Lactobacillus plantarum strains show diversity in biofilm formation under flow conditions.

In many natural and technological applications, microbial biofilms grow under fluid flow. In this project, we investigated the influence of flow on the formation and growth of biofilms produced by gram-positive Lactobacillus plantarum strains WCFS1 and CIP104448. We used an in-house designed device based on a 48-well plate with culture volumes of 0.8 ml, and quantified total biofilm formation under static and flow conditions with flow rates 0.8, 1.6, 3.2 and 4.8 ml/h (with 1, 2, 4 and 6 volume changes per hour) using crystal violet (CV) staining, and determined the number of viable biofilm cells based on plate counts. The amount of total biofilm under flow conditions increased in the CIP 104448 strain, with significantly increased staining at the wall of the wells. However, in the WCFS1 strain, no significant difference in the amount of biofilm formed under flow and static conditions was observed. Plate counts showed that flow caused an increase in the number of viable biofilm cells for both strains. In addition, using enzyme treatment experiments, we found that for WCFS1 in the static condition, the amount of mature biofilm was declined after DNase I and Proteinase K treatment, while for flow conditions, the decline was only observed for DNase I treatment. The CIP104448 biofilms formed under both static and flow conditions only showed a decline in the CV staining after adding Proteinase K, indicating different contributions of extracellular DNA (eDNA) and proteinaceous matrix components to biofilm formation in the tested strains.

Reversibility of membrane permeabilization upon pulsed electric field treatment in Lactobacillus plantarum WCFS1.

Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.

Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: phenotype and transcriptome analysis.

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth were assessed at pH 5.5 without and with 0.75, 1.5 and 3.0 mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from reduced germination and outgrowth rates to complete blockage of germination at 3.0 mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5 mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multidrug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed.