Optimized microscale detection of amino acid decarboxylase for rapid screening of Salmonella in the selective enrichment step

Abstract

To develop a high-throughput spectrophotometric screen for Salmonella contamination, modified amino acid decarboxylase broths with pH indicators supplemented with selective agents were studied in microwell plates. The goal was to develop a rapid, reliable, and efficient procedure for resource-poor settings or food industry with large sample numbers. Detection depends on the broth changing from neutral to acidic pH due to sugar fermentation reflecting cell growth and then to basic due to decarboxylase generating amines, indicating presence of decarboxylase-positive bacteria. The visible absorption spectra of three pH indicators that change color at neutral pH were compared from pH 4.5 to 9. In the modified decarboxylase broth, phenol red showed the largest 550 nm absorbance changes when the pH was shifted from neutral to acidic and basic compared to bromocresol purple and bromothymol blue. Most of tested Salmonella serovars rapidly and specifically metabolized ornithine and lysine and showed positive decarboxylase activity (red broth turning pink) within 6–8 h. All Salmonella serovars possessed arginine decarboxylase, but arginine metabolism took at least 24 h to attain pink broth. Salmonella Typhi and Salmonella Paratyphi A are void of ornithine and lysine decarboxylases, respectively. Therefore, both ornithine and lysine were used as decarboxylase substrates in the selective enrichment media formulated to detect all Salmonella serovars. Three selective inhibitors: magnesium chloride, novobiocin, and magnesium chloride with malachite green (from Rappaport-Vasilliadis soy broth) were chosen to be included in seven new amino acid decarboxylase broths. They were tested for their ability to screen for Salmonella contamination in the selective enrichment step. Collectively, the broth formulations with different amino acid substrates and selective inhibitors not only identified decarboxylase-positive bacteria, but further distinguished between decarboxylase-positive salmonellae and non-salmonellae. The developed screening method was tested on some naturally- and artificially-contaminated food samples of different matrices; homogeneous liquid (pasteurized milk), solid (cooked chicken), and complex food matrix (ready-to-eat food). With proper sample pre-enrichment, this procedure with its seven newly developed broths could reliably and rapidly identify food samples with Salmonella contamination without risking false negative results.