Detection, discrimination and identification of yeasts of the candida genus, microbiological methods and tools therefor

Abstract

The invention relates to microbiological methods and to culture media dedicated to the non-denaturing detection, discrimination and / or identification of yeasts of the Candida genus, in particular C. auris, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. The invention more particularly relates to a method for identifying yeasts of the Candida genus which are optionally present in a biological sample, comprising the following steps: - culturing the yeasts to be identified on or in a culture medium comprising: - a nutrient component that is capable of allowing the development and growth of yeasts of the Candida genus; and - an α-glucosidase inducer and optionally an α-glucosidase repressor; - an acid phosphatase inducer and a phosphatase repressor; - an N-acetyl-β-glucosaminidase inducer; and - detecting any α-glucosidase, acid phosphatase and / or N-acetyl-β-glucosaminidase activities expressed by the yeasts; and - identifying the species to which the yeasts belong on the basis of the α-glucosidase, acid phosphatase and / or N-acetyl-β-glucosaminidase activities actually expressed by the yeasts.

Description

[0001] DETECTION, DISCRIMINATION AND IDENTIFICATION OF YEASTS OF THE GENUS CANDIDA, MICROBIOLOGY METHODS AND TOOLS FOR THIS PURPOSE

[0002] The present invention relates to the detection, discrimination and identification of yeasts of the genus Candida by analytical microbiology methods and tools.

[0003] More specifically, it relates to microbiological methods and culture media dedicated to the non-denaturing detection and identification of microorganisms, in particular yeasts of the genus Candida, possibly present in a biological sample to be analyzed. Once detected / identified with such microbiological methods and culture media, the still viable microorganisms may optionally be recovered / collected, multiplied and / or preserved for the purposes of additional analyses (denaturing or not).

[0004] Yeasts of the genus Candida (or "Candida spp") are ubiquitous microorganisms, widely disseminated in our environment. These yeasts are also found in animals and humans with whom they maintain commensal relationships, populating the skin, mouth, ear canal, respiratory tract, digestive system, genital tract, etc.

[0005] In at-risk individuals (in particular, immunocompromised patients, diabetics, patients receiving broad-spectrum antibiotics and corticosteroids, patients with renal failure, chronic stress, infants, the elderly, pregnant women, etc.), Candida spp can prove to be highly pathogenic opportunists. Their massive and uncontrolled proliferation leads to infections called candidiasis, which can be particularly serious, even fatal.

[0006] When they only affect the mucous membranes and / or skin, these infections are called "superficial" and are generally benign. In the most worrying forms, the infections reach deep organs (called "visceral candidiasis") and / or spread throughout the body (called "invasive candidiasis" or "candidemia").

[0007] From a health perspective, yeasts of the genus Candida are regularly identified as the cause of nosocomial infections, often involving contamination of medical equipment and tools used in invasive procedures (catheters, urinary catheters, etc.). They are believed to be among the leading causes of septicemia in hospitals, which, in 40% of cases, prove fatal within 30 days. While nearly 400 Candida spp have been described to date, only a very small minority are responsible for human pathologies.

[0008] In 2020, the CDC (Control Disease Center, US) reported that globally, 39% of invasive infections linked to Candida spp worldwide were attributed to C. albicans, 28% to C. glabrata (also designated Nakaseomyces glabrata, according to a recently proposed nomenclature reform (Kidd et al., 2023; Open Forum Infectious Diseases (2023): “Fungal Nomenclature: Managing Change is the Name of the Game”)) and 15% to C. parapsilosis.

[0009] At the French level, the French Society of Mycology (SFM) published in 2015 (Reference in Clinical Microbiology, Candidiasis, 5 eme 2015 edition) that approximately 50% of candidiasis (i.e. infections linked to yeasts of the genus Candida) observed in France are attributable to C. albicans. This is followed by C. glabrata (10-20%), C. parapsilosis (10-20%), C. tropicalis (10%) and C. krusei (3%; C. krusei, also designated Pichia kudriavzevii, according to the recently proposed nomenclature reform).

[0010] Other Candida spp are less widespread and their emergence often depends on particular ecologies.

[0011] In the specific case of C. auris, this species, which appeared in Japan in 2009, has since been nicknamed "the Killer Mushroom." In addition to its high level of danger, three other reasons justify the growing attention and concern it is currently attracting:

[0012] - firstly, its ability to spread rapidly in healthcare facilities where it is easily transmitted between patients, healthcare staff and visitors; in the hospital environment (especially on the surfaces of hospital equipment, such as treatment trolleys, infusion pumps, thermometers, blood pressure monitors), C. auris can survive for weeks,

[0013] - also, its multi-resistance to antifungals (for example, fluconazole, itraconazole, voriconazole or amphotericin B), although highly variable from one geographical clade to another, considerably limits the available therapeutic options; similarly, from a health perspective, measures for disinfecting the environment and medical equipment, using conventional quaternary ammoniums, have proven to be of little effectiveness,

[0014] - finally, few analytical and diagnostic tools are currently available for its identification. Due to their prevalence and their dangerousness, the six yeast species mentioned above (namely, C. auris, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. knisei), are referred to here as “major Candida spp” (or “Candida spp of major interest”).

[0015] Because they infect the same types of tissues and organs (mucous membranes, skin, digestive system, respiratory system, genital organs, etc.) and give rise to very similar, even identical, symptoms, a simple clinical examination of the patient does not allow us to identify precisely and with certainty which of these six major Candida spp is actually responsible for a particular case of candidiasis. In addition, given the resistance to antifungals that each has developed, resistance that is very species-dependent, there is no universal / common therapeutic approach to the treatment of candidiasis. No single treatment can therefore be systematically prescribed in cases of candidiasis.Therefore, and in order to assist practitioners in choosing an effective therapeutic treatment, adapted to each patient suffering from candidiasis, it is important to have methods and tools available capable of enabling sufficient discrimination of such yeasts and / or specific identification of each major Candida sp.

[0016] The specific detection and identification of yeast species of the genus Candida is also of vital importance from a health perspective, particularly in the fight against nosocomial diseases where the appearance of any microorganism on the surface of surgical tools and instruments, or in liquids and solutions for medical use, is closely monitored.

[0017] Many analytical techniques already allow detection, discrimination and / or identification of yeast species of the genus Candida. Some are based on PCR amplification technologies of specific nucleic sequences, immunodetection of antigens, mass spectrometry. The present invention focuses instead on analytical microbiology methods and tools which have the advantage of not denaturing the yeasts studied, so that once the identification has been carried out, still viable yeasts can be recovered and possibly subjected to an antifungal test in order to establish an effective therapeutic treatment, adapted to the infected patient. Optionally, the isolated specimens could also be recovered for the purposes of conservation and / or subsequent analyses and studies.More specifically, the present invention relates to analytical microbiology methods which use solid, selective and discriminating microbiological culture media, capable of enabling identification / differentiation of Candida spp. from one another.

[0018] Among the solid, selective and discriminating microbiological culture media, known from the state of the art as intended for the detection and / or identification of yeasts of the genus Candida, the following chromogenic media may be mentioned in particular:

[0019] - CHROMID® Candida agar, developed by the company bioMérieux; this solid culture medium, which not only allows the growth of yeasts in general and particularly C. albicans, C. tropicalis and C. krusei, also allows the identification and differentiation of these three species of yeast thanks to a particular colorimetric marking; this medium does not, however, allow specific detection / identification of C. auris, C. glabrata or C. parapsilosis, '

[0020] - CHROMagar™ Candida media (CHROMagar Microbiology, France), Candi Select™ 4 (BioRad, US) and HiCrome™ Candida differential Agar Base (HiMedia Laboratories, India) allow the detection, identification / differentiation of C. albicans, C. glabrata, C. tropicalis and C. krusei, but they do not allow the detection / identification of C. auris or C. parapsilosis,

[0021] - CHROMagar™ Candida Plus medium (CHROMagar Microbiology, France) allows detection of C. auris as well as other Candida spp, 'but their detection suffers from low specificity (de long et al., 2021; Journal of Clinical Microbiology (2021), 59(4): “Performance of Two Novel Chromogenic Media for the Identification of Multidrug-Resistant Candida auris Compared with Other Commercially Available Formulations”).

