Staphylococcus aureus-specific detection media and identifying and/or counting method using same

• US 7,807,439 B2
• Filed: 03/29/2002
• Issued: 10/05/2010
• Est. Priority Date: 03/30/2001
• Status: Expired due to Fees

• Current Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS)
• Inventors: Re, Andrea, Cotte, Christine, Orenga, Sylvain, Robichon, Denis
• Primary Examiner(s): Ford; Vanessa L.
• Application Number:
  US10/473,835
• Publication Number:
  US 20040121404A1
• Time in Patent Office: 3,112 Days
• Field of Search: 424/184.1, 424/237.1, 435/4, 435/6, 435/7.1, 435/7.2, 435/7.32, 435/7.33
• US Class Current: 435/243
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 2333/31 from Staphylococcus (G)
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)

Abstract

The invention concerns a medium for specific detection of Staphylococcus aureus and/or discrimination between positive-coagulase Staphylococcus and negative-coagulase enabling Staphylococcus to isolate bacteria of the genus staphylococcus and identify the Staphylococcus aureus species, which use at least an enzymatic substrate, preferably a chromogenic or fluorescent agent, and still more preferably consisting of an indoxyl or naphthol base. The invention also concerns a method for identifying, and optionally counting, Staphylococcus aureus using such a medium. It consists of a Staphylococcus aureus culture medium and at least an enzymatic substrate enabling testing of an α-glucosidase activity. The invention is particularly applicable in the field of diagnosis.

Claims

1. A medium for the detection of Staphylococcus aureus and/or of coagulase-positive Staphylococci, which comprises a Staphylococcus aureus culture medium and at least one enzyme substrate for demonstrating α

-glucosidase activity, wherein the enzyme substrate for demonstrating α

-glucosidase activity is a chromogenic or fluorescent agent and is indoxyl-based, umbelliferone-based, or napthol-based, said medium further comprising at least one inhibitor which favors the growth of Staphylococcus aureus and/or of coagulase-positive Staphylococci.

2. The medium according to claim 1, wherein the chromogenic agent is selected from the group consisting of:

6-bromo-3 -indoxyl-α

-D-glucoside,

5-bromo-4-chloro-3-indoxyl-α

-D-glucoside,

5-bromo-4-chloro-3-indoxyl-N-methyl-α

-D-glucoside,

6-chloro-3 -indoxyl-α

-D-glucoside,

4-methylumbelliferyl-α

-D-glucoside, and combinations thereof.

3. The medium according to claim 1, for the detection of Staphylococcus aureus, wherein the chromogenic agent for demonstrating α

-glucosidase activity is naphthol-based.

4. The medium according to claim 1 wherein the chromogenic agent is 2-naphthyl-α

-D-glucopyranoside.

5. The medium according to claim 1 which further comprises at least one enzyme substrate for detecting osidase, esterase, peptidase, or coagulase activity.

6. The medium according to. Claim 5, which comprises two chromogenic agents chosen from the following pairs:

5-bromo-4-chloro-3-indoxyl-N-methyl-α

-D-glucoside combined with

5-bromo-6-chloro-3-indoxyl-β

-D-galactoside,

5-bromo-4-chloro-3-indoxyl-N-methyl-α

-D-glucoside combined with

6-chloro-3-indoxyl-β

-D-glucuronide,

6-chloro-3-indoxyl-α

-D-glucoside combined with 5-bromo-4-chloro-3-indoxyβ

-D-glucuronide, or

6-chloro-3-indoxyl-α

-D-glucoside combined with 5-bromo-4-chloro-3-indoxyβ

-D-glucoside.

7. The medium according to claim 1, wherein the at least one inhibitor is a mixture of the following inhibitors:

lithium chloride,

2,4-diamino-6,7-diisopropylpteridine,

aztreonam, and

amphotericin.

8. The medium according to claim 1, further comprising maltose.

9. A method for identifying Staphylococcus aureus in a sample containing at least one species of bacterium, which method comprises the following steps:

inoculating a medium according to claim 1 with a portion of the sample, and

incubating the inoculated medium so as to produce bacterial colonies of sufficient size to observe the coloration of each chromogenic agent or the fluorescence of each fluorescent agent, which has reacted in said medium.

10. The method according to claim 9, further comprising counting the colonies identified by coloration or a fluorescence related to Staphylococcus aureus.

11. A medium according to claim 5 wherein said other enzyme substrate is for detecting the activity of at least one osidase selected from the group consisting of β

-glucosidase, β

-glucuronidase and β

-galactosidase.

12. A medium according to claim 1, wherein the at least one inhibitor is selected from the group consisting of lithium chloride, sodium azide, colistin, amphotericin, aztreonam, colimicin, sodium chloride and Deferoxamine.

14. The medium according to claim 1, wherein the chromogenic or fluorescent agent is indoxyl-based or umbelliferone-based.

13. A medium for the detection of Staphylococcus aureus and/or of coagulase-positive Staphylococci, which comprises a Staphylococcus aureus culture medium, maltose, and at least one enzyme substrate for demonstrating α

-glucosidase activity, wherein the enzyme substrate for demonstrating α

-glucosidase activity is a chromogenic or fluorescent agent, and wherein the maltose is present at a concentration of between 100 and 300 mg/l.