CN111826296B

CN111826296B - Human staphylococcus and culture method and application thereof

Info

Publication number: CN111826296B
Application number: CN201910309226.2A
Authority: CN
Inventors: 戴文魁; 周潜; 李嘉琪; 黄琳娣; 薛俊免; 周乐天; 栗东芳; 杨振宇; 李寅虎
Original assignee: Shenzhen Wehealth Gene Technology Co ltd
Current assignee: Shenzhen Wehealth Gene Technology Co ltd
Priority date: 2019-04-17
Filing date: 2019-04-17
Publication date: 2022-06-17
Anticipated expiration: 2039-04-17
Also published as: CN111826296A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to human staphylococcus as well as a culture method and application thereof. The staphylococcus hominis is preserved in the Guangdong province microorganism strain preservation center, and the preservation number of the staphylococcus hominis is GDMCC No. 60608. The human staphylococcus has higher safety for treating the pertussis disease, can improve respiratory tract flora, avoids other intestinal diseases, can be applied to infants or adults, has a wider application range, and provides a new selection mode for treating the pertussis disease in the future.

Description

Human staphylococcus and culture method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to human staphylococcus as well as a culture method and application thereof.

Background

Pertussis (Pertussis) is also called mandarin duck cough and epidemic cough in folk, and is an acute respiratory infectious disease caused by bordetella Pertussis. Clinically, the cough is gradually aggravated, and is typically paroxysmal, spasmodic, and a deep chicken cry-like inspiratory roar appears at the end of the cough, and the disease course reaches 2-3 months, so the disease is called whooping cough. The pertussis infection source can be a child with bacteria, and the young has high missed diagnosis rate and unobvious symptoms after infection, and is an important infection source. Pertussis is mainly spread by droplets, symptoms appear after a latent period (about 7 to 10 days), and people who are susceptible to infection and lack specific immunity are susceptible to infection, and the pertussis antibody transmitted by the placenta from the mother is an unprotected antibody and is easy to be infected, so that the newborn infant especially cannot be excluded.

Pertussis is generally caused by infection of bordetella pertussis (abbreviated as bordetella pertussis), and is a gram-negative bacterium, and can produce some pathogenic substances including pertussis toxin (a capsule of smooth bordetella pertussis with high toxicity and a main pathogenic factor), pertussis filamentous hemagglutinin (a common pathogenic adhesion factor of bordetella pertussis, which adheres to the surface of a bacterial membrane), pertussis adhesin (an outer membrane surface-associated protein, which is presumed to be involved in the survival and reproduction of bordetella pertussis in a host), adenylate cyclase (one of the main pathogenic factors of bordetella pertussis, which can cause acute cough in humans), and the like.

In the 50 and 60 decades of the twentieth century, pertussis inactivated whole bacteria, diphtheria toxoid and tetanus toxoid are combined to prepare diphtheria and tetanus vaccine (DTP) in a large scale in the global scope, although the occurrence of pertussis diseases is obviously reduced, few serious adverse reactions and even death cases occur after whole-cell pertussis vaccine (wP) inoculation, and the DTP inoculation rate is obviously reduced once. Subsequently, the application of acellular pertussis vaccine (aP) with stronger immunogenicity and higher safety also causes the problems of insufficient immunity, low memory immune response and easy induction of adaptive variation of pertussis pathogenic bacteria.

Although the global vaccination rate of pertussis vaccines reaches 85%, and some countries even reach over 99%, in recent years, in epidemiology, pertussis begins to die and relive. There are studies that indicate 2400 million cases of pertussis and 16.07 million deaths in children under the age of 5 worldwide in 2014. Even more surprising, the incidence of pertussis has steadily increased over the past 20 years in several countries where immunizations have been performed at all. In addition to the increased morbidity, Erythromycin (EM) resistance is another concern of chinese pertussis infection. 2012, 2013, 5 months, Xian City 87.5% (14/16); 2016 + 2017, month 7, Shanghai 57.4% (81/141); between 6 months 2013 and 2014, 91.4% was even reached in Beijing (91/99).