[0022] Thus, to the Applicant's knowledge, there is currently no microbiological method or tool that allows simple and satisfactory detection and identification of yeasts of the genus Candida. More specifically, for the Applicant, there is currently no tool that is both suitable for the culture of Candida spp, whatever the species, and that allows satisfactory discrimination of yeasts belonging to the six major Candida spp, namely C. auris, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. To the Applicant's knowledge, there is also no microbiological method or tool that is capable of allowing sufficiently specific detection and identification of C. auris.

[0023] In this context and to overcome the deficiencies observed, the present invention aims to propose solid culture media on which it will be possible to grow different Candida spp and to identify them. In particular, the solid culture media proposed must not only be suitable for the culture of the different species of yeast of the genus Candida, in particular C. auris, C. albicans, C. glabrata (Nakaseomyces glabrata , C. tropicalis, C. parapsilosis and C. krusei (P. kudriavzevii), they must also make it possible to differentiate and specifically identify each of these species.

[0024] The present invention thus relates to a solid microbiological culture medium, intended for the detection, discrimination and / or identification of yeasts of the genus Candida (including C. glabrata and C. kruseî), likely to be present in a biological sample to be tested. To do this, said medium comprises:

[0025] - a nutritional component capable of enabling the development and growth of yeasts of the genus Candida, and

[0026] - a discriminating component capable of enabling visual marking of yeast colonies growing on said culture medium, and is characterized in that said discriminating component comprises:

[0027] - an α-glucosidase inducer, a chromogenic and / or fluorogenic α-glucosidase substrate which, under the action of an α-glucosidase, releases a chromophore and / or a fluorophore emitting a first detectable optical signal, as well as optionally an α-glucosidase repressor,

[0028] - an acid phosphatase inducer, a phosphatase repressor, as well as a chromogenic and / or fluorogenic phosphatase substrate which, under the action of a phosphatase, releases a chromophore and / or a fluorophore emitting a second detectable optical signal, said second detectable optical signal being different from said first detectable optical signal,

[0029] - an inducer of N-acetyl-P-glucosaminidase, as well as a chromogenic and / or fluorogenic substrate of N-acetyl-P-glucosaminidase which, under the action of an N-acetyl-P-glucosaminidase, releases a chromophore and / or a fluorophore emitting a third detectable optical signal, said third detectable optical signal being different from said first detectable optical signal and said second detectable optical signal.

[0030] Before going further in the presentation and explanation of the invention, the following definitions are given to allow a better understanding.

[0031] By "biological sample" we mean a clinical sample from a sample of human or animal origin, or a food sample from any type of food, or a sample taken in a particular environment (for example, a hospital environment, an industrial environment, a natural environment, etc.) for example, for health control purposes.

[0032] This biological sample, liquid or solid, may be, for example and without limitation, a clinical sample of blood, plasma, urine or feces, a sample of nose, throat, skin, wound, bronchoalveolar fluid or sputum, a food sample such as milk, fruit juice, a sample of yogurt, meat, egg, vegetable, mayonnaise, cheese, fish, etc., a food sample from feed intended for animals, such as in particular a sample of animal meal. For the purposes of simplifying vocabulary, the expressions "biological sample" and "biological sample" will be used interchangeably.

[0033] By "solid microbiological culture medium" or more simply "solid culture medium" is meant a solid or semi-solid support, in particular of the agar, gelled type, classically used in analytical microbiology to cultivate, isolate and / or identify microorganisms. In contrast to liquid culture media, a solid culture medium incorporates in its composition a gelling, structuring agent which gives it a solid, textured consistency. As a gelling / structuring agent for solid culture media, agar (or agar agar) is widely used; alternatives exist, such as gelatin, agarose, guar gum, xanthan gum. . .

[0034] A culture medium, whether solid or liquid, is mainly composed of an aqueous base (which, in the case of a solid culture medium, is structured using a gelling agent) buffered at an appropriate pH, and a so-called nutrient component, which provides the nutritional intake that the targeted microorganisms need for their development and growth. Very schematically, a nutrient component of a culture medium typically contains: - a carbon and energy source (generally a carbohydrate, in the form of a simple sugar or a complex sugar);

[0035] - a source of calcium (e.g. CaCh.2H2O);

[0036] - a source of nitrogen (for example (NHfhSOf);

[0037] - a source of sulfur (for example (NHfhSOf);

[0038] - a source of magnesium (e.g. MgCh. FLO);

[0039] A nutrient component of a culture medium may also include other nutrients (including amino acids, growth factors, vitamins, minerals, trace elements, iron citrate, sodium citrate, sodium chloride, etc.) to enhance the attributes of the nutrient component. The nutrient component may be provided to the composition of a culture medium in whole or in part by means of isolated, chemically well-identified compounds, and / or compositions whose formulation is perfectly defined, and / or chemically complex compositions (for example, fresh blood, sera, yeast extracts, whole egg, egg yolk, peptones, etc.).

[0040] A number of culture media are listed and described in the Handbook of Microbiological Media - Fourth Edition as suitable for the cultivation of yeasts of the genus Candida. The details of these culture media, in terms of composition (in particular, nutrient component) and preparation, as found in that handbook, are to be considered as an integral part of this description.In a non-exhaustive manner, for the implementation of the present invention, the nutrient components of the following media may be particularly advantageous: AB Y media (“ABY agar - Acid Bismuth Yeast Agar”), BiGGY (“BiGGY agar (Bismuth Sulfite Glucose Glycerin Yeast Extract Agar)”), Candida BCG (“Candida BCG Agar Base - Candida Bromcresol Green Agar Base”), CHROMagar™ Candida Agar (CHROMagar Microbiology, France), CandiSelect™ 4 (BioRad, US), Brillance™ Candida Agar (Thermo Fisher Scientific, US) and HiCrome™ Candida differential Agar Base (HiMedia Laboratories, India).

[0041] Furthermore, in addition to the nutrient component, the solid culture media may also incorporate in their composition a so-called discriminating component, which brings together compounds / ingredients participating in the processes and mechanisms of detection and identification of the target / desired microorganisms. This is the case for the solid culture media according to the invention. In this respect, it should be noted that the particular discriminating component of the solid culture media according to the invention constitutes the very heart of the present invention; it makes possible the specific detection and identification of yeasts belonging to the six major Candida spp., namely C. auris, C. albicans, C. glabrata, C. Iropicalis, C. parapsilosis and C. krusei.

[0042] The specific detection, discrimination and identification of yeasts of the genus Candida implemented by the solid culture media according to the invention are based, in part, on the observation that Candida spp express different levels of α-glucosidase, phosphatase (in particular, acid phosphatase) and N-acetyl-P-glucosaminidase type activities. The manifestation of these enzymatic activities can be advantageously identified through the use of chromogenic and / or fluorogenic substrates specific to these enzymatic activities; the enzymatic hydrolysis of these substrates releases chromophores and / or fluorophores which locally / punctualy tint the surface of the culture medium and the biomass, at the very locations where the targeted enzymatic activities are concentrated and, as a result, produces a visual marking of the colonies of Candida spp expressing these target activities.

