Detection of microbial metabolites

A method of detecting a phosphatidylcholine-specific phospholipase-C enzyme comprising the steps of:

• contacting a sample suspected of containing said phosphatidylcholine-specific phos-pholipase-C enzyme with a substrate containing at least one 3-indoxyl choline phosphate compound of formula (I):
• wherein R is selected from the group consisting of hydrogen and C1-4 alkyl while R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, halogen, cyano, nitro, carboxy, amino, aminomethyl, hydroxy, C1-4 alkoxy, carboxyalkyl in which the alkyl group is a C1-4 alkyl group, sulphonyl, and amino substituted with at least one but not more than two C1-4 alkyl groups, said compound of formula (I) being susceptible to cleavage by said enzyme yielding a dye;
• and
• monitoring for color formation as a consequence of said sample suspected of said phosphatidylcholine-specific phospholipase-C enzyme.

A method of identifying a bacterial microorganism of interest, which is capable of producing a phosphatidylcholine-specific phospholipase-C (PC-PLC) enzyme comprising the steps of:

• (A) providing a test sample suspected of containing said microorganism of interest;
• (B) submitting said test sample to an enrichment broth step if required;
• (C) transferring a portion, at least, of said test sample or the product obtained in step (B) to a medium suitable for culturing said microorganism;
• said medium containing at least one compound of formula (I)
• wherein R is selected from the group consisting of hydrogen and C1-4 alkyl while R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, halogen, cyano, nitro, carboxy, amino, aminomethyl, hydroxy, C1-4 alkoxy, carboxyalkyl in which the alkyl group contains from 1 to 4 carbon atoms, sulphonyl, and amino substituted with at least one but not more than two C1-4 alkyl groups, said compound being capable of producing a color when exposed to said microorganism;
• (D) cultivating said medium with said transferred portion for developing at least one colony exhibiting said color;
• and
• (E) recovering a portion, at least, of said colored colony for final identification.

A method of preparing a chromogenic 3-indoxyl choline phosphate compound of formula (I):

• wherein R is selected from the group consisting of hydrogen and C1-4 alkyl while R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, halogen, cyano, nitro, carboxy, amino, aminomethyl, hydroxy, C1-4 alkoxy, carboxyalkyl in which the alkyl group contains from 1 to 4 carbon atoms, sulphonyl, and amino substituted with at least one but not more than two C1-4 alkyl groups;
• comprising the steps of reacting a corresponding indoxyl-3-dichlorophosphate of formula (II) with a salt of choline in order to obtain an intermediate compound (III) according to the reaction:
• in which A is selected from the group consisting of hydrogen, C1-4 alkyl, and an N-protecting group while X is a counter anion of said salt of choline;
• and removing said N-protecting group from said intermediate of formula (III) when A represents said N-protecting group and, if necessary for obtaining said formula (I) compound, introducing said group R into said intermediate of formula (III).