Screening Enteric Bacteria with Inhibitory Effects Against Pathogenic Clostridium difficile and Its Spore

Abstract

Clostridium difficile is a major cause of C. difficile infection (CDI) and antibiotic-associated diarrhea with the increasing rate of mortality. The standard treatments of CDI are antibiotics. However, patients relapse from the infection within 2 months with the risk of recurrences increasing to 50-65% after two or more episodes of the infection. Recently, alternative treatment for the prevention of CDI and relapse of CDI has included monoclonal antibodies, vaccination, toxin neutralization, fecal microbiota transplantation, and probiotics. Thus, the aims of this study are to identify new enteric bacteria as probiotic against C. difficile and its spore as well as to developed and evaluate the formula of potential probiotics. Nine Enterococcus faecalis including PK1003, PK1201, PK1202, PK1301, PK1302, PK1801, PK2003, PK2004, and PK2502 isolated from breast-fed infant stools could inhibit C. difficile. These strains exhibited characteristics of probiotic including showed higher levels of viability in acid condition (pH 2) as well as tolerance to pepsin and bile salts than Lactobacillus plantarum ATCC 14917 a reference probiotic strain. All isolated strains could effectively adhere to HT-29 human colon cancer cell lines. They were susceptible to ampicillin, penicillin, imipenem, and vancomycin but resistant to gentamicin. In addition, Polymerase chain reaction demonstrated that all isolated strains were not carried vancomycin-resistant genes. Moreover, phenotypic assays showed that none of the isolated strains exhibited hemolysin production, gelatinase activity, and mucin degradation except 3 isolates could produce hemolysin. The effect of virulence factors of E. faecalis were tested by animal model (Galleria mellonella). Nine isolated strains did not affect the five-day survival of G. mellonella larvae when compared to that of negative control. These strains were reduced the Clostridial toxic effect upon HT-29 cell lines. They prevent HT-29 cell rounding, preserving the F-actin microstructure and tight-junctions between adjacent cells. Furthermore, these E. faecalis isolated strains could inhibit the spore germination and sporulation of toxigenic C. difficile strains. Mpreover, the appropriate formula of potential probiotic were determined. Nine E. faecalis suspended with 10% skim milk can prevent cell damage during freeze- drying process and long-term storage. After freeze-drying process, E. faecalis sustain and retain adequate viability in appropriate storage conditions with probiotic properties. Nine E. faecalis exhibit characteristics of probiotic. Overall, all 9 strains of E. faecalis showed potential probiotics that had ability to inhibit C. difficile and its spore. Thus, these probiotic candidates are very attractive for developing novel foods or pharmaceutical products. The efficiency of these potential probiotics to inhibit CDI in vivo and safety should be further evaluation in vivo.