阅读说明：本技术 耐碳青霉烯克雷伯菌显色培养基 (Carbapenem-resistant Klebsiella chromogenic medium ) 是由 卫丽 宗志勇 刘丽娜 乔甫 林�吉 于 2020-08-20 设计创作，主要内容包括：本发明公开了一种耐碳青霉烯克雷伯菌显色培养基,属于疾病微生物培养与鉴定领域。本发明的培养基包括氯化钠4～6重量份、硫酸镁0.1～0.3重量份、磷酸二氢铵0.5～1.5重量份、磷酸氢二钾0.5～1.5重量份、柠檬酸钠4～6重量份、琼脂13～17重量份、溴麝香草酚蓝0.06～1重量份、肌醇9～11重量份、美罗培南0.001～0.003重量份、水900～1100重量份。本发明的培养基特异性好,对耐碳青霉烯克雷伯菌培养快速,有利于医院内耐碳青霉烯克雷伯菌感染防控。(The invention discloses a carbapenem-resistant Klebsiella chromogenic medium, and belongs to the field of disease microorganism culture and identification. The culture medium comprises 4-6 parts by weight of sodium chloride, 0.1-0.3 part by weight of magnesium sulfate, 0.5-1.5 parts by weight of ammonium dihydrogen phosphate, 0.5-1.5 parts by weight of dipotassium hydrogen phosphate, 4-6 parts by weight of sodium citrate, 13-17 parts by weight of agar, 0.06-1 part by weight of bromothymol blue, 9-11 parts by weight of inositol, 0.001-0.003 part by weight of meropenem and 900-1100 parts by weight of water. The culture medium has good specificity, can quickly culture the carbapenem-resistant Klebsiella, and is favorable for preventing and controlling the carbapenem-resistant Klebsiella infection in hospitals.)

1. A carbapenem-resistant Klebsiella chromogenic medium is characterized in that: the preservative comprises 4-6 parts by weight of sodium chloride, 0.1-0.3 part by weight of magnesium sulfate, 0.5-1.5 parts by weight of ammonium dihydrogen phosphate, 0.5-1.5 parts by weight of dipotassium hydrogen phosphate, 4-6 parts by weight of sodium citrate, 13-17 parts by weight of agar, 0.06-1 part by weight of bromothymol blue, 9-11 parts by weight of inositol, 0.001-0.003 part by weight of meropenem and 900-1100 parts by weight of water.

2. The culture medium of claim 1, wherein: comprises 5 weight portions of sodium chloride, 0.2 weight portion of magnesium sulfate, 1 weight portion of ammonium dihydrogen phosphate, 1 weight portion of dipotassium hydrogen phosphate, 5 weight portions of sodium citrate, 15 weight portions of agar, 0.08 weight portion of bromothymol blue, 10 weight portions of inositol, 0.002 weight portion of meropenem and 1000 weight portions of water.

3. Use of the medium according to claim 1 or 2 for the detection of carbapenem-resistant klebsiella spp.

4. A kit for detecting carbapenem-resistant Klebsiella, which is characterized in that: the kit comprises a culture medium in the following proportion:

4-6 parts of sodium chloride, 0.1-0.3 part of magnesium sulfate, 0.5-1.5 parts of ammonium dihydrogen phosphate, 0.5-1.5 parts of dipotassium hydrogen phosphate, 4-6 parts of sodium citrate, 13-17 parts of agar, 0.06-1 part of bromothymol blue, 9-11 parts of inositol, 0.001-0.003 part of meropenem and 900-1100 parts of water.

5. The test kit of claim 4, wherein: the kit comprises a culture medium in the following proportion:

5 parts of sodium chloride, 0.2 part of magnesium sulfate, 1 part of ammonium dihydrogen phosphate, 1 part of dipotassium hydrogen phosphate, 5 parts of sodium citrate, 15 parts of agar, 0.08 part of bromothymol blue, 10 parts of inositol, 0.002 part of meropenem and 1000 parts of water.

6. A method for detecting carbapenem-resistant Klebsiella, which is characterized by comprising the following steps: the sample of non-human or animal origin is placed in the culture medium of claim 1 or 2 to be cultured for 18-24 h, and the carbapenem-resistant Klebsiella can be judged by observing yolk color, round shape, convex and moist bacterial colony.

