What is the difference between e.coli and s.aureus

This triggers synthesis and secretion of immune dampening factors blue crescents surmounting in their effect any proinflammatory stimulation. Moreover, S. This enables pathogen persistence eventually resulting in chronic infection.

The bacterial strains used for udder infusion E. We have used them ever since in many experimental udder infections of healthy first lactating heifers. That particular E. Infusing 10, CFU of S. That S. The latter two strains had been isolated from subclinical cases of mastitis. Strain Z had been classified as causing persistent infections of MEC model cells, while both others were classified as causing transient infections.

These strains have kindly been provided by Prof. Ynte Schukken. The study was designed to compare and discriminate the earliest modulations of the transcriptome of the udder in response to E. It complements our initial description of the infection model including its clinical aspects, sample preparation and the modulated expression of a limited set of candidate genes The trial started by infusing the pathogens into the left hind quarter.

The right front quarter remained untreated and served as baseline control. The fact that SSC values remained largely unchanged indicated by corollary absence of considerable cross-talk between infected and control quarters that shortly after infection. Increased gene expression in the controls causes in tendency to underestimate the infection related extent of induced immune gene expression during E.

Based on that initial profiling of the udder responses we selected for the current study the samples collected from the gland cisterns GC of the infected udders. Infections with both pathogen species had induced in this compartment clear and significant changes in the expression profiles of our candidate genes while little changes had been recorded in proximal locations of the lobulo-alveolar milk-producing parenchyma.

Our previous report about this infection trial 24 included also the statement that the animal experimentation complied with the pertinent ethical standards as outlined in the German Tierschutzgesetz TierSchG and had been approved by the responsible ethics committee of the regional government of Upper Bavaria, Germany No.

In total, the array contains , probe sets representing almost 24, bovine transcripts. Samples containing 5. Quality of the data was controlled with the Expression Console 1.

Background was adjusted with the robust multi-array average RMA algorithm which also served for quantile normalization, and summarization. The annotation used in this study was based on the UMD3.

False discovery rates are listed in the Supplementary Table 1. This parameter was not applied as cut off criterion since it would have been too stringent for some comparisons, eventually resulting in no significantly regulated genes at all.

Sequences of the oligonucleotide primers are given in the Supplementary Table 2. Pre-cultures were started by inoculating a single colony of the pathogen into 1. Plating validated that this was equivalent to approximately 2.

They were infected with 10 6 pathogens, resulting in a multiplicity of infection MOI of Dilutions hereof — were plated to determine the number of internalized pathogens. Controls monitored the adherence of the bacteria to the plastic of the culture plates as well as external adherence to the MAC-T cells. Primary bovine mammary epithelial cells pbMEC were prepared from udder tissue and cultivated RPMI medium on collagen type I coated tissue plates Greiner bio-one as described 9.

The strain S. Bacteria were washed with-and re-suspended in-RPMI prior to applying them for infection. Subsequently, the cells were washed three times with PBS to remove any unbound S. Stress fibers were labelled with the red fluorescing ActinRed ReadyProbes Reagent ThermoFisher following the standard protocol of the manufacturer. Hogeveen, H. Economic aspects of mastitis: new developments.

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Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge.

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Cold Spring Harb Perspect Biol 5 , a Kudryavtseva, E. Wnt signaling genes of murine chromosome 15 are involved in gender-affected pathways of inflammatory arthritis. Subsequently, 4 ml of the supernatant was taken out to measure the OD value at nm to determine the nucleic acid leakage.

Leakage of the soluble protein was detected with the Coomassie Brilliant Blue G dye method according to the description of the previous chapter Total Protein Content of the L. After leakage of the soluble nucleic acids being measured, the remaining supernatant 1 ml was mixed with 5 ml of Coomassie Brilliant Blue G dye and laid for 2 min. The soluble protein leakage was evaluated by measuring the OD value of the mixture at nm.

The standard curve of protein content was drawn as the description of the previous chapter Total Protein Content of the L. Phosphorus metabolism of the indicator bacteria was measured according to the previous method Wang et al. The bacterial suspension 7. The OD value was measured at nm to confirm the phosphorus concentration.

The MRS medium 7. The standard curve of phosphorus content was drawn as follows: the phosphorus standard solution 0. Finally, 2 ml of 2. The indicator bacterial suspension 4 ml, OD , 0. After being washed three times with PBS 0. The gel was dyed with Coomassie Brilliant Blue R for 30 min and decolorized overnight until the background became transparent. The indicator bacterial suspension 10 ml, OD , 0.

