Indoxyl-β-D-glucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples

Abstract

About 97% of Escherichia coli strains produce beta-glucuronidase, but almost all other Enterobacteriaceae lack this enzyme. A D-glucopyranosiduronic acid (glucuronide) possessing a readily detectable beta-linked aglycone should, therefore, constitute a specific reagent for the detection of this organism. For this purpose, the title compound has been synthesized for the first time. The synthesis proceeds in eight steps from readily available D-glucuronolactone, anthranilic acid, and chloroacetic acid and can be carried out on a large scale. The compound has the predicted properties: when included in the standard membrane filter test for the analysis of water, indoxyl-beta-D-glucuronide allows specific detection of E. coli through the formation of blue colonies that are the result of rapid conversion of the liberated aglycone to indigo. The recovery of E. coli is easily measured and almost quantitative.

Enumeration of Enterococcus sp. using a modified mE method

“…Retrospective epidemiological studies have Bacteria growing on media containing an indoxyl-b-D-glydocumented relationships between enterococcal densities and coside and producing the appropriate glycosidase will form swimming-associated gastrointestinal disease in both fresh blue colonies through release of an aglycone that is converted and marine waters (Cabelli et al 1983). Consequently, the US to indigo (Ley et al 1988). Environmental Protection Agency (Anon.…”

Methods for Escherichia coli identification in food, water and clinical samples based on beta‐glucuronidase detection

“…) and indoxyl-p-wglucuronide (IBDG) are chromogenic substrates that have been tested as substitutes for MUG in solid enumeration media(Frampton et al 1988;Ley et al 1988;Watkins et al 1988;Sharpe et al 1989). These substrates release indoxyl (or halogenated indoxyl) which is rapidly oxidized to the insoluble indigo or its analogue, providing a very distinct differentiation of GUD-positive E. colz as blue colonies growing on agar plates or membranes.…”

“…These substrates release indoxyl (or halogenated indoxyl) which is rapidly oxidized to the insoluble indigo or its analogue, providing a very distinct differentiation of GUD-positive E. colz as blue colonies growing on agar plates or membranes. Synthesis of IBDG by a low cost process(Ley et al 1988) may lead to its increased use in G U D detection systems.In addition to the work of individual investigators who combined G U D substrates with various selective and differential media or prepared dried substrates on paper disks or stripsManafi and Rotter 1991 ;, commercial test kits have been developed that are capable of rapidly measuring G U D activity alone or, in conjunction with other enzymes, providing multi-enzyme profiles. Among some of the commercial tests currentlyused are : Rosco tablets (Rosco Diagnostica, Taastrup, Denmark) containing PNPG and evaluated for identifying E. colz in both clinical and environmental isolates (Dibb and Bottolfsen 1984; Pkrez et al 1986); Fluorocult media containing M U G (E. Merck AG, Darmstadt, Germany) also evaluated with clinical isolates (Heizmann et al 1988; Doller et al 1990); the R I M Escherichia colt Kit (Austin Biologicals Laboratories Inc., Austin, TX) evaluated on clinical isolates (Varga and DiPersio 1986; Edberg and Kontnick 1986) includes a tube of buffer plus reagent-.…”

β-glucuronidase activity determination as an indirect estimate of Escherichia coli: development of a miniaturized assay and its application to seawater samples

“…This compound is specifi cally cleaved by E. coli, producing after reaction a waterinsoluble blue-indigo aglycone product that precipitates and is measured at 450 nm wavelength; this makes the observation of the reaction product easy, also under normal daylight [19].…”