阅读说明：本技术 一种耐碳青霉烯类抗生素肠杆菌科细菌的筛查显色平板 (Screening and developing plate for carbapenem-resistant antibiotics enterobacteriaceae bacteria ) 是由 章晓庆 娄斌 于 2021-01-27 设计创作，主要内容包括：本发明属于细菌检测技术领域,具体涉及一种耐碳青霉烯类抗生素肠杆菌科细菌的筛查显色平板。所述显色平板包含特异性显色底物和选择性培养基；其中,所述选择性培养基,包括以下组分：酵母提取物、水杨苷、阿拉伯胶、柠檬酸钠、叶酸、维生素B1、维生素E。该显色平板可用于临床耐碳青霉烯类抗生素耐药菌株的快速筛查,且准确率较高。(The invention belongs to the technical field of bacteria detection, and particularly relates to a screening and developing plate for carbapenem-resistant antibiotics enterobacteriaceae bacteria. The color development plate comprises a specific color development substrate and a selective culture medium; wherein the selective medium comprises the following components: yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E. The color development plate can be used for rapid screening of clinical carbapenem antibiotic resistant strains, and has high accuracy.)

1. A screening chromogenic plate for carbapenem-resistant antibiotics enterobacteriaceae bacteria, which comprises a specific chromogenic substrate and a selective culture medium; wherein the selective medium comprises the following components: yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E.

2. The carbapenem-resistant Enterobacteriaceae bacteria screening and developing plate of claim 1, wherein the selective medium comprises the following components in parts by weight: 5-10 parts of yeast extract, 0.1-2 parts of salicin, 0.5-2 parts of Arabic gum, 1-5 parts of sodium citrate, 0.01-0.05 part of folic acid, 10.01-0.05 part of vitamin B and 0.01-0.05 part of vitamin E.

3. The carbapenem-resistant Enterobacteriaceae bacterium screening color development plate of claim 2, wherein the yeast extract is prepared by a method comprising the steps of:

(1) taking yeast emulsion, carrying out autolysis at constant temperature, and carrying out enzymolysis; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

4. The carbapenem-resistant Enterobacteriaceae bacteria screening color development plate of claim 3, wherein the temperature of the isothermal autolysis is 40-60 ℃; preferably 45 ℃;

the enzyme for enzymolysis is beta-xylosidase, papain and glucanase; the mass ratio of the beta-xylosidase to the papain to the glucanase is 1: 2-3: 1-2; the total mass of the beta-xylosidase, the papain and the glucanase is 0.05-0.1g/L of yeast emulsion; the enzyme activities of the beta-xylosidase, the papain and the glucanase are respectively 5000-20000U/g; the temperature of the enzymolysis is 30-45 ℃, and preferably 35 ℃; the pH value of the enzymolysis is 5-7, and preferably 6.5.

5. The carbapenem-resistant Enterobacteriaceae bacterium screening color development plate according to claim 3,

the autolysis process also comprises adding an autolysis promoter, wherein the promoter is a mixture of citric acid and potassium chloride;

the dosage of the citric acid is 0.05-0.1g/L of the yeast emulsion;

the dosage of the potassium chloride is 0.1-0.2g/L of the yeast emulsion.

6. The carbapenem-resistant Enterobacteriaceae bacterium screening color development plate according to claim 1,

the specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 0.01-5.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 0.01-5.00 g/L.

7. The carbapenem-resistant Enterobacteriaceae bacterium screening color development plate according to claim 1,

the selective culture medium is a culture medium containing carbapenem antibiotics;

the carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of the imipenem is 0.1-50 mg/L; the effective concentration of the meropenem is 0.1-50 mg/L; the effective concentration of ertapenem is 0.1-30 mg/L.

8. The carbapenem-resistant Enterobacteriaceae bacteria screening color development plate of claim 1, wherein the selective medium further comprises the following components: peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water.

9. The carbapenem-resistant Enterobacteriaceae bacterium screening color development plate of claim 8,

the selective culture medium comprises the following components in parts by weight:

5-15 parts of peptone, 5-10 parts of sodium chloride, 1-5 parts of lactose, 5-10 parts of beef extract powder, 10-15 parts of agar powder and 1500 parts of purified water; 5-10 parts of yeast extract, 0.1-2 parts of salicin, 0.5-2 parts of Arabic gum, 1-5 parts of sodium citrate, 0.01-0.05 part of folic acid, 10.01-0.05 part of vitamin B and 0.01-0.05 part of vitamin E.

