Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Abstract

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.

FIG. 1.

Visualization of biofilms formed by B. cereus on LB at 30°C for 48 h at air-liquid interfaces. (A) Biofilms formed in a microtiter plate by B. cereus PAL22 (lane 1), B. cereus PAL27 (lane 2), B. cereus ATCC 14579 (lane 3), B. cereus 72 (lane 4), and B. cereus ATCC 10987 (lane 5). (B) Biofilms formed on stainless steel coupons by B. cereus 72 (lane 1), B. cereus PAL25 (lane 2), and B. cereus ATCC 10987 (lane 3).

FIG. 2.

Biofilm formation by B. cereus on LB (A) and Y1 medium (B) incubated at 30°C for 24 (white bars) and 48 h (light gray bars) and at 20°C for 24 (dark gray bars) and 48 h (black bars). Bars represent average OD₅₉₅ values plus standard errors (error bars).

FIG. 3.

Number of adherent B. cereus spores (light gray bars) to stainless steel coupons, after the addition of 7 log spores per ml, and subsequent biofilm (dark gray bars) formed on LB medium at 30°C after 48 h.

FIG. 4.

Number of adherent B. cereus spores (light gray bars) at 10°C to clean stainless steel coupons (A) or stainless steel coupons with milk pretreatment (B) after the addition of 7 log spores per ml and subsequent biofilm formed (dark gray bars) on LB medium at 10°C after 96 h.

FIG. 5.

Biofilm formation by B. cereus at air-liquid interfaces (light gray bars) and under submerged conditions (dark gray bars) on stainless steel coupons on LB medium at 30°C after 48 h. The left y axis indicates the total log CFU per cm² of stainless steel coupon; the right y axis indicates the total log CFU per stainless steel coupon.