Simple medium that preserves low concentrations of Escherichia coli for use in the water bacteriology proficiency test.

A medium containing (per liter) 6.8 g of sodium acetate trihydrate, 3.4 g of potassium dihydrogen phosphate, and 0.1 g of magnesium sulfate heptahydrate (medium pH, 5.85 plus/minus 0.05) was developed for use in a water bacteriology proficiency test. The medium maintained 80 to 100 % viability of inoculated Escherichia coli at temperatures up to 31 degrees C for at least 12 days, while the concentrations of bacteria in the medium were as low as 20 bacteria per 100 ml. The medium remained stable after a year of storage. It has been used successfully to preserve bacteria in nine statewide bacteriology proficiency tests for potable and nonpotable water and has also been used in a nationwide pilot test. This report presents the results of these tests.     

Simple bacterial preservation medium and its application to proficiency testing in water bacteriology.

A medium composed of nutrient broth, 1.8% boric acid, and 1% sodium chloride at pH 7.0 was shown to maintain the stability of Escherichia coli cultures for up to 10 days at room temperature. By using this preservation medium for preparing a simulated sample a successful proficiency test survey in water bacteriology was conducted.     

An approach to integrated data assessment in a proficiency test on the enumeration of Escherichia coli

Aims: To develop appropriate statistical approaches to plan and evaluate proficiency tests for the enumeration of Escherichia coli, addressing, in particular, a possible but frequently unavoidable lack of test sample homogeneity. Methods and Results: Each of 50 laboratories analysed two samples of a stabilized suspension of E. coli in duplicate, using various media, inoculation methods, and incubation times and conditions. In parallel, the E. coli suspension was tested by the organiser for homogeneity and stability. Escherichia coli counts followed a log‐normal distribution. After eliminating, by Youden analysis, two data sets that were considered outliers and eight data sets for underperformance of the laboratories (substantial lack of repeatability), the standard deviation of the mean was about 0·06 log₁₀ units. There was no evidence of bimodality of the data. Lack of homogeneity of distribution of bacteria had a strong effect on measurement uncertainty, in addition to laboratory bias and method repeatability. The homogeneity decreases during storage of the individual test vials; this effect could be modelled by the known kinetics of inactivation of micro‐organisms. The results were confirmed by Monte Carlo simulations. Conclusions: By a tailored analysis of proficiency testing data, it is possible to distinguish the effect of lack of homogeneity, laboratory bias and method repeatability, on the measurement uncertainty. Significance and Impact of the Study: A statistic tool is provided to solve problems related to lack of stability of microbiological test material and to separate the effects of sample inhomogeneity from the performance of the individual laboratory.     

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.     

Adaptive diversification in genes that regulate resource use in Escherichia coli

While there has been much recent focus on the ecological causes of adaptive diversification, we know less about the genetic nature of the trade-offs in resource use that create and maintain stable, diversified ecotypes. Here we show how a regulatory genetic change can contribute to sympatric diversification caused by differential resource use and maintained by negative frequency-dependent selection in Escherichia coli. During adaptation to sequential use of glucose and acetate, these bacteria differentiate into two ecotypes that differ in their growth profiles. The “slow-switcher” exhibits a long lag when switching to growth on acetate after depletion of glucose, whereas the “fast-switcher” exhibits a short switching lag. We show that the short switching time in the fast-switcher is associated with a failure to down-regulate potentially costly acetate metabolism during growth on glucose. While growing on glucose, the fast-switcher expresses malate synthase A (aceB), a critical gene for acetate metabolism that fails to be properly down-regulated because of a transposon insertion in one of its regulators. Swapping the mutant regulatory allele with the ancestral allele indicated that the transposon is in part responsible for the observed differentiation between ecological types. Our results provide a rare example of a mechanistic integration of diversifying processes at the genetic, physiological, and ecological levels.     

Effects of low concentrations of ampicillin in feed on the intestinal Escherichia coli of chicks

Ampicillin in low concentrations (1.7 and 5 g t^-1) was incorporated in the feed given to 1-d-old chicks for 2 weeks. This was sufficient to select, in the intestinal contents, resistant Escherichia coli strains for which the minimum inhibitory concentration of ampicillin was > 100 μg ml^-1. Different clones of E. coli were identified by their biotype, pattern of resistance to antibacterial agents and plasmid profile (designated P-P types). The experiment was repeated twice and the average proportion of ampicillin-resistant P-P types among 72 isolates of E. coli from chicks given feed containing 0, 1.7 and 5 g ampicillin t^-1 were 10%, 31% and 46% respectively.     

