Rapid selective detection and monitoring culture medium for candida auricula as well as preparation method and application of rapid selective detection and monitoring culture medium

Abstract

The invention discloses a rapid selective detection and monitoring culture medium for candida auricula as well as a preparation method and application of the rapid selective detection and monitoring culture medium, and belongs to the technical field of microbiological detection. The culture medium comprises the following components: peptone, yeast extract powder, glucose, agar, a selective inhibitor, a chromogenic substrate and auxiliary components, the chromogenic substrate comprises the following components: dulcitol and X-Gal, the culture medium disclosed by the invention is added with chloramphenicol and ciclopirox olamine to inhibit infectious microbes, and a chromogenic system of dulcitol and X-Gal realizes specific detection of candida auricula. The culture medium has the advantages of high selectivity, rapid identification and low cost, and is suitable for clinical sample and environment monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a Candida auris rapid and selective detection and monitoring culture medium, and a preparation method and application thereof. Background Art

[0002] Candida auris belongs to the Candida clade of the genus Corynespora. It is primarily transmitted through contact, often in hospital and healthcare settings. Infected individuals may not experience obvious symptoms, but when infected with high-risk individuals, C. auris can cause serious 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 and hospital-acquired transmission risks. Traditional culture methods remain the foundation, but require special conditions. Candida auris can grow on CHROMagar chromogenic medium at 40-42°C, with colonies exhibiting a variety of colors (white, pink, red, or purple). However, these colonies can be easily confused with other Candida species (such as Candida parapsilosis) and require further identification using other 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 misdiagnosis. For example, the color changes of Candida auris on CHROMagar chromogenic medium overlap with those of other strains, requiring confirmation using molecular or mass spectrometry techniques.

[0004] Current testing relies on real-time PCR, MALDI-TOF MS, WGS, and automated drug susceptibility testing systems. These methods are technically demanding, require analytical equipment, and are complex to operate. These methods are neither convenient nor time-consuming, and are expensive. In resource-limited areas, traditional culture and biochemical identification are still relied upon, which carries the risk of missed diagnoses. For example, due to equipment and technical limitations, testing accuracy in county-level hospitals is low.

[0005] Chinese patent CN 115052968 A discloses a screening culture medium and method, utilizing Candida auris to degrade different enzyme substrates to produce distinct colors. The enzyme substrates include raffinose, xylose, N-acetylglucosamine, potassium gluconate, glycerol, and D-glucosamine hydrochloride. This method, in which a specific lineage of Candida auris degrades a specific enzyme substrate to produce a specific color, is cumbersome and can miss detection of undiscovered lineages. Furthermore, the above-mentioned method does not provide a method for clearly distinguishing Candida auris from other Candida species, and cannot avoid interference from other Candida species.

[0006] Therefore, the development of a low-cost, rapid and specific culture medium for selective detection and monitoring of Candida auris is of great clinical significance. Summary of the Invention

[0007] The present invention provides a Candida auris rapid selective detection and monitoring culture medium, a preparation method and an application thereof, and realizes efficient separation and identification of Candida auris by optimizing the combination of a selective inhibitor and a chromogenic substrate.

[0008] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a rapid selective detection and monitoring culture medium for Candida auris, wherein the culture medium components include: Basic culture medium: peptone 10-20g / L, yeast extract powder 5-10g / L, glucose 10-20g / L, agar 15-20g / L; Selective inhibitors: Chloramphenicol 0.5-1.0g / L: inhibits bacterial growth; Ciclopirox 0.05-0.1g / L: inhibits fungi other than Candida auris (such as Candida albicans and Candida glabrata); Chromogenic substrate: dulcitol 5-20g / L, X-Gal 0.1-0.5g / L; C. auris can specifically degrade dulcitol, releasing a chromogenic group, which reacts with X-Gal, turning the C. auris colonies blue; Auxiliary ingredients: ferrous sulfate 0.1-0.5g / L: promotes the growth of Candida auris; sodium chloride 5-10g / L: maintains osmotic pressure.

[0009] In a second aspect, the present invention provides a method for preparing a culture medium for rapid and selective detection of Candida auris, comprising the following steps: 10-20 g of peptone, 5-10 g of yeast extract powder, 10-20 g of glucose, 15-20 g of agar, and auxiliary components are weighed and dissolved in 1000 mL of distilled water, heated until completely dissolved, the pH is adjusted to 6.8-7.2, sterilized at 121° C. for 20 minutes, cooled to 50° C., and then the filter-sterilized selective inhibitor and chromogenic substrate are added. The mixture is mixed, dispensed into culture dishes, and cooled to prepare the culture medium; the auxiliary components include 0.1-0.5 g of ferrous sulfate and 5-10 g of sodium chloride, the selective inhibitor includes 0.5-1.0 g of chloramphenicol and 0.05-0.1 g of ciclopirox olamine, and the chromogenic substrate includes 5-20 g of dulcitol and 0.1-0.5 g of X-Gal.