At present, pertussis treatment is as follows: 1. general therapy; keep the air fresh by isolating according to the respiratory tract, and avoid all factors which can induce spasmodic cough. Intensive care was taken to prevent complications and to pay attention to nutrition. Infants should breathe artificially immediately, supply oxygen, and if necessary, stop convulsion and expel phlegm. Procaine can be administered by intravenous drip to reduce asphyxia or convulsions, taking care of both heart rate and blood pressure. If the blood sugar is low, the disease is treated. The advantages are that: after suffering from pertussis, the traditional Chinese medicine composition has certain help for relieving symptoms such as spasmodic cough and the like. The disadvantages are as follows: only the disease symptoms are relieved, and the disease cannot be cured fundamentally. 2. Antibiotic therapy; can be used in catarrh stage or early stage of spasmodic cough for reducing infectivity, relieving symptoms and shortening course of disease. Erythromycin or roxithromycin is preferred, the treatment course is not less than 10 days, and the compound sulfamethoxazole can also be used. The advantages are that: compared with the common treatment, the traditional Chinese medicine composition has remarkable effects on treating the pertussis (relieving symptoms and shortening the course of disease). The disadvantages are as follows: the medicine has good application effect in the early disease stage, poor curative effect in the spasmodic cough stage and certain side effect.

The prevention method of pertussis comprises the following steps: 1. whole-cell pertussis vaccine (wP); the advantages are that: can obviously inhibit the occurrence of pertussis diseases; ② the manufacturing procedure is relatively simple. The disadvantages are as follows: safety issues: a few serious adverse reactions and even death occur; ② the effective antigen amount is unstable. 2. Acellular pertussis vaccine (aP); the advantages are that: can obviously inhibit the occurrence of pertussis diseases; ② the immunogenicity is stronger than wP; and the safety is higher than wP, and adverse reactions are less likely to occur after inoculation. The disadvantages are that: the immunity generated by aP is attenuated rapidly compared with wP; ② low memory immune response; and adaptive variation of pertussis pathogenic bacteria occurs after the medicine is used. The more virulent or infectious variant strains represent a selective advantage under vaccine pressure.

Disclosure of Invention

The invention aims to provide human staphylococcus and a culture method and application thereof, and aims to solve the technical problems of unsatisfactory effect and great side effect of the existing pertussis drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

a human staphylococcus, wherein the human staphylococcus is preserved in Guangdong provincial microorganism culture collection center, and the preservation number of the human staphylococcus is GDMCC No. 60608.

The human staphylococcus provided by the invention comes from the respiratory tract of healthy children, and the staphylococcus is propagated in a asexual binary fission mode, has strong reproductive capacity, and can generate a large amount of bacteria in a short time. The human staphylococcus has higher safety for treating the pertussis disease, can improve respiratory tract flora and avoid other intestinal diseases, can be applied to infants or adults, has a larger application range, and provides a new selection mode for treating the pertussis disease in the future.

Correspondingly, the invention also provides a culture method of the staphylococcus hominis, which comprises the following steps:

inoculating the staphylococcus hominis into a broth culture medium, and carrying out amplification culture to obtain a seed solution;

and adding the seed solution into a fermentation culture medium, and performing fermentation culture to obtain fermentation liquor.

The invention provides a culture method of the human staphylococcus, which is characterized by low cost and industrial production, and the finally obtained fermentation liquor containing a large amount of the human staphylococcus has certain inhibition effect on bordetella pertussis and can be used for treating pertussis diseases.

Finally, the invention also provides application of the human staphylococcus and/or fermentation liquor obtained by the culture method of the human staphylococcus in preparation of a medicine for treating pertussis.

Drawings

FIG. 1 is a photomicrograph of a human Staphylococcus aureus hominis WHG2019031202 of the present invention;

FIG. 2 is a colony morphology of Staphylococcus aureus hominis WHG2019031202 in blood agar plate medium according to an embodiment of the present invention;

FIG. 3 is a human Staphylococcus aureus hominis WHG2019031202 phylogenetic tree based on the 16S rDNA sequence according to an embodiment of the present invention; wherein, the Latin name in the figure is the number of the corresponding strain in Genbank;

FIG. 4 is a graph showing the results of a plate inhibition experiment of human Staphylococcus aureus hominis WHG2019031202 (pertussis suspension concentration: 0.9 MCF; human Staphylococcus broth concentration: 8 ‰); wherein A is a blank control of a fermentation medium, and B is a fermentation broth of a strain WHG 2019031201; c is supernatant of the strain WHG2019031201 fermentation liquor, and D is filtrate of the supernatant of the strain WHG2019031201 fermentation liquor.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention provides a staphylococcus hominis, which is preserved in Guangdong province microbial strain collection center, and the preservation number of the staphylococcus hominis is GDMCC No. 60608.

