Selective chromogenic culture medium for separation and identification of streptococcus suis and preparation method of selective chromogenic culture medium

Abstract

The invention relates to a selective chromogenic medium for separation and identification of streptococcus suis and a preparation method thereof, and belongs to the field of microbial culture. In the prior art, a streptococcus suis culture medium mainly focuses on increasing the content of streptococcus suis strains, but is not suitable for clinically separating and identifying the streptococcus suis, and other infectious microbes in a clinical sample are difficult to distinguish, so that separation fails. In order to overcome the defects in the prior art, the invention provides a selective chromogenic culture medium for separating and identifying streptococcus suis and a preparation method thereof, the culture medium comprises tryptone, soybean peptone and other components, and when the culture medium is used for separating pathogenic bacteria of a clinical porcine respiratory tract sample, the separation efficiency is greatly improved. The streptococcus suis can be accurately separated and identified according to the specific color development and growth state of the bacterial colony, the influence of other infectious microbes is remarkably reduced, and the method is suitable for being popularized and applied in the porcine respiratory tract sample treatment process.

Description

Technical Field

[0001] The invention relates to a selective culture medium for separating and identifying streptococci of suis and a preparation method thereof, belonging to the field of microbial culture. Summary of the invention

[0002] Streptococcus suis (Ss) is a Gram-positive facultative anaerobic bacterium with a round or oval shape, usually arranged in pairs or short chains, and has a capsule. According to the differences in capsular polysaccharide antigens, Streptococcus suis can be divided into 35 serotypes, of which type 1, type 2, type 7 and type 9 are the main pathogenic types, especially type 2, which can not only infect pigs, but also cause human infection and endanger life. Streptococcus suis infection can be transmitted through the respiratory tract or digestive tract. According to the symptoms and time of onset, it can be divided into acute, subacute and chronic types. Among them, the acute type is mainly manifested as sepsis and meningitis, and the sick pigs die quickly; the chronic type mainly causes pneumonia, endocarditis, arthritis, etc., and the sick pigs have symptoms such as coughing, panting, weight loss, lameness, etc., which can be improved if actively treated. The isolation and identification of Streptococcus suis has become an important step in the effective prevention and treatment of Streptococcus suis infection.

[0003] Streptococcus suis has high nutritional requirements and is usually isolated by direct separation using blood plates. When the sample contains foreign bacteria, selective blood plates containing nalidixic acid and polymyxin B are often used. However, this culture medium has poor inhibitory effects on other foreign bacteria, which affects the separation and identification of Streptococcus suis. Kataoka et al. found in 1991 that a selective culture medium prepared by adding 5% defibrinated sheep blood, 50 mg / L sodium azide, 25 mg / L nalidixic acid, 12.5 mg / L colistin and 2 mg / L crystal violet to Todd-Hewitt broth (THB) can effectively improve the separation efficiency of Streptococcus suis. Luo Longze et al. found in 2007 that a culture medium based on tryptic soy agar with the addition of 1% yeast powder, 2% rabbit serum, 50 mg / L aztreonam, 10 mg / L polymyxin B and 15 mg / L furantoin can effectively separate Streptococcus suis and inhibit the growth of common Gram-negative bacteria and Gram-positive bacteria such as Staphylococcus aureus, beta-hemolytic Streptococcus, and some fecal Streptococci. By adding indicators such as mannitol and neutral red, a transparent indicating selective culture medium (StS culture medium for short) can be made, which greatly improves the detection sensitivity.

[0004] The article "Research on Optimizing Culture Medium Components to Increase the Viable Count of NT15B Strain of Streptococcus suis Type 2" published in the July 2024 issue of "Chinese Journal of Animal Husbandry and Veterinary Medicine" refers to the method of Chen Guanping et al. and improves it to prepare the basic fermentation culture medium. The optimized culture medium can effectively increase the viable count of NT15B strain of Streptococcus suis Type 2, reducing production costs. It is mainly optimized from the aspect of increasing the fermentation yield of Streptococcus suis, and the culture medium is not suitable for the isolation and identification of Streptococcus suis.