[0043] Although it is possible to detect most species of the genus Candida based solely on these three enzymatic / metabolic activities of interest, the level of specificity of such detection is, however, insufficient to allow specific identification of each of the six major Candida spp (C. auris, C. albicans, C. glabrata, C. iropicalis, C. parapsilosis and C. krusei) and / or differentiation of the latter from one another. It was in seeking to resolve this problem of detection and identification specificity that the inventors found that these enzymatic activities of interest, α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase, exhibit a certain sensitivity to compounds that modulate metabolic activity, in particular inducers and / or repressors.This sensitivity of enzymatic activities, in particular α-glucosidase and acid phosphatase activities, to compounds that induce or repress metabolic activity, varies from one Candida sp. to another. This is achieved by ingeniously exploiting:

[0044] - differential and species-dependent expression of α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase enzymatic activities, and

[0045] - the differential and species-dependent modulation of the enzymatic activities of a-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase, by particular inducing compounds and repressor compounds, that the inventors have succeeded in developing analytical microbiology tools, simple and rapid to use, which allow not only the detection of yeasts of various Candida spp, but also to identify / discriminate these yeasts according to the species to which they belong, and in particular C. auris, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

[0046] Thus, essentially thanks to its discriminating component, a solid culture medium according to the invention allows:

[0047] - to create the appropriate conditions to induce, in yeasts of the genus Candida, a particular differential expression of three enzymatic activities of interest (a-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase), thereby generating a particular metabolic activity profile (oriented towards these three enzymatic activities of interest), of high specificity with regard to each Candida sp., and

[0048] - to easily visualize (by colorimetry and / or fluorimetry) each colony of yeast growing on the medium and to precisely deduce the species in question, in particular if it corresponds to C. auris, C. albicans, C. glabrata, C. tropicalis, C. krusei or C. parapsilosis.

[0049] To induce the α-glucosidase activity of yeasts of the genus Candida, more particularly of C. auris, C. albicans, C. tropicalis and C. parapsilosis, a solid culture medium according to the invention advantageously comprises, as α-glucosidase inducer (other than a chromogenic and / or fluorogenic substrate of β-glucosidase), a sugar chosen from maltose and raffinose, at a concentration for example of between 0.05 g / L and 0.5 g / L, preferably 0.3 g / L for maltose and 0.1 g / L for raffinose.

[0050] Optionally, to repress this α-glucosidase activity in yeasts of the genus Candida, galactose and xylose, used at a concentration of between 5 and 30 g / L, preferably of the order of 20 g / L, can be advantageously used.

[0051] According to the invention, to induce acid phosphatase activity in yeasts of the genus Candida, they are placed in an acidic environment. In this regard, a pH buffer makes it possible to stabilize the acid pH of the medium, advantageously at a pH between 5.5 and 6.5. To further reinforce the induction of the acid phosphatase activity of these yeasts, a culture medium according to the invention advantageously contains one or more fermentative sugars, metabolizable by the yeasts. By metabolizing these fermentative sugars, the yeasts produce acidic compounds which are directly accessible to them and which participate in inducing their acid phosphatase activity.

[0052] Advantageously, the culture medium comprises a fermentative sugar metabolizable by the yeasts and a pH buffer capable of maintaining the medium at an acidic pH, preferably between 5.5 and 6.5. Advantageously, the fermentative sugar is glucose, used at a concentration of between 5 and 20 g / L.

[0053] To suppress acid phosphatase activity, the solid culture medium advantageously contains an inorganic phosphate salt which makes it possible to suppress acid phosphatase activity in certain Candida spp, in this case, C. auris, C. albicans and C. parapsilosis. Advantageously and according to the invention, potassium phosphate (KH2PO4) is used for this purpose, preferably at a concentration of between 0.5 g / L and 3 g / L, preferably 2 g / L.

[0054] Finally, the discriminating component of a solid culture medium according to the invention comprises an inducer of N-acetyl-P-glucosaminidase (other than a chromogenic and / or fluorogenic substrate of N-acetyl-P-glucosaminidase), for example a hexosamine, such as for example N-acetyl-glucosamine. Advantageously and according to the invention, N-acetyl-glucosamine at a concentration of between 0.5 g / L and 2 g / L, preferably 1 g / L, is used to induce the N-acetyl-P-glucosaminidase activity of Candida spp, and more particularly of C. albicans and C. tropicalis.

[0055] Thus, under the culture conditions established by a culture medium according to the invention and in which, in a differential and species-dependent manner:

[0056] - an a-glucosidase activity of Candida spp is induced by means of a sugar such as maltose or raffinose,

[0057] - acid phosphatase activity of Candida spp is induced by glucose, and is repressed by potassium phosphate, and

[0058] - an N-acetyl-P-glucosaminidase activity of Candida spp is induced by a hexosamine, the species C. auris, C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis each express a particular metabolic activity profile (based on the observation of the three enzymatic activities of interest) and specific. In this case:

[0059] - C. auris expresses only α-glucosidase activity, - C. albicans expresses only two of the three enzymatic activities of interest, namely α-glucosidase and N-acetyl-P-glucosaminidase,

[0060] - C. tropicalis expresses the three enzymatic activities of interest,

[0061] - C. glabrata and C. krusei both express only acid phosphatase activity but are visually highly recognizable from each other by the morphology of the colonies they form on solid culture media, including a solid culture medium according to the invention; C. glabrata forms colonies with smooth edges while C. krusei forms colonies with jagged, serrated edges, and

[0062] - C. parapsilosis exhibits a low level of α-glucosidase activity (and possibly a residual level of phosphatase activity in the case of partial repression of this activity, reflecting the use of a restricted amount of potassium phosphatase).

[0063] On a solid culture medium according to the invention, each isolated colony is thus associated with a given color which reflects the metabolic activity profile of the species which corresponds to it:

[0064] - the color of C. auris colonies is that of the chromophore / fluorophore of the chromogenic / fluorogenic substrate of a-glucosidase,

[0065] - the color of the colonies of C. glabrata and C. krusei is that of the chromophore / fluorophore of the chromogenic / fluorogenic phosphatase substrate,

[0066] - the color of C. albicans colonies results from the mixing between the chromophores / fluorophores of the chromogenic / fluorogenic substrates of α-glucosidase and N-acetyl-β-glucosaminidase,

[0067] - the color of C. tropicalis colonies results from the mixing between the chromophores / fluorophores of the chromogenic / fluorogenic substrates of α-glucosidase, phosphatase and N-acetyl-P-glucosaminidase,

[0068] - as for the colonies of C. parapsilosis, they appear slightly tinted by the chromophore / fluorophore of the chromogenic / fluorogenic substrate of a-glucosidase (and possibly with a very slight tint reflecting that of the chromophore / fluorophore of the chromogenic / fluorogenic substrate of phosphatase, in the case of partial repression of this activity).

[0069] To implement the present invention, a number of chromogenic and / or fluorogenic substrates applicable to solid culture media and allowing colorimetric and / or fluorometric labeling of enzymatic activities such as α-glucosidase, phosphatase and N-acetyl-P-glucosaminidase, are commercially available and / or have already been described in the literature. The chromophores / fluorophores (and therefore, the chromogenic and / or fluorogenic substrates) representative of the three enzymatic activities of interest will be specially selected for their ability to create a whole palette of color and / or fluorescence, capable of allowing easy visual discrimination of the metabolic activity profiles induced according to the invention.

[0070] According to a particular advantageous embodiment of the invention, the solid culture media are chromogenic and thus contain chromogenic substrates of α-glucosidase, phosphatase and N-acetyl-P-glucosaminidase.

[0071] There are a wide variety of chromogenic substrates for this purpose, including those with chromophore nuclei of the indoxyl, coumarin, hydroxy quinoline type... and derivatives, or substrates from the Aldol® range of the Swiss company BIOSYNTH AG.

