Effect of compatible solutes on the respiratory activity and growth of Escherichia coli K-12 under NaCl stress

The respiratory activity of Escherichia coli K-12 was inhibited by high NaCl concentrations. The addition of compatible solutes such as proline and ectoine led to the recovery of the respiration of E. coli K-12 inhibited by 1 M NaCl to a similar extent as did the addition of glycine betaine. Glucose, an exogenous substrate for the stimulation of respiratory activity, was not taken up by the cells in the presence of 1 M NaCl, but active uptake of glucose was observed following the addition of compatible solutes. As a result, we obtained a good correlation between respiratory activities and glucose uptake rates, suggesting that the glucose uptake activity inhibited by high NaCl concentrations was facilitated by these solutes. The addition of proline did not lead to cell proliferation in the minimal medium containing 1 M NaCl, although the cells took up proline quite efficiently under high osmolarity. On the other hand, cell growth occurred after a lag time in the medium containing glycine betaine or ectoine in place of proline. Similar actions of the compatible solutes mentioned above were observed for E. coli ATCC 9637.     

Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure

Escherichia coli ATCC 25922 in phosphate buffered saline solution (PBS, pH 7.1, 10(8) CFU/ml) was inactivated by high hydrostatic pressure (HHP, 400 to 600 MPa) treatment at 25 degrees C for 10 min. Colonies of E. coli were not detected on non-selective plate count agar immediately after a HHP-treatment of at least 550 MPa. E. coli subjected to at least 500 MPa in PBS were incubated at 4, 25, and 37 degrees C for 120 h. No colonies were detected on plate count agar throughout the 120-h incubation period at 4 or 37 degrees C. In contrast, the number of E. coli during storage at 25 degrees C increased from an undetectable level (< 1 CFU/ml) to the level of initial cell counts regardless of the treatment pressure level. The recovery in PBS required a maximum time of 48 h, while the period during which cell numbers remained at an undetectable level increased from 24 to 72 h as the treatment pressure increased. E. coli treated at 550 and 600 MPa in PBS were inoculated into trypticase soy broth (TSB) and stored at 4, 25, and 37 degrees C for 120 h. No recovery was recorded in TSB during the 120-h storage at 37 degrees C. In contrast, the number of E.coli during storage at 25 degrees C in TSB increased beyond the level of initial cell counts regardless of the treatment pressure level. The recovery of cell numbers observed in TSB was faster than that in PBS samples, as bacterial growth in TSB assisted faster recovery. When the incubation temperature in PBS was shifted to 25 degrees C after 120-h at 4 or 37 degrees C, recovery of E. coli was observed in samples shifted from 4 to 25 degrees C regardless of the treatment pressure. However, the time during which cell numbers remained at an undetectable level was extended by increasing the level of treatment pressure, and recovery required a maximum time of 48 h. On the other hand, no recovery was observed with HHP-treated E. coli subjected to an incubation temperature shift from 37 to 25 degrees C. This study indicates that an appropriate incubation temperature after HHP-treatment is needed to optimize the recovery of HHP-injured bacteria and thus prevent overestimation of the lethal effect of HHP-treatment.     

微生物积累及转运的相容性溶质种类的研究进展

某些微生物在高渗环境下会合成或转运具有自身保护作用物质,这类物质主要为相容性溶质,该物质可以增加细胞渗透耐受性及对不良环境的抵抗能力。然而不同的微生物积累或转运的相容性溶质不同,抗渗能力也不同,综述了微生物合成及转运相容性溶质的种类,以期为研究不同微生物相容性溶质积累的机制提供理论基础。     

