Candida auris rapid selective detection monitoring culture medium, preparation method and application thereof

Abstract

This invention discloses a rapid and selective detection and monitoring culture medium for Candida auris, its preparation method, and its application, belonging to the field of microbial detection technology. The culture medium comprises the following components: peptone, yeast extract, glucose, agar, selectivity inhibitor, chromogenic substrate, and auxiliary components. The chromogenic substrate consists of euonymus alcohol and X-Gal. The culture medium of this invention inhibits contaminating bacteria by adding chloramphenicol and ciclopirox ol, and the chromogenic system of euonymus alcohol and X-Gal achieves specific detection of Candida auris. This culture medium has the advantages of high selectivity, rapid identification, and low cost, and is suitable for clinical samples and environmental monitoring.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a rapid selective detection and monitoring culture medium for Candida auris, its preparation method, and its application. Background Technology

[0002] Candida auris belongs to the Candida clade of the genus Corynebacterium. It is primarily transmitted through contact, often spreading in hospital and nursing care settings. Infected individuals may not exhibit obvious symptoms, but when exposed to high-risk groups, Candida auris can cause severe invasive infections such as bloodstream infections, wound infections, endocarditis, and central nervous system infections, with a mortality rate as high as 30% to 60%.

[0003] Candida auris is a multidrug-resistant fungus with high mortality rates and a risk of nosocomial transmission. Traditional culture methods remain fundamental, but require specific conditions. Candida auris can grow on Kolmargarine chromogenic medium at 40-42°C, producing colonies of various colors (white, pink, red, or purple), but it is easily confused with other Candida species (such as Candida parapsilosis), requiring further identification using additional methods, which can easily lead to misdiagnosis. Traditional culture methods are time-consuming (usually 48-72 hours), and their sensitivity is affected by sample collection and contamination. Traditional biochemical tests (such as sugar assimilation tests) have limited accuracy and are prone to misinterpretation. For example, the color changes of Candida auris on Kolmargarine chromogenic medium overlap with those of other bacterial species, requiring confirmation using molecular or mass spectrometry techniques.

[0004] Current detection methods rely on real-time PCR, MALDI-TOF MS, WGS, and automated antimicrobial susceptibility testing systems. These methods are technically demanding, require specialized equipment, and are complex to operate, making them neither convenient nor time-consuming. In resource-limited areas, reliance on traditional culture and biochemical identification carries the risk of missed diagnoses. For example, county-level hospitals often experience lower testing accuracy due to equipment and technological limitations.

[0005] Chinese patent CN 115052968 A discloses a screening culture medium and screening method, which utilizes *Candida auris* to degrade different enzyme substrates, resulting in different colors. These enzyme substrates include raffinose, xylose, N-acetylglucosamine, potassium gluconate, glycerol, and D-glucosamine hydrochloride. This method, which uses specific lineages of *Candida auris* to degrade specific enzyme substrates and produce specific colors, is cumbersome and prone to missing undetected lineages. Furthermore, the aforementioned technique does not provide a method for clearly distinguishing *Candida auris* from other *Candida* species, and therefore cannot avoid interference from other *Candida* species.

[0006] Therefore, developing a low-cost, rapid, and highly specific selective detection and monitoring culture medium for Candida auris is of significant clinical importance. Summary of the Invention

[0007] This invention provides a rapid selective detection and monitoring culture medium for Candida auris, its preparation method, and its application. By optimizing the combination of selective inhibitors and chromogenic substrates, efficient isolation and identification of Candida auris can be achieved.

[0008] To achieve the objectives of this invention, in a first aspect, this invention provides a rapid and selective detection and monitoring culture medium for Candida auris, the components of which include:

[0009] Basic culture medium: peptone 10-20 g / L, yeast extract 5-10 g / L, glucose 10-20 g / L, agar 15-20 g / L;

[0010] Selective inhibitors: Chloramphenicol 0.5-1.0 g / L: inhibits bacterial growth; Ciclopirox 0.05-0.1 g / L: inhibits non-Candida auris fungi (such as Candida albicans and Candida glabrata);

[0011] Chromogenic substrate: 5-20 g / L of euonymus alcohol and 0.1-0.5 g / L of X-Gal; Candida auris can specifically degrade euonymus alcohol, releasing chromogenic groups. The chromogenic groups react with X-Gal, making the Candida auris colonies turn blue.

