Evaluation of novel beta-ribosidase substrates for the differentiation of Gram-negative bacteria

Abstract

Aims: To synthesize novel substrates for the detection of b-ribosidase and assess their potential for the differentiation of Gram-negative bacteria. Methods and Results: Two novel chromogenic substrates, 3¢,4¢-dihydroxyflavone-4¢-b-D-ribofuranoside (DHFriboside) and 5-bromo-4-chloro-3-indolyl-b-D-ribofuranoside (X-riboside) were evaluated along with a known fluorogenic substrate, 4-methylumbelliferyl-b-D-ribofuranoside (4MU-riboside). A total of 543 Gram-negative bacilli were cultured on media containing either DHF-riboside or X-riboside. Hydrolysis of DHF-riboside or X-riboside resulted in the formation of clearly distinguishable black or blue-green colonies, respectively. Hydrolysis of 4MU-riboside was evaluated in a liquid medium in microtiter trays and yielded blue fluorescence on hydrolysis which was measured using fluorimetry. b-Ribosidase activity was widespread with 75% of strains, including 85AE6% of Enterobacteriaceae, showing activity with at least one substrate. Genera that demonstrated b-ribosidase activity included Aeromonas, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Providencia, Pseudomonas, Salmonella and Shigella. In contrast, strains of Proteus spp., Acinetobacter spp., Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus generally failed to demonstrate b-ribosidase activity. Conclusions: The novel substrates DHF-riboside and X-riboside are effective for the detection of b-ribosidase in agar-based media and may be useful for the differentiation and identification of Gram-negative bacteria. Significance and Impact of the Study: This is the first report describing the application and utility of chromogenic substrates for b-ribosidase. These substrates could be applied in chromogenic media for differentiation of Gram-negative bacteria.

Evaluation of Selective Media Containing Iron Source and Alpha-Glucosidase Substrates for Enterobacter sakazakii (Cronobacter spp.) Detection

“…Hence, E. vulneris did not produce the fluorescent colonies on media as negative reaction [19]. Since Butterworth et al [21] reported that the difference of β-ribosidase activity was due to different rates of substrate uptake through the cell envelop or differences in the substrate affinity of the β-ribosidase enzyme within different species.…”

The application of chromogenic media in clinical microbiology

“…Other chelation‐based substrates applicable to agar media include those based on alizarin and 3′, 4′‐dihydroxyflavone although these have not yet been applied in commercially available chromogenic media (James et al. 2000; Butterworth et al. 2004).…”

“…2007). The development of new media may depend upon the recognition and synthesis of new chromogenic substrates with diagnostic utility (Butterworth et al. 2004).…”

Design and Evaluation of Dihydroxytetrahydro-1H-pyrrolo[2,1-c]- [1,4]benzothiazines as Conformationally Restricted Transition-State Inhibitors of β-Ribosidases

“…The β-ribosidase inhibitory properties of 6 − 8 were evaluated using 3‘,4‘-dihydroxyflavone-4‘-β- d -ribofuranoside 16 (DHF-riboside) as the substrate and bacterial lysates or whole cells as the source of β-ribosidase. The hydrolysis of this chromogenic substrate can be catalyzed by β-ribosidases to release 3‘,4‘-dihydroxyflavone, which forms a highly colored chelate with iron …”