Effects of exogenous compatible solutes on growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Six known compatible solutes as well as twenty L-amino acids were individually added to a glucose minimal medium and their effects on the growth of Sulfolobus solfataricus (DSM 1617) were examined. Among the compatible solutes tested, putrescine, trehalose, and l-glutamate enhanced the growth of S. solfataricus. On the other hand, glycine betaine, choline, and L-proline showed little or no influence on cell growth. When cells were grown in the glucose medium supplemented with trehalose or L-glutamate, S. solfataricus preferentially utilized the compatible solute over glucose. The growth-enhancement effect of L-glutamate was also observed to be dependent on the glucose concentration in the medium: growth enhancement was higher when the concentration of glucose was low and gradually decreased with increasing glucose concentration. Interestingly, the effects of amino acids on cell growth differed markedly depending on the chemical nature of the amino acid added. While acidic amino acids-L-glutamate and L-aspartate-enhanced the growth rate, almost no growth was observed in the presence of glycine, L-leucine, L-valine, L-phenylalanine, L-threonine, L-methionine, or L-cysteine. Among all the low-molecular-weight solutes tested in this study, the growth-stimulation effect was most profound in the presence of L-glutamate. When S. solfataricus cells were grown in a glucose (1.0 g/l) medium supplemented with 3.0 g/l L-glutamate, the maximal cell density and growth rate were about 3.2- and 2.3-fold higher than those obtained without L-glutamate.     

Growth of Escherichia coli and Salmonella typhimurium on Beef Tissue at 25°C

At 25°C, growth of Escherichia coli and Salmonella typhimurium on beef was influenced by type of tissue, pH, gaseous atmosphere, and physiological state of the cells used to inoculate the tissue. These organisms grew after only a short lag period, both aerobically and anaerobically, on beef fatty-tissue, and on high pH muscle (pH ≥ 6). The lag period was considerably extended on low pH muscle (pH ≤ 5.7) incubated aerobically. On low pH lean tissue stored anaerobically at 25°C for 24 hr, cells from aerobically grown broth cultures did not grow whereas cells from anaerobically grown cultures grew after an extended lag. These results suggest that during the cooling of hot-boned meat growth of E. coli and salmonellae is more likely on fatty tissue or muscle of high pH than on lean tissue of low pH.     

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co‐flocculation with soil humates in the presence of Ca²⁺. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate–phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of K_m (2.22 mM ) and activation energy (33.4 kJ mol^?1) similar to those of the free enzyme (2.00 mM and 27.6 kJ mol^?1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80–100 °C, while the free enzyme had its highest activity at 60 °C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry     

Extraction methods for lipopolysaccharides from Escherichia coli ATCC 25922 for quantitative analysis by capillary electrophoresis

Lipopolysaccharides (LPS) are major constituents of the cell wall of Gram-negative bacteria. Capillary zone electrophoresis (CZE) was applied to distinguish between lipopolysaccharides extracted from Escherichia coli ATCC 25922 with various techniques. Extraction methods proposed by Westphal and Jann [Methods Carbohydr. Chem. 5 (1965) 83], Galanos et al. [Eur. J. Biochem. 9 (1969) 245], Ni Eidhin and Mouton [FEMS Microbiol. Lett. 110 (1993) 133] and Nichols [Infect. Immun. 62 (1994) 3753] for LPS preparation were evaluated. Electrophoresis buffers with varying pHs were applied to assess the structure stability of the extracted LPS samples. Variations in structural breakdown were apparent demonstrating that different extraction methods removed different LPS molecules. Furthermore, the results obtained proved the CZE useful as an analytical technique for LPS evaluation. The LPS removed with the Nichols extraction procedure presented a unique electrophorogram that could in future be applied in the rapid identification of Gram-negative foodborne pathogens.     

