EXPRESSION OF CagA GENE IN HELICOBACTER PYLORI ISOLATED FROM DENTAL PLAQUE OF MALE SNUFF USERS

Debnarayan Jana. Helicobacter pylori is recognized as the major etiological agent of chronic gastritis,gastric adenocarcinoma and lymphoma. Stomach lining along withSaliva have some carcinogenic Helicobacter pylori.The present study was carried out to investigate the presence of Helicobacter pylori in dental plaque sample collected from snuff addicted male person (who have been using snuff for >20 years)from Raxaul, Bihar and to study the probable relation between snuff and expression of CagA gene. Here, sixty three bacterial isolates from study group were identified as Helicobacter pylori. Out of them forty two wereCagA positive. Only twenty one bacterial isolates from control group were identified as Helicobacter pylori and two of them possess CagA gene.Result of this study conclude that snuff may stimulate the expression of CagA gene and may one of the potential threat for the development of oral cancer.


Debnarayan Jana.
Helicobacter pylori is recognized as the major etiological agent of chronic gastritis,gastric adenocarcinoma and lymphoma. Stomach lining along withSaliva have some carcinogenic Helicobacter pylori.The present study was carried out to investigate the presence of Helicobacter pylori in dental plaque sample collected from snuff addicted male person (who have been using snuff for >20 years)from Raxaul, Bihar and to study the probable relation between snuff and expression of CagA gene. Here, sixty three bacterial isolates from study group were identified as Helicobacter pylori. Out of them forty two wereCagA positive. Only twenty one bacterial isolates from control group were identified as Helicobacter pylori and two of them possess CagA gene.Result of this study conclude that snuff may stimulate the expression of CagA gene and may one of the potential threat for the development of oral cancer.

Introduction:-
Helicobacter pylori is one of the important gastric pathogenic bacteria,responsible for gastritis, peptic ulcer disease 1 and Stomach Cancer 2 . Helicobacter pylori is a spiral shaped, gram negative, lophotrichous bacterium having 3 µm in length and 0.5µm in diameter 3,4 .The mode of transmission and other epidemiology of Helicobacter pyloriinfection is unclear till today. Though some researchers think that the oral cavity harbors Helicobacter pyloriand may be the source of infection and transmission. Helicobacter pylori shows large diversity of strains. Most of the strain have an approximately 40 KB long Cytotoxic Associated Gene Pathogenicity Island (Cag PAI).This Island has about 30 genes out of these 29 genes are associated with the synthesis of Complex -Type IV secretion system and remains one gene code for a highly virulent protein CagA (1186 amino acid),has capability to cause cancer 5 by deregulating gastric epithelial SHP2 oncoprotein.Helicobacter pylori also contain UreA, UreB and UreC genes in their genome that is responsible for the synthesis of an enzyme Urease 6,7 that neutralize the acidic environment for their survival within stomach lining.Previous research proposed that long term use of snuff are linked with cancer of cheek,gums and inner surface of lips.NelukaFrenando et. al. found that,oral cavity of betel chewers provide more suitable environment for colonization of Helicobacter pylori compared to the oral cavity of non-betel chewers 8 .Thepurposes of this study was to identify the presence ofHelicobacter pylori strain in dental plaque sample from snuff users(have been using snuff for > 20 years)and to identify the expression of CagA gene by isolated Helicobacter pylori.

Materials & Methods:-
Subject selection and collection of sample:-Total 77 male subject were selected randomly in this study from slum area 5km away from Raxual , Bihar with mean age group 47.8 years who were using snuff throughout the working hour. Snuff users for less than 20 years were excluded from our study.From the same area, 52 male subject were selectedwho never took any kind of snuff during their entire life and no report of gastritis or peptic ulcer disease were considered as a control group. The study was carried out from August 2015 to November 2015. In the early morning before washing mouth, the dental plaque were collected by using sterile forceps and sterile cottonand then dipped into Brain heart Infusion(BHI) Broth (Himedia Laboratories Pvt. Ltd. India)supplemented with7% fetal calf serum and 25mM desferrioxamine(sigma) and stored in ice bag for further study in laboratories. All the test tubes with culture broth and sample were placed within a shaking incubator at 37°C for 24 hours then by Quadrant streak Plate Method (KRYSTAL-Biomedical Engineering,Florida Institute of technology) we Seperated different type of bacterial colony on Columbia Agar plate supplemented with 10% horse blood in a micro-aerobic atmosphere at 37° C.

Isolation of Helicobacter pylori by Biochemical tests:-
Bacterial colonies were identified as Helicobacter pylori by biochemical testsincludingGram staining, Urease Reaction Test and Catalase Test.
Gram staining:-Gram staining was carried out by using crystal violet solution,Lugal'siodine,saffranin, iodine acetone solution and PBS solution. At first a loop of bacterial growth were mixed with a droop of PBS solution on a glass slide (for all individual colony used individual slide separately) followed by fixing by low flame burner, crystal violet for 1 min, excess stain removal and covered by lugal's iodine for 1 min,hold in running laminar flow of tap water, covered with saffranin for 30sec and again wash in running tap water. Finally observed under compound light microscope.