[0010] The present invention also provides the Candida auris rapid selective detection and monitoring culture medium for Candida auris ( Candida auris ) identification and cultivation.

[0011] Preferably, the culture temperature is 37°C.

[0012] Preferably, the culture time is 48 hours.

[0013] Method for using the Candida auris rapid selective detection and monitoring culture medium: Candida auris and various bacteria and fungi were inoculated onto the surface of the aforementioned culture medium and incubated aerobically at 37°C for 48 hours. The responses of various strains, including Candida auris, to chromogenic substrates and the inhibitory capacity of selected inhibitors were evaluated.

[0014] The beneficial effects of the present invention are: The present invention provides a low-cost, rapid and highly specific culture medium for selective detection and monitoring of Candida auris, which has the following characteristics: 1. High selectivity: Chloramphenicol inhibits bacteria, and ciclopirox inhibits fungi other than Candida auris. The two work synergistically to significantly reduce background interference.

[0015] 2. Rapid identification: The color development system of dulcitol and X-Gal causes Candida auris to display a characteristic blue color within 48 hours, and after conditions are optimized, the characteristic blue color can be displayed as quickly as 18 hours, without the need for further biochemical identification; the specific auxiliary component combination can promote the growth of Candida auris, and has a synergistic effect with the color development substrate, which can promote the binding of the color development group to X-Gal.

[0016] 3. Low cost: The ingredients are simple, no expensive equipment is required, and it is suitable for grassroots laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 This is the growth and color development results of the strain on the monitoring culture medium in Example 7. DETAILED DESCRIPTION

[0019] The technical solutions of the present invention are further described in detail below in conjunction with the examples. The specific examples described herein are only used to explain the present invention and are not used to limit the present invention. If no specific conditions are specified in the examples, conventional conditions shall be followed.

[0020] In Comparative Examples 1 to 7 and Examples 1 to 7 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0021] A rapid selective detection and monitoring culture medium for Candida auris and a preparation method thereof are as follows: Peptone and yeast extract provide carbon and nitrogen sources, vitamins, and growth factors; glucose and dulcitol are fermentable sugars; chloramphenicol and ciclopirox can inhibit bacteria and non-Candida auris fungi; ferrous sulfate promotes the growth of C. auris, and sodium chloride maintains a balanced osmotic pressure; X-Gal reacts with dulcitol degradation products to turn C. auris colonies blue; and agar is the coagulant of the culture medium.

[0022] The present invention is further described below with reference to the following examples: Examples 1 to 7 illustrate embodiments of the present invention to illustrate a rapid and selective detection and monitoring culture medium for Candida auris. However, the present invention is not limited to the following embodiments.

[0023] The components and content selections of the culture medium for rapid selective detection and monitoring of Candida media in Examples 1 to 7 are shown in Table 1.

[0024] Table 1 Monitoring culture medium formula composition of Examples 1 to 7

[0025] The preparation methods of the monitoring culture medium of Examples 1 to 7 are as follows: (1) Weigh the peptone, yeast extract powder, glucose, agar, ferrous sulfate, and sodium chloride required for the culture medium according to the formula and dissolve them in 1000 mL of distilled water; (2) Heat until completely dissolved, adjust pH to 7.2, and sterilize at 121°C for 20 minutes; (3) When the temperature drops to 50°C, add chloramphenicol, ciclopirox olamine, dulcitol and X-Gal that have been sterilized by filtration, mix well, dispense into culture dishes, and cool to prepare the culture medium for use.

[0026] Comparative Example 1: The colorimetric medium formulation of Chinese patent CN 119061107 A was used, comprising: 1 g / L peptone, 5 g / L beef extract powder, 20 g / L glucose, 3 g / L dipotassium hydrogen phosphate, 0.05 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 2 g / L calcium chloride, 2 mg / L vancomycin hydrochloride, 1 g / L gentamicin, 1.0 g / L chloramphenicol, 2 g / L Tween 80, 0.05 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, and 10 g / L agar powder. The culture medium was prepared according to the preparation method of the patent, wherein the treatment temperature was 100° C. and the treatment time was 10 minutes.

