Growth of Escherichia coli ATCC 9637 through the uptake of compatible solutes at high osmolarity

The growth rate of Escherichia coli ATCC 9637 was determined in a chemically defined (CD) medium with high osmolarity, 1-1.2 M. The addition of ectoine or glycine betaine to the medium resulted in a significant stimulation of growth rate for this strain. In the presence of ectoine derivatives, hydroxyectoine and homoectoine, cell growth was not stimulated to the same extent as when ectoine was added, but it was improved slightly. The acceleration of growth rate of E. coli ATCC 9637 at elevated osmolarity was ascribed to the accumulation in the cells of ectoine or glycine betaine added to the medium, both of which were proved to be genuine osmolytes in cells. Rapid uptake of ectoine by cells was confirmed when ectoine was available in the CD medium with high osmolarity. Since strain ATCC 9637 did not accumulate ectoine in the absence of an energy source, ectoine uptake might take place not only through cellular sensing of the external high osmolarity but through cellular functioning via energization.     

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.     

Estimation of the biofilm formation of Escherichia coli K-12 by the cell number

We developed a method of estimating the biofilm formation of Escherichia coli K-12 strains in microtiter-plate wells by the cell number. Regression lines between the cell number and absorbance of crystal violet that stained the E. coli biofilm consisted of high and low slope lines, respectively.     

Nonthermal inactivation of Escherichia coli K-12 on spinach leaves, using dense phase carbon dioxide

While the use of some chemical sanitizers is approved for inactivation of microbes on the surfaces of fruits and vegetables, these compounds often degrade product quality with limited improvement in product safety. The application of dense phase carbon dioxide (DPCD, or high-pressure CO2) is a nonthermal process for inactivation of foodborne pathogens inoculated into various juices and model solutions. In this work, DPCD was evaluated for its potential to inactivate Escherichia coli K-12 inoculated on fresh spinach leaves. Inoculated leaves were exposed for up to 40 min to DPCD at a subcritical condition (5 MPa, 40 degrees C) and two supercritical conditions (7.5 and 10 MPa, 40 degrees C) at a flow rate of 50 g of CO2/min. E. coli K-12 populations were reduced to nondetectable levels (approximately 5-log reduction) using supercritical treatment conditions at exposure times as short as 10 min; efficacy of DPCD at the subcritical state was limited. This research demonstrates that DPCD has potential as a pasteurization technology for application to leafy green vegetables, although issues with discoloration and other quality measures will need more extensive evaluations.     

Transformation of Escherichia coli K-12 with a high-copy plasmid encoding the green fluorescent protein reduces growth: implications for predictive microbiology

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (mumax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40 degrees C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40 degrees C, mumax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.     

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.     

Simple quantitative analysis of Escherichia coli K-12 internalized in baby spinach using Fourier Transform Infrared spectroscopy

Bacterial contamination continues to be a serious concern for food safety. Although washing fresh produce helps in reducing pathogen levels, pathogen internalization often limits the effectiveness of washing. When pathogens internalize in leafy vegetables, the method of identification and quantitative measurement would be called into question. This study was aimed to use Fourier Transform Infrared (FTIR) spectroscopy integrated with an attenuated total reflectance kit for quantification of Escherichia coli K-12 internalized in baby spinach. The bacteria were inoculated into vascular and intracellar tissues of spinach leaves by syringe injection and the distribution of internalized E. coli K-12 cells was confirmed under scanning electron microscopy (SEM). FTIR measurement following the preparation of bacterial suspension from spinach leaves with high speed pulverizing enabled to detect the absorbance peaks in the amide II region between 1590 and 1490 cm⁻¹ as a fingerprint for the microbes. It was found that the estimated concentrations of E. coli K-12 agreed well with the concentrations determined by plate counting with R² values of 0.98 and 0.97 in peptone water and spinach extracts, respectively. The results demonstrated that FTIR can identify and quantify E. coli K-12 in baby spinach extracts at a limit of detection of approximately 100 CFU/mL in 5 min. The developed method is expected to be suitable for the analysis of pathogenic E. coli strains and other bacterial species in fresh vegetables.     

