Giardia lambelia and yeast. (Table3).

In our study patients with periodontal disease are more likely to infection with protozoa. In female rate of infection with parasite higher than male these result matches with other study [8] (Table 1). In fact, 53% were infected with Entamoeba gingivalis while Trichomonas tenax where 27% (Table 2). This result was higher than another study which refers to infection with parasite was 31.67% of Entamoeba gingivalis while infection with Trichomonas tenax was 35% higher than our study [5]. In the other study rate of E.gingivalis was 35% while T. tenax was 3.3% [8].

In another study in Babylon city rate of infection with E. gingivalis was 42.9 % and T. tenax was 20.6% which closely to our result [16].

Infection of E.gingivalis and T. tenax may be return to periodontitis and have no oral cleanliness [5],or probable these parasites are opportunists particularly lesions gingivalis and periodontal pockets [10].

In this study we found age groups (11-20), (13-40) and (41<) more likely to infected with E.gingivalis, while infected with T. tenax in age group(21-30) and (31-40)(Table 2) .these results were agreement with many studies which say infection with parasites increase with aging [8] Statistical analysis show there was p < 0.05 significant difference.

It has been suggested these protozoans could affect the formation of contribute to the development and progression of periodontal disease. The other researchers state that these protozoans may be opportunistic, since they are capable of proliferating in the microenvironment of the mucobuccal fold affected by periodontal disease [11].

Therefore, if Entamoeba gingivalis helps the development and progression of periodontitis and gingivitis, these diseases increasingly facilitate the proliferation of these protozoa, this vicious circle could explain the increased incidence of these microorganisms in the dental plaque and saliva samples of patients with periodontitis and gingivitis. A suspension of Entamoeba gingivalis was spread on the gingival margins of rats' immunosuppressed with (prednisolone acetate), leading to the development of the clinical signs and inflammatoiy process of periodontal disease much faster than that observed in immunocompetent rats [12].

Protozoa are not the only pathogenic found in patients with periodontal disease, in this study we found wide range of bacteria. The result show that 76% with bacterial infection and present 74.2% female and 80 % male infected with bacteria this result similar the result obtain by [13]. The ratio of infection with parasite and bacteria increased when the age of patient increased. Statistical analysis show there was p < 0.05 significant difference in parasite only.

In this study we found wide range of gram positive, gram negative, aerobic and anaerobic bacteria as shown in table (3) the most bacteria we found was the aerobic bacteria (10 strains) including Bacillus sp. , Staphylococcus sp. , Streptococcus mutans , Micrococcus spp. , Proteus vulgaris, Klebsiella spp. , Echerichia coli and Pseudomonas aeroginosae compared to anaerobic bacteria (4 strains) which include Lactobacillus fermentum, Actinomyces naeslundii , Peptostreptococcus spp. and Bacteroides ovatus (gram positive bacteria more than gram negative bacteria) this results almost similar to [13]

From the 76 patients about 30 patients have streptococcus mutans and 37 patients had Staphylococcus spp. that main Staphylococcus spp. higher than streptococcus mutans which is the opposite of [14]

This wide range of bacteria and protozoa in mouth may cause lose of teeth in younger people [14] further more to that it may cause coronary disease , arteriosclerosis , myocardial infection , pneumonia pre-term births and low birth weight [15,16].

ARTICLE INFO

Article history:

Received 5 August 2015

Accepted 20 September 2015

Available online 30 September 2015

REFERENCES

[1] Loesche, W., 2007. ?Dental caries and periodontitis: contrasting two infections that have medical implications,? Infect. Dis. Clin. of Nor. Ame., 21(2): 471-502.

[2] Rautemaa, R., A. Lauhio, M.P. Cullinan and G.J. Seymour, 2007. ?Oral infections and systemic disease--an emerging problem in medicine," Clin. Microb. and Infec., 13(11): 1041-1047.

[3] White, D.J., K.M. Kozak, R. Gibb, J. Dunavent, M. Klukowska and P.A. Sagel, 2006. A 24-hour dental plaque prevention study with a stannous fluoride dentifrice containing hexametaphosphate. J. Contemp. Dent. Pract., 7(3): 1-11.

[4] Ghabanchi, J., M. Zibaei, A.M Daghigh and A.H. Sarbazie, 2010. Prevalence of oral Entamoeba gingivalis and Trichomonas tenax in patients with periodontal disease and healthy population in Shiraz, southern Iran. Ind. J.of den. res., 21(1): 89-91.

[5] Onyido, A.E., E.S. Amadi, I. Olofin, A.A. Onwumma, I.C. Okoh and C.I. Chikwendu, 2011. Prevalence of Entamoeba gingivalis and Trichomonas tenax among dental patients attending Federal School of Dental Technology and Therapy clinic, Enugu, Nigeria. Nature and Science, 9(9): 59-62.

[6] Quinn, P.J., M.E. Carter, B. Markey, 2004. Clinical Veterinary Microbiology. Mosby 6th ed. Edinburgh. New York. USA; pp: 191-208.

