In Vitro Evaluation of Antimicrobial Activity of Lactic Acid Bacteria against Clostridium difficile

Abstract

Clostridium difficile infection (CDI) has become a significant threat to public health. Although broad-spectrum antibiotic therapy is the primary treatment option for CDI, its use has evident limitations. Probiotics have been proved to be effective in the treatment of CDI and are a promising therapeutic option for CDI. In this study, 4 strains of lactic acid bacteria (LAB), namely, Lactobacillus rhamnosus (LR5), Lactococcuslactis (SL3), Bifidobacterium breve (BR3), and Bifidobacterium lactis (BL3) were evaluated for their anti-C. difficile activity. Co-culture incubation of C. difficile (10(6) and 10(10) CFU/ml) with each strain of LAB indicated that SL3 possessed the highest antimicrobial activity over a 24-hr period. The cell-free supernatants of the 4 LAB strains exhibited MIC50 values between 0.424 mg/ml (SL3) and 1.318 (BR3) mg/ml. These results may provide a basis for alternative therapies for the treatment of C. difficile-associated gut disorders.

Keywords: Clostridium difficile infection; antimicrobial activity; cell-free supernatant; lactic acid bacteria.

Fig. 1.. EC₅₀ of SL3, LR5, BL3 and BR3 co-cultured with Clostridium difficile KCCM 5009 in RCM broth anaerobically for 6 hr at 37℃. SL3 (A), LR5 (B), BL3 (C), and BR3 (D) at 10⁸, 10⁹, or 10¹⁰ CFU/ml were co-cultured with C. difficile at 10⁶ CFU/ml for a simulation of disease prevention and SL3 (E), LR5 (F), BL3 (G), and BR3 (H) at 10⁸, 10⁹, or 10¹⁰ CFU/ml were co-cultured with C. difficile at 10¹⁰ CFU/ml for a simulation of disease treatment.

Fig. 2.. Flow cytometric analysis of C. difficile incubated with ½, 1, and 2 MIC cell-free supernatants of Lc. lactis SL3 for 24 hr. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. Q2: live cell, Q1: dead cell, and Q3: injured cell and debris.

Fig. 3.. Flow cytometric analysis of C. difficile incubated with ½, 1, and 2 MIC cell-free supernatants of L. rhamnosus LR5 for 24 hr. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. Q2: live cell, Q1: dead cell, and Q3: injured cell and debris.

Fig. 4.. Flow cytometric analysis of C. difficile incubated with ½, 1, and 2 MIC cell-free supernatants of B. lactis BL3 for 24 hr. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. Q2: live cell, Q1: dead cell, and Q3: injured cell and debris.

Fig. 5.. Flow cytometric analysis of C. difficile incubated with ½, 1, and 2 MIC cell-free supernatants of B. breve BR3 for 24 hr. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. Q2: live cell, Q1: dead cell, and Q3: injured cell and debris.

Fig. 6.. Fluorescent microscopic images of C. difficile incubated with ½, 1, and 2 MIC cell-free supernatants of Lc. lactis SL3, L. rhamnosus LR5, B. lactis BL3, and B. breve BR3. C. difficile cells were stained with Live/Dead Baclight kit after incubation with the cell-free supernatants. Live cells: green, dead cells: red, yellow and orange: injured.