Hemolysin activities as virulence factor of Enterococcus faecalis isolated from saliva and periapical abscess (gene detection by PCR)

Background: Enterococcus faecalis is a normal flora of the oral cavity, commonly detected in saliva and persistence in endodontic infections. These bacteria have diverse survival and virulence factors. Hemolysin is one of the factor and still had unclear role as a virulence factor of the Enterococcus faecalis to survive in the root canal. Purpose: The purpose of this research was to analyze the presence and activity of hemolysin gene and its activity as a virulence factor isolated from saliva and root canals with periapical abscess. Yet by understanding one of the phenotypes characters which is hemolysin, it is expected a successful endodontic treatment can be provided with the persistent of Enterococcus faecalis bacteria. Methods: Method of the research starting with the identification of Enterococcus faecalis bacteria in isolated saliva and periapical abscess was done in the first part of the study. Then the phenotypes character of Enterococcus faecalis such as gene detection and expression of hemolysin in blood agar cultures of the 60 colonies samples were performed in the later part. results: Not all of the colonies cultured were identified as Enterococcus faecalis. All positive detection on hemolysin gene showed hemolysin expresion in both isolated samples. However, there were samples with hemolysin expression eventough no hemolysin gene detected. Hemolysin expression detection in saliva was higher due to different activation phase of hemolysin in saliva. The study with just one primer could lead to the possibility of undetected hemolysin gene, eventough there were samples that did not have hemolysin gene. The proportion of hemolysin expression in root canals were less than saliva, this could be influenced by environmental factors. However, Hemolysin was considered as important virulence factor, particularly for disease therapy. Conclusion: The conclusion of this research was hemolysin gene discovered in clinical isolated saliva and root canals samples as virulence factor of the Enterococcus faecalis, and hemolysin expression occured from both sources.


introduction
[10]14 Hemolysin has been reported as an imprtant virulene factor especially for disease therapy.This virulence factor can be use for guard antibiotic and corticosteroid combinations of treatment in hemolysin or non-hemolysin strain cases. 15On the other hand, even though hemolysin has been researched in many aspects but still had unclear role as a virulence factor of the Enterococcus faecalis to survive in the root canal, and induce inflammation in the periapical tissues. 16nterococcus faecalis bacteria is a normal flora of the oral cavity, but often found in cases of periapical abscess and endodontic treatment failure cases, therefore it is assumed that there are differences in phenotype activity of hemolysin in saliva and root canals with periapical abscess. 6The involvement of Enterococcus faecalis in periapical tissue infections was not fully understood from literatures.Thus, more detailed data is needed to explain the behavior of the bacterial population in the root canal of the tooth, whether as the pathogen causing the infection of periapical tissues or opportunistic species that become pathogenic because of the conducive micro-environment to their survival.A question needs to be answered through this research because these bacteria are normal flora of the mouth and digestive tract.
The purpose of this research was to analyze the presence and activity of hemolysin gene and its activity as a virulence factor isolated from saliva and root canals with periapical abscess.Yet by understanding one of the phenotypes characters which is hemolysin, it is expected a successful endodontic treatment can be provided with the persistent of Enterococcus faecalis bacteria.

materials and methods
The first part of the study was the identification of Enterococcus faecalis bacteria in isolated clinical saliva and periapical abscess.Then the phenotypes character of Enterococcus faecalis such as gene detection and expression of hemolysin in blood agar cultures were performed in the later part.Samples were taken from six patients with periapical abscess.The samples taken from saliva and root canals were then cultured in chromatogenic agar.Sixty greenish blue color colonies were taken.Extraction of DNA proceeded for PCR preparation.DNA was extracted using the Real Genomic Hi-Yield DNA mini kit.Sequence primers use for 16sRNA, forward: TGGC ATAA GAGT GAAA GGCGC, revers: GGGG ACGT TCAG TTAC TAAC GT.][19] Two PCR reactions were performed.The first one was using 16sRNA to ensure Enterococcus faecalis DNA analyzed, while the second reaction was to confirm the extracted DNA contains hemolysin genes.PCR reaction performed in a total volume of 25µl containing PCR mix dream Tag Fermentation (Real Biotech Co. USA), 3 µl Primer 16sRNA forward 10 µM, 3 µl Primer 16sRNA revers 10 µM, 3 µl Nuclease free water.A total of 3 µl of extracted DNA was added to the reaction mixture.PCR was also performed using a positive control (DNA extracted from the American Type Culture Collection [ATCC] species).For the first amplification, samples were subjected to 22 denaturation cycles at 95° C for 15 min, heating at 94° C for 20 seconds.For the second amplification, PCR reaction conditions were 40 cycles of 67° C for 45 seconds, 72° C for 15 seconds, 72° C for 7 minutes and then 4° C.
Hemolysin gene PCR reaction was performed in the same way.The first temperature was 95° C during 15 minutes as the stage of activation of the DNA denaturation enzyme.Followed with 35 cycles at a temperature of 94° C for 20 seconds, 56° C for 45 seconds and 72° C for 60 seconds.
Then the Enterococcus faecalis phenotypic characters test was done to confirm the hemolysin expression of isolated saliva and root canal samples in blood agar plates.Blood agar medium was made by mixing 40 ml fresh lamb blood into 1 L of blood agar solution.Colony dilution was done (10 -5 ) in order to distribute colonies on blood agar.Hemolysin expression activity will be seen in the form of clear circular nodes (halo) around the bacterial colonies.results About 15% of 70 colonies samples were not Enterococcus faecalis colonies.This suggests the possibility of other types of bacteria isolated in the sample and were not studied further.A total of 60 Enterococcus faecalis colony samples were taken for the next stage in this research (Figure 1).
The proportion of hemolysin gene in the isolated saliva samples were higher compared to root canal but no significant differences (Table 1).The proportion of hemolysin expression in root canal sample is less than in saliva samples, but no significant differences (Table 2 and Figure 2).Table 1 and 2 showed proportion of hemolysin gene and hemolysin expression not compare each other.On positive hemolysin gene detection showed that all of them had hemolysin expression in both clinical isolated samples.However, there were samples with hemolysin expression eventough no hemolysin gene detected (Table 3).The proportion of hemolysin expression without hemolysin gene was 23.1% in root canal samples and 18.2% in saliva samples.