[0005] The "Optimization of Selective Culture Medium for Streptococcus suis" published in the July 2019 issue of the Journal of Preventive Medicine Intelligence uses brain heart broth as the basis, sterilizes at 121°C for 15 minutes, adds 2% rabbit serum and different concentrations of antibacterial substances after cooling, and divides them into test tubes for standby use to prepare a selective enrichment broth. On this basis, it further screens the types and concentrations of antibacterial substances, optimizes the culture medium formula, and further improves the isolation effect of Streptococcus suis.

[0006] Based on this, the present invention is proposed. Summary of the invention

[0008] The culture medium of Streptococcus suis in the prior art mainly focuses on improving the content of Streptococcus suis strains, but is not suitable for clinical separation and identification of Streptococcus suis, and it is difficult to separate other strains clinically. In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a selective culture medium for separation and identification of Streptococcus suis and a preparation method thereof. The culture medium includes components such as tryptone and soytone. When the culture medium is used for the bacterial flora culture of clinical respiratory tissue, Streptococcus suis can be accurately separated and identified, and the influence of other miscellaneous bacteria is significantly reduced, which is suitable for promotion and application in clinical practice.

[0009] The present invention achieves the above technical effects through the following technical solutions:

[0010] A selective culture medium for separating and identifying Streptococcus suis, comprising the following components: 12.0-18.0 g / L of tryptone, 3.0-7.0 g / L of soytone, 4.0-6.0 g / L of sodium chloride, 40-60 ml / L of calf serum, 12.0-18.0 g / L of agar, 15-25 mg / L of polymyxin E, 3-5 mg / L of amphotericin B, 0.14-0.18 g / L of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, and 8-12 g / L of Tween 80, and the volume is fixed to 1 L with pure water.

[0011] As a preferred embodiment, the selective chromogenic medium for isolation and identification of Streptococcus suis is composed of the following components: 15.0 g / L tryptone, 5.0 g / L soytone, 5.0 g / L sodium chloride, 50 ml / L calf serum, 15.0 g / L agar, 20 mg / L polymyxin E, 4 mg / L amphotericin B, 0.16 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, 10 g / L Tween 80, and the volume is made up to 1 L with pure water.

[0012] As another preferred embodiment, the selective chromogenic medium for isolation and identification of Streptococcus suis is composed of the following components: 12.0 g / L tryptone, 7.0 g / L soytone, 4.0 g / L sodium chloride, 60 ml / L calf serum, 12.0 g / L agar, 25 mg / L polymyxin E, 3 mg / L amphotericin B, 0.18 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, 12 g / L Tween 80, and the volume is made up to 1 L with pure water.

[0013] As another preferred embodiment, the selective chromogenic medium for isolation and identification of Streptococcus suis is composed of the following components: 18.0 g / L tryptone, 3.0 g / L soytone, 6.0 g / L sodium chloride, 40 ml / L calf serum, 18.0 g / L agar, 15 mg / L polymyxin E, 5 mg / L amphotericin B, 0.14 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, 8 g / L Tween 80, and the volume is made up to 1 L with pure water.

[0014] The present invention also provides a method for preparing the selective chromogenic culture medium for separation and identification of Streptococcus suis, which comprises the following steps:

[0015] 1) Weigh or measure the prescribed amount of tryptone, soytone, sodium chloride, tween80, and agar, add them to distilled water, stir to dissolve, adjust the pH value to 7.2±0.2, and sterilize at 121°C for 15 minutes to obtain the culture medium mother solution;

[0016] 2) Weigh polymyxin E and dissolve it in pure water to prepare a 20 mg / ml polymyxin E solution; weigh 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and amphotericin B and dissolve them in DMSO to prepare a 0.16 g / ml 5-bromo-6-chloro

[0017] -3-indole-β-D-pyranoglucoside solution and 40 mg / ml amphotericin B solution, filter and sterilize the prepared solution for later use;

[0018] 3) Cool the culture medium to below 55°C, add the prescribed amount of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, polymyxin E solution, amphotericin B solution and calf serum, stir to mix and pour into a plate.

[0019] The present invention also claims the use of the selective chromogenic culture medium for separating and identifying Streptococcus suis. Specifically, the present invention claims the use of the culture medium for separating and identifying Streptococcus suis in lesional respiratory tissues of pigs.

[0020] Preferably, in the use described above, the porcine respiratory tract diseased tissue is lower respiratory tract disease material of porcine respiratory disease or porcine diseased lung tissue material.

