Mona Al-Terehi1, Saadi Shershab2Hadeel Alaa Al-Rrubaei3 and Ali H. Al-Saadi1

 1University of Babylon, College of Science, 2Al-Hilla University College, Dentist Department, 3 University of Babylon, DNA Research Center, Iraq.

Abstract

The microbial community in oral cavity associated with oral infection and disease, variety of microbes have been recorded in oral  cavity with different genetic properties, the present study aims to study some oral cavity isolates using classical methods and PCR for molecular detection O-antigen genes, results show that there were four isolates, three were G- E. coli ; Klebsilla pneumonia and Cardiobacterium  hominis   while  the fourth  species was Staphylococcus aureaus , it had different response to antibiotics E. coli  and  Klebsilla pneumonia were more resistance. The plasmid profile didn’t show any plasmids as well as O- antigen genes it was negative for all isolates. The present study concluded that oral cavity have variety species with different genomic properties , it need more investigation about other genes and bioactivity.

Keywords: Oral cavity, O-antigen, plasmid profile.

Introduction

The oral and dental infection cause by population of Microbial like periodontal diseases, pericoronitis , gingivitis, endodontitis, peri-implantitis, and post extraction infections  each of these   have distinct clinical and microbial features, The r microbial flora in the oral cavity contains  about 1010 of bacterial species ,  more than 500  species yet to now recognized as normal inhabitants in oral cavity ,  However, only 150 microbial species have been isolated and cultured from root canals (Marina and Romana, 2007)

There are  many species related with  oral infections recoded by different studies  like Campylobacter rectus, Prevotella intermedia, Bacteroides forsythus, , Actinobacillus  actinomycetem-comitans, Porphyromonas gingivalis Eubacterium species, Fusobacterium nucleatum also Eikenella corrodens, and Pepto-streptococcus micros. Treponema pallidum-related spirochetes have been associated with acute necrotizing ulcerative gingivitis, Also Porphyromonas endodontalis recorded to be specifically related to endodontic infections (Paster et al., 2000; Paste et al., 2001; Aas et al., 2005;; Dewhirs et al., 2010) it was responsible of two major oral diseases ; caries (tooth decay) and periodontitis (gum diseases) Dahlén et al., 1993; Marsh et al., 1994; Nishihara et al., 2000).

The species  Isolation which causing infection in oral cavity and teeth canal can be  collected by strictly aerobic and anaerobic techniques, also Rapid selective culture and DNA probe have been used in  identification of oral species microbes, however  the detection by Molecular methods  of oral microbial genera or by 16S rRNA genes sequencing improved that the  mouth of human provides a habitat for about 700 species of bacteria, and that between 100 and 200 different species in the healthy mouth of any individual (Paster et al., 2006) . other surveys  suggested that the variety  of bacteria in an individual oral cavity may be slightly higher  by using pyrosequencing technologies, around 500 species (Zaura et al., 2009).

Materials and methods

Sample collection : samples  collected using trans media swaps from dental canal after extraction , all patients were visited porsepa center for dentist clinic, swaps collected by specialist dr. Saadi shershap, then it transfer to lab for culture and analysis.

Culture and diagnosis: aerobic and anaerobic culture, then isolates were re-culture for  purification isolates and diagnostic. Macroscopic and microscopic diagnostic : The features of colonies, culture in differential media and gam stain using to diagnostic isolates then antibiotic sensitivity was detected using disc methods to cefixime, erythromycin, gentamicin, amikacin, meropenem, Nitrofurantoin, ceftazidime, norfloxacin, tetracycline, cefotaxime, aztronam.

DNA extraction and PCR experiment: DNA was extracted and  PCR  performed according to  PCR  colony methods then the following primers were used to amplify game negative isolates   O45 wzx1 (F) 3´-CCG GGT TTC GAT TTG TGA AGG TTG-5´ ,(R) 3´-CAC AAC AGC CAC TAC TAG GCA GAA-5´,  and O45 wzy1 (F) 3´-GAA ATT ATG CCA TCT TGG CGA GCG-5´ , (R) 3-´CAT GTG AAG CCT GAA GGC AAA CTC-5´ (DebRoy et al.., 2004). PCR conditions and size products: PCR experiments performed as a following; pre-denaturation for 5 min at 94ÚC, then 35 cycles ( 30 s at 94ÚC, 30 s at 59ÚC, 30 s at 72ÚC, and finally 10 min at 72ÚC). Electrophoresis was implemented using 1% agarose 0.5 X  for 60 min , 70 V  and 20 mA all testes had control represented by E coli HB101 [pomega ]

Results and discussion

The results of bacterial culture show four species of bacteria three of its was  gram GV-  included E. coli ; Klebsilla pneumonia and Cardiobacterium hominis , the fourth  species was Staphylococcus aureaus.

