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Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile.

ABSTRACT: Lactic acid bacteria, Enterococcus faecium and Lactococcus lactis, previously isolated from Thai fermented sausages were elucidated their probiotic properties especially in the control of Clostridium difficile 630. Both isolates survived in simulated gastric solution at pH 3 followed in simulated intestinal solution at pH 8. The presence of skimmed milk also helped the bacteria to survive through acidic and alkaline in gastrointestinal conditions. The adhesion properties of both isolates were tested using a human colon adenocarcinoma cell line. The result showed that both isolates exhibited desirable probiotic properties which adhered to Caco-2 cells. The neutralized cell-free supernatant of both isolates demonstrated that no cytotoxicity toward Caco-2 cells vice versa cell-free supernatant of C. difficile 630 toward Caco-2 cell demonstrated high toxicity. The immunomodulation effect in response to bacterial neutralized cell-free supernatant and cell-free supernatant was also studied. The expression level of pro-inflammatory cytokine of Caco-2 cell which are tumor necrosis factor-? and interleukin-8 was evaluated using quantitative reverse transcriptase PCR. Both isolates were able to diminish the expression level of TNF-? and IL-8 induced by the cell-free supernatant of C. difficile 630. Hence, these isolates would be able to improve the gut health through counteracting the C. difficile-associated intestinal inflammation in human cell lines. These results may contribute to the development of the isolates using as probiotics.

SUBMITTER: Dowdell P

PROVIDER: S-EPMC7306037 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Project description:BackgroundOral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention.MethodsRecombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay.ResultsAfter the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups.ConclusionRecombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

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Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile.

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