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

Abstract

Glycine, one of the end products of hippurate hydrolysis by microorganisms, was detected by a rapid, specific technique utilizing two-dimensional thin-layer chromatography. A loopful of growth of each organism from its suitable agar medium was washed, suspended, and incubated with 0.1% sodium hippurate for 30 min at 37°C. The supernatant of the incubated suspension from each organism was then dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. Glycine, a product of hippurate hydrolysis, was detected under UV light. This technique does not require prolonged incubation and was found to be more specific and reliable than the standard ninhydrin reaction. In addition, it is inexpensive and can be easily conducted in a clinical microbiological reference laboratory. By this method, 100% (22/22) of Campylobacter jejuni and 0% (0/9) of Campylobacter coli reference strains were positive. In addition, 100% (13/13) of group B streptococci, 100% (24/24) of group D streptococci, and 90% (18/20) of Gardnerella vaginalis clinical isolates were positive for hippurate hydrolysis. This method is useful for the identification to the species level of Campylobacter organisms and the biotyping of Gardnerella organisms and for the detection of hippurate hydrolysis by unknown microorganisms.

Determination of hippurate hydrolysis by gas-liquid chromatography

“…hippuricase activity, as benzoic acid is rarely, if ever, encountered as a bacterial component or metabolite. Thus, the specificity of the GLC benzoate test for hippurate hydrolysis is significantly greater than that with ninhydrin and other reagents (10), which react with glycine or other compounds containing a primary amino group. The GLC benzoate test is particularly useful for those organisms with weak hippuricase activity, as the test can be made with a large cel!…”

“…Methods for detecting benzoate, the other product of hippurate hydrolysis, include a color reaction with ferric chloride (1, 4), a colorimetric test with rhodamine B and uranium acetate (3), and a gas-liquid chromatographic (GLC) procedure in which benzoic acid is measured directly (9). Like the ninhydrin test, the ferric chloride test often gives an equivocal endpoint determination (10). Disadvantages of the reported GLC procedure include the requirement for incubation of the test organisms for 36 h in a hippuratecontaining medium, followed by an overnight incubation period in which the liberated benzoic acid is converted to the methyl ester derivative, before chromatography (9).…”

Gardnerella vaginalis: characteristics, clinical considerations, and controversies.

“…Piot et al (176) found that the pH of the hippurate test solution affected the results; pH 6.4 was optimum. Lin et al (130) discussed these tests for hippurate hydrolysis and described a two-dimensional thin-layer chromatographic technique that was more specific for glycine than the standard ninhydrin reaction. They found 18 of 20 G. vaginalis isolates positive for hippurate hydrolysis by this method.…”