After being grinded with quartz sand and a mortar in an ice-bath for 5 min, the cells were removed into 10 ml of 0. The collected supernatant was used to measure the activity of intracellular enzyme and the content of MDA.

Statistical analysis was implemented using SPSS version A probability level of 0. No antibacterial effect against E. In addition, adjustment of pH to 5. The diameters of inhibition zones around the Oxford cup against E.

In Figure 1 , heat treatment showed a certain negative effect on the antibacterial effect of lacidophilin, and the negative effect increased with heating temperature increased and time prolonged.

Figure 2 shows that different enzymes had different effects on the antibacterial effect of lacidophilin. However, the antibacterial effects of lacidophilin against the two bacteria were both weakened after being treated with papain and amylase.

After being treated with trypsin, lacidophilin lost its antibacterial effect, which indicated that lacidophilin could be degraded completely by trypsin.

In addition, it could be seen from Figure 3 that different types and different concentrations of metal ions had different effects on the antibacterial ability of lacidophilin.

As the concentration of metal ions increased, the antibacterial effect against the two indicator bacteria gradually weakened. Figure 1. The effects of heat on antibacterial activity, diameter mm refers to the diameter of the inhibition zone around the Oxford cup, which displayed the antibacterial activity, and the description of the diameter also applied to the following figure.

The bactericidal effect against E. The photographs of heat treatment effects on antibacterial activity against E. Figure 2. The effects of enzymes on antibacterial activity. The photographs of enzyme treatment on antibacterial activity against E. Figure 3. The effects of metal ions on antibacterial activity against E. In Figure 4 , the dynamic growth of the treated bacteria and the control was similar in the lagged period.

After 1. The control had a high-speed growth trend, whereas the growth of the treated bacteria was significantly inhibited. Figure 5A shows that the untreated E. In the fluorescence spectrum of Figure 5B , the absorption peaks at about nm were both observed in the untreated E.

The result of the fluorescence spectrum further indicated that the cytomembrane had been destroyed. Figure 5. The effect of lacidophilin on the cell membrane integrity of the two indicator bacteria.

Fluorescence micrograph of E. Fluorescence spectrum of E. Figure 6A shows that the inoculum conductivity of the treated E. In Figure 6B , the inoculum absorbance of the treated E. In addition, the absorbance differences between the treated strains and the controls increased significantly with time extension, which showed that the nucleic acid leakage was increasing gradually.

In Figure 6C , S. As time went on, protein leakage both kept increasing. In addition, as could be seen from Figure 6D , the phosphorus contents in the inoculum of the treated bacteria and the controls were all decreased with time prolonged.

However, the phosphorus contents in the inoculum of the two treated bacteria were both higher than those of the two controls throughout the processing, which indicated that lacidophilin inhibited the phosphorus metabolism of the indicator bacteria. Figure 6. Effect of lacidophilin on cell membrane permeability, leakage of electrolyte A , leakage of nucleic acid B , leakage of proteins C , phosphorus metabolism D.

Figure 7 shows that some typical bands of the treated E. For the treated S. The above result illustrated that lacidophilin significantly affected the growth and the protein content of the indicator bacteria. Figure 7. For the two treated indicator bacteria, the contents of MDA in the cells were both higher than those of the controls, and the activities of intracellular enzymes SOD, CAT, and POD also exceeded those of the controls significantly.

The increase of MDA contents indicated that the damage to the cell increased. Thus, it could be used as a preservative in pasteurized food Thirumurugan et al. This was similar to the previous report about the thermal stability of KF1 bacteriocin Zahid, The result of trypsin treatment indicated that lacidophilin might be a protein or peptide that could be completely hydrolyzed by trypsin Lv et al.

The amylase treatment illustrated that the protein or peptide might contain some glycosidic bonds, as amylase mainly hydrolyzes glycosidic bonds. Certain concentration of metal ions weakened the antibacterial activity of lacidophilin Figure 3. Previous research also indicated that divalent cations were antagonists of the bacteriocin Vloten, Thus, it was speculated that certain concentration of metal ions might reduce the antibacterial activity of lacidophilin by changing its spatial conformation, which was associated with antimicrobial activity.

The growth curves of the treated S. Common lacidophilin, such as nisin, only has an antibacterial effect on Gram-positive bacteria and has no antibacterial effect on Gram-negative bacteria Jeong and Ha, , which might ascribe the amyloid formation between the lipopolysaccharide of Gram-negative bacteria cell wall and the antibacterial peptide Wang et al.