Antibiotic resistance the antibiotic sensitivity of isolates were show in table (1)  E  coli was more resistance than other isolates it was resistance to Ceftazidime, Cefotaxime, Cefixime, Aztronam, Amikacin and Tetracycline while it more sensitive to Nitrofurantoin than other isolates. Klebsilla pneumonia isolates was resistance to seven types of antibiotics and it more sensitive  to Tetracycline as well as the other two isolates, Staphylococcus aureaus was resistance to 4 types while Cardiobacterium hominis was resistance to two types only. The resistance to antibiotics types dependent on the  genetic of isolates and its predisposition to acquire a new antibiotic resistance gene by conjugation, transformation and transduction way. The oral cavity was complex ecosystem that consist of hundred different taxa of microorganisms  may be detected founded, some studies suggested that the incoming bacteria to oral cavity like E. coli that don’t  considered as a   part of the normal oral thus can able to attach to extracellular matrix or resident members , and more importantly, overcome the invasion resistance in order to integrate into the community [14], He et al., (2012) concluded that the some bacterial species in oral cavity can be integrated  in this community by  strategy of evasion to development oral microbial community ; which prevents detection by antagonistic oral bacteria and allows integration.

Table 1. Antibiotic sensitivity of bacterial isolates

Bacterial isolates Erythromycin
15mcg
Ceftazidime
30mcg
Nitrofurantoin
300mcg
Cefotaxime
30mcg
Cefixime
5mcg
Norfloxacin
10mcg
Aztronam
30mcg
Amikacin
30mcg
Gentamicin
10mcg
Tetracycline 30 mcg Meropenem
10mcg
 E. coli 17 0 35 0 0 23 0 0 16 0 20
 Klebsilla pneumoniae 0 0 0 23 0 30 0 14 13 33 0
Staphylococcus aureaus 0 0 20 16 0 30 16 15 18 40 0
Cardiobacterium hominis 25 10 30 0 0 27 20 27 23 44 60

The results of DNA profile didn’t show any plasmids in all isolates (figure 1),  also all isolates didn’t had o-antigen gene; the PCR  product show negative results (figure2).

Fig. 1. Plasmid profile of oral bacterial isolates lane 1 positive control E coli Hb101, lane 2-5 oral bacterial isolates  E. coli,  Klebsilla pneumonia, Staphylococcus aureaus, Cardiobacterium hominis  respectively

Fig. 2. Negative Multiplex PCRs targeting the Owzx and Owzy genes Lane 1 M, molecular weight,   lane2-8   bacterial isolates lane 9, E coli HB101. The  Positive multiplex PCR showing the amplified wzx 255 bp and wzy 451 bp

The review of literature about plasmid profile in oral cavity bacterial were little, study performed by Marcia et al., (2003) about plasmid profile in oral Fusobacterium nucleatum, they found three groups of plasmid were classified according to its size. Although of absence  plasmids in present isolates it were had antibiotics resistance, this can be explanation by plasmid curing in oral cavity that resulted from  construction microbial biofilm  initial from adherence to the oral tissues and teeth, cooperation between bacterial species, signaling between the bacteria and its role in pathogenesis, and the transfer of DNA between bacteria by different genetic transfer processes (Kolenbrander et al., 2010).

The Biofilms consider  good environment to exchange  DNA because of a close proximity of cells that able DNA to trapped in extracellular matrix. Roberts et al., (2001) and Warburton et al., (2007) improved horizontal gene transfer between oral streptococci in biofilm communities. Mira (2008) clarified that the DNA transfer between some oral bacteria genera was happen conjugative transposons also t Analysis of the genomes of some oral isolates improved that past horizontal gene transfer happened in about 5% and 45% of genes in different species. DNA transferring between different strains P. gingivalis and Escherichia coli, events by conjugation; its suggested by Koehler et al., (2003). In some genera DNA can be transmitted without direct cells contact as in oral streptococci (Wang et al., 2002).

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