Microorganism detection method comprising at least one alkyl(thio)glycoside

Abstract

A microorganism detection medium, said detection being based on showing the presence of a microbial enzyme activity chosen from esterase and/or osidase and/or peptidase and/or sulfatase and/or phosphatase activities of microorganisms, preferably said microbial enzyme activity being an esterase activity, said medium comprising: at least one chromogenic and/or fluorogenic substrate specific to the enzyme activity sought, preferably specific to an esterase activity, at least one alkyl(thio)glycoside, at least one solvent (S).

Claims

1. A method of obtaining a microorganism detection medium, the method comprising: a) preparing at least one stock solution of at least one chromogenic and/or fluorogenic substrate capable of being hydrolyzed by an enzyme selected from the group consisting of esterase, osidase, peptidase, sulfatase and phosphatase and at least one alkylthioglycoside or at least one alkylglycoside in at least one solvent, wherein the at least one alkylthioglycoside or at least one alkylglycoside corresponds to the general formula R—X-(G).sub.n wherein R is a linear or branched, saturated or unsaturated, aliphatic radical containing from 6 to 12 carbon atoms; X is —O— or —S—; G is a carbohydrate residue; and n is an integer from 1 to 10; b) homogenising the stock solution; and c) mixing the at least one stock solution with a culture medium suitable for growing at least one target microorganism, thereby obtaining said microorganism detection medium.

2. The method of claim 1, further comprising adding at least one additive into the medium.

3. The method of claim 1, wherein the enzyme is esterase.

4. The method of claim 1, wherein X is —O—.

5. The method of claim 1, wherein the at least one alkylthioglycoside or at least one alkylglycoside is an alkylglycoside selected from the group consisting of n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltoside.

6. The method of claim 1, wherein the concentration of the at least one alkylthioglycoside or at least one alkylglycoside in the microorganism detection medium is from 0.5 g/L to 8 g/L.

7. The method of claim 1, wherein the at least one solvent is selected from the group consisting of an alcohol, a polar aprotic solvent, an aqueous solvent, and mixtures thereof.

8. The method of claim 1, wherein the chromogenic and/or fluorogenic substrate comprises a target part capable of being hydrolyzed by the enzyme; and a marker part comprising a chromophore and/or fluorophore.

9. The method of claim 1, wherein the at least one chromogenic and/or fluorogenic substrate comprises indoxyl or derivatives thereof.

10. The method of claim 9, further comprising an agent which promotes the oxidative polymerization of the indoxyl or derivatives thereof.

11. The method of claim 1, wherein the concentration of the at least one chromogenic and/or fluorogenic substrate is from 1 mg/L to 10 g/L.

12. The method of claim 1, wherein the culture medium is selected from the group consisting of MacConkey media, Hektoen selective media, selective chromogenic media for selectively detecting salmonella, chromogenic Salmonella media, Columbia ANC media, PALCAM media, Sabouraud gentamycin-chloramphenicol type media, non-selective Columbia+/−blood media, Trypticase Soy Agar (TSA) media, nutrient agar media, and Sabouraud media.

13. A method of detecting a microorganism by its enzymatic activity, the method comprising: i) seeding a medium with a microorganism to be analyzed, the medium comprising at least one chromogenic and/or fluorogenic substrate capable of being hydrolyzed by an enzyme selected from the group consisting of esterase, osidase, peptidase, sulfatase and phosphatase; at least one alkylthioglycoside or at least one alkylglycoside, wherein the at least one alkylthioglycoside or at least one alkylglycoside corresponds to the general formula R—X-(G).sub.n wherein R is a linear or branched, saturated or unsaturated, aliphatic radical containing from 6 to 12 carbon atoms; X is —O— or —S—; G is a carbohydrate residue; and n is an integer from 1 to 10; at least one solvent; and a culture medium suitable for growing the microorganism to be analyzed; ii) incubating the seeded medium under conditions suitable for growing the microorganism to be analyzed; and iii) detecting colorations and/or fluorescence from colonies formed by the microorganism to be analyzed wherein the colorations and/or fluorescence correspond to the reaction of the at least one chromogenic and/or fluorogenic to the microorganism's activity with the enzyme.

14. The method of claim 13, wherein the enzyme is esterase.

15. The method of claim 13 wherein X is —O—.

16. The method of claim 13, wherein the at least one alkylthioglycoside or at least one alkylglycoside is an alkylglycoside selected from the group consisting of n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltoside.