[0021] Compared with the existing separation culture medium, the present invention has high separation accuracy and can reduce the growth interference of other common respiratory flora. Specifically, it has the following technical advantages compared with the technology:

[0022] 1) The detection effect of different culture media on common bacteria in Example 4 of the present invention shows that respiratory flora such as Escherichia coli, Pasteurella multocida, and Klebsiella pneumoniae still grow well on conventional blood plates and BHI solid culture media, but cannot grow on the selective culture media described in the present invention. The selective culture media described in the present invention can grow Streptococcus suis and Staphylococcus aureus. Compared with conventional blood plates and BHI solid culture media, the color development of the two bacteria on the culture plates is obviously different. Picking the purple-red colonies on the plate can obtain pure culture of Streptococcus suis.

[0023] 2) Example 4 of the present invention shows that the number of strains grown on the selective culture medium of the present invention is significantly reduced compared with the commercially available culture medium, and the accuracy of the color culture determined by sequencing comparison shows that most of the strains are Streptococcus suis. This shows that the selective color culture medium of the present invention can not only significantly reduce the growth interference of other bacteria, but also make Streptococcus suis present specific purple-red colonies, and its accuracy is significantly better than the existing selective culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of streaking culture of lower respiratory tract tracheal swab sample 1 in TSA++ medium and the color development medium of the present invention, where a is the result of culture in TSA++ medium, and b is Figure 1 Results of chromogenic culture medium.

[0025] Figure 2 The results of streaking culture of lower respiratory tract tracheal swab sample 2 in TSA++ medium and the color development medium of the present invention, where a is the result of culture in TSA++ medium, and b is Figure 1 Results of chromogenic culture medium.

[0026] Figure 3 The results of streaking culture of lower respiratory tract tracheal swab sample 3 in TSA++ medium and the chromogenic medium of the present invention, where a is the result of culture in TSA++ medium, and b is Figure 1 Results of chromogenic culture medium.

[0027] Figure 4 The streaking culture results of the diseased lung tissue sample 1 in TSA++ medium and the color development medium of the present invention, wherein a is the culture result of TSA++ medium, b is Figure 1 Results of chromogenic culture medium.

[0028] Figure 5 The results of streaking culture of the diseased lung tissue sample 2 in TSA++ medium and the color development medium of the present invention, wherein a is the result of culture in TSA++ medium, b is Figure 1 Results of chromogenic culture medium.

[0029] Figure 6 The results of streaking culture of the diseased lung tissue sample 3 in TSA++ medium and the color development medium of the present invention, wherein a is the result of culture in TSA++ medium, b is Figure 1 Results of chromogenic culture medium.

[0030] Figure 7 The results of streaking culture of the diseased lung tissue sample 4 in TSA++ medium and the color development medium of the present invention, wherein a is the result of culture in TSA++ medium, b is Figure 1 Results of chromogenic culture medium.

[0031] Figure 8 The streaking culture results of the diseased lung tissue sample 5 in TSA++ medium and the color development medium of the present invention, wherein a is the culture result of TSA++ medium, b is Figure 1 Results of chromogenic culture medium. DETAILED DESCRIPTION

[0032] The present invention is further described below through specific implementations, but those skilled in the art should be aware that the embodiments do not limit the scope of protection of this patent in any way. The scope of patent protection is based on the scope defined by the claims of the present invention, and equivalent replacements for the technical solution of the present invention are included in the scope of protection of the patent of the present invention.

[0033] Example 1 Selective culture medium for separation and identification of Streptococcus suis of the present invention and preparation method thereof

[0034] The selective culture medium for the isolation and identification of Streptococcus suis was prepared according to the following ratio:

[0035]

[0036] The preparation method of the selective culture medium for the isolation and identification of Streptococcus suis is as follows:

[0037] 1. Weigh tryptone, sodium chloride, tween 80 and agar according to the formula, put them into a container and add 950 ml of distilled water, stir to dissolve, adjust the pH value to 7.2±0.2, and sterilize at 121℃ for 15 minutes;

[0038] 2. Weigh 0.02g of polymyxin E, dissolve it in 1ml of pure water to prepare a 20mg / ml polymyxin E solution, filter and sterilize for later use; weigh 0.16g of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, dissolve it in 1ml of DMSO, prepare a 0.16g / ml 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, filter and sterilize for later use; weigh 0.04g of amphotericin B, dissolve it in 1ml of DMSO, prepare a 40mg / ml amphotericin B solution, filter and sterilize for later use;

[0039] 3. When the culture medium is cooled to below 55°C, add 1 ml of the prepared 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, 1 ml of polymyxin E solution, 100 ul of amphotericin B solution, 1 ml of phenol and 950 ml of calf serum to the solid culture medium; mix well and pour into a plate to prepare a thickness of 3-4 mm / dish.

