DETECTION OF ENHANCED EXPRESSION OF PHEROMONES AND QUORUM SENSING SYSTEM AMONG ENTEROCOCCUS FAECALIS FROM DENTAL CARIES BY PCR

As, there has been a paucity in detecting such virulence markers among dental isolates of Enterococcus faecalis in Indian context, we have taken this objective to find out such factors to rule out the relationship between the pathogen and the disease establishment. It indicates that these genes may even play a cardinal role in causing dental caries by E. faecalis.A sum of 20 dental caries enterococcal isolates were detected for expression of pheromones and quorum sensing system genes by PCR. showed positive for enhanced expression of pheromone (eep) and 2/20 (10%) were found to have quorum sensing system (fsr) genes by PCR.It indicates that these genes may even play a cardinal role in causing dental caries by E. faecalis. However, it is important to include more number of isolates to validate the result.


INTRODUCTION
The human dental cavity is colonised with large groups of aerobic and anaerobic bacterial species. faecalis as a nosocomial pathogen can cause serious infections that are frequently isolated (30-90%) from root canal treated patients [1]. Enterococcus faecalis is most commonly found in the faeces.The high prevalence of this species in root canal treated patients evidenced by culturing methods, and molecular detection tools suggested that it may be the reason for most of the endodontic treatment failures virulence genes such as eep (enhanced expression of pheromone) and fsr (quorum sensing system) are playing its critical role in the pathogenesis of E. faecalis infections [3]. The most common cause of root canal treatment failure is due to the microorganismsin the apical portion of the teeth [4,5]. Enterococcus bacteria that are found in the periapical lesion in the root canal treated teeth [6,7]. Thus, this study planned to investigate the presence of enhanced expression of pheromones and quorum sensing system genes by PCR in Enterococcus faecal isolates from dental caries.

DETECTION OF ENHANCED EXPRESSION OF PHEROMONES AND QUORUM SENSING SYSTEM AMONG ENTEROCOCCUS FAECALIS FROM DENTAL CARIES BY PCR
Sarah Banu and Gopinath P Saveetha Dental College and Hospitals, Chennai-600 077

A B S T R A C T
As, there has been a paucity in detecting such virulence markers among dental isolates of Enterococcus faecalis in Indian context, we have taken this objective to find out such factors to rule out the relationship between the pathogen and the disease establishment. It indicates that these genes may even play a cardinal role in causing dental caries by E. faecalis.A sum of 20 dental caries enterococcal isolates were detected for expression of pheromones and quorum sensing system genes by PCR. showed positive for enhanced expression of pheromone (eep) and 2/20 (10%) were found to have quorum sensing system (fsr) genes by PCR.It indicates that these genes may even play a cardinal role in causing dental caries by E. faecalis. However, it is important to include more number of isolates to validate the result.
The human dental cavity is colonised with large groups of bacterial species. Enterococcus pathogen can cause serious infections 90%) from root canal treated Enterococcus faecalis is most commonly found in the faeces.The high prevalence of this species in root canal lturing methods, and molecular detection tools suggested that it may be the reason for most of the endodontic treatment failures [2]. Considering (enhanced expression of (quorum sensing system) are playing its faecalis in dental The most common cause of root canal treatment failure is due to the microorganismsin the apical is the common bacteria that are found in the periapical lesion in the root canal 7]. Thus, this study planned to investigate the enhanced expression of pheromones and quorum sensing system genes by PCR in Enterococcus faecalis repetitive dental caries isolates of were collected included in this study. These isolates were identified by standard biochemical parameters as described by elsewhere. in semi-solid brain heart infusion medium and stored at 4ºC until further use.

Detection of enhanced expression of pheromones (eep) and quorum sensing system (fsr) among by PCR
E. faecalis isolates were detected for the genes by PCR analysis. 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 1mM of MgCl 2 0.2mM of eep and fsr genes with 1U of England Biolabs) in a 1x PCR buffered reaction. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition.

DETECTION OF ENHANCED EXPRESSION OF PHEROMONES AND QUORUM SENSING SYSTEM AMONG ENTEROCOCCUS FAECALIS FROM DENTAL CARIES BY PCR
As, there has been a paucity in detecting such virulence markers among dental isolates of in Indian context, we have taken this objective to find out such factors to rule out the relationship between the pathogen and the disease establishment. It indicates that these genes may even play a cardinal role in causing dental caries by E.
ccal isolates were detected for enhanced expression of pheromones and quorum sensing system genes by PCR.4/20 (20%) were showed positive for enhanced expression of pheromone (eep) and 2/20 (10%) were found rum sensing system (fsr) genes by PCR.It indicates that these genes may even faecalis. However, it is important to These isolates were identified by standard biochemical parameters as described by elsewhere. Isolates were preserved solid brain heart infusion medium and stored at 4ºC

Detection of enhanced expression of pheromones (eep) and quorum sensing system (fsr) among Enterococcus faecalis
isolates were detected for the presence of such genes by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture 0.2mM dNTP mix and 0.8µM genes with 1U of Taq polymerase (New Biolabs) in a 1x PCR buffered reaction. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 98 o C for 6 min and 35 cycles for 30s, 70 o C for 30s and 69 o C for 60s, followed by a final extension of 5 min at 75 o C. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis [9].

Antibiotic susceptibility pattern
We found increased percentage of isolates were shown to be resistant to all the antibiotics used in this study. For ampicillin, amikacin, erythromycin, gentamicin, our isolates were found to resistant between 80-90%. Better sensitivity was observed in linezolid, teicoplanin and vancomycin antibiotics. The detailed results of antibiotic sensitivity patter of Enterococci was given in table 2.

Results for enhanced expression of pheromone (eep) and have quorum sensing system (fsr) genes
Of the 20 dental isolates of Enterococcus faecalis, 4/20 (20%) were showed positive for enhanced expression of pheromone (eep) and 2/20 (10%) were found to have quorum sensing system (fsr) genes by PCR.

DISCUSSION
With increasing resistance to some routinely used antibiotics, enterococcal infections pose a big threat as a nosocomial pathogen. E. faecalis was appeared to cause 90% of the enterococcal infections in humans, and it was frequently found in obturated root canals exhibiting symptoms of chronic apical periodontitis, particularly in post dental monocultures [10,11]. Study conducted by Prashanth and co-workers in 2016, observed different virulence factors in enterococcal isolates from dental caries. Wherein, 48% of the isolates were found to have eep gene indicates that bacterial pheromone secretion is necessary for inducing conjugation, and confirming its role in different dental conditions. It was also reported that eep also involved in biofilm formation [12] and it also provides lysozyme resistance to the host. 81% of isolates also had fsr gene which encodes for pheromone synthesis of this bacteria. In our study, we have observed 20% and 10% of our isolates were found to have eep and fsr gene that codes for enhanced expression of pheromones and quorum sensing system respectively.

CONCLUSION
As, there has been a paucity in detecting such virulence markers among dental isolates of Enterococcus faecalis in Indian context, we have taken this objective to find out such factors to rule out the relationship between the pathogen and the disease establishment. It indicates that these genes may even play a cardinal role in causing dental caries by E. faecalis. However, it is important to include more number of isolates to validate the result. *******