17. The method of claim 13, wherein the concentration of the at least one alkylthioglycoside or at least one alkylglycoside in the medium is from 0.5 g/L to 8 g/L.

18. The method of claim 13, wherein the at least one solvent is selected from the group consisting of an alcohol, a polar aprotic solvent, an aqueous solvent, and mixtures thereof.

19. The method of claim 13, wherein the at least one chromogenic and/or fluorogenic substrate comprises a target part capable of being hydrolyzed by the enzyme; and a marker part comprising a chromophore and/or fluorophore.

20. The method of claim 13, wherein the at least one chromogenic and/or fluorogenic substrate comprises indoxyl or derivatives thereof.

21. The method of claim 20, further comprising an agent which promotes the oxidative polymerization of the indoxyl or derivatives thereof.

22. The method of claim 13, wherein the concentration of the at least one chromogenic and/or fluorogenic substrate is from 1 mg/L to 10 g/L.

23. The method of claim 13, wherein the culture medium is selected from the group consisting of MacConkey media, Hektoen selective media, selective chromogenic media for selectively detecting salmonella, chromogenic Salmonella media, Columbia ANC media, PALCAM media, Sabouraud gentamycin-chloramphenicol type media, non-selective Columbia+/−blood media, Trypticase Soy Agar (TSA) media, nutrient agar media, and Sabouraud media.

24. A method for the detection of at least one target microorganism in a sample, said at least one target microorganism having esterase and/or osidase and/or peptidase and/or sulfatase and/or phosphatase activity, the method comprising: preparing a microorganism detection medium comprising (i) at least one chromogenic and/or fluorogenic substrate capable of being hydrolyzed by an enzyme selected from the group consisting of esterase, osidase, peptidase, sulfatase and phosphatase, (ii) at least one alkylthioglycoside or at least one alkylglycoside, (iii) at least one solvent and (iv) a culture medium suitable for growing the at least one target microorganism, wherein the at least one alkylthioglycoside or at least one alkylglycoside corresponds to the general formula R—X-(G).sub.n wherein R is a linear or branched, saturated or unsaturated, aliphatic radical containing from 6 to 12 carbon atoms; X is —O— or —S—; G is a carbohydrate residue; and n is an integer from 1 to 10; seeding the microorganism detection medium with a sample to be analyzed; incubating the seeded medium under appropriate conditions suitable for growing the at least one target microorganism on the microorganism detection medium; and detecting colorations and/or fluorescence from colonies formed by the at least one target microorganism corresponding to the reaction of the at least one chromogenic and/or fluorogenic substrate to the at least one target microorganism's activity with the enzyme.

25. The method of claim 24, wherein the at least one target microorganism comprises Gram-negative bacteria.

26. The method of claim 25, wherein the Gram-negative bacteria comprises a bacteria of the genus Salmonella.

27. The method of claim 24, wherein the enzyme is esterase.

28. The method of claim 24, wherein X is —O—.

29. The method of claim 24, wherein the at least one alkylthioglycoside or at least one alkylglycoside is an alkylglycoside selected from the group consisting of n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltoside.

30. The method of claim 24, wherein the concentration of the at least one alkylthioglycoside or at least one alkylglycoside in the microorganism detection medium is from 0.5 g/L to 8 g/L.

31. The method of claim 24, wherein the at least one solvent is selected from the group consisting of an alcohol, a polar aprotic solvent, an aqueous solvent, and mixtures thereof.

32. The method of claim 24, wherein the at least one chromogenic and/or fluorogenic substrate comprises a target part capable of being hydrolyzed by the enzyme; and a marker part comprising a chromophore and/or fluorophore.

33. The method of claim 24, wherein the at least one chromogenic and/or fluorogenic substrate comprises indoxyl or derivatives thereof.

34. The method of claim 33, wherein the microorganism detection medium further comprises (v) an agent which promotes the oxidative polymerization of the indoxyl or derivatives thereof.

35. The method of claim 24, wherein the concentration of chromogenic and/or fluorogenic substrate is from 1 mg/L to 10 g/L.

