A Rapid and Specific Method for the Detection of Indole in Complex Biological Samples

Abstract

bIndole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain H10407 produces 3.3 ؎ 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota. Indole is widely distributed in the environment and is a component of diverse important compounds that occur in nature. In the pharmaceutical industry, synthesized indoles and their modified derivatives are popularly known for their medicinal properties. Indole analogs are significant components of a number of products, including vitamin supplements, dye, over-the-counter drugs, flavor enhancers, and perfumery. They are also used in the agricultural and plastics industries. Indole has been shown to play a role in regulating bacterial biofilm formation and virulence and influences diverse physiological processes, including host immune response (1-7).Indole is produced by about 85 bacterial species, including Gram-positive and Gram-negative bacteria, through the enzymatic degradation of tryptophan (8). Once produced, indole can be chemically modified within the same bacterial cell or taken up and modified by non-indole-producing bacteria. The most common naturally occurring indole analog is 3-methylindole (skatole), although other analogs, such as indoxyl sulfate and indole-3-propionic acid, can be found (9-11).Indole production by bacteria is an important phenotypic characteristic that has long been used to differentiate, identify, and diagnose enteric bacterial infections (12). Currently, the Kovács assay (13-17) is the most widely used method for detecting indole-producing bacteria. However, the key component, para-dimethylaminobe...

Functional microbiome deficits associated with ageing: Chronological age threshold

“…Indole was analysed using the Kovács reagent (Sigma‐Aldrich) as described elsewhere (Darkoh, Chappell, Gonzales, & Okhuysen, )). For tryptophan determinations, a series of calibration samples were prepared from a solution of acetonitrile:water (4:1) spiked with different quantities of tryptophan.…”

Fecal Indole as a Biomarker of Susceptibility to Cryptosporidium Infection

“…We could now examine if indole, the product of several common gut bacteria, could have an effect on the susceptibility of individuals to infection and/or illness caused by Cryptosporidium. Previously, healthy subjects showed a wide distribution of FICs, with ϳ75% in the 1 to 4 mM range (15), and 80% of the baseline indole levels in a subset of 50 subjects used in the present study were in the same range. We then went a step further and examined stage 1 FICs with regard to the outcome of a Cryptosporidium challenge (Fig.…”

“…Stool samples collected from subjects were stored at Ϫ80°C. Stage 1 samples were previously tested for FIC (15), while samples from stages 2 and 3 were tested for the present study. All samples were prepared for indole testing immediately after thawing.…”

“…One approach is to use a biomarker such as indole, which represents the 85 known Gram-positive and Gram-negative gut bacteria capable of producing it (reviewed in reference 14). In a recent study, the fecal indole concentrations (FICs) in a majority (75%) of 53 healthy persons ranged from 1.0 to 4.0 mM (15), presumably reflecting differences in the number of indole-producing (IP) bacteria or the regulatory microenvironment controlling indole expression (16,17). Indole's effects in-clude regulation of bacterial motility, epithelial barrier integrity, and biofilm formation (reviewed in references 14 and 18), as well as changes in the virulence and drug resistance of enteric pathogens (19)(20)(21)(22).…”

Biosynthese des enterotoxischen Pyrrolobenzodiazepin‐Naturstoffs Tilivallin

“…Da Stuhlproben von gesunden Menschen üblicherweise 1.0 mm bis 4.0 mm Indol enthalten, [20] vermuten wir, dass 1 und 2 im Darm vorhanden sind und unterschiedliche zytotoxische Funktionen ausüben, die spezifisch fürA AHC und …”