Glycerol metabolism induces Listeria monocytogenes biofilm formation at the air-liquid interface

Listeria monocytogenes is a food-borne pathogen that can grow as a biofilm on surfaces. Biofilm formation in food-processing environments is a big concern for food safety, as it can cause product contamination through the food-processing line. Although motile aerobic bacteria have been described to form biofilms at the air-liquid interface of cell cultures, to our knowledge, this type of biofilm has not been described in L. monocytogenes before. In this study we report L. monocytogenes biofilm formation at the air-liquid interface of aerobically grown cultures, and that this phenotype is specifically induced when the media is supplemented with glycerol as a carbon and energy source. Planktonic growth, metabolic activity assays and HPLC measurements of glycerol consumption over time showed that glycerol utilization in L. monocytogenes is restricted to growth under aerobic conditions. Gene expression analysis showed that genes encoding the glycerol transporter GlpF, the glycerol kinase GlpK and the glycerol 3-phosphate dehydrogenase GlpD were upregulated in the presence of oxygen, and downregulated in absence of oxygen. Additionally, motility assays revealed the induction of aerotaxis in the presence of glycerol. Our results demonstrate that the formation of biofilms at the air-liquid interface is dependent on glycerol-induced aerotaxis towards the surface of the culture, where L. monocytogenes has access to higher concentrations of oxygen, and is therefore able to utilize this compound as a carbon source

Physiological parameters of Bacillus cereus marking the end of acid-induced lag phases

During lag phases microbial cells adapt to their environment and prepare to proliferate. Physiological parameters of B. cereus cells upon exposure to near-growth-boundary acid stress were investigated and markers for the transition between lag phase and growth were identified using fluorescent probes combined with flow cytometry. Determination of cell counts and optical density revealed lag phases of 1 h, 2 h and 5 h, in cultures shifted to pH 7, pH 5.3 (set with lactic acid) and pH 4.9 (set with sulfuric acid), respectively. The obtained lag phases fitted the trends in ATP levels, which were constant during the lag phase and increased after the onset of growth. Both the percentage of PI-stained cells and cells with a significant membrane potential decreased during the lag phase. This points to repair of membrane damage and the loss of membrane potential. However, both trends extended in the growth phase, thus not suitable to mark the onset of growth. The activity of the electron transfer chain and esterases did allow for assessment of transition between lag and growth phase. These activities were generally low during the lag phase and increased after the onset of growth. Our results show that, independent of the duration of the lag phase, for different conditions the same physiological trends could be observed. The change in signal of selected probes can be used as a marker for transition from lag phase to the growth phase and may aid in identification of novel targets interfering with bacterial exit from lag phase

Variability in lag duration of Listeria monocytogenes strains in half Fraser enrichment broth after stress affects the detection efficacy using the ISO 11290-1 method

A collection of 23 Listeria monocytogenes strains of clinical and food origin was tested for their ability to recover and grow out in half Fraser enrichment broth following the ISO 11290-1:2017 protocol. Recovery of sub-lethally heat-injured cells in half Fraser broth was compared to reference cells with no stress pre-treatment. The enrichments were followed over time by plate counts and the growth parameters were estimated with the 3-phase model which described the data best. The reference cells without stress pre-treatment showed a short lag duration, which ranged from 1.4 to 2.7 h. However, significant variation in the ability to recover after 60 °C heat stress was observed among the tested strains and resulted in a lag duration from 4.7 to 15.8 h. A subset of strains was also exposed to low-temperature acid stress, and the lag duration showed to be also stress dependent. Scenario analyses and Monte Carlo simulations were carried out using the growth parameters obtained in the enrichments. This demonstrated that when starting with one cell, the detection threshold for efficient transfer of at least one cell to the secondary enrichment step, i.e. 2 log10 CFU/ml, was not reached by 11 of 23 strains tested (48%) after exposure to 60 °C heat stress. Increasing the incubation time from 24 to 26 h and the transfer volume from 0.1 to 1.0 ml can increase the average probability to transfer at least one cell to the secondary enrichment step from 79.9% to 99.0%. When optimizing enrichment procedures, it is crucial to take strain variability into account as this can have a significant impact on the detection efficacy.</p