10. The method for preparing a screening chromogenic plate for bacteria of the family Enterobacteriaceae resistant to carbapenems as claimed in any of claims 8 to 9, comprising the steps of:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E into a chromogenic substrate, and adding dimethyl formamide or dimethyl sulfoxide; preparing a culture medium,

(2) autoclaving the culture medium;

(3) adding purified water into carbapenem antibiotics, dissolving, and filtering to remove carbapenem antibiotic bacteria to obtain an antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing, and packaging.

Technical Field

The invention belongs to the technical field of bacteria detection, and particularly relates to a screening and developing plate for carbapenem-resistant antibiotics enterobacteriaceae bacteria.

Background

The bacteria belonging to the family Enterobacteriaceae belong to gram-negative bacilli, including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, etc., which are often present in the upper respiratory tract and intestinal tract of the human body and cause infection of various organs and tissues of the whole body when the body resistance is lowered.

Imipenem and meropenem are commonly used carbapenem antibiotics in clinic, have become one of the most important antibacterial drugs for clinically treating serious bacterial infection due to the characteristics of wide antibacterial spectrum, strong antibacterial activity, stability to beta-lactamase, low toxicity and the like, and have been considered as stable beta-lactam antibacterial drugs against ESBLs enzyme and AmpC enzyme, but with the increase of the clinical use of carbapenem antibiotics, the carbapenem antibiotic resistant bacteria are continuously increased. The appearance and rapid propagation of carbapenemase lead to the drug resistance of bacteria to carbapenem antibiotics, and almost break through the last line of defense of clinical anti-infective therapy. Particularly, the coliform bacteria which are ubiquitous in human life are found to carry a drug resistance gene blaNDM-1, and reports indicate that the blaNDM-1 gene and other beta-lactamase genes are frequently coexisted on a transferable genetic element of bacteria, so that multiple drug resistance is widely spread, and bacteria carrying the gene become global public health threats.

There are many methods for detecting coliform bacteria, and conventional bacteriological culture methods, immunological methods, nucleic acid probe techniques, and the like are mainly relied on. The bacteriological culture method needs to be subjected to enrichment culture, selective separation, morphological characteristic observation, physiological and biochemical reaction, serological identification and other processes, generally needs 4 to 7 days, is complex to operate, time-consuming and labor-consuming, has low sensitivity and poor specificity, and cannot detect damaged bacteria and dead bacteria; the immunological method is easy to pollute, and has more serious cross reaction, more false positive and lower sensitivity; the biggest advantage of the nucleic acid probe detection technology is strong specificity, but the probe detection technology has certain problems, such as low sensitivity; a probe is prepared for detecting one bacterium. The paper sheet method is also a commonly used method for rapidly detecting coliform groups; but is easily affected by the properties of the product to be detected, resulting in a decrease in accuracy.

Therefore, the development of a fast and accurate chromogenic plate of Carbapenem-antibiotic-resistant Enterobacteriaceae (CRE) for fast screening of clinical Carbapenem-antibiotic-resistant strains plays an important role.

Disclosure of Invention

In order to overcome the technical problems, the invention provides a screening and developing plate for carbapenem-resistant enterobacteriaceae (CRE), which can be used for rapid screening of clinical carbapenem-resistant antibiotic resistant strains.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

a screening chromogenic plate for carbapenem-resistant antibiotics enterobacteriaceae bacteria, which comprises a specific chromogenic substrate and a selective culture medium; wherein the selective medium comprises the following components: yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E.

Preferably, the selective medium comprises the following components in parts by weight: 5-10 parts of yeast extract, 0.1-2 parts of salicin, 0.5-2 parts of Arabic gum, 1-5 parts of sodium citrate, 0.01-0.05 part of folic acid, 10.01-0.05 part of vitamin B and 0.01-0.05 part of vitamin E.

