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. 2020 Feb 28:11:177.
doi: 10.3389/fmicb.2020.00177. eCollection 2020.

Competitive and/or Cooperative Interactions of Listeria monocytogenes With Bacillus cereus in Dual-Species Biofilm Formation

Vanessa Pereira Perez Alonso  1 , Andréia Miho Morishita Harada  1 , Dirce Yorika Kabuki  1
Affiliations

Competitive and/or Cooperative Interactions of Listeria monocytogenes With Bacillus cereus in Dual-Species Biofilm Formation

Vanessa Pereira Perez Alonso et al. Front Microbiol. .

Abstract

Microorganisms in dairy industries can form monospecies, dual-species, or multispecies biofilms, showing cooperative or competitive behaviors, which might contribute to the reduction of efficiency of cleaning and sanitization processes and eventually turn into a potential source of contamination. This study proposes to evaluate the behavior of Listeria monocytogenes in monospecies biofilms, cocultured with Bacillus cereus. The isolates were of dairy origin, and the selection occurred after studies of competition among species. The biofilm formations on AISI 304 stainless steel at 25°C in a stationary culture were analyzed to observe the cooperative or competitive interactions among species, as well as the effect of pre-adhered cells. Biofilm formation assays were performed in four experiments: Experiment 1: in the presence of strains of antagonistic substance producer B. cereus (+); Experiment 2: extract of the antagonistic substance of B. cereus; Experiment 3: pre-adhered cells of B. cereus; and Experiment 4: pre-adhered cells of L. monocytogenes. Subsequently, cooperative behavior was observed by scanning electron microscopy. The L. monocytogenes monospecies biofilm counts of greater than 5 log colony-forming units (CFU)/cm2 were also observed in dual-species biofilms in the presence of B. cereus (non-producers of antagonist substance), showing cooperative behavior between species. However, in the presence of antagonistic substance produced by B. cereus, the counts were lower, 1.39 and 1.70 log CFU/cm2 (p > 0.05), indicating that the antagonistic substance contributes to competitive interactions. These data are relevant for the development of new studies to control L. monocytogenes in the dairy industry.

Keywords: Bacillus cereus; Listeria monocytogenes; biofilm; cooperative interactions; dual-species biofilms.

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Figures

FIGURE 1
FIGURE 1
Flowchart of dual-species biofilm formation in stationary culture on stainless steel surface in BHI kept at 25°C. B. cereus producer of antagonist substance (+) and B. cereus non-producer antagonist substances (–).
FIGURE 2
FIGURE 2
Counts of L. monocytogenes at 25°C expressed in log CFU/cm2. Experiment 1 (A) in presence of B. cereus producer and non-producer of antagonistic substance; Experiment 2 (B) in presence of antimicrobial extract; Experiment 3 (C) in presence of B. cereus pre-adhered; and Experiment 4 (D) in presence of L. monocytogenes pre-adhered. ∗p < 0.05.
FIGURE 3
FIGURE 3
Image obtained by SEM (JEOL) of microbial cooperative interaction of L. monocytogenes and B. cereus (+). Experiment 1, Group 3, on stainless steel (A); L. monocytogenes (B); and B. cereus (C).
FIGURE 4
FIGURE 4
Image obtained by SEM (JEOL) of the interaction between L. monocytogenes and B. cereus in the biofilm on stainless steel (Experiment 1, Group 3). Agglomerates of dual-species biofilms (A); Biofilm cells (B,C); and low EPS production (D).
See this image and copyright information in PMC

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