Abstract
Clostridioides (Clostridium) difficile infection is implicated as a major cause of antibiotic-associated diarrhea in hospitals worldwide. Probiotics, especially lactic acid bacteria, are the most frequently used alternative treatment. This study aims to identify potential probiotic enterococci strains that act against C. difficile strains and exert a protective effect on colon adenocarcinoma cells (HT-29 cells). To this end, nine Enterococcus strains isolated from the feces of breast-fed infants were investigated. They were identified as E. faecalis by 16s rRNA sequencing and MALDI-TOF. The probiotic properties including their viabilities in simulated gastrointestinal condition, cell adhesion ability, and their safety were evaluated. All strains exhibited more tolerance toward both pepsin and bile salts and adhered more tightly to HT-29 cells compared with the reference probiotic strain Lactobacillus plantarum ATCC 14917. Polymerase chain reaction (PCR) results exhibited that six of nine strains carried at least one virulence determinant gene; however, none exhibited virulence phenotypes or carried transferable antibiotic resistance genes. These strains did not infect Galleria mellonella when compared to pathogenic E. faecalis strain (p < 0.05). Moreover, their antibacterial activities against C. difficile were examined using agar well-diffusion, spore production, and germination tests. The six safe strains inhibited spore germination (100 – 98.20% ± 2.17%) and sporulation, particularly in C. difficile ATCC 630 treated with E. faecalis PK 1302. Furthermore, immunofluorescence assay showed that the cytopathic effects of C. difficile of HT-29 cells were reduced by the treatment with the cell-free supernatant of E. faecalis strains. These strains prevented rounding of HT-29 cells and preserved the F-actin microstructure and tight junctions between adjacent cells, which indicated their ability to reduce the clostridial cytopathic effects. Thus, the study identified six E. faecalis isolates that have anti-C. difficile activity. These could be promising probiotics with potential applications in the prevention of C. difficile colonization and treatment of C. difficile infection.
Highlights
Clostridioides (Clostridium) difficile is a Gram-positive rod, spore-forming, anaerobic, and toxin-producing bacterium
C. difficile infection (CDI) is a major cause of antibiotic-associated diarrhea (AAD) and hospital-acquired diarrhea, and its manifestations range from mild diarrhea to pseudomembranous colitis and death [1]
The results showed that nine E. faecalis strains adhered more strongly to HT-29 cells as compared to L. plantarum American Type Culture Collection (ATCC) 14917
Summary
Clostridioides (Clostridium) difficile is a Gram-positive rod, spore-forming, anaerobic, and toxin-producing bacterium. The environment around CDI patients and the large intestine of patients receiving broad-spectrum antibiotics have been found to be contaminated with the spores of C. difficile. In the absence of normal flora and under appropriate conditions, the spores mature into vegetative cells in the intestines, which eventually leads to CDI [2]. The pathogenesis of CDI is mediated by toxins, such as enterotoxin (TcdA) and cytotoxin (TcdB), which are encoded by tcdA and tcdB genes, respectively [4]. These toxins are major virulence determinants and exhibit both cytopathic and cytotoxic effects on mammalian cells. These effects are mediated by inactivation of the Rho family GTPases, such as Rho, Cdc, and Rac, leading to disrupted and disorganized F-actin cytoskeleton and tight junctions, morphological changes, and subsequent cell death [4, 5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
