Mixed species biofilms of Lactobacillus plantarum and Listeria innocua show facilitated entrance to the VBNC state during chlorine‐induced stress

Abstract

AbstractBiofilms of Lactobacillus plantarum and Listeria innocua were grown under single‐ and mixed‐culture conditions in a bioreactor with either half‐strength brain heart infusion (BHI) medium or BHI medium with sodium dichloroisocyanurate (150 ppm) for 24 hr. A quantitative approach using plate counts and fluorescence in situ hybridization (FISH) was applied, representing the culturable and viable cells, respectively. Species contribution to the single and mixed species biofilms was similar, reaching ca. 6.5 and 7.0 log cfu (cells)/mL for L. plantarum and L. innocua, respectively. In the presence of chlorine, species contribution to the single or mixed species biofilms was 10‐ to 100‐times lower than in the absence of this chemical for L. plantarum and L. innocua, respectively. Notably, a large difference was observed between the culturable and the viable cells in the mixed species biofilms, reaching 1.5 and 2.5 log units for L. plantarum and L. innocua, respectively. L. innocua achieved almost complete nonculturability during an early stage of biofilm growth in the coculture, indicating that the physiological state of cells was significantly affected not only by chlorine challenge but also by interspecies interactions. Importantly, all biofilms consisted of culturable cells during the late stage of growth, which represented a potential source of contamination.Practical ApplicationsFluorescence‐based methods for bacterial viability measurement have remained useful for a wide diversity of applications. In our study, the approach employing FISH in a parallel with plate counting allowed us to enumerate viable and culturable cells of L. plantarum and L. innocua under either single‐ or mixed‐culture conditions. Thus, we recommend it for accurate estimation of viable but nonculturable cells in the experimental set‐up comprising single‐/mixed‐culture sessile community.