In Vitro Evaluation of the Therapeutic Potential of Phage VA7 against Enterotoxigenic Bacteroides fragilis Infection

Abstract

Since the beginning of the 20th century, bacteriophages (phages), i.e., viruses that infect bacteria, have been used as antimicrobial agents for treating various infections. Phage preparations targeting a number of bacterial pathogens are still in use in the post-Soviet states and are experiencing a revival in the Western world. However, phages have never been used to treat diseases caused by Bacteroides fragilis, the leading agent cultured in anaerobic abscesses and postoperative peritonitis. Enterotoxin-producing strains of B. fragilis have been associated with the development of inflammatory diarrhea and colorectal carcinoma. In this study, we evaluated the molecular biosafety and antimicrobial properties of novel phage species vB_BfrS_VA7 (VA7) lysate, as well as its impact on cytokine IL-8 production in an enterotoxigenic B. fragilis (ETBF)-infected colonic epithelial cell (CEC) culture model. Compared to untreated infected cells, the addition of phage VA7 to ETBF-infected CECs led to significantly reduced bacterial counts and IL-8 levels. This in vitro study confirms the potential of phage VA7 as an antibacterial agent for use in prophylaxis or in the treatment of B. fragilis infections and associated colorectal carcinoma.

Keywords: CEC; CRC; ETBF; bacteriophages; colonic epithelial cells; colorectal carcinoma; enterotoxigenic Bacteroides fragilis; in vitro model; phage therapy.

Figure 1

Transmission electron micrograph of B. fragilis-specific phage VA7, exhibiting a siphovirus morphology with an icosahedral head of 60 nm and a tail of 100 nm. Magnification × 250,000.

Figure 2

Scheme of the experimental design to evaluate the effect of phage VA7 lysate on enterotoxigenic B. fragilis (ETBF) infection and IL-8 production in colonic epithelial cell (CEC) cultures. For each arm, two distinct experiments (biological replicates) were performed, and for each of these, four samples (technical replicates) were analyzed. In other words, each data point was the result of eight analyses.

Figure 3

Genome map of the sequenced Bacteroides phage VA7 and comparison using a BLASTn analysis (greyscale) to the closest related phage B124-14. Each arrow represents a coding sequence. In red, genes encoding packaging and lysis-associated proteins are displayed, while green shows the structural proteins and blue the DNA- and metabolism-associated proteins (adapted from EasyFig).

Figure 4

One-step growth curve of phage VA7 in bacterial host A7. The evolution of phage lytic activity (PFU/mL) over time (min) is shown. The data points represent the mean of three experiments with the error bars.

Figure 5

Bactericidal effect (a) and IL-8 modulating effect (b) of VA7 lysate on HCT116 colonic epithelial cell (CEC) cultures infected with enterotoxigenic B. fragilis (ETBF) strain E3. Bacterial counts and IL-8 levels are presented as mean values with error bars representing the 95% confidence intervals (CIs) of the means. A base-10 logarithmic scale is used for the Y-axis of graph a. A statistically significant difference is indicated as * p < 0.05 and ** p < 0.01.