Isolation, characterization and strain-specific detection of canine-derived Lactobacillus acidophilus
Year: 2014
Type: dissertation
Abstract: Lactobacilli are commensal gastrointestinal microbes commonly utilized in probiotic products, as they are believed to bestow multiple beneficial health effects to the host. Most well-studied lactobacilli have been isolated from feces. However, fecal isolates do not reflect the microbiota present in the upper gut, since different niches provide different microbial habitats. The fistulated dog model facilitates investigation of the microbiota in fresh intestinal samples without disturbing the physiology of the canine gut. In this study, jejunal lactobacilli from five permanently fistulated beagles were studied. We found that facultative Lactobacillus strains were abundant in the jejunal microbiota, and L. acidophilus was the dominant species. Repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprint profiles of L. acidophilus isolates revealed one predominant strain, named LAB20. Adhesion is an important factor in bacterial colonization of the host gut. In order to adhere, compete, and dominate within the host, numerous bacterial cell-surface factors are required to interact with the host mucosa. In this study, the protein profile of LAB20 was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and cell structure was observed via transmission electron microscope (TEM). A surface (S) layer protein was revealed from LAB20. S-layer proteins form crystalline arrays of proteinaceous subunits in the outer layer of the cell wall and are involved in mediating bacterial adhesion to host surfaces. Inverse PCR revealed the DNA sequence of the LAB20 S-layer protein, alignment with other lactobacilli S-layer protein genes showed it was a novel one. The discovery of this novel S-layer protein in LAB20 enabled us to develop a strain-specific detection method. Real-time PCR primers targeting the variable region of the S-layer protein gene were used to detect and quantify LAB20 in dog intervention studies. We found that LAB20 persisted in one dog for over 6 weeks after the feeding period (6 × 108 CFU daily for 5 days), whereas the five dogs in the other study maintained high LAB20 numbers only during the feeding period (108 CFU daily for 3 days). Cultivation of fecal samples demonstrated that LAB20 transits through the dog gut and can be identified based on colony morphotype. TEM revealed a putative extracellular polysaccharide (EPS) layer that comprised LAB20’s outermost structure. Using antisense RNA strategy, EPS production was manipulated to investigate its potential impact on the ability of LAB20 to adhere to mucus and epithelial cells. LAB20 displayed significantly higher adhesion in canine cecal mucus relative to the EPS mutant SAA658 and could adhere to Caco-2 and HT-29 epithelial cells. This suggests that wild-type EPS plays an integral role in the adhesion of LAB20 in the host gut. Moreover, LAB20 attenuated lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in HT-29 cells, which indicates that LAB20 could be a probiotic candidate with anti-inflammatory properties. In conclusion, this study investigated the surface structure, persistence, adhesion ability, and probiotic potential of LAB20, the dominant L. acidophilus strain in the canine small intestine. Our results suggest that LAB20 has potential as a canine probiotic.
Source: Työväentutkimus Vuosikirja
Author: Yurui Tang
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2199621001