Escherichia coli K-12 Transcriptomics for Assessing the Mechanism of Action of High-Power Ultrasound.

An investigation into the mechanisms of action on bacteria involving exposure to stress factors was conducted in this study. The effects of ultrasound on Escherichia coli K-12 MG1655 and its isogenic mutant, ∆gadW, under high power ultrasound treatments (26 kHz) were screened and identified by analysing their transcriptome differences between primary and secondary sequential treatments using RNA-Seq. This also helped to assess any developed protection for cells between different generations. According to our results, 1825 genes of all tested conditions were expressed, playing different roles in the cell. The expression of these genes is associated with DNA damage, cell membrane integrity, and also metabolic effects. The studied strains also showed different differential expressed genes (DEGs), with some genes being directly responsible for defence mechanisms, while others play an indirect effect due to cell damage. A gradual decrease in the expression of the genes, as we moved from just one cycle of ultrasound treatment to sequential treatment, was evident from a heat map analysis of the results. Overall, E. coli K-12 builds a self-protection mechanism by increasing the expression of genes involved in the respiration for increased growth, and production of flagellum and pili. It can be concluded that high power ultrasound is a technology that triggers several different defence mechanisms which directly link to E. coli.

The role of temperature and carbon dioxide climatic stress factors on the growth kinetics of Escherichia coli.

AIMS: The global level of carbon dioxide and temperature in the atmosphere is expected to increase, which may affect the survival of the stress-adapted bacteria. In this study, the effect of temperature and dissolved carbon dioxide on the growth rate of Escherichia coli-eGFP tagged strain was studied, thus assessing its response to induced environmental stress factors. METHODS AND RESULTS: A kinetic assay has been performed using a microplate reader with a spectrofluorometer to determine the specific growth rates. Polynomial models were developed to correlate the environmental conditions of temperature and carbon dioxide with Escherichia coli BL21 (DE3) growth in culture media and dairy by-products. At a temperature of 42°C, as the dissolved CO2 increased, a decrease in µmax by 0.76 h-1 was observed. In contrast, at 27°C, this increase led to an increase in µmax by 0.99 h-1. Moreover, a correction factor was added when applying the model to dairy whey samples. CONCLUSIONS: The application of this developed model can be considered a useful tool for predicting the growth of Escherichia coli using climate projections.

Growth behavior of low populations of Listeria monocytogenes on fresh-cut mango, melon and papaya under different storage temperatures.

The growth behavior of Listeria monocytogenes low population (1-4 cells/sample) on fresh-cut mango, melon, papaya and fruit mix stored at 4, 8, 12 and 16 °C was evaluated over 10 days. Mango showed the lowest counts for L. monocytogenes during 10 days regardless of storage temperature (<1.7 log cfu.g-1). Melon supported high bacterial growth over 10 days, reaching 5 log cfu.g-1 at 16 °C. Both the fruit and storage temperature influenced the Listeria low population growth potential (δ). Cumulative frequency distribution of L. monocytogenes showed that after 10 days, 100% of fresh-cut fruits and fruit mix stored at 4 °C remained ≤2 log cfu.g-1, while at 12 and 16 °C 100% of melon, papaya and fruit mix samples exceeded this limit. At 8 °C, 100% of mango and fruit mix samples remained below this limit after 10 days, whereas 100% of melon and papaya reached it after 7 days. Results indicate 4 °C as the ideal to store safely fresh-cut mango, melon, papaya and fruit mix for 10 days. Besides, 8 °C can also be an option, but not for melon and papaya. Findings highlight the ability of L. monocytogenes to survive and grow in fresh-cut fruits even at a very low initial population levels.

Principal component analysis of hyperspectral data for early detection of mould in cheeselets.

The application of non-destructive process analytical technologies in the area of food science got a lot of attention the past years. In this work we used hyperspectral imaging to detect mould on milk agar and cheese. Principal component analysis is applied to hyperspectral data to localise and visualise mycelia on the samples' surface. It is also shown that the PCA loadings obtained from a set of training samples can be applied to hyperspectral data from new test samples to detect the presence of mould on these. For both the agar and cheeselets, the first three principal components contained more than 99 % of the total variance. The spatial projection of the second principal component highlights the presence of mould on cheeselets. The proposed analysis methods can be adopted in industry to detect mould on cheeselets at an early stage and with further testing this application may also be extended to other food products.

Bacterial and fungal contaminants in caprine and ovine cheese: A meta-analysis assessment.

The dairy industry is of great importance to the European economy contributing towards € 8.7 billion of the total trade surplus. Caprine and ovine milk amount to 3.1% of the 152 million tonnes of milk produced in Europe, 95% of which is transformed into dairy products such as cheese. This cheese is mostly produced in small holdings from untreated milk, making it a high-risk dairy product for human consumption. A total of 49 foodborne disease outbreaks caused by dairy products were registered in 2017 in Europe. Therefore, these products remain a serious health risk. This meta-analysis examined 30 studies assessing bacterial or fungal contamination of caprine or ovine milk cheeses. The significantly contaminating microbes were found to be Acremonium spp. (19%), Aspergillus spp. (23%), Bacillus spp. (2%), Brucella spp. (34%), Enterobactericae spp. (36%), Enterococcus spp. (28%), Escherichia spp. (15%), Fusarium spp. (21%), Geotrichum spp. (22%), Listeria spp. (11%), Mucor spp. (15%), Penicillium spp. (25%), Phoma spp. (20%), Rhizopus spp. (15%), Salmonella spp. (3%), Scopulariopsis spp. (19%) and Staphylococcus spp. (25%) in caprine and ovine cheese, indicating a variety of food pathogens as well as spoilers. Raw milk is nutritious hence prone to contamination. However, since traditional cheese is often made from untreated milk, it is important to educate cheesemakers of key safety measures and good manufacturing practice allowing for the safe production of these food items.

Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes.

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle are most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors.

Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin.

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1. The present study tested the hypothesis that inhibiting enzymes in the mevalonate pathway to reduce cellular cholesterol increases the resilience of stromal cells to PLO. We first verified that depleting cellular cholesterol with methyl-ß-cyclodextrin increased the resilience of stromal cells to PLO. We then used siRNA to deplete mevalonate pathway enzyme gene expression, and used pharmaceutical inhibitors, atorvastatin, alendronate or zaragozic acid to inhibit the activity of HMGCR, FDPS and FDFT1, respectively. These approaches successfully reduced cellular cholesterol abundance, but mevalonate pathway enzymes did not affect cellular resilience equally. Inhibiting FDFT1 was most effective, with zaragozic acid reducing the impact of PLO on cell viability. The present study provides evidence that inhibiting FDFT1 increases stromal cell resilience to a cholesterol-dependent cytolysin.

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.

We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.

Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.