However, the lacidophilin in this study had high antibacterial activity against both Gram-positive and Gram-negative bacteria, which further illustrated its potential use as a food preservative. Propionyl iodide is a nucleic acid staining agent that cannot penetrate the entire cytomembrane, but can penetrate the damaged cytomembrane to stain the DNA. FDA could pass through the cytomembrane, and the living cells with FDA staining could emit yellow-green fluorescence Boyd et al.

Therefore, the result of Figure 5 indicated that lacidophilin destroyed the cytomembrane of the bacteria and increased the cytomembrane permeability. Previous study had also suggested that the bacteriocins of bifidocin A produced from B.

Electrolyte, nucleic acid, and proteins are very important for the life of the indicator bacteria. The leakage of electrolyte, nucleic acid, and proteins further illustrated that the cytomembrane of the bacteria was damaged Shen et al.

The leakage might attribute to the formation of the selective pores in the bacteria cytomembrane Liu et al. Phosphorus is an important nutrient required for key biological reactions of cell life, and it is a crucial component of the cytomembrane Razzaque, The metabolic capacity of the cells could be confirmed by detecting phosphorus consumption in the cultivation process Tian and Wang, For the treated bacteria, the content of phosphorus in the inoculum decreased slower than the control, which indicated that lacidophilin inhibited the phosphorus consumption of the indicator bacteria.

Decrease of phosphorus consumption further illustrated that lacidophilin destroyed the metabolic capacity of the cell, inhibited the formation of the cytomembrane, and increased the permeability of the cytomembrane Wang et al.

Proteins are critical parts of bacteria cells, and they are closely related to the metabolism of bacteria cells. Disappearance and fade of typical bands of the cell proteins in SDS-PAGE Figure 7 indicate that lacidophilin had a significant effect on protein and the protein contents of the indicator bacteria.

The SDS-PAGE result further suggested that the cytomembrane was destroyed and the growth of the bacteria was inhibited, resulting in the fade or disappearance of typical bands of cell proteins Cai et al.

In addition, the result also corroborates that lacidophilin damaged the cytomembrane of the indicator bacteria, leading to the leakage of major cell components, which seriously affected the synthesis of protein Meng et al.

Absolute total and peak heats J display fairly linear variations with air volume with better correlation for E. Total and peak values for Escherichia coli average thermograms. Total and peak values for Staphylococcus aureus average thermograms. Physiological saline dilution values for Escherichia coli thermograms. Specific heats are fitted with exponential trendlines, while absolute heats are fitted with linear ones. There are several other regularities in Figure 7 that support both the assumed dissolved — diffused oxygen growth interplay and the actual HVL Peakfit decomposition of the observed thermograms:.

This reflects the hypothetical situation of a calorimetric cell completely filled with bacterial suspension: in this case the whole thermal growth is given only by dissolved oxygen, i. The results presented in Figures 4 , 5 , 6 and 7 consistently support the idea that complex thermal growth patterns as the ones obtained in the present contribution are mainly due to the interplay between dissolved and diffused oxygen.

Truly fermentative growth is not excluded, but its thermal contribution seems to be of minor importance within the growth conditions utilized. The most probable metabolic pathway accounting for bacterial growth of E. As the two MicroDSC instruments utilized in the present study are single-channel, they can run one sample at a time.

Microcalorimetry is very sensitive in detecting small variations in the bacterial density of the inoculum: this is fairly similar to the situation encountered in a busy clinical microbiology laboratory, where each new strain would require rapid processing and analysis. Under such circumstances, even the small variability that takes place in-between experiments needs to be assessed. A series of experiments was performed to evaluate the effect of refrigeration and long-term storage on the CFU viability count, as described in Methods section.

Results are shown in Figure 8 where one may notice a fairly linear decline in CFU count with the time spent in cold storage. Some cells die during cold storage and this lowers the initial concentration of the sealed samples, resulting in longer growth time lags. Variation of viable counts VC with the time spent in cold storage. VC pertain to samples stored in batch cells as detailed in Methods section. There is another natural similarity between the two approaches which involves the well-defined growth conditions, a normal requirement for comparing the growth of different cultures.

In fact, the authors seek for maximum complexity of growth by adjusting the culture medium as a necessary condition for discrimination between species. The present study may be regarded as a start for further, extended investigations for other species and strains.