[0072] As examples of chromogenic substrates that can be considered for the preparation of a solid culture medium according to the invention, the following may be cited:

[0073] - to highlight a-glucosidase activity:

[0074] - Aldol® 455-a-Glu (enzymatic lysis releases a yellow chromophore),

[0075] - Aldol® 518-a-Glu (enzymatic lysis releases a red-tinted chromophore),

[0076] - Aldol® 484-a-Glu (enzymatic lysis releases a red-tinted chromophore),

[0077] - 5-bromo-4-chloro-3-indolyl-aD-glucoside (or Xa-Glu, whose enzymatic lysis releases a blue-tinted chromophore),

[0078] - 5-bromo-6-chloro-3-indolyl-aD-glucosidase (or Mag-a-Glu; enzymatic lysis releases a magenta-red chromophore),

[0079] - 6-chloro-3-indolyl-PD-glucopyranoside (or Rose-a-Glu or Sal-a-Glu; enzymatic lysis releases a salmon-pink chromophore),

[0080] - 5-bromo-4-chloro-3-indolyl-N-methyl-alpha-D-glucoside (or GreenA-a-Glu; enzymatic lysis releases a green / turquoise chromophore), - Alizarin-a-Glu (enzymatic lysis releases a chromophore whose color depends on the metal ion with which it is used and to which it can chelate; for example, this substrate gives a violet color in the presence of iron citrate in the medium, pink / salmon in the presence of aluminum sulfate, and mauve pink in the presence of copper sulfate),

[0081] - phosphatase substrates:

[0082] - 2-naphthyl-phosphate (enzymatic lysis releases a magenta-colored chromophore)

[0083] - 6-bromo-3-indolyl-phosphate (or Red-Phos; enzymatic lysis releases a pink-tinted chromophore)

[0084] - 5-bromo-6-chloro-3-indolyl-phosphate (or Mag-Phos; enzymatic lysis releases a magenta-colored chromophore),

[0085] - 5-bromo-4-chloro-3-indolyl-phosphate (or X-Phos; enzymatic lysis releases a green / turquoise-blue chromophore),

[0086] - 1' Aldol® 470-phosphate (enzymatic lysis releases a yellow chromophore),

[0087] - 1' Aldol® 495-phosphate (enzymatic lysis releases an orange-colored chromophore),

[0088] - 1' Aldol® 515-phosphate (enzymatic lysis releases a red-tinted chromophore), and

[0089] - a PN-acetyl-glucosaminidase substrate:

[0090] - 5-bromo-6-chloro-3-indolyl-|3-N-acetyl-glucosaminide (or Mag-NAG; enzymatic lysis releases a magenta-colored chromophore)

[0091] - 6-chloro-3-indolyl-|3-N-acetyl-glucosaminide (or Rose-NAG or Sal-NAG; enzymatic lysis releases a salmon-pink chromophore),

[0092] - 5-bromo-4-chloro-3-indolyl-N-acetyl-glucosaminide (or X-NAG; enzymatic lysis releases a blue-tinted chromophore).

[0093] The proper functioning of certain chromogenic substrates, for example those with an indoxyl nucleus, may require the presence of a metal salt (such as, for example, iron citrate, aluminum sulfate, copper sulfate or manganese chloride). According to a first preferred embodiment of the invention, to improve the visual discrimination between colonies of C. auris and C. parapsilosis, based on the level of α-glucosidase activity expressed by its two Candida spp, as chromogenic substrate, Aldol®-based indoxyl derivatives are advantageously used, i.e. chromogenic substrates carrying an indoxyl nucleus (1H-indolyl-3-yl) conjugated to the cyclic amine (N-aryl). The structure and preparation of such chromogenic substrates are described in the international PCT application referenced WO 2010 / 128120, filed in the name of Biosynth AG, a Swiss company which also markets such chromogenic substrates itself.

[0094] With such chromogenic α-glucosidase substrates, including Aldol® 455-aD-glucopyranoside (CAS No. 1254798-01-0; Biosynth ref. A-4689_P00), C. auris colonies appear strongly and strongly stained, whereas C. parapsilosis colonies appear weakly stained.

[0095] According to a second preferred embodiment of the invention, which may complement or alternatively serve to improve visual discrimination between colonies of C. auris and C. parapsilosis, the α-glucosidase activity expressed in these two yeast species is modulated by means of a repressor. In this respect, galactose or xylose, used at a concentration of between 5 and 30 g / L, preferably of the order of 20 g / L, makes it possible to strongly repress the α-glucosidase activity of C. parapsilosis and, to a much lesser extent, that of C. auris.

[0096] Thus, under the culture conditions established in accordance with this second preferred embodiment of a culture medium according to the invention and in which:

[0097] - an a-glucosidase activity of Candida spp is induced by means of a sugar such as maltose or raffinose, and repressed by galactose or xylose,

[0098] - acid phosphatase activity of Candida spp is induced by glucose, and is repressed by potassium phosphate, and

[0099] - an N-acetyl-P-glucosaminidase activity of Candida spp is induced by a hexosamine, the species C. auris, C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis each express a particular metabolic activity profile (based on the observation of the three enzymatic activities of interest) and specific. In this case: - C. auris expresses only the a-glucosidase activity,

[0100] - C. albicans expresses only two of the three enzymatic activities of interest, namely α-glucosidase and N-acetyl-β-glucosaminidase,

[0101] - C. tropicalis expresses the three enzymatic activities of interest,

[0102] - C. glabrata and C. krusei both express only acid phosphatase activity but are visually highly recognizable from each other by the morphology of the colonies they form on solid culture media, including a solid culture medium according to the invention; C. glabrata forms colonies with smooth edges while C. krusei forms colonies with jagged, serrated edges, and

[0103] - C. parapsilosis does not express any of the three enzymatic activities of interest (possibly a weak phosphatase activity can be observed in the case of partial repression of this activity due to the use of a restricted quantity of potassium phosphate).

[0104] On a solid culture medium in accordance with this second preferred embodiment of the invention, each isolated colony of particular Candida sp is thus associated with a given color which reflects the metabolic activity profile of the species which corresponds to it:

[0105] - the color of C. auris colonies is that of the chromophore / fluorophore of the chromogenic / fluorogenic substrate of -glucosidase,

[0106] - the color of the colonies of C. glabrata and C. krusei is that of the chromophore / fluorophore of the chromogenic / fluorogenic phosphatase substrate,

[0107] - the color of C. albicans colonies results from the mixing between the chromophores / fluorophores of the chromogenic / fluorogenic substrates of α-glucosidase and N-acetyl-β-glucosaminidase,

[0108] - the color of C. tropicalis colonies results from the mixture between the chromophores / fluorophores of the chromogenic / fluorogenic substrates of α-glucosidase, phosphatase and N-acetyl-P-glucosaminidase,

[0109] - as for the colonies of C. parapsilosis, they appear colorless (and possibly with a very slight tint reflecting that of the chromophore / fluorophore of the chromogenic / fluorogenic phosphatase substrate, in the event of partial repression of this activity). Independently of the preferred embodiment of the invention, advantageously:

[0110] - the α-glucosidase activity is advantageously associated with a yellow chromophore or a similar color (for example, using as chromogenic substrate, Aldol® 455-α-glucoside),

[0111] - the phosphatase activity is advantageously associated with a pink, red or similar chromophore (for example, using as chromogenic substrate, Magenta-phosphate, Aldol® 484-phosphate, Aldol® 518-phosphate, alizarin-phosphate, or Rose-phosphate. . .), and

[0112] - the N-acetyl-P-glucosaminidase activity is advantageously associated with a blue chromophore or a similar color (for example, using as chromogenic substrate, XN-acetyl-|3-glucosaminide).

[0113] Advantageously, a solid culture medium according to the invention is a medium said to be selective for yeasts of the genus Candida. In this respect, in addition to the nutritive component and the discriminating component, a solid culture medium according to the invention also comprises a selective component which promotes the culture of Candida spp rather than that of the accompanying flora possibly present in the biological samples to be tested. In this respect, its composition incorporates a selective component, which comprises one or more compounds having an inhibitory or biocidal effect on “non-Candida” microorganisms. In this case, said selective component comprises one or more of the following antibiotics: amikacin, vancomycin, ceftazidime, chloramphenicol.

[0114] Advantageously, the nutrient component of a culture medium according to the invention comprises a yeast extract content of less than 0.5 g / L. Preferably, it is free of yeast extract.

[0115] The present invention also extends to a method for identifying yeasts of the genus Candida possibly present in a biological sample, comprising the following steps:

[0116] - cultivation of microorganisms to be studied on or in a culture medium comprising:

[0117] - a nutrient component capable of enabling the development and growth of yeasts of the genus Candida, - an inducer of α-glucosidase and possibly an α-glucosidase repressor,

[0118] - an acid phosphatase inducer and a phosphatase repressor, and

[0119] - an inducer of N-acetyl-P-glucosaminidase,

[0120] - detection of possible α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities expressed by yeasts, and

[0121] - identification of the species to which said yeasts belong according to the α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities actually expressed by said yeasts.