Inactivation of Escherichia coli by high hydrostatic pressure at different temperatures in buffer and carrot juice

The inactivation of Escherichia coli MG1655 was studied at 256 different pressure (150-600 MPa)-temperature (5-45 degrees C) combinations under isobaric and isothermal conditions in Hepes-KOH buffer (10 mM, pH 7.0) and in fresh carrot juice. A linear relationship was found between the log10 of inactivation and holding time for all pressure-temperature combinations in carrot juice, with R2-values>or=0.91. Decimal reduction times (D-values), calculated for each pressure-temperature combination, decreased with pressure at constant temperature and with temperature at constant pressure. Further, a linear relationship was found between log10D and pressure and temperature. A first order kinetic model, describing log10D in carrot juice as a function of pressure and temperature was formulated that allows to identify process conditions (pressure, temperature, holding time) resulting in a desired level of inactivation of E. coli. For Hepes-KOH buffer, the Weibull model more accurately described the entire set of inactivation curves of E. coli MG1655 compared to the log-linear or the biphasic model. Several secondary models (first and second order polynomial and Weibull) were evaluated, but all had poor fitting capacities. When the Hepes-KOH dataset was limited to 22 of the 34 pressure-temperature combinations, a first order model was appropriate and enabled us to use the same model structure as for carrot juice, for comparative purposes. The major difference in kinetic behaviour of E. coli in buffer and in carrot juice was that inactivation rate as a function of temperature showed a minimum around 20-30 degrees C in buffer, whereas it increased with temperature over the entire studied temperature range in carrot juice.     

Inactivation of Escherichia coli (ATCC 4157) in diluted apple cider by dense-phase carbon dioxide

Dense-phase carbon dioxide (CO2) treatments in a continuous flow through system were applied to apple cider to inactivate Escherichia coli (ATCC 4157). A response surface design with factors of the CO2/product ratio (0, 70, and 140 g/kg), temperature (25, 35, and 45 degrees C), and pressure (6.9, 27.6, and 48.3 MPa) were used. E. coli was very sensitive to dense CO2 treatment, with a more than 6-log reduction in treatments containing 70 and 140 g/kg CO2, irrespective of temperature and pressure. The CO2/product ratio was the most important factor affecting inactivation rate of E. coli. No effect of temperature and pressure was detected because of high sensitivity of the cells to dense CO2. Dense CO2 could be an alternative pasteurization treatment for apple cider. Further studies dealing with the organoleptic quality of the product are needed.     

Growth of Escherichia coli in milk from endotoxin-induced mastitic quarters and the course of subsequent experimental Escherichia coli mastitis in the cow

The objective of this study was to assess growth of Escherichia coli in milk from endotoxin-induced mastitic quarters and to relate the in vitro findings to the course of experimental E. coli mastitis. Whole and skim milks from 24 rear quarters of 12 cows were inoculated with E. coli 0:157 and incubated at 38 degrees C. Growth of E. coli 0:157 was not inhibited in milk collected from rear quarters immediately prior to endotoxin infusion. However, growth inhibition occurred in all but one whole mastitic milk samples collected from mastitic quarters 18 h after infusion of .1 mg of endotoxin. Skim milk samples from mastitic quarters were bactericidal in four cows (7 quarters), whereas growth occurred in skim mastitic milk from 17 quarters of nine cows. Rear quarters of all cows were inoculated with 10(4) cfu of E. coli 0:157 19 h after the quarters had been infused with endotoxin. Clinical parameters and milk production were monitored during 36 h and 21 d, respectively. None of the inoculated quarters developed signs of inflammation, and secreta from inoculated quarters were bacteriologically negative after 48 h. Therefore, growth-inhibitory property of skim milk from endotoxin-induced quarters was apparently not a suitable parameter to monitor differences in susceptibility to E. coli mastitis.     

大肠杆菌高密度培养发酵L-色氨酸

为实现高水平的大肠杆菌高密度培养,提升发酵法产L-色氨酸的生产效率,以大肠杆菌TRTH为供试菌株,在初始发酵工艺的基础上,先后对发酵接种量和底物KH_2PO_4添加量各设置4个梯度进行发酵对比试验,并着重考察了底物KH_2PO_4添加量对菌体生长、产酸、磷酸盐消耗、糖酸转化率及副产物积累的影响。试验结果表明,在接种量为20%(体积分数),底物KH_2PO_4添加量为10 g/L时,最高菌体密度达到65. 39 g/L,最终L-色氨酸产量为59. 55 g/L,分别较优化前提高了45. 99%和31. 17%,发酵延滞期明显缩短,菌体生长迅速,原料利用率大幅提高,主要副产物积累量较低,发酵总体水平达到最优。为大肠杆菌高密度培养在L-色氨酸发酵中的应用提供了一个成本低、可行性高的新策略,同时为L-色氨酸发酵生产中底物磷酸盐的调控提供了参考。     

