Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-06-22
2001-05-08
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S018000, C435S029000, C435S004000, C435S404000, C435S405000
Reexamination Certificate
active
06228606
ABSTRACT:
The present invention relates to a culture medium allowing the investigation, the isolation, the counting and the direct identification of pathogenic bacteria of the genus Listeria as well as a method employing this medium.
The isolation and the identification of the bacterium
Listeria monocytogenes
is a major problem in the monitoring of agrifood hygiene and of medical bacteriology. Among the bacteria of the genus Listeria spp only the species
monocytogenes
is known to be pathogenic for man. The other species of Listeria are not pathogenic or are pathogenic only for animals. This is the case, especially, for
Listeria ivanovii
. It is additionally known that is possible to inhibit the growth of
Listeria monocytogenes
by using bacteriocins. For example, the Patent Application EP 326 062 describes a method for inhibition of
Listeria monocytogenes
which employs a bacteriocin originating from
Pediococcus acidilactici.
The route of contamination by
Listeria monocytogenes
is most often of food origin. Thus, it is necessary to provide for the detection of
Listeria monocytogenes
all along the agrifood pathway, from raw materials passing through the environment of production facilities up to the finished product intended for consumption.
Within the context of diagnosis of bacterial conditions in man, it is likewise important to distinguish
Listeria monocytogenes
from among the other bacteria of the genus Listeria spp. which are not pathogens.
The detection and the isolation of Listeria spp. are conventionally carried out using selective culture media. The Oxford (Curtis et al.,
Lett. Appl. Microbiol
. (1989), 8, pp. 85-98) and Palcam (Van Netten et al.,
J. Food Microbiol
. (1988), 6, pp. 187-188) selective media are the most currently used. These media allow the detection of all the species of the genus Listeria spp. Thus, the typical colonies observed must be subjected to supplementary identification tests, such as microscopic and/or biochemical and/or immunological and/or genetic tests, so as to establish membership of the
monocytogenes
species. However, the supplementary manipulations necessary for the identification of
Listeria monocytogenes
increase the length and the cost of the analyses. They require a vast number of reagents and the use of qualified personnel. In addition, the withdrawal of the colonies subjected to the identification being uncertain, the supplementary manipulations are often a source of error or at least the cause of lower precision and reliability. This is the case especially when the colonies of
Listeria monocytogenes
on the isolation medium are very minor with respect to the colonies formed by the other species of Listeria.
The Patent Application EP 496 680 describes a bacteriological analysis method to differentiate
Listeria monocytogenes
from other bacteria of the genus Listeria spp.
According to this method, an identification medium is used comprising a chromogenic or fluorogenic substrate capable of being hydrolysed by glycine aminopeptidase. The medium used can likewise possibly contain a fermentation substrate and/or a reducible substrate and/or a substrate which is hydrolysable enzymatically (such as the substrate of &agr;-mannosidase) whose chemical transformation allows the species Listeria present in the sample to be analysed to be characterized.
It is additionally known that it is possible to discriminate pathogenic species of
Listeria, Listeria monocytogenes
and
Listeria ivanovii
from other Listeria by demonstrating the specific phosphatidylinositol activity of specific phospholipase C of phosphatidylinositol (PIPLC).
In fact, it has been shown that PIPLC is secreted into the culture medium of pathogenic species of the genus Listeria such as
Listeria monocytogenes
and
Listeria invanovii
(Leimeister-Wächter et al.,
Mol. Microbiol
. (1991) 5(2), pp. 361-366; J. Mengaud et al.,
Mol. Microbiol
. (1991) 5(2), pp. 367-372 and Goldfine et al.,
Infection and Immunity
(1992) 60(10), pp. 4059-4067). It is likewise known that it is possible to identify these two pathogenic species by means of indirect methods (Notermans et al.,
App. and Env. Microbiology
(1991), Vol. 57 No. 9, pp. 2666-2670). According to the method proposed by Notermans et al., the strain of Listeria to be tested, which has previously been isolated, is inoculated as a spot onto the surface of a TY (tryptone yeast extract) agar. After 24 to 48 hours' incubation at 37° C., a second agar layer is poured into the Petri dishes. This second layer contains agarose, chloramphenicol and L-&agr;-phosphatidylinositol, the natural substrate of PIPLC. The dishes are then incubated again at 37° C. and observed for 5 days. This method thus requires several steps, such as the isolation of the strain, the inoculation as a spot onto agar and the distribution of a second layer of agar containing the PIPLC substrate. In addition, this method does not allow
Listeria monocytogenes
bacteria, which are pathogenic for man, or
Listeria ivanovii
bacteria to be distinguished which, as indicated previously, are not pathogenic for man.
The subject of the invention is a medium allowing the identification of pathogenic bacteria of the genus Listeria in a single step.
The present invention relates more especially to a specific culture medium allowing the investigation, the isolation, the counting and the direct identification of pathogenic species of the genus Listeria, the said medium containing a synthetic chromogenic substrate specifically cleaved by PIPLC, especially 5-bromo-4-chloro-3-indolylphosphatidyl-myoinositol. This chromogenic substrate is preferably used in salt form, for example in sodium, potassium or ammonium salt form. Among the salts preferred is ammonium 5-bromo-4-chloro-3-indolylmyoinositol-1-yl-phosphate.
The medium according to the invention allows direct distinction between, on the one hand,
Listeria monocytogenes
and
Listeria ivanovii
which form coloured colonies and, on the other hand, the other species of Listeria whose colonies are not coloured.
According to a preferred method of carrying out the invention, the use of 5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol allows the detection of the species
Listeria monocytogenes
and
Listeria ivanovii
which form blue colonies, the other species of Listeria remaining non-coloured.
Preferentially, the nutritive agar culture medium according to the invention contains 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol in free form or in salt form, at a concentration of 100 to 500 mg/l, preferably at a concentration of 150 to 300 mg/l.
The nutritive agar culture medium used in the invention must allow the growth of Listeria spp. It is, for example, possible to use Columbia agar, made up of peptones, starch, sodium chloride and agar and well known to the person skilled in the art.
The present invention also relates to the use of 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts for the preparation of a specific culture medium allowing the investigation, the isolation, the counting and the direct identification of pathogenic species of the genus Listeria and especially of the bacterium
Listeria monocytogenes.
Surprisingly, it has also been found that the addition of blood or of its derivatives, such as, for example, plasma or serum, to the culture medium allows positive PIPLC colonies having a very clear colour to be obtained.
The present invention likewise relates to a specific nutritive agar culture medium, allowing the investigation, the isolation, the counting and the direct identification of pathogenic species of the genus Listeria and especially of the bacterium
Listeria monocytogenes
, said medium containing 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts and blood or one of its derivatives, preferably serum.
The proportion of blood or of its constituents in the medium according to the invention can be between 20 and 80 ml, more especially between 40 and 60 ml, preferably 50 ml per liter of culture medium. Culture media are preferred comprising 5-
Facon Jean-Pierre
Simon Frederic
Jacobson Price Holman & Stern PLLC
Leary Louise N.
Pasteur Sanofi Diagnostics
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