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。     

Demonstration of the applicability of the Weibull-log-logistic survival model to the isothermal and nonisothermal inactivation of Escherichia coli K-12 MG1655

Published isothermal semilogarithmic survival curves of Escherichia coli K-12 MG1655, in the range of 49.8 to 60.6 degrees C, all had noticeable downward concavity. They could be described by the model log S(t) = -b(T)t n, where S(t) = N(t)/N0, N(t) and N0 being the momentary and initial number of organisms, respectively; b(T) is a temperature-dependent rate parameter; and n is a constant found to be about 1.5. The temperature dependence of b(T) could be described by the log-logistic model, b(T) = ln[1 + exp[k(T - Tc)]], which had an almost perfect fit, with k = 0.88 degrees C(-1) and Tc = 60.5 degrees C. The constants, n, k, and Tc were considered the organism's survival parameters in the particular medium. They were incorporated into a rate equation on the assumption that in nonisothermal heating, the momentary inactivation rate is the isothermal rate at the momentary temperature at a time that corresponds to the momentary survival ratio. This model's estimates matched the actual survival curves obtained in the same work under two different nonisothermal heating profiles, lending support to the notion that the Weibull-log-logistic model combination can be used not only to describe isothermal inactivation mathematically, but also to predict survival patterns under nonisothermal conditions.     

Effect of tannins on the in vitro growth of Escherichia coli O157:H7 and in vivo growth of generic Escherichia coli excreted from steers

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37 degrees C, was reduced (P < 0.05) with as little as 400 microg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 microg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 microg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 microg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.     

Membrane damage and viability loss of Escherichia coli K-12 in apple juice treated with radio frequency electric field

The need for a nonthermal intervention technology that can achieve microbial safety without altering nutritional quality of liquid foods led to the development of a radio frequency electric fields (RFEF) process. In order to understand the mechanism of inactivation of bacteria by RFEF, apple juice purchased from a wholesale distributor was inoculated with Escherichia coli K-12 at 7.8 log CFU/ml and then treated with RFEF. The inoculated apple juice was passed through an RFEF chamber operated at 20 kHz, 15 kV/cm for 170 micros at a flow rate of 540 ml/min. Treatment condition was periodically adjusted to achieve outlet temperatures of 40, 45, 50, 55, and 60 degrees C. Samples at each outlet temperature were plated (0.1 ml) and the number of CFU per milliliter determined on nonselective and selective agar media was used to calculate the viability loss. Bacterial inactivation and viability loss occurred at all temperatures tested with 55 degrees C treatment, leading to 4-log reductions. No significant effect was observed on bacterial population in control samples treated at 55 degrees C with a low-RFEF (0.15 kV/cm) field strength. These observations suggest that the 4-log reduction in samples treated at 15 kV/cm was entirely due to nonthermal effect. RFEF treatment resulted in membrane damage of the bacteria, leading to the efflux of intracellular ATP and UV-absorbing materials. Populations of injured bacteria recovered immediately (<30 min) from the treated apple juice averaged 0.43 log and were below detection after 1 h of RFEF treatment and determination using selective plates (tryptic soy agar containing 5% sodium chloride). The results of this study suggest that mechanism of inactivation of RFEF is by disruption of the bacterial surface structure leading to the damage and leakage of intracellular biological active compounds.     

