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http://dx.doi.org/10.4014/jmb.1705.05055

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting  

Yang, Zhen-quan (College of Food Science and Engineering, Yangzhou University)
Xue, Yu (College of Food Science and Engineering, Yangzhou University)
Rao, Sheng-qi (College of Food Science and Engineering, Yangzhou University)
Zhang, Mi (College of Food Science and Engineering, Yangzhou University)
Gao, Lu (College of Food Science and Engineering, Yangzhou University)
Yin, Yong-qi (College of Food Science and Engineering, Yangzhou University)
Chen, Da-wei (Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University)
Zhou, Xiao-hui (Department of Pathobiology and Veterinary Science, University of Connecticut)
Jiao, Xin-an (Jiangsu Key Laboratory of Zoonosis)
Publication Information
Journal of Microbiology and Biotechnology / v.27, no.11, 2017 , pp. 1971-1982 More about this Journal
Abstract
Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.
Keywords
Lactobacillus rhamnosus; pilus subunit; polyclonal antibody; colony immunoblotting;
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