Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-05-25
2004-05-11
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S016000, C435S029000
Reexamination Certificate
active
06733986
ABSTRACT:
This application claims priority to application PCT/FR98/02380 filed Nov. 6, 1998 which claims priority to France 97 14191 filed Nov. 6, 1997.
The present invention relates to a method for detecting and identifying and/or quantifying an enzymatic activity such as deaminase activity in a culture medium for microorganisms, to the compounds and detection agents which are suitable for this method, to the method for preparing these compounds and detection agents, as well as to the culture media for implementing said method.
The detection and identification of microorganisms are very important in particular in medicine, in the agrifoods industry or for environmental control (water, etc.). Microorganisms may be sought for their pathogenicity, as contamination indicators, for monitoring manufacturing methods, and the like.
The techniques for detecting and identifying microorganisms are currently based on the search for characteristic nucleotide sequences, the search for antigens or antibodies, culturing in selective or nonselective medium, or alternatively the search for metabolic and in particular enzymatic activities (for example osidase, esterase, peptidase, oxidase, etc. activities).
Usually, the methods for detecting and identifying and/or quantifying microorganisms combine several of these techniques. Culturing is thus used to multiply and select the desired microorganisms. In order to simply their detection, it has been proposed to demonstrate biochemical activities by introducing molecules which produce a coloration or a fluorescence, directly into the culture medium. Such media are referred to as detection media. The biochemical activities can be demonstrated by various methods, such as:
physicochemical modification of the medium: change in pH in the presence of a colored or fluorescent indicator (methylumbelliferone, etc.),
change in the redox potential, revealed with the aid of a colored indicator (tetrazolium salts, etc.) or a fluorescent indicator (EPO-424 293),
hydrolysis of molecules which release a colored compound or a fluorescent compound (indoxyl, naphthol, coumarin, etc.),
reaction of a molecule which is produced by the microorganisms with a compound which is present in the medium and which results in a coloration (detection of indole, James et al., 1986).
It is known that gelled (or solid) media are particularly suitable for culturing and isolating microorganisms from a sample, as well as for detecting “target” microorganisms in a mixture of microorganism taxa.
A method for differentiating bacteria of the Proteus group from other enterobacteria is known (Sverre Dick Henriksen, State Institute for Public Health, Bacteriological Department, Oslo, Norway, Jun. 6 1950). This method uses an equal amount, in gelled medium, of D- and L-phenylalanine in the presence of iron salt. In comparison with the urease assay, the green colored reaction obtained, although positive for identifying the Proteus group, is presented as being an assay which is laborious to use.
The document by GIAMMANCO & PIGNATO (“Rapid identification of microorganisms from urinary tract infections by beta-glucuronidase, phenylalanine deaminase, cytochrome oxidase and indole tests on isolation media”, JOURNAL OF MEDICAL MICROBIOLOGY, vol. 41, No. 6, December 1994, pages 389-392) is also known, which discloses, for detecting a deaminase activity of bacteria of the Proteeae group, the use of natural amino acids, such as tryptophan and phenylalanine, in combination with an iron salt (FeCl
3
).
The document by MANAFI & ROTTER (“A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae”, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 14, No. 2, November 1991, pages 127-134) is also known, which discloses, for detecting a deaminase activity of bacteria of the Proteeae group, the use of tryptophan alone with iron salt (ammoniacal iron citrate). This document mentions, for other markers of enzymatic activity (4-methylumbelliferone, nitrophenyl), that a diffusion of these markers is detrimental. The only solution for minimizing the effect thereof is to incubate the media for a shorter time.
The document by PELOUX & LEFORT (“Lysine deaminase of the Proteus-Providencia group by means of the Edwards and Fife lysine-iron medium. Practical value of this medium for differentiating enterobacteria”, FEUILL. BIOL., vol. 13, No. 68, 1972, pages 37-42) is also known, which compares the activity of three amino acids (tryptophan, phenylalanine and lysine), for detecting deaminase, in combination with an iron salt.
The document by SIVOLODSKII (“Modification of a method for the determination of tryptophan deaminase and phenylalanine deaminase content in bacteria”, LAB. DELO., No. 3, 1982, pages 166-168) is also known, which proposes adding enzymatic hydrolysates of proteins, consisting of mixtures of natural amino acids, of peptides and of other compounds, in order to detect tryptophan and phenylalanine deaminase activities.
Patent application WO-A-92/00068 is also known, which discloses substituted histidine compounds which act as antagonists of angiotensin II receptors. These compounds, according to the formula (I) and the examples, comprise, inter alia, the substitution of the amine or carboxyl group.
U.S. Pat. Nos. 5,643,743 and 5,411,867 are also known, which disclose tryptophan for detecting tryptophanase, this enzyme being different from the deaminases.
U.S. Pat. No. 4,603,108 is also known, which discloses D,L-beta(p-nitrophenyl)alanine as a substrate for detecting phenylalanine deaminase. The reading of the reaction is carried out either at 480 nm, without adding a revelator, for phenylalanine deaminase, or by assaying ammonia for leucine deaminase.
Finally, U.S. Pat. No. 5,541,802 is known, which discloses, for detecting deaminases, the amino acids phenylalanine and tryptophan, which make it possible to obtain an orangey color which diffuses in the medium.
Either these documents disclose the use of natural amino acids, which is incompatible with a limitation of the diffusion of alpha-keto acid. The only solution proposed by certain of these documents comes down to limiting the incubation time, which is very prejudicial to the quality of the detection. However, the limitation of diffusion is evidence of differentiation of several microbial colonies present in the same medium. Or these documents disclose the use of artificial and modified amino acids which do not satisfy formula (I) of the invention, and which can, in addition, provide a different application, such as for example antagonism with angiotensin II receptors.
Despite all the biochemical assays currently on the market, it turns out that currently there are no means available, which are particularly well-suited and easy to implement, in particular in gelled medium, for detecting and identifying and/or quantifying, in a multimicrobial culture, an enzymatic activity such as a deaminase activity of microorganisms.
The present invention thus intends to resolve this problem.
A first subject of the invention is a method for detecting and identifying and/or quantifying an enzymatic activity such as deaminase activity of a microorganism, according to which an inoculum which is capable of containing a microorganism with a deaminase activity is brought into contact with a culture medium for microorganisms, the culture medium comprising at least one detection agent for demonstrating, by forming a colored product with a revealing agent, an enzymatic activity such as deaminase activity; said detection agent is a cyclic L-amino acid of following general formula (I):
in which:
R represents a cyclic amino acid radical, substituted with 1 to 3 groups X, which are identical or different,
X represents a group which limits the diffusion of the &agr;-keto acid produced by the deamination of the cyclic amino acid,
the compound of formula (I) being able to be substituted with various groups which do not interfere with the function of the group X.
“Group which limits the diffusion” is intended to mean:
any group of hydrophobic type which li
Armstrong Lyle
James Arthur
Orenga Sylvain
Bio Merieux
Gitomer Ralph
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