Assessing the Adhesion Properties of Isolated Probiotic Bacillus subtilis to Human Colonic Epithelial Cells

M. Krishna moorthy¹*, Bijaya Kumar Nayak², Anima Nanda¹, Anbarasi. P³

¹Department of Biomedical Engineering, Sathyabama University, Chennai, Tamil Nadu, India. 600 119

²Department of Plant Science & Plant Biotechnology, KMCPGS, Airport Road, Lawspet, Pondicherry, India. 605 008

³Avanz Bio Private Limited, 12 MES Road, East Tambaram, Chennai, Tamil nadu, India 600059

*Corresponding Author E-mail: krish2285@gmail.com

ABSTRACT:

Key criterion for selection of a Probiotic should have the adherence property of bacteria to human epithelial cells and mucosal surface. In this study we assess the adhesion property of selected indigenous probiotic Bacillus subtilis strain by the ability to adhere to human epithelial cells. (Yudhishtir, et, al. 2010)

Bacillus subtilis is isolated from fermented plant beverage type coconut toddy, among 137 organisms Bacillus subtilis showed good probiotic characteristics further to its characterization it’s studied for its adhesion property to human epithelial cells.

Colonization potential is tested by using CaCo₂ and HT29 colonic adenocarcinomal human intestinal epithelial cell lines. Bacillus subtilis strain that adhered to prescribed cell lines was also examined by plating in to the suitable medium by plating them after trypsinization. This colony shows that the isolated Bacillus subtilis has their ability to adhere to epithelial cells and thus this could have better prospects to colonize the gut with extended transit.

KEYWORDS: Adhesion, CaCo2, HT29, Bacillus subtilis, probiotic.

1. INTRODUCTION:

Microbes inhabit in enormous numbers and in great diversity in intestinal tract, urogenital tract, skin, oral cavities, nasal cavities, and the exposed part of the body to the outside environment which favors the bacterial colonization. Around 10¹⁴ cells on the human body is identified as human commensals (Drasar and Hill 1974). Although major part of the microbial population resides in human GUT region but they are largely got hindered due to the oral drug and dietary intake. Thus a selective probiotic bacterium should be extremely effective to colonize in the extreme conditions and they should to the human intestinal cells. CaCo₂ and HT29 cell lines are generally expressing the structural and functional property of normal human enterocytic function and these cells are generally used for the in vitro study (Wang B. 2008 and Chauviere G. 1992). The present study was done with objective to identify the adherence potential of Bacillus subtilis isolated from coconut toddy using CaCo₂ and HT29 cell lines.

2. MATERIALS AND METHODS:

2.1 Human Adenoma epithellial cell line:

The cell lines (CaCo₂ and HT29) were obtained from National Centre for Cell Science (NCCS), Pune, India. The cells were grown in T25 culture flasks containing DMEM supplemented with 10% Fetal Bovine Serum (FBS), L-Glutamine and antibiotic antimycotic solution. The cells were maintained in a 5% CO₂ incubator at 37°C. Cells were observed everyday on its morphology, sterility and vacuole formation. Cells should be maintained till it reaches 80% confluence. Cells are cryopreserved and stored in liquid nitrogen for long term storage (ATCC, 2010).

2.2 Adhesion Assay:

Adhesion assay was carried out after 45-80 passages for HT-29 and 30-60 for CaCo₂ cell lines. Adhesion of the Lactobacillus cultures was measured.

The cell suspension with 1X10⁵ cells prepared in 4 ml complete DMEM medium was transferred to each well of six-well tissue culture plates.

The medium was changed every alternate day. When cells reached 80 per cent confluency, the medium was replenished each day consecutively for 20 days for both the cell lines.

2.3 Transfection of cell line:

After the incubation period the spent medium was completely removed 24 h before adhesion assay and cells were fed with DMEM medium lacking antibiotics. The cells were then washed twice with phosphate-buffered saline (PBS).

An aliquot of two ml of DMEM (without serum and antibiotics) was added to each well and incubated at 37°C for 30 min. Different cultures suspended in 1 ml serum free DMEM medium were added to different wells.

The plates were incubated at 37°C in 5 per cent CO₂-95 per cent air for 2 h. The monolayers were washed five times with sterile PBS. The adhesion score was measured by enumerating adhered bacteria per 20 different microscopic fields.

Serial Dilution and Plating:

Cells from the monolayer are detached by trypsinization and they are aspirated to make a Homogenous suspension. The cell lines are taken and they are mixed in 0.89% Saline. They are serially diluted using the saline and from each dilution plating is done using Nutrient agar medium and they are incubated at 37 °C for 24-48hrs.

3. RESULTS AND DISCUSSION:

ADHESION ASSAY:

The bacterial culture was added to the monolayer cells and incubated. Further, they were serially diluted and plated on agar media. Over an incubation of 24-48hours, only the transfected cells were found to grow on the agar media. The number of colonies grown were counted and recorded. When the transfected cells were analysed on SEM, the interaction and binding affinity between cells and bacterial isolate is very prominent.

SEM Image of Transfection of SEM image of Transfection of Bacillus

Bacillus subtilis on CaCo2 cell line subtilis on HT29 cell line

PLATING METHOD:

After trypsinization process the grown monolayer are serially diluted and they are plated on nutrient agar and this clearly shows the growth of Bacillus subtilis on the transfected cell lines this shows that the test organism Bacillus subtilis has a good adhesive property to human epithelial cell lines.

Nutrient Agar showing Bacillus subtilis

4. CONCLUSION:

The Bacillus subtilis strains isolated from the coconut toddy shows better adherence property to human epithelial cells under in vitro conditions. Although the in vitro adherence assay for assessing the adherence potential of isolated strain may not exactly the same as human intestinal GUT flora although this can be further justified by establishing their functional efficacy in human subjects in subsequent clinical studies

5. REFERENCE:

1- Chauviere G, Coconnier MH, Kerneis S, Fourniat J, Servin AL. (1992), Adhesion of Lactobacillus acidophilus strain LB to human enterocytes – like Caco2 cells. J Gen Microbol; 138 : 1689-96

2- Drasar, B.S. and Hill, M.J. (1974) Human intestinal flora. Academic Press, New York, NY.

3- M. Krishna Moorthy; Bijaya Kumar Nayak ; Anima Nanda. J Chem and Pharm Res 2015. 7(3), 95-101.

4- Mary Ellen Sanders, Considerations for use of probiotic bacteria to modulate human health, Dairy of Food Culture Technologies, Littleton, CO 80122- 2526.

5- Raj Kumar Duary, Yudhishthir Singh Rajput, Virender Kumar Batish and Sunita Grover (2011), Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells; Indian J Med Res 134 : 664-671

6- Wang B, Wei H, Yuan J, Li Q, Li Y, Li N, et al, (2008), Identification of a surface protein from Lactobacillus reuteri JCM1081 that adhere to porcine gastric mucin and human enterocytes like HT29 cells. Crr Microbiol; 57 : 33-8

Received on 03.03.2016 Modified on 01.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 394-396

DOI: 10.5958/0974-360X.2016.00071.8