Preferably, the preparation method of the yeast extract comprises the following steps:

(1) taking yeast emulsion, carrying out autolysis at constant temperature, and carrying out enzymolysis; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

Preferably, the effective content of the yeast emulsion is 5-10%;

preferably, the temperature of the constant-temperature autolysis is 40-60 ℃; preferably 45 ℃;

preferably, the enzyme for enzymolysis is beta-xylosidase, papain and glucanase;

preferably, the mass ratio of the beta-xylosidase to the papain to the glucanase is 1: 2-3: 1-2;

preferably, the total mass of the beta-xylosidase, the papain and the glucanase is 0.05-0.1g/L of yeast emulsion;

preferably, the enzyme activities of the beta-xylosidase, the papain and the glucanase are 5000-;

preferably, the temperature of the enzymolysis is 30-45 ℃, and preferably 35 ℃;

preferably, the pH value of the enzymolysis is 5-7, preferably 6.5;

preferably, the autolysis process further comprises adding an autolysis promoter, wherein the promoter is a mixture of citric acid and potassium chloride;

preferably, the amount of the citric acid is 0.05-0.1g/L of the yeast emulsion;

preferably, the amount of potassium chloride is 0.1-0.2g/L of the yeast emulsion.

Preferably, the specific chromogenic substrates are 5-bromo-6-chloro-3-indole-beta-glucoside and 5-bromo-4-chloro-3-indole-beta-galactoside.

Preferably, the specific chromogenic substrate has a concentration of 5-bromo-6-chloro-3-indole-beta-glucoside and a concentration of 5-bromo-4-chloro-3-indole-beta-galactoside of 0.01-5.00 g/L.

Preferably, the selective medium is a medium containing carbapenem antibiotics.

Preferably, the carbapenem antibiotic is one or more of imipenem, meropenem and ertapenem;

preferably, the carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

preferably, the effective concentration of the imipenem is 0.1-50 mg/L; the effective concentration of the meropenem is 0.1-50 mg/L; the effective concentration of ertapenem is 0.1-30 mg/L.

Preferably, the selective medium further comprises the following components:

peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water;

preferably, the selective medium comprises the following components in parts by weight:

5-15 parts of peptone, 5-10 parts of sodium chloride, 1-5 parts of lactose, 5-10 parts of beef extract powder, 10-15 parts of agar powder and 1000 parts of purified water plus 1500.

Preferably, the selective medium comprises the following components in parts by weight:

5-15 parts of peptone, 5-10 parts of sodium chloride, 1-5 parts of lactose, 5-10 parts of beef extract powder, 10-15 parts of agar powder and 1500 parts of purified water; 5-10 parts of yeast extract, 0.1-2 parts of salicin, 0.5-2 parts of Arabic gum, 1-5 parts of sodium citrate, 0.01-0.05 part of folic acid, 10.01-0.05 part of vitamin B and 0.01-0.05 part of vitamin E.

Another object of the present invention is to provide a method for preparing the color developing plate, comprising the steps of:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E into a chromogenic substrate, and adding dimethyl formamide or dimethyl sulfoxide; preparing a culture medium,

(2) autoclaving the culture medium;

(3) adding purified water into carbapenem antibiotics, dissolving, and filtering to remove carbapenem antibiotic bacteria to obtain an antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing, and packaging.

Preferably, in the step (1), the addition amount of the dimethylformamide or the dimethyl sulfoxide is 0.1-0.5ml/L of the culture medium;

preferably, in the step (2), the pressure of the autoclaving is 0.15 to 0.25 MPa.

Preferably, in step (3), the filtration is performed using a 0.2-0.5 μm filter membrane;

preferably, in step (4), the temperature of the mixing is 45-50 ℃.

Compared with the prior art, the invention has the technical advantages that:

(1) the invention aims to artificially synthesize specific chromogenic substrates suitable for escherichia coli and coliform groups by using the principle of a chromogenic culture medium and a means of combining drug sensitivity tests by utilizing the characteristics that different bacteria have different specific enzymes, when the escherichia coli and the coliform bacteria can generate different enzymes in the growth process, the different enzymes respectively act on the chromogenic substrates to decompose the chromogenic substrates into chromophores and sugars, the sugars are utilized by the bacteria, the chromophores dye bacterial colonies of the bacteria into different colors, and meanwhile, carbapenem antibiotics are added into the culture medium, so that only the bacteria with carbapenemase in the culture medium can grow, and the bacteria without carbapenemase are inhibited.

(2) The whole detection process is completed within 24 hours, and the separation, identification and drug resistance detection can be realized in one step.

(3) The chromogenic plate provided by the invention has better sensitivity and specificity for identifying the carbapenem-resistant antibiotic enterobacteriaceae bacteria.