[0040] Example 2 Selective culture medium for separation and identification of Streptococcus suis of the present invention and preparation method thereof

[0041] The selective culture medium for the isolation and identification of Streptococcus suis was prepared according to the following ratio

[0042]

[0043] The preparation method of the above selective culture medium is the same as that in Example 1.

[0044] Example 3 Selective culture medium for separation and identification of Streptococcus suis of the present invention and preparation method thereof

[0045] The selective culture medium for the isolation and identification of Streptococcus suis was prepared according to the following ratio

[0046]

[0047] The preparation method of the above selective culture medium is the same as that in Example 1.

[0048] Example 4 Comparison of the selective chromogenic medium for the isolation and identification of Streptococcus suis of the present invention and other common culture media for the cultivation of swine respiratory tract infection materials

[0049] The culture medium of the present invention was used to culture common respiratory bacteria, with blood agar and BHI solid culture medium as controls, at 37°C for 18-24 hours. The blood agar and BHI solid culture medium used were purchased from Qingdao Haibo Biotechnology Co., Ltd. and prepared according to their instructions. The comparison of the detection results of common bacteria of pig respiratory tract infection by different culture media is shown in Table 1.

[0050] Table 1 Comparison of the detection effects of different culture media on common bacteria in pig respiratory tract infection materials

[0051]

[0052] The number of "+" indicates bacterial growth, "++++" indicates good growth, and 1-3 "+" indicates that the growth is inhibited to varying degrees. "-" indicates no growth. The strains listed in the table were all isolated from pathological lung tissue samples of pigs with respiratory diseases.

[0053] From the detection effects of the above-mentioned different culture media on common bacteria, it can be seen that respiratory flora such as Escherichia coli, Pasteurella multocida, and Klebsiella pneumoniae still grow well on conventional blood plates and BHI solid culture media, but cannot grow on the selective culture media described in the present invention. The selective color development culture media described in the present invention can grow Streptococcus suis, Staphylococcus aureus, and Staphylococcus warfarinii. Compared with conventional blood plates and BHI solid culture media, the three bacteria have obvious differences in color development on the selective color development culture media described in the present invention, which shows that the culture media described in the present invention can significantly distinguish common pathogens in porcine respiratory bacterial diseases from Streptococcus suis, effectively separate and identify Streptococcus suis, and pick the purple-red colonies on the plate to obtain pure culture of Streptococcus suis.

[0054] Example 5 Isolation of Streptococcus suis from clinical samples using TSA++ medium and the selective medium of the present invention

[0055] Actual sample detection: Take clinically collected pig lower respiratory tract tracheal swabs (3) and diseased lung tissue samples (5), rinse the swabs or diseased tissues thoroughly with 500ul sterile saline, take 20ul and drop it on the embodiment color development medium, and inoculate it in 3-4 zones, culture at 37°C for 18-24h, and observe the growth of colonies; pick red colonies, expand the culture, use bacterial 16S rRNA gene universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), PCR amplify the purified isolated bacteria, send the amplicon to the sequencing company for sequencing, and after obtaining the sequence, conduct sequence comparison online to identify the bacterial species and compare the accuracy of the color development culture. At the same time, a culture medium commonly used for isolating porcine respiratory pathogens (TSA + 5% calf serum + 0.003% NAD +, TSA++) was used as a control culture medium. The TSA++ basic culture medium used was purchased from Qingdao Haibo Biotechnology Co., Ltd. and prepared according to its instructions.

[0056] Table 2 Comparison of detection effects of the selective culture medium of the present invention and commercially available culture medium in clinical samples

[0057]

[0058] "-" indicates no growth. One colony of each morphology was selected and species identification was performed using 16s rRNA sequencing.