Inactivation of Escherichia coli K-12 exposed to pressures in excess of 300 MPa in a high-pressure homogenizer

Homogenization is used widely in the dairy industry to improve product stability and quality. High-pressure homogenization (HPH) of fluid foods up to pressures of 300 MPa has demonstrated excellent potential for microbial inactivation. Microbial inactivation can be enhanced during HPH with the inclusion of antimicrobial compounds. Escherichia coli K-12 cells, grown statically or in chemostat, were exposed to HPH processing pressures of 50 to 350 MPa in the absence or presence of the antimicrobial nisin. Valve temperature was regulated by a water bath and pressure, and temperature data were recorded continuously after process initiation. Survivors were enumerated via plating on nonselective growth media. Pressure and temperature at the valve outlet port exhibited a quadratic relationship (R(2) = 0.9617, P < 0.05). Significant HPH-induced inactivation of the gram-negative microorganism was observed in the range of 100 to 250 MPa. Above 300 MPa, heat was the main factor promoting microbial inactivation, regardless of whether cells were grown in chemostat or statically. Chemostat-grown cells were significantly (P < 0.05) more resistant to HPH processing than were statically grown cells. Data indicate potential synergistic effects of nisin and HPH on the inactivation of bacterial contaminants. This study represents the first report of inactivation of a bacterium with HPH pressures in excess of 300 MPa in the presence and absence of an antimicrobial.     

Effect of sodium hypochlorite (NaClO) sanitizer-induced stress on growth kinetics and morphological changes in Escherichia coli and Bacillus cereus spores

Sanitizer-induced stress on the growth kinetics of Escherichia coli and Bacillus cereus spores was investigated. Morphologies of B. cereus vegetative cells and spores were also compared. Nonpathogenic E. coli and pathogenic E. coli O157:H7 and B. cereus spores were treated with 100 ppm sodium hypochlorite in inoculated broth and stored at 13, 18, 24, 30, and 36°C. The lag time (LT) of stressed E. coli was extended more at low temperatures, regardless of pathogenicity. E. coli and B. cereus vegetative cells were sensitive to a sanitizer treatment with NaClO. Stressed strains grew more rapidly than unstressed strains. However, sanitizer stress did not significantly (p>0.05) affect the LT and specific growth rate (SGR) of B. cereus spores, regardless of storage temperature. Transmission electron microscopy analysis also revealed that stress induced using the NaClO sanitizer caused morphological damage to B. cereus vegetative cells, but not to B. cereus spores.     

Interaction between the respiratory burst activity of neutrophil leukocytes and experimentally induced Escherichia coli mastitis in cows

The respiratory burst activity of neutrophil leukocytes from bovine peripheral blood was studied before and during an experimentally induced Escherichia coli mastitis. The competence of neutrophils to generate reactive oxygen species following stimulation with opsonized particles prior to infection was negatively correlated with severity of subsequently induced E. coli mastitis. In the presence of the soluble activator, phorbol myristate acetate, no such correlation was obtained. However, combination of blood neutrophil numbers with phorbol myristate acetate induced respiratory burst competence, called reactive oxygen species-generating capacity, displayed a negative correlation with the intensity of a subsequent inflammation of the bovine mammary gland. At the onset of mastitis, a concomitant reduction in blood neutrophil numbers, a strong shift in cell types, and a substantial decrease in production of reactive oxygen species occurred. Reestablishment and even enhancement of the respiratory burst activity coincided with the reappearance of mature neutrophils. Possible stimulatory effects on neutrophil superoxide generation are discussed. Data suggest that generation of reactive oxygen species by mature neutrophils may be of primary importance for microbial killing during the onset and recovery from mastitis.     

Survival of staphylococcus aureus and Escherichia coli as affected by ethanol and NaCl

Growth of three strains of Staphylococcus aureus and two strains of Escherichia coli on nutrient agar (NA) supplemented with ethanol and NaCl was investigated. S. aureus did not grow on NA containing > or =10% ethanol (wt/wt) combined with > or =0% NaCl (wt/wt), or 7.5% ethanol combined with 7.5% NaCl. Neither E. coli nor E. coli O157:H7 grew on NA containing > or =7.5% ethanol combined with > or =0% NaCl, 5% ethanol combined with > or =2.5% NaCl, or > or =5% NaCl combined with > or =0% ethanol. It is apparent that NaCl enhanced the inhibitory effect of ethanol on growth of S. aureus and E. coli When cells were suspended in nutrient broth containing 12.5, 20, or 40% ethanol combined with NaCl, viable cells decreased with an increase of ethanol concentration. Ethanol sensitivity among strains and between genera varied in a limited range. When the cells were exposed to 20% ethanol in combination with 5% NaCl, S. aureus and E. coli lost viability after 30 and 10 min, respectively. When treated with 40% ethanol combined with > or =0% NaCl, all test strains lost viability within 5 min.     