(4) During the preparation process of the yeast extract, the potassium chloride is added to promote the autolysis of the yeast on one hand and promote the activity of the enzyme on the other hand. The yeast extract has larger influence on the detection sensitivity of bacteria, and the reasonable selection of the preparation method of the yeast extract has important significance on improving the detection sensitivity of the bacteria.

(5) The chromogenic medium provided by the invention can inhibit the growth of other unrelated bacteria, and improves the selectivity; meanwhile, some unrelated bacteria can not grow because of lack of nutrition by adjusting the nutrition in the culture medium, and the nutrition of the target bacteria can be satisfied in the chromogenic culture medium, so that the target bacteria can grow.

Detailed Description

The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1

A preparation method of yeast extract comprises the following steps:

(1) taking yeast emulsion with effective content of 6%, adding 0.06g/L citric acid and 0.15g/L potassium chloride at 45 ℃, carrying out constant-temperature autolysis, adding 0.06g/L yeast emulsion according to the mass ratio of 1: 3: 1 (enzyme activity is 6000, 6000 and 10000U/g) at 35 ℃ and pH value of 6.5; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 3.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 1.00 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 10 mg/L; the effective concentration of the meropenem is 5 mg/L; the effective concentration of ertapenem is 10 mg/L.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 1 part of salicin, 0.9 part of Arabic gum, 3 parts of sodium citrate, 0.02 part of folic acid, 10.03 parts of vitamin B and 0.01 part of vitamin E.

The preparation method of the color development plate comprises the following steps:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E into a chromogenic substrate, and adding 0.3ml/L dimethylformamide; preparing a culture medium,

(2) sterilizing the culture medium under 0.20 Mpa;

(3) dissolving carbapenem antibiotics in purified water, and filtering with 0.3 μm filter membrane to remove carbapenem antibiotics to obtain antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing at 45 deg.C, and packaging.

Example 2

(1) Taking yeast emulsion with the effective content of 10%, adding 0.05g/L of citric acid and 0.2g/L of potassium chloride at 40 ℃, carrying out constant-temperature autolysis, adding 0.05g/L of yeast emulsion according to the mass ratio of 1: 2: 2 (the enzyme activity is 5000, 5000 and 20000U/g respectively), and the beta-xylosidase, the papain and the glucanase are subjected to enzymolysis at the temperature of 30 ℃ and the pH value of 5; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 0.01g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 5.00 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 0.1 mg/L; the effective concentration of the meropenem is 50 mg/L; the effective concentration of ertapenem is 0.1 mg/L.

The selective culture medium comprises the following components in parts by weight:

5 parts of peptone, 10 parts of sodium chloride, 1 part of lactose, 10 parts of beef extract powder, 10 parts of agar powder and 1500 parts of purified water; 5 parts of yeast extract, 2 parts of salicin, 0.5 part of Arabic gum, 5 parts of sodium citrate, 0.01 part of folic acid, 10.01 parts of vitamin B and 0.05 part of vitamin E.

The preparation method of the color development plate comprises the following steps:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E into a chromogenic substrate, and adding 0.1ml/L dimethyl sulfoxide; preparing a culture medium,

(2) sterilizing the culture medium under 0.15 Mpa;

(3) dissolving carbapenem antibiotics in purified water, and filtering with 0.5 μm filter membrane to remove carbapenem antibiotics to obtain antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing at 50 deg.C, and packaging.

Example 3

(1) Taking yeast emulsion with effective content of 5%, adding 0.1g/L citric acid and 0.1g/L potassium chloride at 60 ℃, carrying out constant-temperature autolysis, adding 0.1g/L yeast emulsion according to the mass ratio of 1: 3: 1 (enzyme activity is 6000, 5000 and 8000U/g), at 45 deg.C and pH 7; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 5.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 0.01 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 50 mg/L; the effective concentration of the meropenem is 0.1 mg/L; the effective concentration of ertapenem is 30 mg/L.

Preferably, the selective medium comprises the following components in parts by weight:

15 parts of peptone, 5 parts of sodium chloride, 5 parts of lactose, 5 parts of beef extract powder, 15 parts of agar powder and 1000 parts of purified water; 10 parts of yeast extract, 0.1 part of salicin, 2 parts of Arabic gum, 1 part of sodium citrate, 0.05 part of folic acid, 10.05 parts of vitamin B and 0.01 part of vitamin E.