Optimization of the advanced procedure for different thermal data is straightforward. Bacterial populations of Staphylococcus aureus and Escherichia coli exhibit different microcalorimetric growth patterns in both qualitative and quantitative assessments. The devised experimental routine based on thermograms obtained from samples kept in cold storage, sealed in the measuring batch cells[ 7 ] is sufficiently reproducible and accurate.

The use of the 3 most reliable parameters defined for both raw and volume-normalized thermograms t 0. The first parameter t 0. The discrimination method advanced in the present contribution has its limitations. The assumption that it can be used for S. For samples with same initial bacterial concentration but different volumes variability encountered within the same strain is smaller than the differences between the studied strains, allowing for discrimination.

Variation of the initial bacterial concentration also requires supplementary investigation, as this is known to markedly influence the growth time lag and thus the proposed time parameters. As microcalorimetric data on bacterial growth is accumulating, interest in this method is expected to result in standardization of the optimal bacterial concentration and sample volume involving different research centers.

For the time being, this method is not intended to be used in clinical practice with raw biological products sputum, blood as there is no control on bacterial sample concentration and other cell populations that could contaminate the thermogram. Extension of the microcalorimetric growth pattern characteristics to other bacterial populations, with the eventual build-up of a database, may prove to be sufficiently accurate for bacterial strains discrimination.

The information presented within this contribution may complement recent attempts to evaluate antimicrobial[ 5 , 6 , 29 — 31 ], antiparasitic[ 32 ], or antifungal[ 33 ] action on microcalorimetry monitored growth of various strains. Peakfit decomposition of the thermograms obtained within specified conditions of this study and the quantitative analysis of thermal effects advanced herein point to an oxygen-controlled bacterial growth, at least in its thermal manifestation.

There is an interplay between dissolved and cell headspace diffused oxygen: their contribution to the observed thermal behavior may be accounted for in terms of Peakfit decomposition of the overall thermogram.

A systematic Peakfit analysis of such complex thermal growth patterns seems to be mandatory for the determination of the optimal growth conditions required for standardization and essential for the extensive use of microcalorimetry in clinical applications. Both instruments were Joule effect factory calibrated and periodically checked with the factory naphthalene standard.

Each calorimeter had an outer thermostatic loop provided by a Julabo FHE device operating in standard mode. The Calisto v1. This included baseline integration end export in Excel with equally spaced time increments. Heat values obtained were further analyzed in Excel and Origin 8. Exported baselines were further processed in Peakfit.

Data exported from Calisto were processed in Peakfit by means of previously reported routines[ 16 , 17 ]. Calisto-generated baselines were imported and subtracted from the heat flow HF, mW signal. This procedure brings all thermograms to a common X time scale, but definitely excludes any analysis of the growth lag time.

This brings all thermograms to a common Y NHF scale, with the advantage that areas of the component peaks represent their fraction to the overall thermal effect. All subsequent peak fitting involved the NHF — time thermograms. This function resulted in both the best statistical criteria r 2 , F-statistic, standard errors, etc. The medium was autoclaved before use and was microbiologically pure. For viability counts, preparation of isolated colonies for inoculation and random sample check of aseptic technique, we used plates with Tryptic Soy Agar TSA, Oxoid, UK ; this solid medium has the same basic composition as TSB.

This value was subtracted from further measurements to obtain the true nephelometric value of the growing inoculum. Isolated colonies were picked-up with an inoculation loop and aseptically passed into a sterile tube containing 5 ml of TSB. This sample was grown until it reached a value of 0. The nominal volume of a batch calorimetric cell is 1 ml. However, in practice the maximum volume available for liquid sample filling for the o-ring sealed cell was 0.

The cell headspace air volume was calculated as 1 — V sample ml for all runs. The experiments required three types of sample preparations:. The microcalorimetric cells were filled with the required volume of sample at room temperature inside a laminar flow biosecurity hood and were hermetically sealed with their silicon o-ring covers.

The time required to fill the cells was under 5 minutes, so significant thermogram differences are not expected to arise from the time needed to accomplish this procedure.

Physiological saline was added to the calorimetric cells filled with bacterial suspension, as described above. Sterile mineral paraffin oil Sigma, DE was carefully added at the air-fluid interface of the simple culture media sample, resulting in a three-phase sample: air, oil meant as a barrier to oxygen diffusion and bacterial culture.

The experiments were performed at 1 day intervals using these samples. Before each microcalorimetric run, the cell content was thoroughly homogenized, and the excess sample was removed from the cell. The experiments were performed at 1 day intervals using samples kept in cold storage. Working temperature was reached by ramp heating with 0.

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