[0059] The results of streaking culture of lower respiratory tract tracheal swab sample 1, lower respiratory tract tracheal swab sample 2, lower respiratory tract tracheal swab sample 3, diseased lung tissue material 1, diseased lung tissue material 2, diseased lung tissue material 3, diseased lung tissue material 4, and diseased lung tissue material 4 in TSA++ medium and the chromogenic medium of the present invention are as follows: Figure 1-Figure 8 As shown, a is the result of TSA++ culture medium, and b is the result of chromogenic culture medium culture in Example 1. As shown. The experimental results show that the number of morphological strains grown on the culture medium using the selective culture medium of the present invention is significantly reduced compared with the commercially available culture medium, and the accuracy of the chromogenic culture determined by sequencing comparison shows that most of the strains are Streptococcus suis. This shows that the selective chromogenic culture medium of the present invention can not only significantly reduce the growth interference of other bacteria, but also can significantly distinguish between common pathogens in porcine respiratory bacterial diseases and Streptococcus suis, and its accuracy is significantly better than the existing selective culture medium.

[0060] The selective color development culture medium of the present invention in Example 4 and Example 5 can be replaced with the culture medium described in Example 2 or Example 3, which can achieve the same technical effect.

[0061] In summary, the selective chromogenic culture medium of the present invention can effectively inhibit the growth of miscellaneous bacteria and distinguish the target bacteria-Streptococcus suis through specific color development, which can improve the diagnosis of Streptococcus suis infection in clinical samples and its separation and identification.

Claims

1. A selective culture medium for the isolation and identification of Streptococcus suis, comprising the following components: Tryptone 12.0-18.0g / L, soytone 3.0-7.0g / L, sodium chloride 4.0-6.0g / L, calf serum 40-60ml / L, agar 12.0-18.0g / L, polymyxin E 15-25mg / L, amphotericin B 3-5mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.14-0.18g / L, Tween 80 8-12g / L, make up to 1L with pure water.

2. The selective culture medium for separation and identification of Streptococcus suis according to claim 1, characterized in that: It is composed of the following components: tryptone 15.0 g / L, soytone 5.0 g / L, sodium chloride 5.0 g / L, calf serum 50 ml / L, agar 15.0 g / L, polymyxin E 20 mg / L, amphotericin B 4 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.16 g / L, Tween 80 10 g / L, and the volume is made up to 1 L with pure water.

3. The selective culture medium for separation and identification of Streptococcus suis according to claim 1, characterized in that: It is composed of the following components: tryptone 12.0 g / L, soytone 7.0 g / L, sodium chloride 4.0 g / L, calf serum 60 ml / L, agar 12.0 g / L, polymyxin E 25 mg / L, amphotericin B 3 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.18 g / L, Tween 80 12 g / L, and the volume is made up to 1 L with pure water.

4. The selective culture medium for separation and identification of Streptococcus suis according to claim 1, characterized in that: It is composed of the following components: tryptone 18.0 g / L, soytone 3.0 g / L, sodium chloride 6.0 g / L, calf serum 40 ml / L, agar 18.0 g / L, polymyxin E 15 mg / L, amphotericin B 5 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.14 g / L, Tween 80 8 g / L, and the volume is made up to 1 L with pure water.

5. A method for preparing a selective culture medium for separation and identification of Streptococcus suis according to any one of claims 1 to 4, comprising the following steps: 1) Weigh or measure the prescribed amount of tryptone, soytone, sodium chloride, tween80, and agar and add them to distilled water. After stirring and dissolving, the pH value was adjusted to 7.2±0.2, and sterilized at 121°C for 15 minutes to obtain a culture medium mother solution; 2) Weigh polymyxin E and dissolve it in pure water to prepare a 20 mg / ml polymyxin E solution, weigh 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and amphotericin B and dissolve them in DMSO to obtain a 0.16 g / ml 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution and a 40 mg / ml amphotericin B solution, and filter and sterilize the prepared solution for later use; 3) Cool the culture medium to below 55°C, add the prescribed amount of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, polymyxin E solution, amphotericin B solution and calf serum, stir to mix and pour into a plate.

6. Use of the selective culture medium according to any one of claims 1 to 4 in isolating and identifying Streptococcus suis in lesional tissues of the respiratory tract of pigs.

7. The use of the selective culture medium according to claim 6, characterized in that The respiratory tract diseased tissue is lower respiratory tract disease material of pig respiratory disease or pig diseased lung tissue material.

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