7. A preparation method of a carbapenem-resistant Klebsiella chromogenic medium is characterized by comprising the following steps: the method comprises the following steps:

1) dissolving 4-6 parts by weight of sodium chloride, 0.1-0.3 part by weight of magnesium sulfate, 0.5-1.5 parts by weight of ammonium dihydrogen phosphate, 0.5-1.5 parts by weight of dipotassium hydrogen phosphate, 4-6 parts by weight of sodium citrate, 13-17 parts by weight of agar and 0.06-1 part by weight of bromothymol blue in 700-900 parts by weight of water, sterilizing at 121 ℃ under high pressure, cooling to 50-55 ℃, and preserving heat for later use;

2) dissolving 9-11 parts by weight of inositol in 100-300 parts by weight of water, filtering with a filter membrane for sterilization, and adding the mixture obtained in the step 1);

3) adding 0.001-0.003 parts by weight of meropenem into the mixture obtained in the step (2);

4) cooling and solidifying the mixture obtained in the step 3) in a culture dish.

8. The method of claim 7, wherein:

the step 1) is as follows: dissolving 5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 1 part by weight of ammonium dihydrogen phosphate, 1 part by weight of dipotassium hydrogen phosphate, 5 parts by weight of sodium citrate, 15 parts by weight of agar, 0.08 part by weight of bromothymol blue, 10 parts by weight of inositol and 0.002 part by weight of meropenem in 900 parts by weight of water, sterilizing at 121 ℃ under high pressure, cooling to 50-55 ℃, and preserving heat for later use.

9. The method of claim 7 or 8, wherein:

the step 2) is as follows:

dissolving 10 parts by weight of inositol in 100 parts by weight of water, filtering and sterilizing by using a filter membrane, and adding the mixture obtained in the step 1).

10. The method of claim 7 or 8, wherein:

the step 3) is as follows:

0.002 part by weight of meropenem was added to the mixture obtained in step (2).

Technical Field

The invention belongs to the field of disease microorganism culture and identification.

Background

Klebsiella, belonging to the family Enterobacteriaceae, is a gram-negative Brevibacterium crassimum. Single or short chain, no movement, obvious capsule. The Klebsiella has strong resistance to the outside world and is susceptible to drug resistance to most antibiotics.

Over the past 10 years, carbapenem-resistant drugs have been considered as the last line of defense in the treatment of drug-resistant gram-negative bacteria. With the rapid increase of the detection rate of carbapenem drug-resistant strains, particularly Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, the method becomes a difficult problem of clinical anti-infection treatment at present. According to recent bacterial resistance monitoring of CHINET, the first three isolates of 8274 carbapenem-resistant enterobacteriaceae bacteria were klebsiella pneumoniae (68.6%), escherichia coli (10.8%) and enterobacter cloacae (6.9%) in this order. The drug resistance rates of Klebsiella pneumoniae on imipenem and meropenem are respectively increased from 3.0% and 2.9% in 2005 to 25.0% and 26.3% in 2018, and the increase range is more than 8 times.

Therefore, the method has important significance for continuously doing the monitoring work of bacterial drug resistance, identifying the carbapenem-resistant Klebsiella bacteria in early stage, particularly the carbapenem-resistant Klebsiella pneumoniae, strengthening the infection prevention and control measures in hospitals and reducing the nosocomial spread of drug-resistant strains.

At present, the microorganism laboratory of clinical laboratory mostly adopts an automatic instrument method to identify the bacteria of the genus Klebsiella and carry out drug sensitivity test, and the general report time is more than 72 h. The selective chromogenic culture method is an ideal method for rapidly screening target bacteria, and generally only takes 18-24 hours. However, the existing chromogenic culture methods on the market do not specially screen for carbapenem-resistant Klebsiella bacteria, and the closest chromogenic culture medium is urethra flora-localized chromogenic culture medium (CHROMagar, France). The suspicious colonies are metallic blue or grey blue, and comprise bacteria of Klebsiella, Enterobacter, Citrobacter and Serratia, and the carbapenem-resistant Klebsiella cannot be effectively screened through the colony color.

Therefore, in order to cope with the rapidly growing carbapenem-resistant Klebsiella, especially carbapenem-resistant Klebsiella pneumoniae, a rapid screening chromogenic medium is urgently needed.

Disclosure of Invention

The invention aims to solve the problems that: provides a carbapenem-resistant Klebsiella pneumoniae chromogenic medium.