[0122] The present invention also extends to a method for identifying C. auris, possibly present in a biological sample, comprising the following steps:

[0123] - cultivation of microorganisms to be studied on or in a culture medium comprising:

[0124] - a nutritional component capable of enabling the development and growth of yeasts of the genus Candida,

[0125] - an α-glucosidase inducer and possibly an α-glucosidase repressor,

[0126] - an acid phosphatase inducer and a phosphatase repressor,

[0127] - an inducer of N-acetyl-P-glucosaminidase,

[0128] - detection of possible α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities expressed by yeasts, and

[0129] - identification as C. auris, yeasts which, under the said culture conditions, express an α-glucosidase activity and express neither an acid phosphatase activity nor an N-acetyl-P-glucosaminidase activity.

[0130] Advantageously, in a method for identifying yeasts of the genus Candida according to the invention as well as in a method for identifying C. auris according to the invention, said α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities, possibly expressed, are detected (or demonstrated) by means of chromogenic and / or fluorogenic substrates. Advantageously and according to the invention, said method for identifying yeasts of the genus Candida and said method for detecting and / or identifying C. auris according to the invention are also characterized by all or some of the following technical characteristics:

[0131] - the α-glucosidase inducer is a sugar chosen from maltose and raffinose, at a concentration for example between 0.05 g / L and 0.5 g / L, preferably a concentration of 0.3 g / L for maltose and 0.1 g / L for raffinose;

[0132] - optionally, the α-glucosidase activity expressed in certain Candida spp is repressed by galactose or xylose, used at a concentration between 5 g / L and 30 g / L, in particular of the order of 20 g / L;

[0133] - acid phosphatase activity is induced by placing the yeasts in an acidic environment, buffered at a pH between 5.5 and 6.5, and in the presence of one or more fermentative sugars, such as glucose at a concentration between 5 g / L and 20 g / L;

[0134] - the repression of phosphatase activity is achieved by means of an inorganic phosphate salt, such as for example potassium phosphate (KH2PO4) which, used at a concentration of between 0.5 g / L and 3 g / L, preferably 2 g / L, has demonstrated a repressive effect on phosphatase activity in certain Candida spp, in this case, C. auris, C. albicans and C. parapsilosis, ' and

[0135] - the induction of N-acetyl-P-glucosaminidase activity is obtained by using a hexosamine, such as for example N-acetyl-glucosamine, at a concentration between 0.5 g / L and 2 g / L, preferably 1 g / L.

[0136] Advantageously and according to the invention, said method for identifying yeasts of the genus Candida according to the invention and said method for detecting and / or identifying C. auris are implemented by means of a solid culture medium as previously described.

[0137] Other aims, characteristics and advantages of the invention will become apparent from the following description and the examples developed below, which are intended to facilitate the understanding of the invention and its implementation. These examples are given for explanatory purposes and do not limit the scope of the invention. The examples reported below only partially reproduce the work carried out by the inventors in the context of the development and design of the present invention, and relate to the data considered relevant for the assessment of the merit of the invention and that of the inventors.

[0138] EXAMPLES

[0139] 1 / - Evaluation of the metabolic activities of a-glucosidase, acid phosphatase and N-acetyl-B-glucosaminidase, expressed by the six major Candida spp.

[0140] The tests carried out within the framework of the present invention and as set out below concern 38 pure strains of yeasts of the genus Candida, covering six different species, namely:

[0141] - 9 strains of C. auris, from clades I, II, III and IV,

[0142] - 7 strains of C. albicans,

[0143] - 5 strains of C. glabrata

[0144] - 6 strains of C. tropicalis,

[0145] - 6 strains of C. krusei, and

[0146] - 5 strains of C. parapsilosis.

[0147] These 38 yeast strains all come from bioMérieux's private collection. These strains have been collected over the years from various organizations such as:

[0148] - the CDC (“Centers for Disease Control and Prevention”), in the United States;

[0149] - the CBS (“Centraal Bureau voor Schimmelcultures”), in the Netherlands;

[0150] - the BCCM (“Belgian Coordinated Collections of Microorganisms”), in Belgium;

[0151] - the Leibniz Institute DSMZ, in Germany

[0152] - the CHUV (Vaud University Hospital Center), in Switzerland;

[0153] - the Charité University Hospital in Berlin (“Charité - Universitatsmedizin Berlin”), in Germany;

[0154] - the Faculty of Pharmacy of Montpellier, France;

[0155] - the Bellevue Hospital Center in Saint-Etienne, France;

[0156] - Michallon de la Tronche Hospital, in France; and

[0157] - the University Hospital of Lille, France. These six yeast species were evaluated for their ability to express different metabolic activities under specific conditions, established on a solid culture medium having as its basic composition that of an SDA medium (“Sabouraud Dextrose Agar”).

[0158] It should be noted that before being able to retain the three metabolic activities of α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase, as enzymatic activities of interest for the detection and identification of yeasts of the genus Candida, the inventors considered and studied nearly eighty enzymatic activities including osidases (α-glucosidase, P-glucosidase, α-maltosidase, ribosidase...), phosphatases, esterases, sulfatases, phospholipases, peptidases and nitro-reductases. Ultimately, the combination of α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase was selected as having promising potential for good differentiation / discrimination of species of the genus Candida, and good characterization of C. auris.

[0159] Only work closely linked to α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities is reported below, and that aimed at highlighting a possible impact of metabolic activity modulators (in particular, galactose, xylose, potassium phosphate) on these three metabolic activities.

[0160] 1.1 / - α-glucosidase activity and optional repression

[0161] Among the metabolic activities possibly expressed by Candida spp, the inventors were interested in α-glucosidase activity.

[0162] It was thus found that α-glucosidase activity was indeed expressed in C. auris, C. albicans, C. tropicalis and C. parapsilosis. Conversely, no α-glucosidase activity could be detected in C. glabrata and C. krusei.

[0163] In order to further optimize the discriminatory power of α-glucosidase activity with respect to Candida spp, different repressors were evaluated, including various sugars and, in particular, simple sugars. A repressor effect on α-glucosidase activity expressed by C. parapsilosis was thus observed, both with galactose and xylose. Conversely, the latter have no effect on the α-glucosidase activity of C. auris, C. albicans and C. tropicalis. Table 1 below very briefly summarizes the results of the work on the detection of α-glucosidase activity carried out on the six major Candida spp, in the presence or absence of a repressor (in this case, galactose or xylose) of this particular metabolic activity. Detection of α-glucosidase activity was performed using 5-bromo-4-chloro-3-indolyl-alpha-D-glucoside (or Xa-Glu).

[0164] The (+) sign indicates that the enzymatic activity of interest was actually observed and identified by a clear coloration of the colonies. The (-) sign indicates that colonies did indeed appear on the culture medium but that they remained colorless; this attests to an enzymatic activity of interest not detected, under the repression conditions tested. The (+ / -) sign indicates a less pronounced enzymatic activity of interest and colonies with a less intense, relatively pale coloration. More specifically, regarding the chromogenic labeling of the α-glucosidase activity of Candida spp, chromogenic substrates of different structures were tested, in particular with a view to selecting the chromophores allowing good visual differentiation of C. auris colonies and C. parapsilosis colonies, which all express α-glucosidase activity, but at different levels.

[0165] Table 2 below summarizes the results of work on some chromogens tested in this regard. The observed coloration and intensity are shown schematically in this table. The higher the numerical value indicated, the more intense the coloration.

[0166] Table 2

[0167] With Aldol®-based indoxyl-derived chromogenic substrates, such as Aldol® 455-a-Glu, the inventors achieved excellent visual discrimination between C. auris and C. parapsilosis, NQC, OR galactose-free colonies by a significant difference in staining intensity between the two species.

[0168] 1.2 / - Acid phosphatase activity, and influence of glucose and KH2PO4

[0169] Acid phosphatase activity is a second metabolic activity of yeasts of the genus Candida that the inventors were interested in.