重组大肠杆菌高细胞密度发酵研究进展

重组大肠杆菌高细胞密度发酵(high cell-density fermentation,HCDF)是获得高外源蛋白产率的一种重要策略,文中就影响重组大肠杆菌高细胞密度发酵主要因素的研究进展进行了综述,包括宿主菌、接种量、培养条件、代谢副产物、培养方式等.     

乳铁蛋白对大肠杆菌生长的抑制与促进研究

研究不同浓度(2、4、6、8、10 mg/m L)乳铁蛋白对大肠杆菌生长促进或抑制的情况。结果表明,浓度≥6 mg/m L时,乳铁蛋白在初期发挥抑菌作用,其后表现出良好的营养促进剂的作用,大肠杆菌的生长比对照组更加旺盛;浓度≤4 mg/m L时,乳铁蛋白并不抑制大肠杆菌的生长,表现出明显的营养作用。     

Effect of negative air ions on Escherichia coli ATCC 25922 inoculated onto mung bean seed and apple fruit

The effect of negative air ions on the reduction of Escherichia coli ATCC 25922 inoculated onto mung bean sprout seed and whole or fresh-cut apple fruit was studied. Mung bean seeds, whole Gala apples, and Gala apple slices were inoculated with E. coli ATCC 25922 before being exposed to negative air ions for up to 18 h at room temperature (-23 degrees C). Results revealed a less than 0.5-log reduction of E. coli on mung bean seed even after 18 h of exposure. The reduction of E. coli on the surface of whole apples increased with increasing exposure time from 0.5 to 3 h, but the maximum reduction was less than 1 log CFU/g. Increasing exposure time from 3 to 18 h did not lead to increased treatment efficacy. No reduction of E. coli was observed on apple slices after 3 h of treatment. When the negative air ion system was applied with acetic acid vapor, no additive or synergistic effect of negative ions on the reduction of E. coli was found. These results suggest that negative air ions have a very limited effect on the population of E. coli on mung bean seed and apples.     

Behaviour of log-phase Escherichia coli at temperatures near the minimum for growth

The behaviour of cold-adapted, log-phase Escherichia coli in broth cultures incubated at temperatures between 7 and 15 degrees C was examined by determinations of numbers of colonies recovered on plate count agar (PCA); absorbance at 600 nm (A600); cell lengths from photomicrographs; and cell size distributions by flow cytometry. Cultures incubated between 7 and 10 degrees C were evaluated for 8 days or until A600 values approached 1.0. Cultures incubated at > or =12 degrees C were subcultured to maintain them in the log phase for up to 8 days. Numbers of colonies recovered declined when cultures were incubated at 7 degrees C, but increased when cultures were incubated at higher temperatures. However, A600 values increased during incubation at all temperatures. The mean lengths of cells doubled during incubation at 7 degrees C for 8 days, but remained constant during incubation at 10 degrees C for 1.25 days. Forward angle light scatter (FALS) measurements obtained by flow cytometry indicated that the mean length of cells increased at < or = 8 degrees C, but not at 10 degrees C. A reference value at the 90th percentile of FALS measurements on day 0 was used to determine changes in the distribution of the lengths of cells. About 80% or 17% of the cells were above the reference value after 5 days of incubation at 7 degrees C or 1.25 days of incubation at 10 degrees C, respectively. Cultures that were maintained in the log phase at 12 degrees C became increasingly heterogeneous in cell size after 2 days, but cultures that were maintained at 13 degrees C remained constant in cell size for 8 days. The observations have implications for the prediction of mesophile proliferation at temperatures that approach their minima for growth.     

