In vitro adhesion assay of lactic acid bacteria, Escherichia coli and Salmonella sp. by microbiological and PCR methods

• Main Authors: Didier Montet, Gerard Loiseau, Penkhae Wanchaitanawong, Sunee Nitisinprasert, Nuntaporn Pungsungworn
• Format: Article
• Language: English
• Published: Prince of Songkla University 2006-03-01
• Series: Songklanakarin Journal of Science and Technology (SJST)
• Subjects: adhesion assay; microbiology assay; Lactobacillus; PCR; Escherichia coli; Salmonella sp.
• Online Access: http://www.sjst.psu.ac.th/sjst.psu.ac.th/journal/28_suppl1_pdf/13_acid_bacteria.pdf

In vitro adhesion assay using Lactobacillus reuteri KUB-AC5 as a test strain has been studied by applying simple PCR reaction together with image analysis and plate count techniques. Critical factor affecting the PCR method was quality and quantity of DNA. The cell lysis technique was modified to optimize this method. Thus, lysozyme and proteinase K were added to lyse the cells, followed by SDS solution to obtain a complete cell lysis. Only PCR products from total cells (TC) were obtained, with low consistency, but none from cells bound to mucus (BC) at either 0.1 or 0.5 mg/mL concentration. It was hypothesized that the attached cells might not be extracted into the cell suspension. Therefore, 1% SDS solution and 0.1M NaOH were used directly in the extraction. As expected, PCR products were observed when both TC and BC were used as a DNA template. Adhesion appeared at a wide range of 0-45%, with low consistency. Therefore, a simple microbiological method (plate count) was used. The extraction of bound cells into cell suspension was critical in this method. Extraction times of 20, 60, 120 and 150 min were tried. Results showed that maximum cell number was obtained with 120 min extraction. L. reuteri KUB-AC5, L. reuteri KUB-AC16, L. reuteri KUB-AC20, L. salivarius KUB-AC21, L. acidophilus KV-1, Escherichia coli E010, Salmonella sp. S003, E. coli ATCC8739, and S. typhimurium ATCC 13311 exhibited adhesion activity of 21.6%, 0.8%, 5.7%, 1.1%, 23.1%, 10.7%, 10.3%, 4.4% and 3.2%, respectively. Among the 9 types of microorganisms tested L. acidophilus KV-1 and L. reuteri KUB-AC5 showed higher adhesion activity than the others.