The technical scheme of the invention is as follows:

a carbapenem-resistant Klebsiella coloration culture medium comprises, by weight, 4-6 parts of sodium chloride, 0.1-0.3 part of magnesium sulfate, 0.5-1.5 parts of ammonium dihydrogen phosphate, 0.5-1.5 parts of dipotassium hydrogen phosphate, 4-6 parts of sodium citrate, 13-17 parts of agar, 0.06-1 part of bromothymol blue, 9-11 parts of inositol, 0.001-0.003 part of meropenem, and 900-1100 parts of water.

The culture medium comprises 5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 1 part by weight of ammonium dihydrogen phosphate, 1 part by weight of dipotassium hydrogen phosphate, 5 parts by weight of sodium citrate, 15 parts by weight of agar, 0.08 part by weight of bromothymol blue, 10 parts by weight of inositol, 0.002 part by weight of meropenem and 1000 parts by weight of water.

Use of the aforementioned culture medium for the detection of carbapenem-resistant Klebsiella for non-disease diagnostic purposes.

A kit for detecting carbapenem-resistant Klebsiella, which comprises a culture medium with the following mixture ratio:

4-6 parts of sodium chloride, 0.1-0.3 part of magnesium sulfate, 0.5-1.5 parts of ammonium dihydrogen phosphate, 0.5-1.5 parts of dipotassium hydrogen phosphate, 4-6 parts of sodium citrate, 13-17 parts of agar, 0.06-1 part of bromothymol blue, 9-11 parts of inositol, 0.001-0.003 part of meropenem and 900-1100 parts of water.

The detection kit comprises the following culture media in proportion:

5 parts of sodium chloride, 0.2 part of magnesium sulfate, 1 part of ammonium dihydrogen phosphate, 1 part of dipotassium hydrogen phosphate, 5 parts of sodium citrate, 15 parts of agar, 0.08 part of bromothymol blue, 10 parts of inositol, 0.002 part of meropenem and 1000 parts of water.

A method for detecting carbapenem-resistant Klebsiella comprises the steps of placing a sample from a non-human body or an animal body in the culture medium for culturing for 18-24 hours, and observing a bacterial colony which is yolk-colored, round, convex and moist, so that the carbapenem-resistant Klebsiella can be judged.

A preparation method of a carbapenem-resistant Klebsiella chromogenic medium comprises the following steps:

1) dissolving 4-6 parts by weight of sodium chloride, 0.1-0.3 part by weight of magnesium sulfate, 0.5-1.5 parts by weight of ammonium dihydrogen phosphate, 0.5-1.5 parts by weight of dipotassium hydrogen phosphate, 4-6 parts by weight of sodium citrate, 13-17 parts by weight of agar and 0.06-1 part by weight of bromothymol blue in 700-900 parts by weight of water, sterilizing at 121 ℃ under high pressure, cooling to 50-55 ℃, and preserving heat for later use;

2) dissolving 9-11 parts by weight of inositol in 100-300 parts by weight of water, filtering with a filter membrane for sterilization, and adding the mixture obtained in the step 1);

3) adding 0.001-0.003 parts by weight of meropenem into the mixture obtained in the step (2);

4) cooling and solidifying the mixture obtained in the step 3) and a culture dish.

The preparation method comprises the following steps of 1): dissolving 5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 1 part by weight of ammonium dihydrogen phosphate, 1 part by weight of dipotassium hydrogen phosphate, 5 parts by weight of sodium citrate, 15 parts by weight of agar, 0.08 part by weight of bromothymol blue, 10 parts by weight of inositol and 0.002 part by weight of meropenem in 900 parts by weight of water, sterilizing at 121 ℃ under high pressure, cooling to 50-55 ℃, and preserving heat for later use.

The preparation method comprises the following steps of 2):

dissolving 10 parts by weight of inositol in 100 parts by weight of water, filtering and sterilizing by using a filter membrane, and adding the mixture obtained in the step 1).

The preparation method comprises the following steps of 3):

0.002 part by weight of meropenem was added to the mixture obtained in step (2).

The culture medium can be used for rapidly and accurately detecting the carbapenem-resistant Klebsiella, the false positive rate is close to 0 in the detection of thousands of samples in hospitals, and the early identification of the carbapenem-resistant Klebsiella is facilitated.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: appearance of carbapenem-resistant Klebsiella on the media of the invention.

Detailed Description