Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-05-17
2003-05-06
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S968000
Reexamination Certificate
active
06558917
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel and potentially fluorogenic compounds which become fluorogenic and, hence fluoroscopically detectable upon contact with certain microorganisms or substances produced by such microorganisms, as well as to the use of such compounds for detection and identification of various bacteria. Additionally, the present invention relates to new and improved plating media comprising at least one of the fluorogenic compounds noted above.
PRIOR ART
Conventional assay procedures for identification of bacteria rely on traditional reactions, such as specific characteristics of acid production from particular carbohydrates, esculinase production, pH indicators, and gene-ration of hydrogen sulfide. Such methods tend to be laborious and time consuming and, hence, are costly.
It is known that the enzyme termed “phosphatidylinositol-specific phospholipase C” i.e. 1-phosphatidyl-D-myo-ino-sitol also known as inositolphosphohydrolase, also termed PI-PLC herein for short; enzyme classification EC 3.1.4.10) can be found in culture supernatants of various bacteria including
Bacillus thuringiensis
as well as some pathogenic bacteria such as
Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Staphylococcus aureus
and
Clostridium novyi
(cf. S. G. Rhee et al, Science 244 (1989) 546 ff).
More recently, Notermans et al. (Applied and Environmental Microbiology 57 (1991), 2666) have reported an assay method based upon analyzing for PI-PLC by overlaying
Listeria monocytogenes
colonies with a particular substrate, L-&agr;-phosphatidyl-inositol, and examining for turbid halos around the colony indicating the presence of the enzyme. This method is a discontinuous one.
A prior art continuous assay for PI-PLC is based upon the use of 2-naphthyl myoinositol-1-phosphate as a substrate for fluorometric measurement of PI-PLC activity (c.f. M. S. Shashidhar, J. J. Volwerk, J. F. W. Keana, O. H. Griffith; Anal. Biochem. 198 (1991), 10). This substrate has two major disadvantages, however: while 2-naphthol has its maximum fluorescence intensity at pH 10.4, PI-PLC has an optimal pH at about pH 7.4 and is not active above pH 9.0.
OBJECTS AND SUMMARY OF THE INVENTION
Accordingly it is a primary object of the present invention to provide for novel and potentially fluorogenic compounds which avoid the disadvantages of prior art.
The term “potentially fluorogenic” as used herein with reference to the novel compounds according to the invention indicates the capacity of these compounds to become “fluorogenic”—i.e. fluoroscopically active and detectable by fluoroscopic methods—upon interaction with PI-PLC.
Further objects of the invention include improved means for detecting and identifying various pathological bacterias, such as Listeria sp.
The above and further objects and advantages as apparent from the specification will be achieved according to a first embodiment of the invention by potentially fluorogenic compounds of formula (I)
in which R
1
, R
2
, R
3
, R
4
and R
5
are independently selected from the group consisting of hydrogen and chromogenic substituents, while X is selected from the group consisting of hydroxyl; OR
6
wherein R
7
is selected from the group consisting of C
1
-C
4
alkyl; and O—Me
+
wherein Me
+
is a cation derived from an organic or inorganic base.
While no theoretical limitation is intended, the effectiveness of compounds of formula (I) as substrates for PI-PLC detection is believed to reside in the fact that cleavage of a compound according to the invention by bacterial PI-PLC results mainly in the formation of inositol 1,2-cyclic phosphate and 4-methylumbelliferone which is fluorogenic.
According to a second embodiment, the invention provides for a method of detecting microbial activity in a sample, e.g. in the manner of a screening test, by combining the sample with a compound of formula (I) or a salt thereof and inspecting the sample combined with the formula (I) compound or the salt thereof, preferably as its resulting mixture, by fluoroscopic means.
According to a third embodiment, the invention provides for a method of identifying a bacterial microorganism of interest which is capable of producing a phosphatidyl-inositol-specific phospholipase C enzyme by:
(A) providing a test sample suspected of containing the microorganism of interest;
(B) submitting the test sample to a pre-enrichment step;
(C) combining a portion, at least, of the product obtained in step (B) with a compound of formula (I) to provide a screening sample which exhibits a positive fluoroscopic response when the microorganism of interest or the PI-PLC it produced is present in the test sample;
(D) if the screening sample shows a positive fluoroscopic response in step (B) a portion, at least, of the product obtained in step (B) is transferred to a medium suitable for culturing the microorganism; the medium contains at least one compound capable of producing a colour when exposed to the microorganism;
(E) cultivating the medium with the transferred portion for developing at least one colony exhibiting color; and
(F) recovering a portion, at least, of the coloured colony for final identification.
According to a third embodiment, the invention provides for a substrate for detecting PI-PLC as an indication of bacterial activity; the substrate contains at least one compound of formula (I) in a suitable medium, such as aqueous agar-agar.
According to a fourth object, the invention provides for a kit for detecting PI-PLC as an indication of bacterial activity; the kit includes at least one compound of formula (I).
According to yet a further object, the invention provides for a method of producing a compound of Formula (I) by converting a compound of formula (IIIA) into the compound of formula (IIIB) according to the reaction
in which “Ins” represents inositol and G represents a HO-protecting group on each hydroxyl of the inositol except the 1-hydroxy, and wherein X and R
1
-R
5
have the meaning indicated above for formula (I); removing the HO-protecting groups; and optionally forming a salt by reaction with an organic or inorganic base when X represents hydroxyl.
PREFERRED EMBODIMENTS OF THE INVENTION
Compounds of formula (I) and (II)—also termed “substrates” herein in view of their interaction with an enzyme—may be obtained and used in racemic form and such mixtures can be resolved to obtain the enantiomers. It is to be expected, however, that no substantial advantages will normally be obtained with the enantiomers.
Accordingly, use of racemic mixtures of formula (I) and (II) compounds will be a preferred form of the invention.
Examples of suitable organic and inorganic bases preferred for use according to the invention in its various embodiments including but not restricted to formula (I) when X is hydroxy, or O
−
Me
+
, respectively, are sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, diethylamine, triethylamine, tetramethylammonium hydroxide, tetraethylammonium hydroxide, cyclohexylamine, pyridine, piperidine, pyrrolidine, morpholine, N-methyl-morpholine, N-ethyl-morpholine and p-toluidine.
A preferred group of compounds of formula (I) are those wherein the chromogenic substituents are: C
1
-C
4
alkyl groups, i.e. methyl, ethyl, propyl and butyl including the isomeric forms, optionally containing an oxygen atom in the alkyl chain; C
1
-C
4
alkoxy; nitro; carboxy, C
1
-C
4
carboxyalkyl; and cyano; optionally, the alkyl groups just mentioned may include one or more halogen atoms, preferably fluorine, chlorine and bromine, as substituents; the trifluoromethyl group is a specific example of a preferred halo-substituted alkyl for use as chromogenic substituent in formula (I).
An even more preferred group of formula (I) compounds includes those wherein R
3
is a lower alkyl optionally containing one or more halogen atoms, X is hydroxyl, and R
1
, R
2
, R
4
and R
5
are hydrogen while R
3
is a lower alkyl or alkoxy group.
A preferred specific novel compound of formula (I) is 4-methyl umbelliferyl my
Biosynth AG
Blank Rome LLP
Leary Louise N.
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