Isolation, Partial Characterization and Application of Bacteriophages in Eradicating Biofilm Formation by Bacillus cereus on Stainless Steel Surfaces in Food Processing Facilities

Abstract

The Bacillus cereus (B. cereus) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log₁₀ UFC/cm² have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log₁₀ UFC/cm². These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems.

Keywords: B. cereus group; bacteriophage; biofilm; characterization; eradication; inhibition; sewage; stainless steel.

Figure 1

(A–G) Plaque morphology on agar plates of isolated phages. (A) Ø1BC478, (B) Ø1BC3990, (C) Ø2BC4663, (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 2

(A–G) Transmission electron micrographs of seven B. cereus- specific bacteriophages isolated in this study. (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663. The virions were negatively stained with uranyl acetate and visualized at a magnification of 100,000× (B,D,F), 120,000× (C,G) and 150,000× (A,E). The scale bar indicates 100 nm.

Figure 3

SDS-PAGE analysis of phage structural proteins. Lane 1; 5; 8 and 10: Precision Plus Protein^TM unstained standards (Biorads). Lane 2; 3; 4; 6; 7; 9 and 11: Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663; respectively.

Figure 4

Restriction enzyme digestion pattern analysis of isolated bacteriophages DNAs. Lane 2 to Lane 8: TaqI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lane 10 to Lane 16: Hin6I digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1), Ø4BC4663(2) and Ø1BC4663, respectively. Lane 18 to Lane 24: EcoRI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lanes 1, 9, 17 and 25: Molecular marker (SmartLadder 10 kb).

Figure 5

(A–G) Effect of phage post-treatments at different titrations (10⁵–10⁸ PFU/mL) on pre-formed biofilms of 24, 48 and 72 h and the reduction percentages of biofilm in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 6

(A–G): Effect of phage treatments at different titrations (10⁵–10⁸ PFU/mL) on biofilm formation during incubation periods of 24, 48 and 72 h and the inhibition percentages of biofilm formation in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.