Insight in lag phase of Listeria monocytogenes during enrichment through proteomic and transcriptomic responses.

The dynamics of the enrichment-based detection procedure of the foodborne pathogen Listeria monocytogenes from food still remains poorly understood. This enrichment is crucial in the reliable detection of this pathogen and more insight into the recovery mechanism during this step is important to advance our understanding of lag phase behaviour during enrichment. In this study we combined transcriptomic and proteomic analyses to better understand the physiological processes within the lag phase of L. monocytogenes during enrichment. Upon transfer of BHI-cultured stationary phase L. monocytogenes cells to half-Fraser enrichment broth (HFB), motility-associated genes and proteins were downregulated, while expression of metal uptake transporters, resuscitation-promoting factors that stimulate growth from dormancy, antibiotic efflux pumps and oxidative stress proteins were upregulated. Next to this, when cells with a heat stress history were cultured in enrichment broth, proteins necessary for recovery were upregulated with functions in DNA-damage repair, protein refolding, cell-wall repair, and zinc transport. Proteomic results pointed to possible factors that support shortening the lag duration, including the addition of 10 µM zinc and the addition of spent HFB containing presumed concentrations of resuscitation-promoting factors. However, these interventions did not lead to biologically relevant reduction of lag phase. Also, when cells were enriched in spent HFB, final cell concentrations were similar to enrichments in fresh HFB, indicating that the enrichment broth seems not to lack critical substrates. Concludingly, this study gives insight into the proteomic changes in the lag phase during enrichment and shows that supplementation of HFB is not the best strategy to optimize the current enrichment method.

Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and impact of culturing conditions.

In this study, different methods were evaluated for enumeration of spores of G. stearothermophilus, different sporulation methods were assessed for yields and wet heat resistances of obtained spores, and subsequently, the variation in heat resistances of spores was determined. Overall, tryptone soya agar (TSA) was the most suitable medium for enumeration of spores of this thermophilic bacterium. Sporulation on different media both at 55 and at 61 °C led to considerable variation in spore heat resistance. The heat resistance of spores was highest upon sporulation on medium supplemented with free ions of calcium, potassium, magnesium and manganese (CaKMgMn). For 18 different G. stearothermophilus strains that were isolated from various sources, spores were subsequently produced on nutrient agar supplemented with CaKMgMn at 55 °C. Strain ATCC 12980T, also known as 9A20, which is commonly used in steam sterilization tests was included. The survival of spores of all strains was assessed at 125 °C and 130 °C using two independent spore batches per strain. The mean D125°C for spores of the 18 strains was 1.1 min (95% PI 0.48-2.3 min) and the mean D130°C was 0.37 min (95% PI 0.17-0.82 min). For spore inactivation of these 18 strains, a z-value of 11.1 °C was estimated, resulting in an estimated D-value of 2.4 min (95% PI 1.1-5.2) at the reference temperature 121.1 °C. Based on the data sets obtained in this study, it was found that the variability in spore heat resistance could largely be attributed to strain variability and conditions used during sporulation (especially the sporulation medium); reproduction and experimental variabilities were much smaller. The established variabilities were compared with the overall variability in spore heat resistance of G. stearothermophilus based on a meta-analysis of reported D-values. The data presented indicate that strain variability and history of sporulation each account for approximately half of the overall variability observed with respect to the heat resistance of spores of G. stearothermophilus. The findings presented in this study allow for optimal recovery of G. stearothermophilus spores from foods and a better understanding of factors that determine the heat resistance properties of spores of G. stearothermophilus. Moreover, this study once more underlines the limited effects of heat treatments used in the food industry on inactivation of spores of this bacterium.

Performance of stress resistant variants of Listeria monocytogenes in mixed species biofilms with Lactobacillus plantarum.

Population diversity and the ability to adapt to changing environments allow Listeria monocytogenes to grow and survive under a wide range of environmental conditions. In this study, we aimed to evaluate the performance of a set of acid resistant L. monocytogenes variants in mixed-species biofilms with Lactobacillus plantarum as well as their benzalkonium chloride (BAC) resistance in these biofilms. L. monocytogenes LO28 wild type and acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Homolactic fermentation of glucose by L. plantarum created an acidic environment with pH values below the growth boundary of L. monocytogenes. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, indicating differences in adaptive behaviour between the variants. Two variants that showed a high level of acid adaptation when grown in liquid cultures, showed also better performance in mixed species biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. It can be concluded that acid resistant variants of L. monocytogenes show diversity in their adaptation to acidic conditions and their capacity to survive in mixed cultures and biofilms with L. plantarum.

First characterization of bioactive components in soybean tempe that protect human and animal intestinal cells against enterotoxigenic Escherichia coli (ETEC) infection.

Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modifications.