36. The method of claim 24, wherein the culture medium is selected from the group consisting of MacConkey media, Hektoen selective media, selective chromogenic media for selectively detecting salmonella, chromogenic Salmonella media, Columbia ANC media, PALCAM media, Sabouraud gentamycin-chloramphenicol type media, non-selective Columbia+/−blood media, Trypticase Soy Agar (TSA) media, nutrient agar media, and Sabouraud media.

Description

DETAILED DESCRIPTION

Example 1: Medium for Detecting Salmonellae—Improvement of the Detection Sensitivity

(1) 1.1 Preparation of the Media—Solubilisation of the Enzyme Substrates

(2) A stock solution of the enzyme substrate 5-bromo-6-chloro-3-indoxyl caprylate (magenta-C8) is made in a DMSO-type solvent. A volume of this stock solution which makes it possible to obtain a final substrate concentration of 360 mg/L is then added into flasks respectively containing Tween 20 (final concentration 6 g/L), n-octyl-β-D-glucopyranoside (OG) to final concentrations of 0.7, 2 and 6 g/L, and n-dodecyl-β-D-maltoside (DM) to the same concentrations. The different solutions are agitated and the volumes are introduced into supercooled agar media (chromID® Salmonella media). The protocol is presented in table 1 below:

(3) TABLE-US-00001 TABLE 1 Ref. Medium T 1 2 3 4 5 6 Final substrate 0.36 0.36 0.36 0.36 0.36 0.36 0.36 concentration in the medium (g/L) Final Tween 20 6 0 0 0 0 0 0 concentration in the medium (g/L) Final OG 0 0.7 2 6 0 0 0 concentration in the medium (g/L) Final DM 0 0 0 0 0.7 2 6 concentration in the medium (g/L)

(4) The final Tween 20 concentration of 6 g/L was chosen because it is optimal in this context of solubilisation/microbiological activity.

(5) 1.2 Preparation of the Media

(6) Microorganisms of the genus Salmonella from the Applicant's collection were seeded on each of the aforementioned media in accordance with the three-dial technique from bacterial suspensions calibrated to 0.5 McF. The strains were selected for their low to average expression of esterase activity, giving low to average violet coloration intensities on the chromID® Salmonella medium marketed by bioMérieux (under the references 43621 and 43629).

(7) The dishes were incubated for 24 h at 37° C. Then the colonies which formed were examined visually after 24 h of incubation. The coloration intensities were recorded. The results are lodged in Table 2 below.

(8) TABLE-US-00002 TABLE 2 T 1 2 3 4 5 6 S. enteritidis 2 1.5 3 4 4 4 3 0107018 S. agona 2 1.5 3 4 4 4 3 0008024 S. dublin 0 1 2 2 1 1 0.1 0008035 S. infantis 2 1.5 3 4 4 4 3 0904097 S. tennessee 1.5 1.5 3 4 4 3 2.5 0904084 S. typhimurium 2 1.5 3 4 4 4 3 0011049 S. typhimurium 2 1.5 2.5 4 4 4 3 0107036 S. tennessee 2 2 3 4 4 3 2.5 0111019 S. enteritidis 2 2 3 4 4 4 3 0107020 S. infantis 2 1.5 2.5 4 4 4 3 0008041 S. panama 2 1.5 3 4 4 4 3 0008050 S. enteritidis 1.5 1.5 2.5 3 4 4 3 0107017 S. panama 2.5 1.5 3.5 4 4 4 2.5 9009020 Coloration intensity: scale from 0 to 4, respectively no coloration to highly intense coloration; 0 = no coloration 0.1 = trace of coloration 0.5 = very pale coloration 1 = distinct low-intensity coloration, 2 = clear medium-intensity coloration 3 = intense coloration 4 = highly intense coloration NB: The n.5s (for example 1.5, 2.5, 3.5) correspond to intermediate coloration intensities
1.3 Conclusion

(9) Media 2 (OG 2 g/L), 3 (OG 6 g/L), 4 (DM 0.7 g/L), 5 (DM 2 g/L) and 6 (DM 6 g/L) make it possible to obtain higher coloration intensities than the control medium T (Tween 20, 6 g/L) for all of the strains. Media 1 (OG 0.7 g/L), 2 (OG 2 g/L), 3 (OG 6 g/L), 4 (DM 0.7 g/L) and 5 (DM 2 g/L) make it possible to detect all the salmonellae tested including S. dublin. The media offering the best performances are media 2 (OG 2 g/L), 3 (OG 6 g/L), 4 (DM 0.7 g/L) and 5 (DM 2 g/L).