[0170] Their work led in particular to the following observations:

[0171] - C. auris, C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis all express acid phosphatase activity;

[0172] - in C. auris, C. albicans and C. parapsilosis, this metabolic activity can be repressed by an inorganic phosphate salt, for example potassium phosphate (KH2PO4), notably used at a concentration of 0.5 g / L to 3 g / L; this effect of repression of phosphatase activity has not, however, been observed in C. glabrata, C. tropicalis and C. krusei.

[0173] Table 3 below very briefly summarizes the results of the work detecting acid phosphatase activity in the six major Candida spp, in the presence or absence of a repressor such as potassium phosphate.

[0174] The (+) sign indicates that the enzymatic activity of interest was indeed observed. The detection of phosphatase activity was carried out using 5-bromo-6-chloro-3-indolyl-phosphate (or Mag-Phos). The (-) sign indicates that colonies did appear on the culture medium but that they remained colorless; this attests to an enzymatic activity of interest not detected, under the repression conditions tested.

[0175] Table 3

[0176] 1.3 / - N-acetyl-P-glucosaminidase activity

[0177] Finally, the inventors selected N-acetyl-P-glucosaminidase activity for its discriminating power against yeasts of the genus Candida. This metabolic activity was thus observed to be expressed in C. albicans and C. tropicalis. However, no N-acetyl-P-glucosaminidase activity was observed in C. auris, C. parapsilosis, C. glabrata and C. krusei.

[0178] For this purpose, the yeasts were stimulated with N-acetyl-P-glucosamine (at a final concentration in the medium of 1 g / L).

[0179] Table 4 below very briefly summarizes the results of the work to detect N-acetyl-P-glucosaminidase activity in the six major species of the genus Candida. The detection of phosphatase activity was carried out using 5-bromo-4-chloro-3-indolyl-N-acetyl-glucosaminide (or X-NAG). The (+) sign indicates that the enzymatic activity of interest was indeed observed. The (-) sign indicates that colonies did appear on the culture medium but that they remained colorless.

[0180] Table 4

[0181] Furthermore, the inventors have found that glucose exerts a concentration-dependent repressing effect on N-acetyl-P-glucosaminidase activity. Therefore, in the event that, concomitantly with the detection of N-acetyl-P-glucosaminidase activity, the use of glucose is envisaged, for example to stimulate glucosidase activity (α- and / or P-glucosidase) and / or acid phosphatase activity (in this case, glucose is used as a fermentation sugar making it possible to establish an acidic environment in the immediate vicinity of the colonies formed), its concentration in the medium must be chosen to be sufficiently low to prevent it from interfering with the expression of N-acetyl-P-glucosaminidase. A glucose concentration of less than or equal to 20 g / L, preferably of the order of 5 g / L, has been established as appropriate to avoid this interference with the N-acetyl-P-glucosaminidase activity of C. albicans and C. iropicalis.under the conditions of implementation of the present invention.

[0182] Unlike glucose, galactose does not appear to have an impact on the expression of N-acetyl-P-glucosaminidase in C. albicans and C. iropicalis. Therefore, concomitantly with labeling C. albicans and / or C. iropicalis with N-acetyl-P-glucosaminidase activity, repression of α-glucosidase expression in C. parapsilosis can be achieved on the same culture medium, without it being necessary to take special precautions regarding the choice of the concentration of galactose to use so as not to block N-acetyl-P-glucosaminidase activity.

[0183] 1.4 / - Three metabolic activities to discriminate the six major Candida spp.

[0184] 1.4.1 / - According to the first preferred embodiment of the invention Table 5 below summarizes very schematically and synthetically the results presented above, according to the first embodiment of the invention, that is to say without differential repression of the α-glucosidase activity and in which an indoxyl derivative based on Aldol®, in this case Aldol® 455-αD-glucopyranoside, is advantageously used as a chromogenic substrate for α-glucosidase.

[0185] The (+) sign indicates that the enzymatic activity of interest was actually observed and identified by a clear coloration of the colonies. The (-) sign indicates that colonies did indeed appear on the culture medium but that they remained colorless; this attests to an enzymatic activity of interest not detected, under the repression conditions tested. The (+ / -) sign indicates a notably less pronounced enzymatic activity of interest, reflected by a significantly less intense coloration.

[0186] Table 5

[0187] 1.4.2 / - According to the second preferred embodiment of the invention

[0188] Table 6 below summarizes very schematically and synthetically the results presented above, according to the second preferred embodiment of the invention, that is to say with differential repression of the α-glucosidase activity (by means of galactose or xylose, added to the culture medium).

[0189] The (+) sign indicates that the enzymatic activity of interest was actually observed and identified by a clear coloration of the colonies. The (-) sign indicates that colonies did indeed appear on the culture medium but that they remained colorless; this attests to an enzymatic activity of interest not detected, under the repression conditions tested.

[0190] Table 6

[0191] 1.4.3 / - Balance sheet

[0192] It emerges from the results presented that, based on the detection of three particular metabolic activities, which are the activities:

[0193] - a-glucosidase,

[0194] - acid phosphatase and

[0195] - N-acetyl-P-glucosaminidase, it is possible to discriminate yeasts of the genus Candida, according to the species to which they belong, and this in a relatively simple way. To do this, it is necessary to place the yeasts to be identified in particular culture conditions, in this case by placing them in the presence of differential modulators (inducers and repressors) of metabolic activity skillfully chosen. In doing so, on the basis of the expression of only three reference enzymatic activities, a great diversity of metabolic activity profiles is generated, which is relatively well representative of the diversity of Candida spp. Each metabolic activity profile thus identified can be associated with a few particular species of yeast of the genus Candida.

[0196] By applying this principle to the six major yeast species of the genus Candida (i.e., C. auris, C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis), five specific metabolic activity profiles were established (see Table 5 and Table 6). Four of these profiles exhibit high specificity, each being associated with a particular yeast species of the genus Candida (i.e., C. auris, C. albicans, C. tropicalis, or C. parapsilosis).

[0197] With regard to C. glabrata and C. krusei, these two species share the same metabolic activity profile according to the invention; both express only acid phosphatase activity under the operating conditions of the present invention. Culturing these two species on a solid culture medium makes it very easy to distinguish them. Indeed, on solid culture media, yeasts belonging to the species C. glabrata typically form colonies with rather smooth edges, whereas yeasts belonging to the species C. krusei form colonies with serrated edges.

[0198] It should also be noted that based on the detection of a metabolic activity profile, combining only α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities, and in the absence of differential repressors, it is not possible to obtain such perfect discrimination of Candida spp. It is also not possible to obtain identification of C. auris with such good specificity. Table 7 below very briefly and synthetically shows an attempt to discriminate Candida spp under operating conditions very similar to the invention, but in the absence of differential repressors, such as galactose or xylose to repress α-glucosidase activity, and KH2PO4 to repress acid phosphatase activity.

[0199] The (+) sign indicates that the enzymatic activity of interest was actually observed and identified by a clear coloration of the colonies. The (-) sign indicates that colonies did appear on the culture medium but that they remained colorless; this attests to an enzymatic activity of interest not detected under the repression conditions tested. The (+ / -) sign indicates a somewhat less pronounced enzymatic activity of interest, identified by a less intense coloration of the colonies.

[0200] Table 7

[0201] 2 / - Development of solid culture media for the purpose of detecting the discriminating metabolic activity profile of yeasts of the genus Candida, for identification according to the species Through the design of a solid and chromogenic culture medium, the inventors have found a simple and rapid way of applying the principle demonstrated above, and allowing visual identification and discrimination of Candida spp on a colorimetric recognition of a particular metabolic activity profile induced by established operating conditions.The inventors thus worked to develop a solid culture medium: establishing the culture conditions appropriate to the yeasts to be studied and conducive to directing the metabolic activity of said yeasts studied towards well-established metabolic activity profiles of interest, and allowing direct reading of the results, namely an identification of the yeast species developing on the surface of said solid culture medium, based on the color of the colonies formed, possibly also on their morphology.