[0012] Auxiliary ingredients: Ferrous sulfate 0.1-0.5g / L: promotes the growth of Candida auris; Sodium chloride 5-10g / L: maintains osmotic pressure.

[0013] Secondly, the present invention provides a method for preparing a rapid selective detection and monitoring culture medium for Candida auris, comprising the following steps:

[0014] Weigh 10-20g peptone, 5-10g yeast extract, 10-20g glucose, 15-20g agar, and auxiliary components, dissolve them in 1000mL distilled water, heat until completely dissolved, adjust the pH to 6.8-7.2, sterilize at 121℃ for 20 minutes, cool to 50℃, add the filtered and sterilized selective inhibitor and chromogenic substrate, mix well, dispense into petri dishes, cool, and prepare the culture medium; the auxiliary components include 0.1-0.5g ferrous sulfate and 5-10g sodium chloride, the selective inhibitor includes 0.5-1.0g chloramphenicol and 0.05-0.1g ciclopirox olamine, and the chromogenic substrate includes 5-20g euonymus alcohol and 0.1-0.5g X-Gal.

[0015] This invention also provides the aforementioned rapid selective detection and monitoring culture medium for Candida auris (…). Candida auris Identification and cultivation of ).

[0016] Preferably, the culture temperature is 37℃.

[0017] Preferably, the incubation time is 48 hours.

[0018] Method of using the rapid selective detection and monitoring culture medium for Candida auris:

[0019] Candida auris and various bacteria and fungi were inoculated onto the surface of the above-mentioned culture medium and cultured at 37°C in an aerobic environment for 48 hours. The responsiveness of various strains, including Candida auris, to chromogenic substrates and their inhibitory activity against selective inhibitors were investigated.

[0020] The beneficial effects of this invention are:

[0021] The present invention provides a low-cost, rapid, and highly specific selective detection and monitoring culture medium for Candida auris, which has the following characteristics:

[0022] 1. High selectivity:

[0023] Chloramphenicol inhibits bacteria, while ciclopirox olamine inhibits non-Candida fungi. The two work synergistically to significantly reduce background interference.

[0024] 2. Rapid identification:

[0025] The chromogenic system of eugenol and X-Gal enables Candida auris to exhibit a characteristic blue color within 48 hours, and with optimized conditions, the characteristic blue color can be observed as early as 18 hours, without the need for further biochemical identification; specific auxiliary components can promote the growth of Candida auris and have a synergistic effect with the chromogenic substrate, promoting the binding of the chromogenic group to X-Gal.

[0026] 3. Low cost:

[0027] It has simple ingredients, requires no expensive equipment, and is suitable for primary laboratories. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 The results of detecting and monitoring the growth and color development of the strains on the culture medium in Example 7 are shown. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Unless otherwise specified in the embodiments, conventional conditions shall apply.

[0031] In Comparative Examples 1-7 and Examples 1-7 of this invention, all raw materials and reagents used can be purchased from the market.

[0032] A rapid selective detection and monitoring culture medium for Candida auris and its preparation method are as follows:

[0033] Peptone and yeast extract provide carbon and nitrogen sources, vitamins, and growth factors; glucose and eugenol are fermentable sugars; chloramphenicol and ciclopirox ol inhibit bacteria and non-Candida auris fungi; ferrous sulfate promotes the growth of Candida auris, and sodium chloride maintains balanced osmotic pressure; X-Gal reacts with eugenol degradation products to give Candida auris colonies a blue color; agar is the solidifying agent of the culture medium.

[0034] The present invention will be further illustrated below with reference to the embodiments, wherein embodiments 1 to 7 illustrate the implementation of a rapid selective detection and monitoring culture medium for Candida auris provided by the present invention. However, the present invention is not limited to the following embodiments.

[0035] The components and contents of the monitoring culture medium for rapid selective detection of Candida auris in Examples 1 to 7 are shown in Table 1.

