Propionibacterium acnes selective culture medium and preparation method thereof

Abstract

The invention relates to a propionibacterium acnes selective culture medium and a preparation method thereof, the culture medium comprises casein pancreatin digest, meat and stomach enzyme digest, heart and pancreatin digest, yeast extract powder, corn starch, sodium chloride, agar and defiberized sheep blood, and the volume is fixed with distilled water. Compared with an existing culture medium, the culture medium has the advantages of being reliable in detection and high in propionibacterium acnes separation rate and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a selective culture medium for Propionibacterium acnes and its preparation method. Background Technology

[0002] Acne is a chronic inflammatory disease of the pilosebaceous unit characterized by comedones, papules, and pustules, with a high incidence and a tendency to recur. Besides being related to androgen levels, increased sebum secretion, and abnormal keratinization of cells around the hair follicles, the inflammatory response caused by the excessive proliferation of Propionibacterium acnes is also a major cause of acne. Therefore, topical or systemic application of antibacterial drugs is one of the treatment methods for moderate to severe acne. In addition, Propionibacterium acnes can also cause serious infections such as endocarditis, arthritis, and meningitis, and has also been reported to be closely related to the pathogenesis of atopic dermatitis.

[0003] Propionibacterium acnes is an obligate anaerobic bacterium that primarily resides within the sebaceous glands of the skin, while normal flora also colonize the sebaceous glands and the skin surface. Currently, clinical microbiology laboratories primarily rely on antibiotic-free Columbia blood agar plates for the isolation and culture of Propionibacterium acnes, or on Columbia blood agar medium supplemented with colistin and metronidazole. Commercially available culture media, such as Ruichu Bio's Propionibacterium acnes solid medium (antibiotic-free), are also used. However, these conventional culture methods generally suffer from low detection rates, possibly due to competitive inhibition by dominant skin flora or insufficient nutrient conditions in the culture medium to adequately support the growth of this bacterium.

[0004] CN120442747A discloses a specific culture medium for Propionibacterium acnes, but its application is limited to cosmetic testing. According to the "Cosmetic Safety Technical Specifications" (2015 edition), China stipulates that microbial testing of cosmetics includes five indicators: total bacterial count, thermotolerant coliforms, Staphylococcus aureus, Pseudomonas aeruginosa, molds, and yeasts. The limit for total bacterial count in eye, lip, and children's cosmetics is ≤500 CFU / g(ml), and for other products it is ≤1000 CFU / g(ml). Pathogenic bacteria must not be detected. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a selective culture medium for Propionibacterium acnes and its preparation method. Compared with existing culture media, it has the characteristics of reliable detection and high isolation rate of Propionibacterium acnes, and has good application prospects.

[0006] The present invention also provides a selective culture medium for Propionibacterium acnes, the culture medium comprising casein trypsin digest, meat stomach enzyme digest, cardiac pancreatic enzyme digest, yeast extract, corn starch, sodium chloride, agar and defibrinated sheep blood, and diluted with distilled water.

[0007] Preferably, the concentration of the casein pancreatic enzyme digest is 10 g / L, the meat stomach enzyme digest is 5 g / L, the cardiac pancreatic enzyme digest is 3 g / L, the yeast extract is 10 g / L, the corn starch is 1 g / L, the sodium chloride is 5 g / L, the agar is 15 g / L, and the defibrinated sheep blood is 50 mL / L.

[0008] Furthermore, the culture medium also includes 10 mg / L of gentamicin sulfate.

[0009] Furthermore, the culture medium also includes Tween 80 1 mL / L.

[0010] This invention also provides a method for preparing a selective culture medium for Propionibacterium acnes, comprising the following steps:

[0011] (1) Take casein pancreatic enzyme digest, meat stomach enzyme digest, cardiac pancreatic enzyme digest, yeast extract powder, corn starch, sodium chloride and agar, mix them, distilled water to make up the volume, add Tween 80, adjust the pH value to 7.2-7.3, and autoclave at 121℃ for 45 min.

[0012] (2) Cool down to 45-50℃, add defibrinated sheep blood and gentamicin sulfate, mix well, pour into sterile petri dishes, and set aside.

[0013] In this invention, casein pancreatic enzyme digests, meat gastric enzyme digests, cardiac pancreatic enzyme digests, yeast extract, and corn starch provide carbon and nitrogen sources, vitamins, and growth factors; sheep blood is an excellent nutrient for bacterial growth and reproduction; adding blood to the basal culture medium at 45–50°C preserves certain heat-sensitive growth factors in the blood without destroying blood cells. Tween 80 can stimulate the growth of Propionibacterium acnes. Appropriate concentrations of gentamicin sulfate can inhibit the growth of Gram-negative bacteria such as Staphylococcus, Streptococcus, and Enterobacteriaceae, but will not affect the growth of Propionibacterium acnes.

[0014] Beneficial effects

[0015] Compared with existing culture media, this invention has the advantages of reliable detection and high isolation rate of Propionibacterium acnes, and has good application prospects. Attached Figure Description

[0016] Figure 1 Original plates after acne specimens were inoculated with the experimental group (a), control group 1 (b), and control group 2 (c) culture media from the same acne patient. Detailed Implementation

[0017] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0018] Example 1

[0019] 1. Objects and methods.