The preparation method of the color development plate comprises the following steps:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, folic acid, vitamin B1 and vitamin E into a chromogenic substrate, and adding 0.5ml/L dimethylformamide; preparing a culture medium,

(2) sterilizing the culture medium under 0.25 Mpa;

(3) dissolving carbapenem antibiotics in purified water, and filtering with 0.2 μm filter membrane to remove carbapenem antibiotics to obtain antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing at 45 deg.C, and packaging.

Comparative example 1

The difference compared to example 1 is the different preparation method of the yeast extract.

A preparation method of yeast extract comprises the following steps:

(1) taking yeast emulsion with effective content of 6%, carrying out constant-temperature autolysis at 45 ℃, adding 0.06g/L yeast emulsion with mass ratio of 1: 3: 1 (enzyme activity is 6000, 6000 and 10000U/g) at 35 ℃ and pH value of 6.5; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 3.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 1.00 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 10 mg/L; the effective concentration of the meropenem is 5 mg/L; the effective concentration of ertapenem is 10 mg/L.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 1 part of salicin, 0.9 part of Arabic gum, 3 parts of sodium citrate, 0.02 part of folic acid, 10.03 parts of vitamin B and 0.01 part of vitamin E.

The procedure of the color plate was the same as in example 1.

Comparative example 2

The difference compared to example 1 is the different preparation method of the yeast extract.

A preparation method of yeast extract comprises the following steps:

(1) taking yeast emulsion with effective content of 6%, adding 0.06g/L citric acid at 45 ℃, carrying out constant-temperature autolysis, adding 0.06g/L citric acid according to the mass ratio of 1: 3: 1 (enzyme activity is 6000, 6000 and 10000U/g) at 35 ℃ and pH value of 6.5; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 3.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 1.00 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 10 mg/L; the effective concentration of the meropenem is 5 mg/L; the effective concentration of ertapenem is 10 mg/L.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 1 part of salicin, 0.9 part of Arabic gum, 3 parts of sodium citrate, 0.02 part of folic acid, 10.03 parts of vitamin B and 0.01 part of vitamin E.

The procedure of the color plate was the same as in example 1.

Comparative example 3

The difference compared to example 1 is the different preparation method of the yeast extract.

A preparation method of yeast extract comprises the following steps:

(1) taking yeast emulsion with effective content of 6%, adding 0.15g/L potassium chloride at 45 ℃, carrying out constant-temperature autolysis, adding 0.06g/L yeast emulsion, wherein the mass ratio is 1: 3: 1 (enzyme activity is 6000, 6000 and 10000U/g) at 35 ℃ and pH value of 6.5; preparing enzymolysis liquid;

(2) and (4) taking the supernatant of the enzymolysis liquid, and concentrating under reduced pressure to obtain a yeast extract.

The specific chromogenic substrate comprises 5-bromo-6-chloro-3-indole-beta-glucoside with the concentration of 3.00g/L and 5-bromo-4-chloro-3-indole-beta-galactoside with the concentration of 1.00 g/L.

The carbapenem antibiotic is a mixture of imipenem, meropenem and ertapenem;

wherein the effective concentration of imipenem is 10 mg/L; the effective concentration of the meropenem is 5 mg/L; the effective concentration of ertapenem is 10 mg/L.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 1 part of salicin, 0.9 part of Arabic gum, 3 parts of sodium citrate, 0.02 part of folic acid, 10.03 parts of vitamin B and 0.01 part of vitamin E.

The procedure of the color plate was the same as in example 1.

Comparative example 4

The difference compared to example 1 is that the selective medium composition is different, replacing salicin with sodium citrate.

A method for producing a yeast extract is the same as in example 1.

The specific chromogenic substrate was the same as in example 1.

The carbapenem antibiotic is the same as in example 1.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 0.9 part of Arabic gum, 4 parts of sodium citrate, 0.02 part of folic acid, 10.03 parts of vitamin B and 0.01 part of vitamin E.

The preparation method of the color development plate comprises the following steps:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, acacia, sodium citrate, folic acid, vitamin B1, vitamin E, chromogenic substrate, and 0.3ml/L dimethylformamide; preparing a culture medium,

(2) sterilizing the culture medium under 0.20 Mpa;

(3) dissolving carbapenem antibiotics in purified water, and filtering with 0.3 μm filter membrane to remove carbapenem antibiotics to obtain antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing at 45 deg.C, and packaging.