[0036] Table 1. Composition of monitoring culture medium formulations for Examples 1 to 7

[0037]

[0038] The methods for preparing the monitoring culture media in Examples 1 to 7 are as follows:

[0039] (1) Weigh out the required amount of peptone, yeast extract, glucose, agar, ferrous sulfate and sodium chloride according to the formula composition of the culture medium, and dissolve them in 1000 mL of distilled water;

[0040] (2) Heat until completely dissolved, adjust pH to 7.2, and sterilize at 121°C for 20 minutes;

[0041] (3) When the temperature drops to 50°C, add the filtered and sterilized chloramphenicol, ciclopirox ol, eugenol and X-Gal, mix well, dispense into petri dishes, cool, and prepare the culture medium for use.

[0042] Comparative Example 1: The chromogenic culture medium formula in Chinese Patent CN 119061107 A was adopted, and the components included: peptone 1g / L, beef extract powder 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 0.05g / L, magnesium sulfate 1.5g / L, calcium chloride 2g / L, vancomycin hydrochloride 2mg / L, gentamicin 1g / L, chloramphenicol 1.0g / L, Tween 80 2g / L, 5-bromo-4-chloro-3-indole-N-acetylglucosamine 0.05g / L, 5-bromo-6-chloro-3-indole-phosphate 0.05g / L, 5-bromo-4-chloro-3-indole-octyl ester 0.05g / L, and agar powder 10g / L;

[0043] The culture medium was prepared according to the preparation method of the patent, wherein the treatment temperature was 100℃ and the treatment time was 10min.

[0044] Comparative Example 2: Peptone 10 g / L, yeast extract 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, amphotericin B 0.1 g / L, eurythromycin 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L;

[0045] The culture medium was prepared according to the preparation method of the present invention, except that amphotericin B was used instead of ciclopirox olamine in this comparative example.

[0046] Comparative Example 3: Peptone 10 g / L, yeast extract 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, actinomycin 0.1 g / L, eurythrin 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L;

[0047] The culture medium was prepared according to the preparation method of the present invention, except that in this comparative example, actinomycete ketone was used instead of ciclopirox amine.

[0048] Comparative Example 4: Peptone 10 g / L, yeast extract 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, nystatin 1000 U / L, euonymus alcohol 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L;

[0049] The culture medium was prepared according to the preparation method of the present invention, except that nystatin was used instead of ciclopirox olamine in the comparative example.

[0050] Comparative Example 5: Peptone 10 g / L, yeast extract 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, ciclopirox olamine 0.03 g / L, 5-bromo-4-chloro-3-indole-N-acetylglucosamine 0.05 g / L, 5-bromo-6-chloro-3-indole-phosphate 0.05 g / L, 5-bromo-4-chloro-3-indole-octyl ester 0.05 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L;

[0051] The culture medium was prepared according to the preparation method of the present invention, except that in this comparative example, 5-bromo-4-chloro-3-indole-N-acetylglucosamine, 5-bromo-6-chloro-3-indole-phosphate, and 5-bromo-4-chloro-3-indole-octyl ester were used instead of euonymus alcohol and X-Gal.

[0052] Comparative Example 6: Peptone 10 g / L, yeast extract 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, ciclopirox olamine 0.03 g / L, raffinose 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L;

[0053] The culture medium was prepared according to the preparation method of the present invention, except that raffinose was used instead of euonymus alcohol in this comparative example.

[0054] Comparative Example 7: Peptone 10 g / L, Beef extract powder 5 g / L, Glucose 15 g / L, Dipotassium hydrogen phosphate 3 g / L, Potassium dihydrogen phosphate 0.05 g / L, Magnesium sulfate 1.5 g / L, Calcium chloride 2 g / L, Chloramphenicol 0.5 g / L, Ciclopirox olamine 0.03 g / L, Euonymus 10 g / L, X-Gal 0.3 g / L, Agar 17 g / L.

[0055] The culture medium was prepared according to the preparation method of the present invention, except that in this comparative example, beef extract powder, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride were used instead of yeast extract powder, ferrous sulfate, and sodium chloride.