discussion
The identification of Enterococcus faecalis bacteria was performed using chromatogenic agar as medium.This medium was used for positive Gram bacteria.A more distinctive color display showed bacteria colonies in chromatogenic agar, greenish blue color colonies suspected as Enterococcus faecalis or Enterococcus faecium.PCR 16sRNA was used to confirm the Enterococcus faecium colonies. 20The results showed that 85% of the samples were Enterococcus faecalis colonies and the remaining 15% were not.This suggests the possibility of other types of bacteria isolated in the samples and were not studied further.
The proportion of hemolysin gene in isolated root canal was 56.7%, smaller than the proportion of saliva which was 63.3%, there were no significant difference (p>0.05).In this study, cytolisin A primers were used to detect the presence of hemolysin gene, while hemolysin gene can detect using cytolisin A, B or M primers.The production and activation of hemolysin has several stages.Lysis Precursor factors (CylL) synthesized in ribosome, modified and undergo a process of translation by CylM, secreted from the cell by CylB, then activated by CylA. 15CylA primers selection was based on the final stage.Hemolysin gene was dicovered more in saliva than in the root canal, although not significant, due to the activation of extracellular stage of hemolysin in saliva, while in root canal was still in the initial stage where bacteria grow.One primers was used in this test (CylA), with only CylA might cause other types of hemolysin gene undetected.
The results of this study found that not all of the samples had hemolysin gene.A total of 43% of root canal samples and 36.7% saliva samples showed negative hemolysin gene.The literature showed that most of the strains of Enterococcus faecalis were non-haemolytic. 7,9Several studies supported the finding. 18Nevertheless, primers of all hemolysin phases should be used to ensure complete detection of all stages in the formation of hemolysin gene.
Hemolysin expression of root canal samples was less than saliva samples, this could be caused by silent Cyl genes; hemolytic activity on blood agar was not likely to happen because of environmental factors.The infected surrounding environment would be activated during the expression.It found that 6 of the 31 samples had CylA Enterococcus faecalis gene but none expressed hemolysin activity. 17Because of the small percentage, the role of this protein as a virulence factor considered very small.However, recent findings indicated that negative phenotype profile could be activated due to environmental factors to finally express hemolysin.
Researches using animal models demonstrated that Hemolysin as an important virulence factor.Antibiotic and corticosteroid combinations of treatment showed effective results in non-hemolysin strain cases, and has the effect to decrease tissue damage in endophthalmitis cases, while not useful in the case of strains that produce hemolysin. 15t was found that positive hemolysin gene indicates expression of hemolysin (100%) in both isolated clinical sources (root canal and saliva).However, there were samples that showed hemolysin expression without hemolysin gene existed.In root canal samples, the proportion of hemolysin expression with negative hemolysin gene was 23.1%, and 18.2% in the saliva samples.Only CylA was used in this study, this might showed negative result but perhaps other types of hemolysin gene was involved.
This research showed that hemolysin is one of the virulence factor of Enterococcus faecalis isolated from saliva and root canal.All samples detected hemolysin gene indicates expression of hemolysin.On the other hand, there were samples that had hemolysin expression without hemolysin gene.It can occur because the research used only one primers, in fact, there are three stage of formation hemolysin gene.Highly suggestion to use all of the kind of primers to detect all of the hemolysin gene for the further researches.

acknowledgments
This research was funded by the Research Fund DRPM UI Budget Year 2012.Directorate of Research and Community Service, University of Indonesia.

table 2 .
Hemolysin expression of clinical isolated samples * Chi Square Test figure 1. PCR 16sRN Result from 60 samples using a 100 bp DNA Ladder (GeneRuler, CA).

table 1 .
Hemolysin gene distribution of clinical isolated samples

table 3 .
Hemolysin expression presence distribution with hemolysin gene of clinical isolated samples