[0202] In this context, the inventors developed and tested different solid culture media, varying in particular: the basic nutrient composition, the nature and final concentration in the medium, inducers and repressors of metabolic activity, the chromogenic substrates for each of the enzymatic activities of interest.

[0203] For each culture medium developed, the tests were carried out by inoculating 10 pL of a yeast suspension concentrated at 10 6 CFU / mL. Once inoculated, the culture media were incubated at 35°C (± 2°C). The results were read after 24 hours (± 4 hours) or after 48 hours (± 6 hours) of incubation. Culture performance was then analyzed, in terms of colony density and size, as well as the color tone and intensity of these colonies and, possibly, in terms of colony morphology.

[0204] The following description partly reproduces the results obtained and corresponds to data specifically selected with the aim of facilitating understanding of the development of chromogenic solid culture media according to the invention.

[0205] 2.1 / - Solid culture medium and basic nutrient component To produce a solid culture medium according to the invention, it is possible to start from a large number of compositions comprising a basic nutrient component suitable for the culture of yeasts of the genus Candida.

[0206] The following examples refer to two culture media of relatively basic composition, suitable for the cultivation of yeasts of the genus Candida, and from which it is sufficient to add a few additional suitable ingredients (such as chromogenic and / or fluorogenic substrates, inducers and repressors of metabolic activity) to obtain solid culture media according to the invention.

[0207] Table 8 below details the composition of these two environments, noted Environment 1 and Environment 2.

[0208] Table 8

[0209] These two media, Medium 1 and Medium 2, differ from each other in their nutrient component (a mixture of yeast extract and malt extract for one, and a mixture of gelatin peptones and casein for the other).

[0210] Of course, other nutrient components suitable for yeast culture exist and can very well be considered for the implementation of the present invention (for example other mixtures of yeast extract and / or peptones and / or casein hydrolysate and / or pancreatic hydrolysate and / or meat extract; see for example the few compositions of culture media for yeasts of the genus Candida, as listed in the work "Handbook of Microbiological Media - Fourth Edition"). 2.2 / - Selection of chromogenic substrates

[0211] To visually mark the target enzymatic activities expressed by yeasts cultured on solid culture medium, a number of chromogenic substrates are available on the market, offering a rich palette of colors. In this regard and as an example, we can cite the chromogenic substrates of the indoxyl family, among which:

[0212] - chromogens carrying a 5-bromo-4-chloro-3-indolyl group release a blue-tinted chromophore,

[0213] - those carrying a 3-indolyl group also release a blue-tinted chromophore,

[0214] - those carrying a 5-bromo-6-chloro-3-indolyl group release a magenta-red chromophore,

[0215] - those carrying a 6-chloro-3-indolyl group release a salmon-colored chromophore,

[0216] - those carrying a 5-iodo-3-indoxyl group release a purple-tinted chromophore,

[0217] - those carrying a 5-bromo-4-chloro-3-indolyl-N-methyl group release a green chromophore.

[0218] These chromogens are divided into various chromogenic substrates specific to a particular enzymatic activity, and in particular into:

[0219] - chromogenic substrates of a-glucosidase, such as

[0220] 5-bromo-4-chloro-3-indolyl-a-glucoside (or Xa-Glu),

[0221] - chromogenic phosphatase substrates, such as

[0222] 6-chloro-3-indolyl-phosphate (or Rose-Phos or Sal-Phos), and

[0223] - chromogenic substrates of N-acetyl-P-glucosaminidase, such as 5-bromo-6-chloro-3-indolyl-|3-N-acetyl-glucosaminide (or Mag-NAG).

[0224] Besides the chromogenic substrates of the indoxyl type, many other families exist and are commercially available, for example substrates carrying a coumarin nucleus, a hydroxyquinoline nucleus, the indoxyl derivative substrates based on Aldol® from the Swiss company BIOSYNTH AG, and from which it is possible to select the substrates of α-glucosidase, phosphatase and N-acetyl-P-glucosaminidase in order to implement the present invention. For each of the three metabolic activities of interest, the inventors selected and evaluated different chromogenic substrates in order to identify the most appropriate for use in solid culture media, and whose use in combination with other selected substrates could offer a satisfactory contrast for good discrimination of the metabolic activity profiles induced according to the invention.

[0225] Also, the inventors studied the possible interactions between the selected chromogenic substrates and the nutritive elements which enter into the composition of the solid culture support.

[0226] 2.2.1 / - Chromogenic a-glucosidase substrates

[0227] For α-glucosidase activity, chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-α-glucoside (or Xa-Glu), 1' Aldol® 455-α-glucoside (or Aldol® 455-α-Glu), 1' Aldol® 484-α-glucoside (Aldol® 484-α-Glu) and Alizarin-α-glucoside were evaluated.

[0228] Tests carried out, for example, with C. auris resulted in the following findings, after 24 hours of culture:

[0229] - 5-bromo-4-chloro-3-indolyl-alpha-glucoside (or Xa-Glu), used for example at a concentration of 0.1 g / L, dyes colonies of clade I, III and IV strains blue, and colonies of clade II strains a less intense blue; it should be noted that the color of the colonies can tend more towards green when the medium is more strongly acidic,

[0230] - Aldol® 455-alpha-glucoside (or Aldol® 455-a-Glu), used for example at 0.1 g / L, colors colonies of clades I, III and IV yellow; but this coloring is not visible with isolated colonies of clade II, of low density;

[0231] - 1' Aldol® 484-alpha-glucoside (Aldol® 484-a-Glu), used at a concentration of 0.1 g / L, gives the colonies a low intensity orange coloration, regardless of the clade;

[0232] - Alizarin-a-glucoside, for example used at 0.05 g / L in combination with iron citrate at 0.05 g / L, colors colonies of clades I, III and IV purple, and colonies of clade II lighter purple.

[0233] 2.2.2 / - Chromogenic phosphatase substrates, and induction of acid phosphatase activity. Since acid phosphatases are characterized by an acidic pH of optimal activity, their detection requires placing the yeasts in an acidic environment. To do this, the culture media are supplemented with a fermentative sugar (e.g. glucose) and a buffer system adapted to maintain the medium at a pH of around 5.5 to 6.5 (e.g. a phosphate buffer). The fermentation of this sugar by the yeasts locally releases acidic entities, which benefit the yeasts by stimulating their acid-dependent phosphatase activity.

[0234] The possible phosphatase activity (both acid and alkaline) expressed by yeasts of the genus Candida has been demonstrated using chromogenic phosphatase substrates. For this purpose, various chromogenic substrates are available on the market. For example, those whose hydrolysis releases a chromophore 6-chloro-3-indolyl nucleus give a pink tint to the colonies and those releasing a 5-bromo-6-chloro-3-indolyl nucleus give a magenta tint.

[0235] Also, the influence of an inorganic phosphate salt on this acid phosphatase activity was also evaluated.

[0236] Each yeast strain tested was thus cultivated on a solid agar medium (notably of composition such as Medium 1 or Medium 2) buffered and containing, among other things:

[0237] - glucose at different final concentrations in the medium, in this case, a final concentration of less than 20 g / L, in particular from 1 g / L to 20 g / L, preferably 5 g / L;

[0238] - KH2PO4 at different final concentrations in the medium, in this case at final concentrations ranging from 0.5 g / L to 3 g / L, preferably 2 g / L;

[0239] - a chromogenic substrate specific to phosphatases (such as 5-bromo-6-chloro-3-indolyl-phosphate (Mag-Phos), 1' Aldol® 470-phosphate, 1' Aldol® 495-phosphate, 1' Aldol® 515-phosphate).

[0240] After 48 hours of culture at 35°C, it was observed that with most chromogenic substrates (with the exception of the Aldol® 515-phosphate substrate):

[0241] - in C. auris, C. albicans and C. parapsilosis, phosphatase activity is expressed, it does not appear to be glucose dependent and can be repressed with the addition of KH2PO4; - C. glabrata expresses acid phosphatase activity only in the presence of glucose, regardless of the KH2PO4 concentrations in the medium.

