Gene Expression of Biofilm Regulatory Protein A (Brp A) in Streptococcus mutans Isolated from Oral Cavity

A total of 100 oral swabs were collected from patients  suffering from dental caries and priodontal infections, ranging from 15-65 years old of both genders whom admitted to the Al-Diwaniyah Teaching Special Center of Dentistry within the period from December 2012 to March 2013, in order to  quantify the gene expression of biofim regulatory protein (Brp A) in Streptococcus mutans (S. mutans) isolates by using the Real Time polymerase chain reaction. The gene expression of biofilm regulatory protein A(Brp A) was quantified in comparison with housekeeping gene 16s rRNA as a reference gene of biofilm S.mutans by using reverse transcriptase polymerase chain reaction (RT-PCR).The results revealed that the expression (absolute) of BrpA gene is decreased in the test treatment (S.mutans with 1% glucose+ 50% curcumine) in comparison with control  (S.mutans grown in 1% glucose only). 
In conclusion, the present study established the role of Brp A gene in biofilm formation and recommended to possibility of using the curcumine  as adddative material in composition of toothpaste due to their inhibitory and reduction effect on biofilm S.mutans isolates.


Gene Expression of Biofilm Regulatory Protein A (Brp A) in Streptococcus mutans Isolated from Oral Cavity
In conclusion, the present study established the role of Brp A gene in biofilm formation and recommended to possibility of using the curcumine as adddative material in composition of toothpaste due to their inhibitory and reduction effect on biofilm S.mutans isolates.

Introduction:
Dental plaque is a complex microbial biofilm , it is considered as the primary etiologic in development of dental caries and periodontal disease . The bacteria form (60% -70%) of its volume, may reach to 300 -500 cell. These accumulation subjected teeth and the periodontium to high concentration of bacterial metabolites , which result in dental infection . Plaque bacteria produce two forms of toxin which work as extracellular enzymes and endotoxin (1). S.mutans are primary colonizer of oral hard and soft tissue . It is highly cariogenic because it ferment dietary sugar (sucrose) to lactic acid , which is dissolving tooth structures and lead to cavity (2).
Virulence factors of S. mutans help to protect the bacterium against possible defenses and maintain its ecological niche in the oral cavity , acid tolerance , protease production and production of glucosyltransferases and intracellular polysaccharides ( 3 ) .Beyond initial adherence, it appears that a variety of genes are required for the adaptation of S. mutans and other oral streptococci in biofilms.These include genes associated with intercellular communication systems and environmental sensing systems, regulators of carbohydrate metabolism, and adhesion-promoting genes (4). Among the genes were brpA (1YtR), comDE and vicR encoding regulatory proteins (5), A number of studies have indicated that expression of the genes responsible for biofilm formation depends on environmental conditions (6), and is also genetically regulated (7). It was therefore of interest to identify transcriptional changes for several genes that accompany the formation of an in vitro biofilm by the most important pathogens in the development of dental caries, such as S.mutans. To assess the expression of genes known to be involved in biofilm formation by S.mutans, for comparison with the expression of genes in planktonic cells, real time PCR was used.
Previous studies have shown that BrpA play a major role in acid and oxidative stress tolerance and biofilm formation by S.mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity (8).

Aim of the study:
In the present study we attempt to quantify the intensity of biofilm formation by S. mutans clinical strains by measuring biofilm volume and further analyze how expression differ between various isolated S.mutans strains that showed either high or low levels of ability to form biofilm, and we compared the results with those regarding biofilm formation ability by using reverse transcriptase Polymerase Chain Reaction (PCR) method.

Methods
Specimens collection: A total of 100 swabs were taken by inserting into the middle of cavity in case of carious tooth, while swabs were taken from dental plaque in case of periodontitis and gingivitis over the tooth surfaces. Swabs for culture were placed in tubes containing transport media to maintain the bacterial swab vital until being taken to the laboratory. Each specimen was immediately inoculated on blood agar plates, nutrients agar and chocolate agar. All plates were incubated aerobically and anaerobically by candle jar at 37 C o for 24-48 hrs. Isolation and identification: Each transport media swab was inoculated into universal tube containing 5ml of nutrient broth and incubated at 37 C o for 18-24 hrs, a loop full of broth was streaked on surface of nutrient agar, blood agar, MacConkey agar which then incubated at the same conditions. Colonies had been primarily identified depending on morphological colonies characteristics were recorded on the media used in this study for primary identification of microorganisms and microscopic properties. Gram , s stained slide was done for each individual isolate of microorganism recovered from the different agar media to study the microscopic properties of genera (9) and the biochemical tests done by using VITEK 2 system.