Comparative example 5

The difference compared to example 1 is that the selective medium composition is different, replacing folic acid with vitamin B1.

A method for producing a yeast extract is the same as in example 1.

The specific chromogenic substrate was the same as in example 1.

The carbapenem antibiotic is the same as in example 1.

The selective culture medium comprises the following components in parts by weight:

10 parts of peptone, 7 parts of sodium chloride, 4 parts of lactose, 6 parts of beef extract powder, 13 parts of agar powder and 1000 parts of purified water; 8 parts of yeast extract, 1 part of salicin, 0.9 part of Arabic gum, 3 parts of sodium citrate, 10.05 parts of vitamin B and 0.01 part of vitamin E.

The preparation method of the color development plate comprises the following steps:

(1) adding peptone, sodium chloride, lactose, beef extract powder, agar powder and purified water into the purified water; adding yeast extract, salicin, Arabic gum, sodium citrate, vitamin B1 and vitamin E into a chromogenic substrate, and adding 0.3ml/L dimethylformamide; preparing a culture medium,

(2) sterilizing the culture medium under 0.20 Mpa;

(3) dissolving carbapenem antibiotics in purified water, and filtering with 0.3 μm filter membrane to remove carbapenem antibiotics to obtain antibiotic solution;

(4) adding antibiotic solution into culture medium, mixing at 45 deg.C, and packaging.

Comparative example 6

The difference compared to example 1 is the composition of the carbapenem antibiotic.

A method for producing a yeast extract is the same as in example 1.

The specific chromogenic substrate was the same as in example 1.

The carbapenem antibiotic is a mixture of imipenem and ertapenem;

wherein the effective concentration of imipenem is 15 mg/L; the effective concentration of ertapenem is 10 mg/L.

The composition of the selective medium was the same as in example 1 in terms of parts by weight.

The procedure of the color plate was the same as in example 1.

Examples of effects

1. Color development identification

Respectively carrying out streak inoculation on escherichia coli and klebsiella pneumoniae of carbapenem antibiotics on a flat plate, culturing for 18-24 hours at the temperature of 35 ℃, and observing the result; colibacillus grows well, and bacterial colony shows mauve, and Klebsiella pneumoniae grows well and bacterial colony shows blue.

2. Sensitivity and specificity test

The strain source is clinical case inspection urine separation sample, which is identified by VITEK-2compact full-automatic bacteria identifier and GN identification card, and 207 CRE strains in total: respectively consisting of 95 Klebsiella pneumoniae strains and 112 Escherichia coli strains; 86 non-CRE strains: respectively, Escherichia coli 59 strain, Pseudomonas aeruginosa 21 strain and Enterobacter cloacae 6 strain sensitive to carbapenem antibiotic drugs, and freezing and storing at-80 deg.C.

The frozen 207 CRE strains and 86 non-CRE strains are inoculated on blood plates, single colonies are picked, a bacterial suspension with the concentration of 0.5 McLee is prepared by normal saline, 10 mu L of the bacterial suspension is respectively inoculated on the chromogenic plates prepared in examples 1-3 and comparative examples 1-6, then the plates are placed in an incubator at 35 ℃ for culture, and the coloration of the colonies is observed after 24 h. The sensitivity and specificity of the chromogenic plate for identifying Escherichia coli in examples 1-3 and comparative examples 1-6 were calculated using the identification result of the VITEK-2compact automatic bacteria identifier as a standard.

Sensitivity-number of growing and developing CRE strains/number of all CRE strains × 100%;

specificity ═ number of non-CRE strains not grown/number of all non-CRE strains × 100%.

Wherein growth and color development is positive, and non-growth is negative. The results are shown in Table 1.

TABLE 1 sensitivity and specificity parameters

Therefore, the chromogenic plate provided by the invention has higher sensitivity and specificity, and can be used for quickly and accurately detecting the carbapenem antibiotic-resistant strains, particularly Escherichia coli and Klebsiella pneumoniae. Meanwhile, the chromogenic medium provided by the invention has a large influence on the selectivity of target bacteria, can inhibit the growth of other unrelated bacteria, and improves the selectivity of the target bacteria; further improving the sensitivity and specificity of bacteria detection.

The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.