Increasing the water uptake of wood veneers through plasma treatment at atmospheric pressure

In order to provide a database which documents the influence of plasma treatment on water uptake of wood veneers, veneers of 27 wood species underwent immersion tests in untreated and plasma-treated states. Plasma treatment was executed using an air driven dielectric barrier discharge at atmospheric pressure. The results showed that plasma treatment led to significantly improved water uptake for most of the wood species, but some wood species remained unaffected after plasma treatment.     

Multi-pulsed high hydrostatic pressure treatment for inactivation and injury of Escherichia coli

Escherichia coli cells in peptone water were pressurized at 300?MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300?s for single- (300?s?×?1 pulse) and multi-pulsed (150?s?×?2 pulses, 100?s?×?3 pulses, 75?s?×?4 pulses, 60?s?×?5 pulses, 50?s?×?6 pulses and 30?s?×?10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log₁₀ more reduction on selective medium than the non-selective medium indicating that more than 95?% of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4?°C revealed that there was less recovery from injury for multi-pulsed HHP treatment.     

The Effects of High-Pressure Processing at Low and Subzero Temperatures on Inactivation of Microorganisms in Frozen and Unfrozen Beef Mince Inoculated with Escherichia coli Strain ATCC 25922

Frozen and unfrozen beef mince inoculated with Escherichia coli strain ATCC 25922 were exposed to a pressure of 300 MPa for 5 min at different temperatures (?10, ?5, 0, 10 and 20 °C). A maximum reduction of 1.5 log in total aerobic count (TAC) was obtained in unfrozen samples at ?5 °C, whereas at 20 °C, the reduction was only 0.6 log. Microbial inactivation in beef mince was enhanced by freezing the beef mince prior to pressurization. An average log reduction of 3.0 (SD?=?0.2) in both E. coli and TAC was obtained in frozen beef mince treated at ?5 °C. The highest bacterial reductions were observed in frozen samples. The extent of bacterial injury was substantially less in frozen samples than unfrozen samples, indicating that the damage inflicted on microorganisms in frozen beef mince by high pressure was irreversible. Lightness (L ^*), redness (a ^*) and yellowness (b ^*) values measured in accordance of Commission Internationale de l’Eclairage (CIE) for all the pressure-treated frozen and unfrozen samples differed slightly from unfrozen control samples (average total colour change, ΔE?=?6.1, SD?=?1.1). Water-holding capacity (WHC) measured by “high-pressure expressed moisture”, a new method proposed in this study, showed that freezing the samples prior to pressurization could increase WHC of minced beef. The results suggest that high-pressure processing could be used to make safer traditional raw minced meat products, such as steak tartare and cig kofte, a traditional Turkish dish made with minced beef, bulgur and spices.     

Growth and survival of Escherichia coli O157:H7 on fresh-cut apples in modified atmospheres at abusive temperatures

The effects of reduced-O2 and elevated-CO2 modified atmospheres (MAs) and abusive temperatures on the growth and survival of E. coli O157:H7, yeast, and molds and on changes in the visual quality of fresh-cut apples were evaluated. High-CO1 and low-O2 (> or = 15% and < 1%, respectively) atmospheres inhibited the growth of the pathogen on apple slices at 15 and 20 degrees C. However, the population of the pathogen increased by 1 log cycle after 2 weeks of storage in air. The high-CO2 MA resulted in the inhibition of yeast and mold growth, less browning, and better visual quality than did air and ambient-CO2 atmospheres. The results of this study confirm that E. coli O157:H7 can grow on apple slices in air. These results also show that these organisms survive but are inhibited in MAs with high CO2 levels at abusive temperatures. An MA can increase the shelf life of fresh-cut apples by improving retention of visual quality and inhibiting yeast and molds. Thus, contamination of minimally processed apples with E. coli O157:H7 can be a safety issue for both air- and MA-packaged cut apples.     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.