Membrane damage and viability loss of Escherichia coli K-12 and Salmonella enteritidis in liquid egg by thermal death time disk treatment

Bacterial injury, including leakage of intracellular substance and viability loss, of Escherichia coli K-12 (ATCC 23716) and Salmonella Enteritidis (ATCC 13076) inoculated in liquid egg white and liquid whole egg was determined by thermal death time disk. E. coli K-12 and Salmonella Enteritidis were inoculated in liquid egg white and liquid whole egg to a final count of 7.8 log CFU/ml and were thermally treated with thermal death time disks at room temperature (23"C), 54, 56, 58, and 60 degrees C from 0 to 240 s. Sublethal injury, leakage of intracellular substances, and viability loss of E. coli K-12 and Salmonella Enteritidis was investigated by plating 0.1 ml on selective trypticase soy agar containing 3% NaCl, 5% NaCl, sorbitol MacConky agar, and xylose lysine sodium tetradecylsulfate and nonselective trypticase soy agar. No significant (P > 0.05) differences on percent injury or viability loss for E. coli K-12 and Salmonella populations were determined in all samples treated at 23 degrees C. Sublethal injury occurred in E. coli and Salmonella populations at 54 degrees C or above for 120 s. Viability losses for both bacteria averaged 5 log at 54 degrees C or above for 180 s, and the surviving populations were below detection (<10 CFU/ml). Thermal treatment at 40 degrees C and above led to membrane damage, leakage, and accumulation of intracellular ATP from 2 to 2.5 log fg/ml and UV-absorbing substances of 0.1 to 0.39 in the treated samples. These results indicate similar thermal injury/damage on both E. coli and Salmonella membranes as determined by the amount of inactivation, viability loss, and leakage of intracellular substances of bacteria.     

Modelling the combined temperature and salt (NaCl) limits for growth of a pathogenic Escherichia coli strain using nonlinear logistic regression

A broth-based method is used to determine if exponential phase Escherichia coli R31, an STEC, is able to grow within 50 days under various combinations of sub-optimal temperatures and salt concentrations. From these data, the growth limits for combinations of temperature (7.7-37.0 degrees C) and water activity (0.943-0.987; NaCl as humectant) are defined and modelled using a nonlinear logistic regression model. That form of model is able to predict the combinations of salt concentration/water activity and temperature that will prevent the growth of E. coli R31 with selected levels of confidence. The model fitted the data with an approximate concordance rate of 97.3%. The minimum water activity that permitted growth occurred in the range 25-30 degrees C, the temperature range which optimises cell yield. At temperatures below this range the minimum water activity which allowed growth increased with decreasing temperature.     

Effect of growth rate reduction and genetic modifications on acetate accumulation and biomass yields in Escherichia coli

Although acetate biosynthesis in Escherichia coli provides an important intermediary for ATP synthesis, its accumulation inhibits both cell growth and protein production. Since pyruvate provides the largest flux to acetate and is central to the problem of acetate production, acetate accumulation could be reduced or abolished if the pyruvate pool for the TCA cycle was reduced. To examine this possibility, various pyruvate kinase (pyk) and phosphotransferase system (pts) mutants were tested for acetate production in batch cultures with glucose as the only carbon source. The pykA pykF mutant exhibited significant reductions in the specific growth rate and acetate production compared with the wild-type strain. Interestingly, in the case of pts and pts pyk mutants in which increased biomass yields were observed in comparison with the wild-type strain, no acetate production was detected. Therefore, these mutants are potentially useful for higher production of recombinant proteins. The results from the continuous cultivation performed using the wild-type strain at various dilution rates, suggest acetate reduction as a consequence of both genetic changes and growth rate diminutions.