COMPARISON OF PERIODONTOPATHOGENS AND DETECTION OF TETRACYCLINE RESISTANCE GENES BY PCR

Periodontal disease is the result of an imbalance in the microbial ecology of the and it also depends on host susceptibility.[1] Studies have shown a close association between periodontitis and the presence of a specific group of microorganisms, mainly Porphyromonasgingivalis, Aggregatibacter, Actinomycetemcomitans denticola and Tannerella forsythia.Thus, this study is to compare the and to detect the tetracycline resistance genes by PCR among patients with different periodontal conditions. In our study, T. forsythia was the most frequently det pathogen in 5/5 (100%) chronic periodontitis and 4/5 (80%) aggressive periodontitis patients. A. actinomycetemcomitans was the least detected pathogen as it detected only one sample of chronic periodontitis case. Upon subjected them to tetracycline r (TetM gene), all were negative. T. forsythia and A. actinomycetemcomitans were the most frequently and least detected periodontic pathogens respectively. Tetracycline resistance was not observed in our isolates. This study reveals the knowle periopathogens and tetracycline resistance gene in our region.


INTRODUCTION
Periodontal disease is the result of an imbalance in the microbial ecology of the oral cavity, and it also depends on host susceptibility. [1] Studies have shown a close association between periodontitis and the presence of a specific group of microorganisms, mainly Porphyromonasgingivalis Aggregatibacter, Actinomycetemcomitans denticola and Tannerella forsythia [1,2]. Recent studies have reported periodontal pathogens resistant to different antibiotics, with percentages that vary according to populations studied, this could be due to the lack of control over antibiotic use and to poor patient treatment compliance in certain countries [3]. Tetracycline and some of its derivatives have been widely used as adjuvants in the treatment of periodontitis, both topically and systemically. [4,5]. Thus, this study is to compare the periodontopathogens and to detect the tetracycline resistance genes by PCR among patients with different periodontal conditions.

MATERIALS AND METHODS
Five subgingivalsamples from each periodonticcases includes chronic periodontitis and aggressive periodontitis as well as healthy population were subjected to 16s