[0242] - in C. tropicalis and C. krusei, phosphatase activity is expressed in a sustained manner and does not appear to be glucose-dependent; nor does it appear to be repressed by KH2PO4.

[0243] 2.2.3 / - Chromogenic substrates of N-acetyl-P-glucosaminidase

[0244] For N-acetyl-P-glucosaminidase activity, different chromogenic substrates were tested. No particular behavior was observed with the tested substrates.

[0245] 2.3 / - Examples of some possible effects of the nutrient component on the metabolic activity profiles induced according to the invention

[0246] 2.3.1 / - Incidence of yeast extracts

[0247] Table 9 below compiles some specific operating conditions that reveal possible impacts that certain nutrient components of the culture medium, in particular nutrient components containing yeast extracts, may have on the detection of metabolic activities of interest according to the invention.

[0248] Table 9

[0249] With Medium 1, an absence of expression of acid phosphatase activity is observed in C. glabrata. It has been shown that the yeast extract present in the nutrient component of this medium acts as an inhibitor on the acid phosphatase activity of this species (see Table 10 below).

[0250] Yeast extract from this medium, combined with a high concentration of glucose (20 g / L), also inhibits acid phosphatase activity in C. auris (thus acting competitively or additionally with KH2PO4).

[0251] With Medium 2, free of any yeast extract, this phenomenon of inhibition of acid phosphatase activity in C. glabrata and C. auris is no longer observed.

[0252] Table 10

[0253] 2.3.2 / - Implementation of the invention with the Medium 2

[0254] Tables 11 and 12 below illustrate the implementation, in Medium 2, of the appropriate culture conditions to induce, in yeasts of the genus Candida (with or without galactose), a particular differential expression of the three enzymatic activities of interest (α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase) and making possible a visual discrimination of C. auris, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

[0255] Table 11 3 / - Optimization of the nutritive component of a solid culture medium composition according to the invention

[0256] As previously explained, the composition of Medium 2 corresponds to a particular embodiment of a nutritive component of a solid culture medium according to the invention. This has demonstrated particularly interesting and perfectly acceptable results in terms of fertility with regard to yeasts of the genus Candida and, more particularly, C. auris. C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

[0257] As indicated above, other nutrient component compositions conducive to the development and growth of Candida spp may be considered for implementing the present invention. These other nutrient component compositions are part of the state of the art, or even the general knowledge of those skilled in the art, in particular they may be found in the manual "Handbook of Microbiological Media - Fourth Edition" or derived therefrom.

[0258] However, as seen previously with Medium 1, nutrient components containing yeast extracts should be avoided. Thus, for the preparation of a solid culture medium according to the invention. It is advisable to use nutrient components for Candida spp, which are low in yeast extract (or even completely free of yeast extract) or else provide for a substitution, at least partial, of yeast extracts by nutritional alternatives, more or less complex, such as for example meat extracts, peptones, malt extracts. . .

[0259] Starting from the composition of Medium 2 (see Table 8), the inventors investigated various points of improvement, different approaches, with a view to optimizing its performance, in particular fertility. In this regard, additional work focused, among other things, on: the evaluation of various peptones from different suppliers and, in particular, alternatives to gelatin peptone, such as potato peptone, lupin peptone, bioSoyase (a papain peptone from soybeans), malt extracts, casein; the replacement of glucose with sodium pyruvate; the optimization of the acid pH of the medium, and the appropriate buffer system.

[0260] Table 13 below gives an example of optimization of Medium 2.

[0261]

[0262] Table 13

Claims

CLAIMS 1) Solid microbiological culture medium, for the detection, discrimination and / or identification of yeasts of the genus Candida likely to be present in a biological sample to be tested, said medium comprising: - a nutritional component capable of enabling the development and growth of yeasts of the genus Candida, and - a discriminating component capable of enabling visual marking of yeast colonies growing on said culture medium, and being characterized in that said discriminating component comprises: - an α-glucosidase inducer and a chromogenic and / or fluorogenic α-glucosidase substrate which, under the action of an α-glucosidase, releases a chromophore and / or a fluorophore emitting a first detectable optical signal, - an acid phosphatase inducer, a phosphatase repressor and a chromogenic and / or fluorogenic phosphatase substrate which, under the action of a phosphatase, releases a chromophore and / or a fluorophore emitting a second detectable optical signal, said second detectable optical signal being different from said first detectable optical signal, - an inducer of N-acetyl-P-glucosaminidase and a chromogenic and / or fluorogenic substrate of N-acetyl-P-glucosaminidase which, under the action of an N-acetyl-P-glucosaminidase, releases a chromophore and / or a fluorophore emitting a third detectable optical signal, said third detectable optical signal being different from said first detectable optical signal and said second detectable optical signal. 2) Culture medium according to claim 1, which comprises, as an α-glucosidase inducer, a sugar chosen from maltose and raffinose, at a concentration of between 0.05 g / L and 0.5 g / L. 3) Culture medium according to claim 1 or 2, which comprises, as acid phosphatase inducer, a fermentative sugar metabolizable by yeasts and a pH buffer capable of maintaining said medium at an acid pH. 4) Culture medium according to claim 3, which comprises a pH buffer maintaining the pH between 5.5 and 6.

5. 5) Culture medium according to claim 3 or 4, which comprises, as fermentation sugar, glucose at a concentration of between 5 g / L and 20 g / L. 6) Culture medium according to any one of claims 1 to 5, which comprises, as phosphatase repressor, an inorganic phosphate salt, preferably potassium phosphate at a concentration of between 0.5 g / L and 3 g / L, preferably 2 g / L. 7) Culture medium according to any one of claims 1 to 6, which comprises, as an N-acetyl-P-glucosaminidase inducer, a hexosamine, preferably N-acetyl-P-glucosamine. 8) Culture medium according to any one of claims 1 to 7, wherein said α-glucosidase substrate is a chromogenic substrate carrying an indoxyl (1H-indolyl-3-yl) nucleus conjugated to the cyclic amine. 9) Culture medium according to any one of claims 1 to 8, which comprises an α-glucosidase repressor, chosen between galactose and xylose, at a concentration of between 5 g / L and 30 g / L. 10) Culture medium according to any one of claims 1 to 9, in which the nutrient component is free of yeast extract. 11) Method for identifying yeasts of the genus Candida possibly present in a biological sample, comprising the following steps: - culturing the yeasts to be identified on or in a culture medium comprising: a nutrient component capable of allowing the development and growth of yeasts of the genus Candida, an α-glucosidase inducer, an acid phosphatase inducer and a phosphatase repressor, and an N-acetyl-P-glucosaminidase inducer, - detection of possible α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities expressed by yeasts, and - identification of the species to which said yeasts belong according to the α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities actually expressed by said yeasts. 12) Identification method according to claim 11, wherein the detection of said α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities is carried out using chromogenic and / or fluorogenic substrates. 13) Identification method according to claim 11 or 12, wherein the detection of said α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities is carried out using a solid microbiological culture medium as defined by any one of claims 1 to 10. 14) Method for detecting and / or identifying C. auris possibly present in a biological sample, comprising the following steps: - cultivation of any yeasts to be detected and / or identified on or in a culture medium comprising: a nutrient component capable of allowing the development and growth of yeasts of the genus Candida, an α-glucosidase inducer, an acid phosphatase inducer and a phosphatase repressor, and an N-acetyl-P-glucosaminidase inducer, - detection of possible α-glucosidase, acid phosphatase and / or N-acetyl-P-glucosaminidase activities expressed by yeasts, and - identification as C. auris, yeasts which express α-glucosidase activity and express neither acid phosphatase activity nor N-acetyl-P-glucosaminidase activity. 15) Detection and / or identification method according to claim 14, wherein said α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities are detected using chromogenic and / or fluorogenic substrates. 16) Detection and / or identification method according to claim 14 or 15, wherein the detection of said α-glucosidase, acid phosphatase and N-acetyl-P-glucosaminidase activities is carried out using a solid microbiological culture medium as defined by any one of claims 1 to 10.

Images