Enhanced biofilm formation in dual-species culture of Listeria monocytogenes and Ralstonia insidiosa

Abstract

In the natural environments microorganisms coexist in communities as biofilms. Since foodborne pathogens have varying abilities to form biofilms, investigation of bacterial interactions in biofilm formation may enhance our understanding of the persistence of these foodborne pathogens in the environment. Thus the objective of this study was to investigate the interactions between Listeria monocytogenes and Ralstonia insidiosa in dual species biofilms. Biofilm development after 24 h was measured using crystal violet in 96-well microtiter plate. Scanning electron microscopy and cell enumeration were employed after growth on stainless steel coupons. When compared with their single species counterparts, the dual species biofilms exhibited a significant increase in biofilm biomass. The number of L. monocytogenes in co-culture biofilms on stainless steel also increased significantly. However, there was no effect on the biofilm formation of L. monocytogenes when cultured with R. insidiosa separated by a semi-permeable membrane-linked compartment or cultured in R. insidiosa cell-free supernatant, indicating that direct cell-cell contact is critical for this interaction.

Keywords: Listeria monocytogenes; Ralstonia insidiosa; biofilm.

Figure 1.. Biofilm formation of L. monocytogenes strains in monocultures and in mixed cultures with R. insidiosa measured by the crystal violet staining assay.

Figure 2.. Biofilm formation of L. monocytogenes and R. insidiosa in physically separated cultures. Biofilm formation was measured by crystal violet staining of biomass in the base compartment. Letter in the parentheses indicate Listeria or Ralstonia cells in the insert compartment. Neg: Negative control, L: biofilm formation of L. monocytogenes, L(R): effect of R. insidiosa (gown in the insert compartment) on biofilm formation of L. monocytogenes (Grown in the base compartment), R: biofilm formation of R. insidiosa, R(L): effect of L. monocytogenes (Insert compartment) on biofilm formation of R. insidiosa (Base compartment), LR: dual-species biofilm formation in mixed culture in the base compartment.

Figure 3.. SEM observations of biofilm structure on stainless steel coupons. (A) L. monocytogenes, (B) R. insidiosa, and (C) dual-species. Arrows points to the observed short EPS filaments in the biofilms of the mixed culture.