Identification of Shiga toxigenic Escherichia coli seropathotypes A and B by multiplex PCR.

A multiplex PCR assay was developed to identify the six clinically important enterohemorrhagic Escherichia coli (EHEC) serotypes classified in seropathotypes A and B and to differentiate these from Shiga toxigenic E. coli. The assay simultaneously detects genes for Shiga toxin (stx) and intimin (eae), including allelic variants of both genes, 16S internal amplification control, as well as unique sequences in the wzx genes that are specific for serotypes O157, O26, O111, O103, O121 and O145. PCR analysis of 40 representative strains showed that the assay correctly identified the virulence genes, if present, and the respective O antigen type of all the strains, including some atypical EHEC, as well as enteropathogenic E. coli and E. coli strains examined.

Genetic and evolutionary analysis of mutations in the gusA gene that cause the absence of beta-glucuronidase activity in Escherichia coli O157:H7.

Escherichia coli serotype O157:H7 do not exhibit beta-glucuronidase (GUD) activity but carry the gusA gene (uidA) that encodes for GUD. In trans-complementation, the gusA gene cloned from the GUD-positive variant strain 493-89 effectively restored GUD activity in O157:H7 strain 35150. Comparison of gusA sequences from the GUD-negative 35150 strain to that of 493-89 revealed several base mutations, including a guanosine (G) dinucleotide insertion that caused a frameshift in the 35150 gusA gene and introduced a predicted premature termination codon. This explains the absence of GUD activity in O157:H7. A 35150 gusA construct from which the G-G insertion was deleted restored activity in GUD-negative O157:H7 transformants. The G-G insertion was present in all GUD-negative O157:H7 strains but was absent in their GUD-positive variants. The G-G insertion that produced the characteristic GUD-negative phenotype to O157:H7 strains appeared later than the other gusA mutations in the evolutionary emergence of O157:H7.

Enzymatic synthesis of 4-amino-3,5-diethylphenyl sulfate, a rodent metabolite of alachlor.

Rat liver tissue homogenates were utilized for in vitro enzymatic conversion of 2,6-diethylaniline (DEA) to the important alachlor metabolite 4-amino-3,5-diethylphenyl sulfate (ADEPS), which was also generated as a radiolabeled standard for use in metabolism studies. ADEPS formation in rodents is associated with the production of other reactive metabolites implicated in alachlor rodent carcinogenesis, making dependable access to an ADEPS standard highly desirable. (14)C-DEA was oxidized by rat liver microsomes to (14)C-4-amino-3,5-diethylphenol, which was further converted to ADEPS via addition of the phosphosulfate transferase cofactor adenosine-3'-phosphate-5'-phosphosulfate. Microprobe NMR was used in conjunction with high-resolution mass spectrometry to fully characterize the resulting (14)C-ADEPS and confirm its structure. Because microgram quantities sufficed for full characterization, the enzymatic transformation provides a viable alternative to radiosynthesis of (14)C-ADEPS.

Nuclear magnetic resonance timecourse studies of glyphosate metabolism by microbial soil isolates.

1. Triple Resonance Isotope EDited nmr spectroscopy (TRIED) has been developed to detect and examine minute levels of glyphosate metabolites in microbial soil isolates. Using stable isotopic labelling (13C and 15N), TRIED allows the simultaneous detection of multiple metabolites in crude matrices at submicrogram levels. An improvement over earlier techniques where milligrams are needed, TRIED can detect 500 ng of triply labelled compound in a crude sample (1:14,000 mass ratio) in just hours. 2. TRIED is used here to compare the kinetics and metabolic pathways of glyphosate metabolism by two strains of Ochrobactrum anthropi, LBAA and S5. Both LBAA and S5 appear to metabolize glyphosate primarily via the aminomethylphosphonate (AMPA) pathway, since no detectable levels of glycine or sarcosine are observed in the media or lysates of either microbe. The formation of N-methylAMPA is common to the metabolism of both microorganisms, but N-acetylAMPA is observed only in LBAA. N-methylacetamide is detected predominantly in media and lysates of S5, although some evidence also points to the formation of this metabolite in LBAA. 3. Results are consistent with conventional radioactive tracer studies. TRIED nmr provides more specific structural information complementary to radiolabel methods. Both nmr and radioactivity studies show S5 glyphosate metabolism to be much slower than that of LBAA.

Metabolic deactivation of the herbicide thiazopyr by animal liver esterases.

1. Thiazopyr was hydrolysed in vitro to its corresponding acid by rabbit and porcine liver esterases. 2. A wide range of thiazopyr esterase activity was observed in extracts from liver acetone powders from 15 animal species with bovine, rabbit and pigeon showing the highest activities. 3. Using soybean tissue culture cells and Arabidopsis seedlings, the acidic metabolite was shown to possess < 1% of the herbicidal activity of thiazopyr. 4. We propose that biotransformation of thiazopyr to the acid is a critical pathway of metabolism in animals and plants.