(10) OG and DM therefore make it possible to improve the esterase activity detection sensitivity in Salmonella, seemingly via an improvement of this enzyme expression. Furthermore, these alkylglycosides also make it possible to detect all of the salmonellae strains tested, including S. dublin.

Example 2: Detection of P. aeruginosa and A. baumannii Via the Expression of a C9 Esterase Activity

(11) 2.1 Preparation of the Media—Solubilisation of the Enzyme Substrates

(12) Two stock solutions at 25 g/L of ALDOL™ 470-nonanoate (ALDOL 470-C9, developed by Biosynth AG, Rietlisstrasse 4,9422 Staad, Switzerland) and of ALDOL™ 495-nonanoate (ALDOL 495-C9, also developed by Biosynth AG, Rietlisstrasse 4,9422 Staad, Switzerland) are made in a DMSO-type organic solvent. Then a volume corresponding to a final concentration of enzyme substrates of 200 mg/L is added into flasks containing respectively: Tween 20 (final concentration in the medium of 6 g/L), and DM (final concentrations of 0.7, 2 and 6 g/L). The different flasks are vigorously agitated, then all of the contents are added into the supercooled agar media: TSA (trypticase soy agar) base. The composition of the different media is presented in table 3 below:

(13) TABLE-US-00003 TABLE 3 Media 1 2 3 4 5 6 7 8 ALDOL 470-C9 0.2 0.2 0.2 0.2 0 0 0 0 g/L ALDOL 495-C9 0 0 0 0 0.2 0.2 0.2 0.2 g/L Tween 20 6 0 0 0 6 0 0 0 g/L DM 0 0.7 2 6 0 0.7 2 6 g/L
2.2 Biological Activity

(14) Bacteria from the Applicant's collection were seeded on each of the aforementioned media in accordance with the three-dial technique from bacterial suspensions calibrated to 0.5 McF. The dishes were incubated for 24 h at 37° C. and the colonies formed were analysed visually. Thus, the colorations and the coloration intensities were recorded. The results are set out in table 4 below.

(15) TABLE-US-00004 TABLE 4 1 2 3 4 5 6 7 8 P. aeruginosa 0 1.5 1.5 2 0 0.5 1.5 1.5 0001001 P. aeruginosa 0.5 0.5 0.5 1.5 0 0 0 0.5 0002019 P. aeruginosa 0.5 1 1.5 1.5 0.5 1 1 1.5 0110078 A. baumannii 0 1.5 1.5 2 0 1 2 2 0509060 A. baumannii 0 0.5 0.5 0.5 0 0 0 0.5 0202018 A. baumannii 0.5 2 2 2 0 1.5 2 2.5 0409007 A. baumannii 0 1 1.5 1.5 0 1 2 2.5 9811074 A. baumannii 0 0.5 0.5 0.5 0 0.5 1 1 9809057 Coloration intensity: scale from 0 to 4, respectively no coloration to highly intense coloration; 0 = no coloration 0.1 = trace of coloration 0.5 = very pale coloration 1 = distinct low-intensity coloration, 2 = clear average-intensity coloration 3 = intense coloration 4 = highly intense coloration NB: The n.5s (for example 1.5, 2.5, 3.5) correspond to intermediate coloration intensities
2.3 Conclusion

(16) P. aeruginosa and A. baumannii are bacteria known as being esterase-positive. Control media 1 and 5 do not make it possible to reflect this characteristic. Indeed, the coloration intensities are low (0.5) or zero for the majority of the strains tested—with these 2 ALDOL™-based enzyme substrates (containing Tween 20 as a surface active agent).

(17) The replacement of the Tween 20 at 6 g/L by DM at the same concentration unexpectedly makes it possible to improve the expression of esterase activity. Indeed, this substitution makes it possible to detect all of the strains tested, with decent coloration intensities whatever the enzyme substrate tested. A very strong impact of DM is noted on the expression of esterase activity when the substrate is ALDOL™-based.

(18) The substitution of Tween 20 by n-dodecyl-β-D-maltoside (DM) improves the detection of the esterase activity of the microorganisms, seemingly due to better biological activity of the esterase substrates, which is manifested by a more intense coloration of the target microorganism.