Escherichia coli O157 can grow in natural freshwater at low carbon concentrations

Whereas much information on the die-off of Escherichia coli in the aquatic environment is available, only few data support its growth under such conditions. We therefore investigated batch growth in microcosms containing different types of sterile freshwater. The water samples were inoculated with low starting cell concentrations of E. coli O157 (3 × 10³ cells ml⁻¹) and growth was followed using nucleic acid staining combined with flow cytometry. We demonstrated that E. coli O157 is able to grow in sterile freshwater at low carbon concentrations, which is against the common view that cell numbers decline over time when added to freshwater samples. A correlation between apparent assimilable organic carbon (AOC_app) concentration and the final cell concentration reached by E. coli O157 was established ( P  <  0.01). A considerable fraction of the AOC_app (34 ± 13%) was used by E. coli O157 but the numerical cell yield was about five-times lower in comparison with the bacterial AOC-test community, which originated from natural freshwater. On average, the maximum specific growth rate ( μ _max) of E. coli O157 growing in sterile freshwater at 30°C was 0.19 ± 0.07 h⁻¹. Batch growth assays at five different temperatures revealed a positive influence of temperature on μ _max of E. coli O157. The results give new information on the behaviour of this common pathogen in the aquatic environment and contribute to microbial risk assessment in order to prevent spreading of water-borne diseases.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Design of a system for the control of low dissolved oxygen concentrations: critical oxygen concentrations for Azotobacter vinelandii and Escherichia coli

The physiological activity of microorganisms in environments with low dissolved oxygen concentrations often differs from the metabolic activity of the same cells growing under fully aerobic or anaerobic conditions. This article describes a laboratory-scale system for the control of dissolved oxygen at low levels while maintaining other parameters, such as agitator speed, gas flowrate, position of sparger outlet, and temperature at fixed values. Thus, it is possible to attribute in dilute nonviscous fermentations all physiologic changes solely to changes in dissolved oxygen. Experiments were conducted with Azotobacter vinelandii and Escherichia coli. Critical oxygen concentrations for growth (that value of oxygen allowing growth at 97% of mu max) were measured as 0.35 +/- 0.03 mg/L for A. vinelandii and 0.12 +/- 0.03 mg/L for E. coli. These values are significantly different from the commonly quoted values for critical oxygen concentrations based on respiration rates. Because of the superior dissolved oxygen control system and an improved experimental protocol preventing CO2 limitation, we believe that the values reported in this work more closely represent reality.     

A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking water

A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5.4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial.     

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH₄⁺ as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L⁻¹ product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Morphological changes in Escherichia coli cells exposed to low or high concentrations of hydrogen peroxide

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (less than 2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (less than 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (greater than 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

An improved medium for preparation of filterable Escherichia coli hemolysin

We found that Tween 80 has potent activity in enhancing passage of E. coli hemolysin through a membrane filter and, on the basis of this finding, we devised a medium, heart infusion broth supplemented with 0.5% Tween 80 and 0.1% glucose (HI-TG) for preparation of culture filtrates containing fairly large amounts of hemolysin. When 27 strains of hemolytic E. coli were cultivated to late logarithmic growth phase in the HI-TG broth at 37°C, the culture filtrates of most strains contained 64 to 128 HU₅₀ (50% hemolytic unit) per ml. From these and other results, we established a routine method for partially purifying E. coli hemolysin.     

Simple phagemid-based system for generating allele replacements in Escherichia coli.

We describe a phagemid-based system for rapidly generating chromosomal allele replacements in two steps. The system utilizes simple vector-determined selection procedures for each step and requires only that the target strain be male and sup+ (nonsuppressing for amber mutations).     

Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli.

Cysteine, in concentrations down to 0.04 micrograms/ml, induces transient amino acid starvation in Escherichia coli growing in minimal medium. The duration depends on the concentration and is 5 min at 2 micrograms of cysteine per ml. At low cysteine concentrations, threonine and isoleucine almost completely abolish the starvation.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。