Urease test:-
For each set four tube were taken and marked as S, UC, BC, B(S=sample,UC=Urea control,BC=bacteria control, B=blank). The S and UC marked tubes were 1/3 filled with media(Brain heart Infusion (BHI) Broth supplemented with7% fetal calf serum and 25mM desferrioxamine)contain urea and BC & B marked tubes were 1/3 filled with only media without urea and allow them for solidification. OnlyBC and S marked tubes inoculated with bacterial sample. Urea hydrolysing bacteria shows red or pink coloration on S marked test tube and other than urea hydrolysing bacteria shows no changes of colouration on S marked test tube.

Catalase test:-
Catalase test was carried out by using a specific protocol evolved by "Jackie Reynolds, Richland College, BIOL 2421"

Determination of virulence properties of isolated H. pylori:-
To detect the virulence property of Helicobacter pyloriwe performed Motility test Protease test by using a specific protocol evolved by "Jackie Reynolds, Richland College, BIOL 2421" Bio-film Assay:-Biofilm assay 9 were performed by method used by C. Ghosh and S. Biswas et al 10

DNA isolation & Detection of CagA gene:-
Bacterial culture of Helicobacter pylori was harvested in PBS. Then cells were pelleted in 300 µl SET (25% sucrose, 1 mM EDTA, 10 Mm Tris HCL) ,lysozyme (10 mg/ml, dissolved in SET),EDTA (0.5M), proteinase K (10 mg/ml), 10% Sodium dodecylsulphate and mixture was incubated at 65° C for 1-2 hours. Then DNA was extracted using phenol-chloroform, Na-acetate,ethanol.A total of 63 DNA samples from study group and 21 DNA samples from control group were amplified through PCR with upstream primer: 5' ATGACTAACGAAGCCATT 3' and downstream primer: 5'TTAAGATTTTTGGAAACC 3' to detect presence of CagA as a virulence marker. PCR reaction mixture was carried out in 25 µl volume using 10 ng of genomic DNA, 1U of Taq polymerase, and 10 pmol of each primer, 0.25 mM (each)deoxynucleotide triphosphate, and 2-3 mM MgCl 2 in PCR buffer. PCR amplification reactions were performed in a thermal cycler with a programme consisting of initial denaturation at 94 • C for 1min and 35 cycles of denaturation at 94 • C for 1 min, annealing at 59 • C for 1 min, extension step at 72 • C for 1.3 min and final extension step at 72 • C for 20 mins. Amplified PCR products were analyzed by 1% agarose gel containing Ethidium Bromide (EtBr) in Tris Acetate buffer.

Results & Discussion:-
We identified the isolates from study group and control group as H. pylori by using Comulbia Agar media with specific microaerobic environment and by selective biochemical tests(Gram staining, Urease Reaction Test, Catalase test).In study group out of 77 isolates 63 were found as gram-ve bacteria. Morphologically they were Round opaque (74.02%) and Round translucent(9.09%). Only 3.9% were found as gram +ve Rugose colony. In control group out of 52 isolates 21 were found as gram -ve, and morphologically 17.3% were round opaque , 11.5% were round translucent and 71.15% were found as rugose colony.On the basis of protease and motility test we did not find any significant difference of virulence property in between round opaque and round translucent colony.Phenotypic appearance has a great importance for pathogenicity in bacterial life cycle as it was reported earlier that opaque colony is more virulent than the translucent one 11 and several recent studies reports that H. pylori form Biofilm either in vitro 12 or in vivo 13 . In our study round opaque colonies were more adherent to the hydrophobic glass surface compared to round translucent colonies among the study group. In control group the adherent property of both type of bacterial colonies were less compared to the study group (Table1.1). In study group and control group opaque colonies were found to be more adherent compared to round translucent colonies. Adherent property of bacteria is one of the determining factor for virulence. We found Round Opaque colony posses more virulent property(Table1.2).  14,15 . Previous studies suggest that CagA positive strains can significantly increase the risk for developing severe gastritisand gastric carcinoma compared with CagA negative Helicobacter pylori strains 16,17,18 .Helicobacter pylorihas only a transient presence in the oral cavity as growth of the organism is inhibited by the antagonist effects of oral microflora 19 . It is also evident that both chewing tobacco reduces salivation and alter the normal oral microflora 20 . Some other reports revealed that tobacco was significantly associated with presence of Helicobacter pylori, as tobacco modulates the periodontal defences and thus favours the colonization of this organism in buccal cavity 21 . On the basis of these prior reports we predict that similarunderlying reasons may be responsible for significant occurrence of Helicobacter pylori isolates in dental plaque of male snuff users study group incompared to snuff non-using control malewho did not demonstrate significant presence of Helicobacter pylori in their dental plaque sample.

Conclusion:-
From this study we conclude that CagA gene is significantly express in male snuff users but we did not conduct any studies to demonstrate the direct role of snuff in CagA gene expression and wepredict that these CagA positive Helicobacter pyloriisolates from dental plaque of male snuff users may be significant for increasing chance to develope oral infection as well as carcinoma in future.