(19) The alkylglycoside according to the invention offers improved detection sensitivity and even makes it possible to detect certain colourless strains in the presence of Tween.

(20) Furthermore, since the coloration or fluorescence intensities are higher (due to said alkylglycosides), the enzyme substrate concentration can thus be reduced, which represents an advantage in economic terms.

Example 3: Medium for Detecting Salmonellae Via the Use of an Esterase (Lipase) Substrate with 16 Carbon Atoms—Improvement of the Detection Sensitivity

(21) 3.1 Preparation of the Media—Solubilisation of the Enzyme Substrate

(22) A stock solution of the enzyme substrate 5-bromo-4-chloro-3-indoxyl palmitate (X—C16) is made in a DMSO-type solvent. A volume of this stock solution which makes it possible to obtain a final substrate concentration of 100 mg/L is then added into flasks respectively containing Tween 20 (final concentrations of 0.1%, 0.2% and 0.6% by volume), n-octyl-β-D-glucopyranoside (OG) to final concentrations of 2 g/L, an OG (2 g/L) and Tween 20 mixture (0.1% volume), and n-octyl-β-D-thioglucopyranoside (OTG) to a final concentration in the medium of 2 g/L The different solutions are agitated and the volumes are introduced into supercooled agar media (chromID® Salmonella media). A summary of the composition of the various media is given in table 5 below.

(23) TABLE-US-00005 TABLE 5 Medium ref. 1 2 3 4 5 Final concentration 0.1 0.1 0.1 0.1 0.1 of X-C16 in the medium (g/L) Final concentration 0.1 0.2 0 0.1 0 of Tween 20 in the medium (% vol.) Final concentration 0 2 2 2 0 of OG in the medium (g/L) Final concentration 0 0 0 0 2 of OTG in the medium (g/L)
3.2 Seeding of the Media

(24) Microorganisms of the genus Salmonella, from the Applicant's collection were seeded on each of the aforementioned media in accordance with the three-dial technique from bacterial suspensions calibrated to 0.5 McF. The strains were chosen for their different level of expression of the esterase activity. A strain corresponding to the strain from collection ATCC 25922 of E. coli serves as negative control for the expression of such an enzyme activity.

(25) The dishes were incubated for 24 h at 37° C. Then the colonies formed were examined visually after 24 h of incubation. The coloration intensities were recorded. The results are lodged in table 6 below.

(26) TABLE-US-00006 TABLE 6 1 2 3 4 5 S. infantis 1 1.5 2.5 2.5 2.5 0904097 S. enteritidis 1 1 2.5 2.5 2 0107017 S. typhimurium 1 1 2.5 2 2 0011049 S. panama 1.5 1.5 2.5 2 2 0008050 S. panama 1 1.5 2.5 2 2.5 9009020 S. typhimurium 1.5 1.5 2.5 2.5 2 0107036 S. enteritidis 1 1 2.5 2 2 0107020 S. enteritidis 1 1.5 2.5 2 2 0107018 S. tennessee 1 1.5 2.5 2 2 0904084 S. infantis 1.5 1.5 2.5 2.5 2.5 0008041 S. dublin 0 0 2 1.5 0.1 0008035 S. agona 1 1 2.5 2 2 0008024 S. dublin 0 0 2.5 1.5 0 0204046 S. tennessee 1 1.5 2.5 2 2 0111019 E. coli 0 0 0 0 0 1105059 Coloration intensities: scale from 0 to 4, respectively no coloration to highly intense coloration; 0 = no coloration 0.1 = trace of coloration 0.5 = very pale coloration 1 = distinct low-intensity coloration, 2 = clear medium-intensity coloration 3 = intense coloration 4 = highly intense coloration Note: the intensities denoted n.5 (for example 1.5 and 2.5) represent intermediate intensities.
3.3 Conclusion

(27) Media 3, 4 and 5 make it possible to obtain higher coloration intensities than on the control medium (medium 1), comprising neither OG, nor OTG.

(28) It is further noted that the media containing OG, alone or in mixture with Tween 20 (in particular especially when OG is used alone as in medium 3), make it possible to obtain the best performance: 100% detection sensitivity on these media with high coloration intensities for all of the strains, including S. dublin.

(29) The E. coli strain, esterase-negative, did not appear positive, which indicates that the specificity of detection is preserved.