[0027] 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, dulcitol 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L; A culture medium was prepared according to the preparation method of an embodiment of the present invention, except that amphotericin B was used instead of ciclopirox olamine in this comparative example.

[0028] 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, cycloheximide 0.1 g / L, dulcitol 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L; A culture medium was prepared according to the preparation method of an embodiment of the present invention, except that cycloheximide was used instead of ciclopirox olamine in this comparative example.

[0029] 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, dulcitol 10 g / L, X-Gal 0.3 g / L, ferrous sulfate 0.3 g / L, sodium chloride 7 g / L; A culture medium was prepared according to the preparation method of an embodiment of the present invention, except that nystatin was used instead of ciclopirox olamine in this comparative example.

[0030] Comparative Example 5: peptone 10 g / L, yeast extract powder 5 g / L, glucose 15 g / L, agar 17 g / L, chloramphenicol 0.5 g / L, ciclopirox 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; The culture medium was prepared according to the preparation method of the embodiment of the present invention, except that 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 dulcitol and X-Gal in this comparative example.

[0031] 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; The culture medium was prepared according to the preparation method of the embodiment of the present invention, except that raffinose was used instead of dulcitol in this comparative example.

[0032] 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, dulcitol 10 g / L, X-Gal 0.3 g / L, agar 17 g / L.

[0033] The culture medium was prepared according to the preparation method of the embodiment of the present invention, except that 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 in this comparative example.

[0034] Detection method of strain growth status and color development: Under a sterile environment, the working bacterial solution of each strain was diluted with 0.9% physiological saline to an appropriate concentration of bacterial suspension (≤100 cfu / 0.1 mL), and each bacterial suspension was inoculated on a comparative example 1 to comparative example 7 culture medium plate and an example 1 to example 7 monitoring culture medium plate, and two parallel experiments were performed. The pouring method was used to synchronize the inoculation operation using tryptic soy agar medium. The inoculated culture medium was placed at 37 ° C and cultured for 48 hours. The color development results were observed, and the recovery rate was analyzed using tryptic soy agar medium.

[0035] 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, wherein the growth conditions 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 medium of Comparative Examples 1 to 7 is basically within 36-48 hours.

[0036] Table 2 Growth and color development of bacterial colonies in culture medium of Comparative Examples 1 to 7

[0037] Table 3 Growth and color development of each bacterial colony in the culture medium of Examples 1 to 7

[0038] As shown in Table 2, when comparing Comparative Examples 1 to 4 with Example 4, the combination of vancomycin hydrochloride, gentamicin, and chloramphenicol inhibitors cannot inhibit Candida, the combination of chloramphenicol and amphotericin B inhibitor, chloramphenicol and cycloheximide inhibitor, and the combination of chloramphenicol and nystatin inhibitor cannot inhibit all Candida, and there is a significant inhibition on the growth of Candida auris, indicating that the combination of chloramphenicol and ciclopirox olamine inhibitor in the present invention can specifically inhibit bacteria and non-Candida auris fungi without affecting the growth of Candida auris, thereby enabling selective cultivation of Candida auris.

[0039] As shown in Table 2, when comparing Comparative Examples 5 to 6 with Example 4, the mixed developer 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 had a color development phenomenon on some Candida auris strains, and the color development phenomenon was inconsistent; the mixed developer components raffinose and X-Gal did not show a unique color development phenomenon on Candida auris, and no color reaction occurred with X-Gal, all of which were white colonies. Therefore, if the content of the selective inhibitor in the culture medium is changed to the content in Example 1 or Example 2, it will cause Candida auris to be unable to be distinguished from Candida. Therefore, both mixed developers cannot be applied to the selective identification of all Candida auris, indicating that the Candida auris of the present invention can specifically degrade dulcitol and react with X-Gal to develop a blue color, which can quickly screen out Candida auris. Other Candida species are completely inhibited from growing on the culture medium or may grow in white, such as Candida albicans and Candida glabrata. The Candida auris of the present invention develops blue colonies and can be clearly distinguished from other Candida species.

[0040] As shown in Table 2, Comparative Example 7 is compared with Example 4. When peptone, beef extract powder, magnesium sulfate, calcium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate are compounded, the color of the Candida auris colony is different, gray or gray-blue, while when yeast extract powder, ferrous sulfate, and sodium chloride are compounded in the present invention, the Candida auris colony is uniformly light blue, indicating that the present invention selects specific nutrients and auxiliary ingredients to promote the growth of Candida auris, and has a synergistic effect with the chromogenic substrate, which can promote the binding of the chromogenic group to X-Gal.

