Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-09-07
2003-11-18
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S029000, C435S033000, C435S034000
Reexamination Certificate
active
06649365
ABSTRACT:
The present invention relates to the use of at least one compound based on an indolamine derivative for revealing an aminopeptidase or peptidase activity in a microorganism culture, as well as to a method and a culture medium for detecting such an enzymatic activity or for identifying microorganisms which express or do not express this activity.
“Aminopeptidase” is the term generally given to an enzyme which is capable of cleaving, by hydrolysis, the amide group formed between an acyl amino acid and a primary amine, and “peptidase” is the term given to an enzyme which is capable of cleaving, by hydrolysis, the amide group formed between the acyl residue of a peptide and a primary amine. In the present application, the term “peptidase” may refer to, depending on the cases, both a peptidase as defined above and an aminopeptidase.
The detection and identification of microorganisms are very important in particular in medicine, in the agrifoods industry or for monitoring the environment (for example controlling water). Microorganisms may be sought for their pathogenicity, as contamination indicators, or for monitoring manufacturing methods.
Techniques for detecting and identifying microorganisms are currently based on searching for characteristic nucleotide sequences, on searching for antigens or antibodies, on culturing in selective or nonselective medium, or on searching for metabolic and in particular enzymatic activities (for example osidase, esterase, peptidase, oxidase, etc. activities).
Usually, the methods for detecting and identifying microorganisms combine several of these techniques. Culturing is thus used to multiply and select the desired microorganisms. In order to simplify their detection, it has been proposed to reveal biochemical activities by introducing, directly into the culture medium, molecules which produce a coloration or a fluorescence. Such media are termed detection media. The biochemical activities sought can be revealed by diverse methods such as:
physicochemically modifying the medium: for example changing pH, revealed with the aid of a coloured or fluorescent indicator (methyl-umbelliferone),
changing the redox potential, revealed with the aid of a coloured (tetrazolium salt) or fluorescent indicator,
reacting a molecule produced by the microorganisms with a compound present in the medium, which leads to a coloration,
or alternatively hydrolysing molecules which release a coloured or fluorescent compound (naphthol, coumarin).
The hydrolyses detected are generally the result of the reaction of an enzyme produced by the microorganism with a natural or synthetic enzymatic substrate. These enzymatic activities are for example those of the following enzymes: esterases (for example lipases, phosphatases), osidases (&bgr;-galactosidase, &bgr;-glucuronidase, N-acetylhexosaminidase), peptidases (alanine aminopeptidase, trypsinase, gelatinase), DNAses, decarboxylases, deaminases, ureases, tryptophanases, oxidases, catalases, etc.
It is known moreover that gelled media are particularly well suited to culturing and isolating microorganisms from a sample, as well as to detecting “target” microorganisms in a mixture of microorganisms. On these media, microorganisms form colonies which can be detected with the naked eye, and it is highly desirable that the products of the biochemical activities sought remain localized on their site of production. This in fact makes it possible to distinguish a colony from its neighbours if they do not express the same activities. Various methods can thus be used which detect, for example, changes in pH (FR-A-2,671,100), esterase activities (FR-2,457,323), osidase activities (FR-A-2,684,110), etc. It is of course possible to use several of these methods together in order to detect several species or strains, and/or in order to increase the sensitivity and/or specificity of the detection.
Patents U.S. Pat. No. 5,210,022 and U.S. Pat. No. 5,358,854 describe chromogenic substrates which are sensitive to the action of &bgr;-galactosidase. These 3-hydroxyindole-derived substrates are, for example, indolyl-&bgr;-D-galactosides, the indole nucleus of which can carry halogen substituents in positions 4, 5, 6 or 7.
Document EP-A-0,224,830 describes a method for detecting Gram-negative bacteria in a urine sample, in which a natural extract originating from certain species of crab is added to the sample, and then, after incubation, the sample is brought into contact with a support containing a peptide substrate which comprises a chromogenic leaving group. The natural extract used contains a proenzyme, and if the sample contains Gram-negative bacteria in sufficient number, the endotoxins of these bacteria activate the proenzyme into an enzyme which is capable of cleaving the peptide substrate with formation of a coloured product on the support.
There is currently no means available, which is suitable for gelled media, for detecting microorganism aminopeptidase and peptidase activities. Specifically, the enzymatic substrates used so far have the drawback of releasing coloured or fluorescent molecules which diffuse in the gelled media, or which are revealed only with U.V irradiation (in the case of naphthylamine or of aminocoumarin), or which require the addition of reagents (in the case of naphthylamine), or the coloration of which is of relatively poor contrast in the reaction media used in microbiology (in the case of nitroaniline).
In the present application, a term such as (amino acid)-BIA or (peptide)-BIA refers to a compound such as 3-acylamino-5-bromoindole, the acyl of which is that of said amino acid or said peptide.
It is known that L-leucine-aminopeptidase has been demonstrated in mammalian histological sections by means of an enzymatic substrate, 3-L-leucylamino-5-bromoindole, also termed L-leucine-3-(5-bromo-indolamine), known as L-Leu-BIA for short, which produces a coloured compound after hydrolysis; see Pearson et al., 1963, Lab. Invest., 12: 712. In 1967, Yarborough et al., J. Reticuloendoth. Soc., 4: 390 repeated the technique of Pearson et al. in similar applications (histological sections). They specify that adding a mixture of potassium ferri- or ferrocyanide or copper sulphate inhibits the reaction.
In 1975, Lojda and Havrankova, Histochemistry, 43: 355 proposed to improve the method using the substrate L-Leu-BIA by adding a mixture of tetrazolium salt and phenazine methosulphate, the coloured reaction observed being derived, in this case, from reduction of the tetrazolium salt to formazan.
Document WO 98/04735, which was published after the priority date from which the present application benefits, describes the use of a 5-bromoindolamine acylation derivative, the acyl being chosen from leucyl and alanyl residues, as a revealing agent for demonstrating, by formation of a coloured product, a peptidase activity in a microorganism culture.
In the course of the studies which lead to the present invention, an investigation was, carried out as to whether it was possible to use this enzymatic substrate for detecting microorganisms cultured, in particular, on gelled media. During preliminary tests, L-Leu-BIA or L-Pro-BIA was added to a medium currently used to search for osidases, which is described in Example 1 below. It was not possible to demonstrate a leucine-aminopeptidase or proline-aminopeptidase activity, whatever the microorganism cultured in this medium (in particular genera Escherichia, Klebsiella, Citrobacter, Pseudomonas, Enterococcus, Staphylococcus and Streptococcus). Addition of the reagents proposed by Lojda and Havrankova was reflected by a more or less total inhibition of the growth of the microorganisms without allowing the revelation of a peptidase activity with L-Leu-BIA or L-Pro-BIA.
Conversely, if 7-L-leucylamino-4-methylcoumarin (L-Leu-AMC) or 7-L-prolylamino-4-methylcoumarin (L-Pro-AMC) is added to the same medium from Example 1, a fluorescence (and thus a leucine-aminopeptidase or proline-aminopeptidase activity) is detected with some of these same microorganisms (see Example 1). Similarly, in this mediu
Bio Merieux
Gitomer Ralph
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