[7] Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, R.H. Yolken, 2003. Manual of clinical microbiology. 8th ed., Washington, D.C., American Society for Microbiology Press, 1.

[8] Ibrahim, S. and R. Abbas, 2012. Evaluation of Entamoeba gingivalis and Trichomonas tenax in patients with periodontitis and gingivitis and its correlation with some risk factors. J. Bagh. College Dentistry, 24(3): 59-62.

[9] Talaro, K.P. and A. Talaro, 2002. Foudation in Microbiology. 4th Ed. McGraw Hill Companies Inc New York, pp: 697.

[10] El-Azzoumi, M. and A.M. Al-Brady, 1994. Frequency of Entamoeba gingivalis among periodontal and patients under chemotherapy. J. Egypt Soc. Paras, 24(3): 649-55.

[11] Al-Saeed, W.M., 2003. Pathogenic effect of Entamoeba gingivalis on gingival tissue of rats. Al-Rafidain Dent. J. 3(1): 70-73.

[12] Daniluk, T., G. Tokajuk, D. Cylwik-Rokicka, D. Rozkiewicz, M.L. Zaremba and W. Stokowska, 2006. Aerobic and anaerobic bacteria in subgingival and supragingival plaques of adult patients with periodontal disease. Adv. in med. Sci., 51: 81-85.

[13] Mohapatra, S.B., M. Pattnaik and P. Ray, 2012. Microbial association of dental caries. Asian J. Exp. Biol. Sci., 3(2): 360-367.

[14] Darvean, R.P., A. Tanner and R.C. Page, 2000. The microbial challenge in periodontitis. Periodontology. 14: 12-32.

[15] Al-hamiary, A.K., M.Y. Kezar and Y.A. Al-Khafaji, 2011. Prevalence of Oral Protozoa in Periodontitis and Gingivitis Patients Whose Attended to Clinics Periodontics, Dentistry College\Babylon Univ. Magazin of al-kufa university for bio., 1: 3.

Sura I. A. Jabuk, Rafla'a S. H. Hussien, Zahraa M. Altaee , Hawraa M. R. Najam, Noor M. Naji

University of Babylon, College of Science, Department of Biology, Hilla, Iraq

Corresponding Author: Sura I. A. Jabuk, Hawraa M. R. Najam, Noor M. Naji University of Babylon, College of Science, Department of Biology, Hilla, Iraq
Table 1: The number and percentage of positive and negative samples
isolated from female and male

Total   Number of samples contain parasite

        Female               Male

        N.(%)     P.(%)      N.(%)      P.(%)

100     3(4.2)    34(48.5)   7(10)      5(16.6)
100     6(8.5)    27(38.5)   4(13.3)    14(46.6)
200     9(12.8)   61(87.1)   11(36.6)   19(63.3)

Total   Number of samples contain bacteria

        Female                Male

        N.(%)      P.(%)      N.(%)     P.(%)

100     9(12.8)    24(34.2)   4(13.3)   14(46.6)
100     9(12.8)    28(40)     2(6.6)    10(33.3)
200     18(25.7)   52(74.2)   6(20)     24(80)

Total   Nature of samples

100     Teeth decay
100     Periodonta l
200     Total

p < 0.05 significant difference

Table 2: relationships between parasite infection and age

Age     No. of   Entamoeba    Trichomonas
        sample   gingivalis   tenax

                 No.   %      No.   %

1-10    14       5     35.7   4     28.5
11-20   46       25    54.3   9     36
21-30   30       15    50     10    66.6
31-40   5        4     80     4     100
41<     5        4     80     0     0
Total   100      53           27

p < 0.05 significant difference

Table 3: relationships between bacteria infection and age

Age     No. of sample   No. of positive sample (%)

1-10    14              9(64.3)
11-20   46              32(69.5)
21-30   30              24(80)
31-40   5               4(80)
41<     5               5(100)
Total   100             76

p > 0.05 no significant difference

Table 4: Number and type of microorganism isolated from periodontal
disease

Type of microorganism        Number of microorganism

1-bacteria
a-aerobic
1-Grams positive
Bacillus cereus              12
Bacillus subtilis            15
Staphylococcus aureus        25
Streptococcus mutans         30
Micrococcus spp.             5
Proteus vulgaris             17
Staphylococcus epidermidis   12
2-Grams negative
Klebsiella spp.              9
Escherichia coli             8
Pseudomonas aeroginosae      1
b-Anearobic
1-Grams positive
Lactobacillus fermentum      10
Actinomyces naeslundii       4
Peptostreptococcus spp.      16
2-Grams negative
Bacteroides ovatus           3
2-parasite
Entamoeba gingivalis         53
Trichomonas tenax            27
Entamoeba coli               2
Iodamoeba butschlii          1
Chilomastix mesnili          1
Giardia lambelia             1
3-Yeast
Candida albicans             34
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Article Details
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Author:Jabuk, Sura I.A.; Hussien, Rafla'a S.H.; Altaee, Zahraa M.; Najam, Hawraa M.R.; Naji, Noor M.
Publication:Advances in Environmental Biology
Article Type:Report
Date:Sep 1, 2015
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