[0041] As shown in Table 3, in Example 1 to Example 3, by adjusting the amount of inhibitor added, other bacteria such as Bacillus subtilis, Escherichia coli, Staphylococcus aureus, etc., and non-Auris Candida fungi such as Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Saccharomyces cerevisiae, Aspergillus niger, etc. can be inhibited. Even if the amount of antibacterial agent added cannot completely inhibit the growth of Candida albicans and Candida glabrata in Candida, the colonies are white and can still be distinguished from the blue color reaction of Candida auris-specific degradation of dulcitol and X-Gal, and Candida auris can be quickly screened out. Therefore, the inhibitors chloramphenicol and ciclopirox olamine added in the present invention can preferably inhibit the growth of bacteria and non-Auris Candida fungi, further improve the selective inhibition of the culture medium, significantly reduce background interference, and achieve rapid selection and identification of Candida auris without further biochemical identification.

[0042] As shown in Table 3, in Examples 4 to 7, by adjusting the nutrient components of the culture medium, the growth rate of Candida auris is increased and the degree of blueness of the colonies is accelerated. Example 7 is the best example of the present invention. Figure 1As shown, all strains of Candida auris showed blue in the chromogenic reaction of dalcidol degradation, and obvious blue colonies could be formed within 18 hours at the fastest. The recovery rate of all Candida auris (shown in the bottom line of Table 3) was increased from 70% to 96%. The added inhibitor had no inhibitory effect on the growth of Candida auris and did not interfere with the culture and identification of Candida auris. This shows that the Candida auris rapid selective detection and monitoring culture medium of the present invention has a good growth-promoting ability for Candida auris, and the purpose of culturing and identifying Candida auris can be achieved by the color of the colonies grown on the plate culture medium of the present invention.

[0043] In summary, the present invention provides a rapid and selective detection and monitoring culture medium for Candida auris, which promotes the growth of Candida auris by adjusting the types of nutrients, adds chloramphenicol to inhibit the growth of bacteria, and adds ciclopirox to inhibit the growth of non-Candida auris fungi. The addition of dulcitol to react with X-Gal makes Candida auris appear as blue colonies, thereby quickly selecting and identifying Candida auris. The culture medium has high practicality and application promotion value.

[0044] The present invention can obtain a Candida auris rapid and selective detection and monitoring culture medium and culture method, which can simply and quickly screen the presence of Candida auris.

[0045] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather represents only selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A rapid selective detection and monitoring culture medium for Candida auris, characterized in that: The invention comprises the following ingredients: peptone, yeast extract powder, glucose, agar, selective inhibitor, chromogenic substrate, and auxiliary ingredients; the chromogenic substrate ingredients are: dulcitol and X-Gal; The selective inhibitor ingredients 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 10-20g / L, yeast extract powder 5-10g / L, glucose 10-20g / L, agar 15-20g / L, chloramphenicol 0.5-1.0g / L, ciclopirox olamine 0.05-0.1g / L, dulcitol 5-20g / L, X-Gal 0.1-0.5g / L, ferrous sulfate 0.1-0.5g / L, and sodium chloride 5-10g / L.

3. A method for preparing a culture medium for rapid selective detection and monitoring of Candida auris according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: weighing 10-20 g of peptone, 5-10 g of yeast extract powder, 10-20 g of glucose, 15-20 g of agar, and auxiliary components, dissolving them in 1000 mL of distilled water, heating until completely dissolved, adjusting the pH to 6.8-7.2, sterilizing at 121° C. for 20 minutes, cooling to 50° C., adding a filter-sterilized selective inhibitor and a chromogenic substrate, mixing, dispensing into culture dishes, and cooling to prepare the culture medium; the auxiliary components comprise 0.1-0.5 g of ferrous sulfate and 5-10 g of sodium chloride, the selective inhibitor comprises 0.5-1.0 g of chloramphenicol and 0.05-0.1 g of ciclopirox olamine, and the chromogenic substrate comprises 5-20 g of dulcitol and 0.1-0.5 g of X-Gal.

4. Use of the Candida auris rapid selective detection and monitoring culture medium according to any one of claims 1 to 2 in the identification and cultivation of Candida auris.

5. Use of the Candida auris rapid selective detection and monitoring culture medium according to claim 4 in the identification and cultivation of Candida auris, characterized in that: The culture temperature of the Candida auris rapid selective detection monitoring culture medium is 37°C.

6. Use of the Candida auris rapid selective detection and monitoring culture medium according to claim 4 in the identification and cultivation of Candida auris, characterized in that: The culture time of the Candida auris rapid selective detection and monitoring culture medium is 48 hours.

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