Rapid method for simultaneous detection of the arginine dihydrolase system and amino acid decarboxylases in microorganisms

Abstract

A specific procedure has been developed for the detection of the first two enzymes involved in the arginine dihydrolase system and the detection of the decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine. A loopful of growth of each organism from dihydrolase-decarboxylase induction agar medium (or broth) was washed and incubated separately with 0.2-ml samples of three test media supplemented with different amino acids. Each spent test medium was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products (citrulline, ornithine, y-amino-n-butyric acid, and amines) produced during incubation were estimated by comparing the fluorescent intensities of end products from the spent test media and of the corresponding parent amino acids from test medium controls after thin-layer chromatography. The method is reproducible, requiring incubation of an organism in three test media for 1 h for simultaneous detection of the first two enzymes involved in the arginine dihydrolase system and of eight amino acid decarboxylases. This method has been successfully applied to gram-positive and gramnegative microorganisms and also to Mycoplasmatales. It could simplify and improve the accuracy of the corresponding biochemical tests performed in clinical laboratories for the identification and differentiation of microorganisms, and it may prove particularly useful for the differentiation of species of Pseudomonas and Mycoplasma. Enzymes involved in the arginine dihydrolase system and the amino acid decarboxylases are widely distributed in microorganisms (1, 2, 8). The microbial arginine dihydrolase system which converts arginine to ornithine via citrulline consists of three enzyme reactions: arginine + H20-* citrulline + NH3 by arginine deiminase; citrulline + Pi ornithine + carbamyl

Rapid, inexpensive method for specific detection of microbial beta-lactamases by detection of fluorescent end products

“…were grown anaerobically on Columbia base agar (BBL) with supplements as described previously (21). The other microorganisms described previously (2) were grown aerobically on GC medium base (Difco Laboratories, Detroit, Mich.) containing 1% defined supplement (22) at 37°C overnight, except for Neisseria gonorrhoeae, which was grown in a CO2 incubator (19). Portions (50 ,lI) of each 3-lactam antibiotic were separately placed in a microcentrifuge tube (200 ,lI; Stockwell Scientific, Monterey Park, Calif.).…”

Two-dimensional thin-layer chromatography for the specific detection of hippurate hydrolysis by microorganisms

“…We previously reported rapid procedures for the simultaneous detection of eight bacterial amino acid decarboxylases and for the detection of the arginine dihydrolase system by using two-dimensional TLC (2-D TLC) on polyamide sheets (4). In the present study, we modified the 2-D TLC procedures for the rapid, inexpensive, and specific detection of glycine resulting from hippurate hydrolysis.…”