In vitro biotransformation of thiazopyr by rat liver microsomes: oxidative cleavage of a carboxylic methylester by monooxygenases.

1. Thiazopyr was metabolized by liver microsomes from male Sprague-Dawley rats to a previously unidentified metabolite. 2. The new metabolite was identified by coelution with an authentic standard in hplc and by electrospray lc/ms as the corresponding carboxylic acid. 3. Formation of the carboxylic acid metabolite was inhibited in the presence of mono-oxygenase inhibitors including piperonyl butoxide, 1-aminobenzotriazole, metyrapone and tetcyclacis. 4. Transformation of thiazopyr to its carboxylic acid by rat liver microsomes is mediated by mono-oxygenases and not hydrolases.

In vitro transformation of dithiopyr by rat liver enzymes: conversion of methylthioesters to acids by oxygenases.

1. Transformation of dithiopyr by rat liver enzymes in vitro produced the corresponding monoacids as the predominant metabolites. 2. Transformations of the methylthioester functional groups in dithiopyr to the monoacids were mediated via rat liver microsomal oxygenases, and not via esterases. 3. Based on the formation of a dithiopyr-glutathione conjugate, the mechanism of monoacid formation is believed to proceed through an initial sulphur oxidation of the methylthioester group and a subsequent nucleophilic displacement reaction.

Metabolism of alachlor by rat and mouse liver and nasal turbinate tissues.

The metabolism of alachlor was studied using in vitro incubations with microsomal fractions prepared from liver and nasal turbinates of rats and mice. Specifically, the transformation of alachlor to 3,5-diethylbenzoquinone-4-imine was examined. A key intermediate in this pathway was identified as 2,6-diethylaniline, the formation of which required catalysis by microsomal arylamidases. 2,6-Diethylaniline was oxidized to 4-amino-3,5-diethylphenol and the electrophilic 3,5-diethylbenzoquinone 4-imine. Rat nasal tissue possessed high enzymatic activity which can promote the formation of the reactive quinone imine. Whole body autoradiographic analysis demonstrated localization of radioactivity in the rat nasal tissue following oral administration of alachlor. A methylsulfide metabolite of alachlor was shown to be a precursor to 2,6-diethylaniline. The deposition of radioactivity in the rat nasal tissue was more pronounced following oral administration of the methylsulfide metabolite of alachlor.

Synthesis and analysis of glucuronides of N-hydroxyamides.

Reactions of five N-hydroxyacetylarylamines (N-hydroxyphenacetin, N-hydroxyacetanilide, N-hydroxy-p-chloroacetanilide, N-hydroxy-2-acetylaminonaphthalene, and N-hydroxy-2-acetylaminofluorene) with uridine 5'-diphosphoglucuronic acid catalyzed by immobilized glucuronyl transferase enzyme resulted in the formation of glucuronides conjugated at the N-hydroxyl group. Chromatographic properties of these compounds were examined by using thin layer, gas liquid, and high pressure liquid chromatography. A variety of mass spectrometric techniques were also evaluated for characterization of compounds of this class, including fast atom bombardment, chemical ionization, and electron impact. Electron impact spectra of the trimethylsilyl derivatives contain class characteristic ions and fragmentation pathways that would permit the special nature of the site of conjugation of this important class of compounds to be recognized in glucuronides of undetermined structure. Molecular ion species were not reliably detected in the electron impact spectra; however, these were readily distinguishable by the other two techniques.

Stable isotope analysis by fast-atom bombardment labeling of UDP-glucuronic acid.

Fast-atom bombardment mass spectrometry is found to provide a method for analysis of isotopes in the enzyme cofactor uridine-5'-diphosphoglucuronic acid, heretofore unsusceptible to mass-spectral characterization. This technique was used to determine optimal conditions for the introduction of 18O by acid-catalyzed exchange in H218O and to evaluate the loss of the isotope when labeled cofactor is used in enzymatic incubations. Fast-atom bombardment mass spectrometry provided a quantitative assessment of various isotopic species and also permitted the location of the isotopes in the molecule to be determined.

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).

Metabolism of clofazimine in leprosy patients.

We have identified two metabolites of clofazimine (B663; Lamprene; 3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine) in our initial investigation of its metabolism in leprosy patients. Based on mass, ultraviolet, and visible spectrometry, we characterized an unconjugated (metabolite I, 3-(p-hydroxyanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine ) and a conjugated (metabolite II, 3-(beta-D-glucopyranosiduronic acid)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine) metabolite from the urine of patients. Both metabolites were red in color, similar to clofazimine; however, both were considerably more polar than the parent drug. We suggest that metabolite I was formed by a hydrolytic dehalogenation reaction, and metabolite II by hydrolytic deamination followed by glucuronidation.