[0020] 1.1 Culture medium preparation:

[0021] Experimental group: The culture medium consisted of 10g casein pancreatic enzyme digest, 5g meat stomach enzyme digest, 3g cardiac pancreatic enzyme digest, 10g yeast extract, 1g corn starch, 5g sodium chloride, 15g agar, 50mL defibrinated sheep blood, 10mg gentamicin sulfate, 1mL Tween 80, and distilled water to 1000mL.

[0022] The preparation method includes the following steps:

[0023] (1) Take casein pancreatic enzyme digest, meat stomach enzyme digest, cardiac pancreatic enzyme digest, yeast extract powder, corn starch, sodium chloride and agar, mix them together, distilled water to make up the volume, add Tween 80, adjust the pH value to 7.2-7.3, and autoclave at 121℃ for 45 min.

[0024] (2) Cool down to 45-50℃, add defibrinated sheep blood and gentamicin sulfate, mix well, pour into sterile petri dishes, and set aside.

[0025] Control group 1: Columbia blood agar medium from Wenzhou Kangtai Biotechnology Co., Ltd.

[0026] Control group 2: Propionibacterium acnes solid culture medium from Ruichu Biotechnology, containing per liter: 10g tryptone, 10g beef extract, 5g glucose, 3g yeast extract, 5g sodium chloride, 3g sodium acetate, 1g soluble starch, 0.5g L-cysteine ​​hydrochloride, 15g agar, pH 6.6-7.0. Prepared according to instructions, poured into plates, allowed to solidify, and ready for use.

[0027] 1.2 Acne Specimen Processing and Culture: For outpatients or inpatients with acne, the facial lesions were disinfected with 75% ethanol. The contents of the acne were then extracted using a sterile needle. A sterile swab was moistened with sterile saline and used to collect the acne contents, which were immediately sent for testing. Specimens were inoculated onto three groups of plates (experimental group and control group), streaked in three areas, and then incubated in an anaerobic environment for 48 hours at 37°C. When colonies grew on the plates, 4-5 round, raised, smooth-edged, grayish-white to milky-white colonies (1-2 mm in size) were picked and directly smeared for Gram staining. If the colonies were Gram-positive bacilli, slightly curved, rod-shaped, without spores or capsules, they were re-inoculated onto routine blood agar plates and incubated in both aerobic and anaerobic environments for 48 hours. Those with negative anaerobic test results were identified using MALDI-TOFMS.

[0028] 1.3 Statistical analysis: SPSS 17.0 was used for statistical analysis. P < 0.05 was considered statistically significant.

[0029] 2. Results:

[0030] Of the 100 inoculated specimens, 95 were identified as *Propionibacterium acnes* by bacterial culture in the experimental group (positive rate 65%, 95% CI 55.5%-74.5%). Control group 1 had 65 cases (positive rate 65%), and control group 2 had 87 cases (positive rate 87%, 95% CI 80.3%-93.7%). Statistical analysis showed that the positive rates in both the experimental and control groups were significantly higher than those in the control groups (P<0.05).

[0031] Figure 1 The results show that after inoculating acne specimens, the culture medium of this invention has significantly fewer contaminating bacteria and exhibits better colony growth. Control group 1, Columbia blood agar medium, although a classic anaerobic culture medium, lacks selectivity, allowing contaminating bacteria to become the dominant species. Control group 2, Propionibacterium acnes solid culture medium, is easy to prepare but has poor nutritional value, resulting in smaller colonies. However, because no antibiotics are added, it cannot inhibit the growth of colonizing bacteria on the skin surface. Therefore, the culture medium of this invention can improve the detection rate of Propionibacterium acnes, providing assistance for clinical diagnosis and treatment and scientific research.

Claims

1. A selective culture medium for Propionibacterium acnes, characterized in that, The culture medium comprises casein pancreatic digest, meat pancreatic digest, cardiac pancreatic digest, yeast extract, corn starch, sodium chloride, agar, and defibrinated sheep blood, and is brought to a final volume with distilled water.

2. The selective culture medium for Propionibacterium acnes according to claim 1, characterized in that, The concentrations of the casein pancreatic enzyme digests were 10 g / L, the meat pancreatic enzyme digests were 5 g / L, the cardiac pancreatic enzyme digests were 3 g / L, the yeast extract was 10 g / L, the corn starch was 1 g / L, the sodium chloride was 5 g / L, the agar was 15 g / L, and the defibrinated sheep blood was 50 mL / L.

3. The selective culture medium for Propionibacterium acnes according to claim 1, characterized in that, The culture medium also includes 10 mg / L of gentamicin sulfate.

4. The selective culture medium for Propionibacterium acnes according to claim 1, characterized in that, The culture medium also includes Tween 80 1 mL / L.

5. A method for preparing a selective culture medium for Propionibacterium acnes as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Take casein pancreatic enzyme digest, meat stomach enzyme digest, cardiac pancreatic enzyme digest, yeast extract powder, corn starch, sodium chloride and agar, mix them, distilled water to make up the volume, add Tween 80, adjust the pH value to 7.2-7.3, and autoclave at 121℃ for 45 min. (2) Cool down to 45-50℃, add defibrinated sheep blood and gentamicin sulfate, mix well, pour into a sterile petri dish, and set aside.

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