[0056] Methods for detecting strain growth and color development:

[0057] Under aseptic conditions, the working bacterial suspensions of each strain were diluted with 0.9% physiological saline to a suitable concentration (≤100 CFU / 0.1 mL). Each bacterial suspension was inoculated onto the culture plates of Comparative Examples 1 to 7 and the monitoring culture plates of Examples 1 to 7, and two parallel experiments were conducted. The inoculation was performed simultaneously using tryptic soy agar medium via pouring. The inoculated culture media were placed at 37°C and incubated for 48 hours. The colorimetric results were observed, and the recovery rate was analyzed using tryptic soy agar medium.

[0058] The experimental results of Comparative Examples 1 to 7 are shown in Table 2, and the experimental results of Examples 1 to 7 are shown in Table 3. The growth status and color development of each strain in Example 7 are shown in Table 3. Figure 1 As shown, the color development time of the culture media for Comparative Examples 1 to 7 was basically within 36-48 hours.

[0059] Table 2. Growth and color development of bacterial colonies on the culture media of Comparative Examples 1 to 7.

[0060]

[0061] Table 3. Growth and color development of each colony in the culture medium of Examples 1-7.

[0062]

[0063] As shown in Table 2, Comparative Examples 1 to 4 were compared with Example 4. The combination of vancomycin hydrochloride, gentamicin, and chloramphenicol inhibitors could not inhibit Candida. The combination of chloramphenicol and amphotericin B inhibitor, chloramphenicol and actinomycin B inhibitor, and chloramphenicol and nystatin inhibitor could not inhibit all Candida species, but showed a significant inhibitory effect on the growth of Candida auris. This indicates that the combination of chloramphenicol and ciclopirox olamine inhibitor in this invention can specifically inhibit bacteria and non-Candida auris fungi, while not affecting the growth of Candida auris, thereby enabling selective culture of Candida auris.

[0064] As shown in Table 2, comparing Comparative Examples 5-6 with Example 4, the mixed chromogenic agent components 5-bromo-4-chloro-3-indole-N-acetylglucosamine, 5-bromo-6-chloro-3-indole-phosphate, and 5-bromo-4-chloro-3-indole-octyl ester only showed color development for some Candida auris strains, and the color development was inconsistent. The mixed chromogenic agent components raffinose and X-Gal did not show unique color development for Candida auris, and did not react with X-Gal, resulting in white colonies. Therefore, if the content of the selective inhibitor in the culture medium is changed to that in Example 1 or Example 2, Candida auris will not be distinguishable from Candida. Thus, neither of the two mixed chromogenic agents can be used for the selective identification of all Candida auris strains, indicating that in this invention, Candida auris can specifically degrade euonymus and react with X-Gal to produce a blue color development, allowing for rapid screening of Candida auris. Other Candida species are completely inhibited or may grow white on the culture medium, such as Candida albicans and Candida glabrata. The Candida auris of the present invention shows blue colonies, which can be clearly distinguished from other Candida species.

[0065] As shown in Table 2, when Comparative Example 7 and Example 4 are combined, the color of Candida auris colonies varies, appearing gray or grayish-blue, when peptone, beef extract powder, magnesium sulfate, calcium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate are mixed. However, when yeast extract powder, ferrous sulfate, and sodium chloride are mixed in this invention, the Candida auris colonies are uniformly light blue. This indicates that the specific nutrients and auxiliary components selected in this invention can promote the growth of Candida auris and have a synergistic effect with the chromogenic substrate, promoting the binding of chromogenic groups to X-Gal.

[0066] As shown in Table 3, in Examples 1 to 3, by adjusting the amount of inhibitor added, the growth of other bacteria, such as Bacillus subtilis, Escherichia coli, and Staphylococcus aureus, as well as non-Candida fungi, such as Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Saccharomyces cerevisiae, and Aspergillus niger, can be inhibited. Even if the amount of antibacterial agent added cannot completely inhibit the growth of Candida albicans and Candida glabrata, the colonies are white and can still be distinguished from the blue color reaction of euonymus alcohol and X-Gal, which specifically degrades Candida auris, allowing for rapid screening of Candida auris. Therefore, the inhibitors chloramphenicol and ciclopirox olamine added in this invention can effectively inhibit the growth of bacteria and non-Candida auris fungi, further improving the selective inhibition of the culture medium, significantly reducing background interference, and achieving rapid selection and identification of Candida auris without further biochemical identification.

