Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-01-28
2003-04-15
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S014000, C435S883000
Reexamination Certificate
active
06548268
ABSTRACT:
The present invention relates to a novel chromogenic medium intended to reveal
Staphylococcus aureus.
Staphylococcus aureus
is a bacterium, the detection of which is proving to be increasingly advantageous as it relates to bacteria which are often carried by patients who have to be subjected to traumatic care, syringes, catheter or various operations. In this case, there is a very great danger sooner or later of infection immediately these patients enter into care.
It is therefore a pathogenic bacterium which is increasingly implicated in nocosomial infections in hospital environments, for example.
In fact, epidemiological surveillance of
Staphylococcus aureus
is becoming increasingly necessary and widespread.
The conventional medium for the detection of
Staphylococcus aureus
in the clinical field is a mannitol-salt medium which is based on the characteristics of resistance to salt and of acidification in the presence of mannitol. This test is not very satisfactory, in particular as regards sensitivity and specificity, this highly selective medium results in an excessively high number of false negatives and, furthermore, also gives false positives. Moreover, the staining of the colonies originating from the acidification spreads into the medium, which does not facilitate the reading when the sample is not a pure culture and when isolated colonies are close to one another.
The prior art has already suggested the use of alternative media, in particular using phosphatase as feature for the detection of
Staphylococcus aureus
(Stevens D. L. and Jones C: Use of trehalose-mannitol-phosphatase agar to differentiate
Staphylococcus aureus
and
Staphylococcus saprophyticus.
J. Clin. Microbiol., 20, 977-980, 1984) with, for example, a medium comprising the phenolphthalein phosphate indicator. However, this medium exhibits the failing of giving a staining which can spread, which presents a problem when the sample is not a pure culture and, as in the preceding case, when the isolated colonies are close to one another.
The present invention is based on the use of a chromogenic medium comprising two chromogenic agents, making it possible to obtain a sensitive medium giving a virtually no false negatives for
S. aureus
and making it possible in addition to differentiate
S. aureus
from the other species, such as Streptococcus.
When it is indicated that a medium “does not give” or “gives virtually no” false positives or false negatives, it should always be understood that this is with respect to the strains which have been tested, it is not possible to be totally affirmative as atypical or mutant strains can appear every day.
More particularly, the invention relates to a medium for the detection of
Staphylococcus aureus
comprising, as chromogenic agent, at least one compound chosen from:
5-bromo-6-chloro-3-indoxyl phosphate and
5-bromo-4-chloro-3-indoxyl glucoside,
in a culture medium, and additionally comprising deferoxamine.
Preferably, both chromogenic compounds will be used. When 5-bromo-6-chloro-3-indoxyl phosphate is used without the 5-bromo-4-chloro-3-indoxyl glucoside derivative, then use will preferably be made of a combination with a chromogenic substrate of glucosidase, generally of indoxyl glucoside type.
When 5-bromo-4-chloro-3-indoxyl glucoside is used without 5-bromo-6-chloro-3-indoxyl phosphate, use will preferably be made of a combination with a chromogenic substrate of phosphatase, generally of indoxyl phosphate type.
S. aureus
culture media are known and are described in particular in the manual “Oxoïd Unipath Limited”, Wade Road, Basingstoke, Hampshire, RG24 0PW, England. It can be, for example, “Nutrient Agar Oxoïd CM3”, a medium based essentially on extracts of yeast, peptone and agar.
The term “chromogenic agent” is understood to denote a compound which changes color in the presence of a specific strain, in particular under the effect of the enzymatic system of said strain.
The use of the medium according to the invention makes it possible to observe a mauve staining of 5-bromo-6-chloro-3-indoxyl phosphate in the presence of
S. aureus,
whereas 5-bromo-4-chloro-3-indoxyl glucoside stains blue a large number of Streptococcus strains without staining
Staphylococcus aureus.
This is entirely unexpected as the literature indicates that
S. aureus
is &bgr;-glucosidase positive, like numerous Streptococcus strains, this can be confirmed by using, for example, para-nitrophenyl &bgr;-glucoside. In point of fact, the surprising observation has been made that 5-bromo-4-chloro-3-indoxyl glucoside stains blue Streptococcus strains without staining
Staphylococcus aureus
strains, this has been confirmed with respect to a large number of
S. aureus
strains found in hospitals.
Furthermore, the presence of deferoxamine in the culture medium according to the present invention makes it possible to inhibit the growth of
Staphylococcus epidermis
without inhibiting
Staphylococcus aureus,
which makes it possible to distinguish these two organisms. The concentration used will preferably be between 0.010 and 0.100 g/l.
The medium according to the present invention can also be improved by adding one or both of the following chromogens:
5-bromo-4-chloro-3-indoxyl galactoside and
5-bromo-4-chloro-3-indoxyl glucuronide,
which are two features negative for
Staphylococcus aureus
and thus make it possible to differentiate it from the strains positive for these chromogens.
The media according to the present invention will comprise preferably from 0.01 to 0.500 g/l, in particular from 0.050 to 0.150 g/l, of 5-bromo-6-chloro-3-indoxyl phosphate, preferably from 0.010 to 0.200 g/l of 5-bromo-4-chloro-3-indoxyl glucoside, preferably from 0.010 to 0.200 g/l of 5-bromo-4-chloro-3-indoxyl galactoside and preferably from 0.010 to 0.200 g/l of 5-bromo-4-chloro-3-indoxyl glucuronide.
A preferred medium according to the invention, which makes it possible to differentiate
Staphylococcus aureus
microorganisms by the mauve staining of the colonies, whereas other strains are inhibited or give colorless or blue colonies, is as follows:
Peptone and yeast extract
50
g/l
5-bromo-6-chloro-3-indoxyl phosphate
0.100
g/l
5-bromo-4-chloro-3-indoxyl glucoside
0.050
g/l
5-bromo-4-chloro-3-indoxyl galactoside
0.050
g/l
5-bromo-4-chloro-3-indoxyl glucuronide
0.050
g/l
Deferoxamine
0.050
g/l
Agar
15
g/l
REFERENCES:
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patent: WO 9212259 (1992-07-01), None
patent: WO 9504157 (1995-02-01), None
patent: WO 9520674 (1995-08-01), None
patent: WO 9832874 (1998-07-01), None
patent: WO 00/53799 (2000-09-01), None
Lindsay J. A; Aravena-Roman M A; Riley TV: “Identification of Staphylococcus epidermidis and Staphyloccus hominis from blood cultures by testing susceptibility to desferrioxamine” European Journal of Clinical Microbiology & Infectious Diseases, vol. 12, 1993, pp. 127-131.
Heuck Dagmar; Witte Wolfgang; Braulke Christine; Reissbrodt Rolf: “Susceptibility to desferrioxamines and other chelators of coagulase-negative staphylocci” Zentralblatt Fuer Bakteriologie, vol. 280, 1994, pp. 304-311, XP000921273 tableux 2,3.
Mulder J G: “A simple and inexpensive method for the identification of Staphylococcus epidermidis and Staphyloccus hominis”European Journal of Clinical Microbiology & Infectious Diseases, vol. 14, 1995, pp. 1052-1056, XP000921270.
DL Stevens, C Jones: “Use of trehalose-mannitol-phosphatase agar to differentiate Staphyloccus aureus and Staphyloccus saprophyticus”, Journal of Clinical Microbiology, vol. 20, 1984, pp. 977-980, XP000856420.
Blakely & Sokoloff, Taylor & Zafman
Leary Louise N.
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