Quantitative Reverse Transcriptase Real-Time PCR:
Quantitative Reverse Transcriptase Real-Time PCR technique was performed for measurement of absolute quantification (mRNA copy numbers) and relative quantification (gene expression analysis) for Biofilm regulation protein A gene in treatment and control S. mutans isolates. The Biofilm S. mutans plate design for Real-time PCR is included: 1-Treatment (T) S.mutans isolates which grown in trypticase soya broth with 1% glucose and curcumine(50%) then incubated at 37 0 C for 18 hours. 2-Control (C) S.mutans isolates which grown in trypticase soya broth with 1% glucose only then incubated at 37 0 C for 18 hours. This technique was done according to the method described by (10)  qPCR results was performed for measurement of absolute quantification (mRNA copy numbers) and relative quantification (gene expression analysis) for Biofilm regulation protein A gene and housekeeping gene 16S rRNA in treatment and control of S.mutans isolates. This assay was performed by using AccuPower TM Green Star Real-Time PCR kit that depended on SYBER Green dye detection of gene amplification in Real-Time PCR system as follow: Absolute quantification: RT-qPCR absolute quantification analysis was included in the measurement of mRNA copy numbers of target gene (brpA) and compared with housekeeping gene (16S rRNA) in test and control by using Dilution of genomic DNA standard curve for (1x10 8 1x10 6 , 1x10 4 , 1x10 2 copy number) as follow; Genomic DNA standard curve for S. mutans standard strain was prepared by estimation the concentration of extracted DNA by Nanodrop spectrophotometer ng/µL and whole genomic sequence of S.mutans, then used DNA copy number calculation formula online. The (1x10 8 copy number) that obtained by DNA copy number formula was diluted for 10 fold as (1x10 8 , 1x10 7 , 1x10 6 , 1x10 5 1x10 4 ,1x10 3 , 1x10 2 , 1x10 1 ) and only (1x10 8 , 1x10 6 , 1x10 4 , 1x10 2 ) genomic DNA dilution was used for preparation of standard curve for amplification of brpA( Table 1) and 16S rRNA genes ( Table  2):  After that, qPCR master mix components mentioned above loaded on Accopwer Green star qPCR premix standard plate tubes that contain the SYBER Green dye and other PCR amplification components, then the plate mixed by Exispin vortex centrifuge for 3 minutes, than placed in Exicycler Real-Time PCR system, for thermocycling.

Results and Discussion:
The S. mutans was comprised the high ratio (40.47%) in comparison with the other streptococcus species (S. sangius 11.90%, S. mitis 10.70%, S.intermedius 10.70%, S.oralis 9.50%, S.salivarius 5.95%, S.pneumoniae 4.76%, S.pyogenes 3.57%, S.sorbinus 2.38%). No isolate of S.mutans were isolated from the healthy control. This explains that S.mutans represent the main causative agent of dental diseases, especially dental caries and to a lesser degree the periodontal diseases. All S. mutans colonies showed αhemolysis (green coloration on blood agar plates ) which was not evident after 24 hrs. of incubation and required further incubation. Gram staining were adopted and showed Gram positive cocci arranged in chains. Biochemical and physiological characteristics of all S. mutans isolates showed negative reactions with catalase, oxidase tests and positive for sugars(glucose. lactose, sorbitol. manitol, and raffinose).
Bacterial identification was specified by using VITEK 2 compact system. Based on the results of VITEK 2, S. mutans was the most frequent species of streptococcus.
Biofilm formation can be considered as a developmental process, which is characterized through the identification of structural and regulatory genes required at the various steps of the process (11). To gain an insight into molecular estimation differences in gene expression of biofilm regulation protein A gene in test and control of S. mutans isolates, the researcher used quantitative Reverse Transcription Real-Time PCR (RT-qPCR) was performed .This assay also used by Shemesh (12) whom used real-time RT-PCR in differential gene expression profiling of S. mutans cultured under biofilm and planktonic conditions.RT-qPCR absolute quantification analysis included the measurement of mRNA copy numbers of target gene (brpA) and compared with housekeeping gene (16S rRNA) in test and control groups by using Dilution of genomic DNA standard curve for (1x10 8 1x10 6 , 1x10 4 , 1x10 2 copy number). The results of DNA standard curve were shown in Real-Time PCR amplification plot (Fig. 1) and Real-Time PCR standard curve (Fig. 2).  The results of housekeeping gene (16S rRNA) are shown in Real-Time PCR amplification plot (Fig. 3) and Real-Time PCR standard curve (Fig. 4).    Absolute quantification of mRNA transcript levels in brp A gene and house keeping gene (16S rRNA) were obtained by compared threshold cycle numbers of these genes with threshold cycles of genomic DNA standard curve in Real Time PCR system. This procedure is consistent with Bustin ( 2 0 ) who indicated that when appropriate standard curves are used, absolute copy numbers of mRNA can easily be calculated.
The absolute quantification results of Biofilm regulation protein A gene showed a clear decrease in mRNA copy numbers in test isolates (4.12E+03± 1705.22074) compared with control isolates (2.05E+04± 1035.28311) . The statistical analysis of absolute quantification of brpA gene found significant differences (P ≤ 0.05) in test isolates compared with control. Whereas, the absolute quantification results of housekeeping gene (16S rRNA) revealed no differences in mRNA copy numbers in test isolates (4.76E+03±1012.04414) compared with control groups (4.42E+03±260.34166).
The statistical analysis of absolute quantification of 16SrRNA gene was found no significant differences (P≥0.05) in test isolates compared with control (Fig. 6).
The absolute quantification results of housekeeping gene (16S rRNA) showed no significant differences (P≥0.05) in mRNA copy numbers in test isolates. These results interpreted that house keeping gene (16S rRNA) is suitable as reference gene for all S. mutans isolates that used in the present study, and these findings consistent with Paule & White ( 4 0 ) whom evaluated rRNAs, which constitute 85-90% of total cellular RNA, are useful internal controls, has expression stability when exposed to different stress conditions. Quantification of mRNA transcription can be either relative or absolute, relative quantification determines the changes in transcription of a gene and is often adequate. During the RT-PCR assay, the target Ct is compared directly with the calibrator Ct and is recorded as containing either more or less mRNA. In contrast, absolute quantification of transcription allows the precise determination of copy number per cell, total RNA concentration, or unit mass of tissue. It requires the construction of an absolute standard curve for each individual amplicon to ensure accurate reverse transcription and PCR amplification profiles (10). Therefore, absolute quantification was used in this study.