[0067] As shown in Table 3, in Examples 4 to 7, by adjusting the nutrient composition of the culture medium, the growth rate of *Candida auris* increased and the blue color of the colonies accelerated. Example 7 is the optimal example of this invention. Figure 1 As shown, all strains of Candida auris turned blue in the euonymus alcohol degradation colorimetric reaction, and formed distinct blue colonies within 18 hours at the fastest. The recovery rate of Candida auris (shown at the bottom of Table 3) increased from 70% to 96%. The added inhibitor did not inhibit the growth of Candida auris and did not interfere with the culture and identification of Candida auris. This indicates that the rapid selective detection and monitoring medium for Candida auris of the present invention has a good growth-promoting ability for Candida auris. The color of the colonies growing on the plate medium of the present invention can achieve the purpose of culturing and identifying Candida auris.

[0068] In summary, the present invention provides a rapid selective detection and monitoring culture medium for Candida auris. By adjusting the types of nutrients to promote the growth of Candida auris, adding chloramphenicol to inhibit bacterial growth, adding ciclopirox olamine to inhibit the growth of non-Candida auris fungi, and adding eugenol to react with X-Gal to make Candida auris colonies appear blue, thereby rapidly selecting and identifying Candida auris. This culture medium has high practicality and application promotion value.

[0069] This invention provides a rapid and selective detection and monitoring culture medium and culture method for Candida auris, which can easily and quickly screen for the presence of Candida auris.

[0070] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but only to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A rapid and selective detection and monitoring culture medium for Candida auris, characterized in that, It contains the following ingredients: peptone, yeast extract, glucose, agar, selective inhibitor, chromogenic substrate, and auxiliary ingredients; the chromogenic substrate consists of: euonymus alcohol and X-Gal; The selective inhibitors are: chloramphenicol and ciclopirox olamine; The auxiliary components are: ferrous sulfate and sodium chloride.

2. The rapid selective detection and monitoring culture medium for Candida auris according to claim 1, characterized in that, It contains the following ingredients: peptone 10-20 g / L, yeast extract 5-10 g / L, glucose 10-20 g / L, agar 15-20 g / L, chloramphenicol 0.5-1.0 g / L, ciclopirox ol 0.05-0.1 g / L, eurythromycin 5-20 g / L, X-Gal 0.1-0.5 g / L, ferrous sulfate 0.1-0.5 g / L, and sodium chloride 5-10 g / L.

3. A method for preparing a rapid selective detection and monitoring culture medium for Candida auris as described in any one of claims 1-2, characterized in that, The process includes the following steps: Weigh 10-20g peptone, 5-10g yeast extract, 10-20g glucose, 15-20g agar, and auxiliary components, dissolve them in 1000mL distilled water, heat until completely dissolved, adjust the pH to 6.8-7.2, sterilize at 121℃ for 20 minutes, cool to 50℃, add the filtered and sterilized selective inhibitor and chromogenic substrate, mix well, dispense into petri dishes, cool, and prepare the culture medium; the auxiliary components include 0.1-0.5g ferrous sulfate and 5-10g sodium chloride, the selective inhibitor includes 0.5-1.0g chloramphenicol and 0.05-0.1g ciclopirox olamine, and the chromogenic substrate includes 5-20g euonymus alcohol and 0.1-0.5g X-Gal.

4. The application of the rapid selective detection and monitoring culture medium for Candida auris as described in any one of claims 1-2 in the identification and culture of Candida auris for non-disease diagnostic purposes.

5. The application of the rapid selective detection and monitoring culture medium for Candida auris according to claim 4 in the identification and culture of Candida auris for non-disease diagnostic purposes, characterized in that, The culture temperature of the rapid selective detection monitoring medium for Candida auris is 37°C.

6. The application of the rapid selective detection and monitoring culture medium for Candida auris according to claim 4 in the identification and culture of Candida auris for non-disease diagnostic purposes, characterized in that, The culture time for the rapid selective detection monitoring medium for Candida auris was 48 hours.

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