Microquantitative determination of glucose in solutions using microorganisms as the test system

A technique for determining small quantities of glucose in solutions and biological liquids is proposed. The assay can be performed in the presence of some other carbohydrates, except those which are transported into E. coli cells by means of the phosphoenol-transferase system. The method is based on pH-metric regictration of H+-ions which are released from bacteria as a result of glucose consumption. The suspension of cells of E coli st. M-17 was used as a test system. We can also recommend a commercial preparation of colibacterin, which contains lyophilized bacteria E. coli st. M-17 in the sucrose-gelatin medium. The sensitivity of the method is about 1 microgram of glucose in 5-10 microliter.     

Membrane filter method for enumerating Escherichia coli.

A membrane filter procedure for enumerating Escherichia coli was developed and evaluated. The method quantifies E. coli within 24 h without requiring subculture and identification of isolates. It incorporates a primary selective-differential medium for gram-negative, lactose-fermenting bacteria; resuscitation of weakened organisms by incubation for 2 h at 35 degrees C before incubation at 44.5 degrees C for 18 to 22 h; and an in situ urease test to differentiate E. coli from other thermotolerant, lactose-positive organisms. The recovery of E. coli from marine, estuarine, and freshwater samples exceeded 90%. Of the presumptively positive colonies, 91% were verified as E. coli. Less than 1% of all of the verified E. coli colonies failed to react typically.     

A new selective and differential agar medium for Escherichia coli and coliform organisms

W right , R.C. 1984. A new selective and differential agar medium for Escherichia coli and coliform organisms. Journal of Applied Bacteriology 56 , 381–388.
An enriched lauryl sulphate-aniline blue agar medium which is selective for Escherichia coli and coliform organisms is described. From faecal samples, the medium gave higher counts of colonies producing acid from lactose than media containing bile salts. From contaminated water and food samples, the medium gave comparable or higher counts of colonies identified as E. coli than standard media. Colonies of E. coli were more readily differentiated from those of other coliform organisms.     

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity.

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

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.     

Estimation of Escherichia coli in raw ground beef.

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Estimation of Escherichia coli in raw ground beef

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

IMPROVEMENT OF SELECTIVITY FOR ISOLATION OF ESCHERICHIA COLI O157:H7 FROM GENERIC ESCHERICHIA COLI

A modified selective medium was developed to increase selectivity for isolation of Escherichia coli O157 from generic E. coli based on the knowledge that E. coli O157:H7 has more resistance against HCl condition than E. coli. As a preliminary experiment, four strains of E. coli O157:H7 (ATCC 35150, 43889, 43890, and 43894) were tested to determine the maximum concentration of 6N HCl (from 0 to 250 μL) added to 50 mL of MacConkey agar medium (MAC). The maximum level was 125 μL/50 mL (6N HCl/MAC), which E. coli O157:H7 strains could tolerate against the HCl concentration. After determination, comparative growth of 15 isolates of E. coli O157 and generic E. coli were evaluated on modified selective medium (HCl-MAC; with the addition of 125 μL/50 mL) and conventional MAC, respectively. All tested strains of E. coli O157 were grown on both media, whereas 9 out of 15 generic E. coli (60% of tested strains) were strongly inhibited on HCl-MAC. For selective isolation of E. coli O157 from generic E. coli, HCl-MAC has an effective potential for an implemental use. This information can extend as a baseline for use of HCl to conventional medium for successful isolation E. coli O157 from generic E. coli.     

Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts

A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7.     

A new selective and differential agar medium for Escherichia coli and coliform organisms

An enriched lauryl sulphate-aniline blue agar medium which is selective for Escherichia coli and coliform organisms is described. From faecal samples, the medium gave higher counts of colonies producing acid from lactose than media containing bile salts. From contaminated water and food samples, the medium gave comparable or higher counts of colonies identified as E. coli than standard media. Colonies of E. coli were more readily differentiated from those of other coliform organisms.     

大肠杆菌O54 O-抗原基因簇的破译及进化分析

破译了大肠杆菌O5 4O 抗原基因簇的序列 ,序列全长 1 4 0 6 2bp。用生物信息学方法分析序列并鉴定基因 ,共确定 1 0个基因 ,包括鼠李糖合成酶基因BDA和C(rmlBDA和rmlC) ,糖基转移酶基因 ,O 抗原转运酶基因 ,O 抗原聚合酶基因和合成磷酸丝氨酸侧链的基因及 1个不能确定功能的开放阅读框。对rmlC的 (G C) %含量 ,稀有密码子含量及进化分析都表明大肠杆菌O5 4O 抗原基因簇是在近期通过rmlC介导的重组形成 ,而且大肠杆菌O5 4和鲍氏志贺氏菌 9型的亲缘关系很近。对UTP 葡萄糖 1 磷酸 尿苷转移酶基因 (galF)和 6 磷酸葡萄糖脱氢酶基因(gnd)的进化分析揭示志贺氏菌属与大肠杆菌属在进化上属于同一个属。用PCR方法筛选出了针对大肠杆菌O5 4的特异基因 ,用于基因芯片或PCR方法对大肠杆菌O5 4的快速检测。     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).     

Adaptation of E. coli cell method for micro-scale nitrate measurement with the Griess reaction in culture media

The E. coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E. coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions. Therefore, the E. coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes. Furthermore, it was found that for the Griess reaction the pH values of samples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached. It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate. The adapted E. coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by measuring the standard curve of a dilution of each individual medium component. It was found that many organic medium components contain traces of nitrate, while none of them contain detectable nitrite. Among these, the extract of meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively. MRS broth contains nitrate from 0.3 to 0.6 mg N/l depending on the batch numbers of the product. The adapted E. coli can also be used for nitrate measurement in other matrices.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.