A B S T R A C T
Periodontal disease is the result of an imbalance in the microbial ecology of the and it also depends on host susceptibility. [1] Studies have shown a close association between periodontitis and the presence of a specific group of microorganisms, mainly Porphyromonasgingivalis, Aggregatibacter, Actinomycetemcomitans denticola and Tannerella forsythia.Thus, this study is to compare the and to detect the tetracycline resistance genes by PCR among patients with different periodontal conditions. In our study, T. forsythia was the most frequently det pathogen in 5/5 (100%) chronic periodontitis and 4/5 (80%) aggressive periodontitis patients. A. actinomycetemcomitans was the least detected pathogen as it detected only one sample of chronic periodontitis case. Upon subjected them to tetracycline r (TetM gene), all were negative. T. forsythia and A. actinomycetemcomitans were the most frequently and least detected periodontic pathogens respectively. Tetracycline resistance was not observed in our isolates. This study reveals the knowle periopathogens and tetracycline resistance gene in our region.
Periodontal disease is the result of an imbalance in the microbial ecology of the oral cavity, and it also depends on host susceptibility. [1] Studies have shown a close association between periodontitis and the presence of a specific group of Porphyromonasgingivalis, Aggregatibacter, Actinomycetemcomitans, Treponema Recent studies have reported periodontal pathogens resistant to different antibiotics, with percentages that vary according to the populations studied, this could be due to the lack of control over antibiotic use and to poor patient treatment compliance Tetracycline and some of its derivatives have been widely used as adjuvants in the ntitis, both topically and systemically. periodontopathogens and to detect the tetracycline resistance genes by PCR among patients with different periodontal conditions. subgingivalsamples from each periodonticcases which includes chronic periodontitis and aggressive periodontitis as well as healthy population were subjected to 16s rDNA analysis for P. gingivalis, T. denticola, T. forsy actinomycetemcomitans, Eikenellacorrodens followed by the detection of tetracycline resistance genes (TetM) by PCR as per the following cyclic condition. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 2mM of Mgcl 2 0.2mM dNTP mix and 0.5µM of can gene Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of S. aureus with cna gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 97 for 30s, 54 o C for 1min and 74 o extension of 10 min at 72 o C. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis. [6] Five subgingivalsamples from each periodonticcaseswhich includes chronic periodontitis and aggressive periodontitis as well as healthy population were subjected to 16s rDNAanalysis for P. gingivalis, T. denticola, T. forsy actinomycetemcomitans, Eikenellacorrodens followed by the detection of tetracycline resistance genes (TetM) by PCR as described elsewhere. The amplicons were resolved in 4% agarose gel electrophoresis and findings were compared.
Periodontal disease is the result of an imbalance in the microbial ecology of the oral cavity, and it also depends on host susceptibility. [1] Studies have shown a close association between periodontitis and the presence of a specific group of microorganisms, Aggregatibacter, Actinomycetemcomitans, Treponema Thus, this study is to compare the periodontopathogens and to detect the tetracycline resistance genes by PCR among patients with different In our study, T. forsythia was the most frequently detected pathogen in 5/5 (100%) chronic periodontitis and 4/5 (80%) aggressive periodontitis patients. A. actinomycetemcomitans was the least detected pathogen as it detected only one sample of chronic periodontitis case. Upon subjected them to tetracycline resistance gene (TetM gene), all were negative. T. forsythia and A. actinomycetemcomitans were the most frequently and least detected periodontic pathogens respectively. Tetracycline resistance was not observed in our isolates. This study reveals the knowledge on the prevalence of periopathogens and tetracycline resistance gene in our region. analysis for P. gingivalis, T. denticola, T. forsythia, A. actinomycetemcomitans, P. intermedia and Eikenellacorrodens followed by the detection of tetracycline resistance genes (TetM) by PCR as per the following cyclic of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 2mM of 0.2mM dNTP mix and 0.5µM of can gene with IU of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of S. aureus with cna gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling dition. Initial denaturation at 97 o C for 1 min and 35 cycles o C for 1min, followed by a final C. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel Five subgingivalsamples from each periodonticcaseswhich includes chronic periodontitis and aggressive periodontitis as well as healthy population were subjected to 16s rDNAanalysis for P. gingivalis, T. denticola, T. forsythia, A. P. intermedia and Eikenellacorrodens followed by the detection of tetracycline resistance genes (TetM) by PCR as described elsewhere. The amplicons were resolved in 4% agarose gel electrophoresis

Review Article
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RESULTS
In our study, T. forsythia was the most frequently detected pathogen in 5/5 (100%) chronic periodontitisand 4/5 (80%) aggressive periodontitis patients. A. actinomycetemcomitans was the least detected pathogen as it detected only one sample of chronic periodontitis case. Upon subjected them to tetracycline resistance gene (TetM gene), all were negative.

DISCUSSION
In this cross-sectional study, we used PCR to determine the prevalence of nine periodontopathogens in the subgingival microbiota of patients diagnosed as healthy gingivitis, CP and AgP. Study conducted by Collins and coworkers in 2016 have demonstrated that, 63.6% of P. micra, 54.5% of T. forsythia, 45.4% F. nucleatum and E. corrodens and followed by other periopathogens isolated from healthy population. Whereas, in case of chronic periodontitis cases, T. forsythia (96.7%) scored the first predominant pathogen and in case of aggressive periodontitis cases, Both F. nucleatum and T. denticola were the most identified bacteria. [6] In contrast to their study, we found T. forsythia was the most frequently detected pathogen in 5/5 (100%) chronic periodontitis and 4/5 (80%) aggressive periodontitis patients. A. actinomycetemcomitans was the least detected pathogen as it detected only one sample of chronic periodontitis case.

CONCLUSION
T. forsythia and A. actinomycetemcomitans were the most frequently and least detected periodontic pathogens respectively. Tetracycline resistance was not observed in our isolates. This study reveals the knowledge on the prevalence of periopathogens and tetracycline resistance gene in our region.