The human Staphylococcus (classified name Staphylococcus hominis) provided by the invention is preserved in Guangdong province microbial culture collection center (GDMCC) in 2019, 3 months and 13 days, and the preservation addresses are as follows: "building 5", Guangzhou microbial research institute, Zhongluo 100, Mr. 59, Guangdong province; the microbial preservation number is as follows: GDMCC No. 60608; the embodiment of the invention is named as WHG 2019031202. As shown in figure 1, the human Staphylococcus aureus hominis WHG2019031202 is gram-positive coccoid, has a thallus diameter of about 0.8 μm, is round or oval, is arranged like grape, and is non-capsule, non-spore, non-flagellate and non-motile. As shown in FIG. 2, after the human Staphylococcus aureus hominis WHG2019031202 grows for 24 hours on a blood agar culture medium, the colony is gray-white and round and convex, the surface is wet and smooth, the colony is opaque, and the edge is neat. The physiological and biochemical characteristics of the human Staphylococcus aureus hominis WHG2019031202 are as follows: 1) the urease test is positive; 2) d-mannose, D-maltose, lactose and sucrose can be utilized for fermentation to produce acid; 3) 6.5% NaCl tolerates growth, O/129 (diaminopteridine) tolerance, neomycin tolerance, alprotoxin tolerance.

The human staphylococcus WHG2019031202 is derived from respiratory tract of healthy children, is propagated in asexual binary fission mode, has strong reproductive capacity, and can produce a large amount of thallus in a short time. The raw materials have wide sources and low production cost. The preparation taking the human staphylococcus WHG2019031202 thallus as the main component has lower side effect compared with antibiotic therapy, can improve respiratory tract flora simultaneously, can be applied to infants or adults, and has a wider application range. In the embodiment of the invention, according to the related experiment results, the thallus with different concentrations has certain inhibiting effect on the bordetella pertussis with the same concentration, and the inhibiting effect is enhanced along with the increase of the thallus concentration in a certain range. Therefore, the human staphylococcus WHG2019031202 has strong ability of inhibiting growth of Bordetella pertussis.

Furthermore, the identification primers of the staphylococcus hominis in the embodiment of the invention are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table. PCR amplification is carried out on human staphylococcus by the identification primer, and the product is sequenced by an ABI-3730xl sequencer, so that the sequence can be logged in an NCBI gene bank to carry out homologous comparison, and a phylogenetic tree is constructed by the sequence and the sequence with homology for identification, as shown in figure 3. Based on phylogenetic tree analysis, the strain human Staphylococcus hominis WHG2019031202 is clustered with Staphylococcus hominis (NR036956.1, human Staphylococcus), Staphylococcus hominis subsp. novobiosepticus (NR041323.1, human Staphylococcus) in a large branch, and the self-development value reaches more than 90, so that the strain can be proved to be human Staphylococcus.

Correspondingly, the embodiment of the invention also provides a culture method of staphylococcus hominis, which comprises the following steps:

s01: inoculating the staphylococcus hominis in the embodiment of the invention into a broth culture medium, and carrying out amplification culture to obtain a seed solution;

s02: and adding the seed solution into a fermentation culture medium, and performing fermentation culture to obtain fermentation liquor.

The embodiment of the invention provides a culture method of the staphylococcus hominis, which has low cost and can be industrially produced, and the finally obtained fermentation liquor containing a large amount of the staphylococcus hominis has a certain inhibition effect on bordetella pertussis and can be used for treating pertussis diseases.

Specifically, in step S01, the broth includes peptone, beef extract, and NaCl. In one embodiment, the broth medium is: 10.0g of peptone, 3.0g of beef extract, 15.0g of NaCl15 and 1L of distilled water. And the pH of the broth medium is 7.0-7.2. The components and the pH of the broth culture medium can better perform scale-up culture on the human staphylococcus to obtain a high-activity seed solution.

Further, the temperature of the amplification culture is 35 ℃; the culture time of the amplification culture is 22-26 h. In one embodiment, the scale-up culture is performed in a constant temperature incubator at 35 ℃ for 24 hours.

Specifically, in step S02, the fermentation medium includes glucose, tryptone, NaCl, KH₂PO₄And K₂HPO₄(ii) a In one embodiment, the fermentation medium is: glucose 20.0g, tryptone 20.0g, NaCl5.0g, KH₂PO₄1.0g，K₂HPO₄2.0g, 1L of distilled water. And the pH of the fermentation medium is 7.0-7.2. The components and the pH value of the fermentation culture medium can better perform fermentation culture on the seed liquid to obtain staphylococcus hominis with more activity.

Further, the temperature of the fermentation culture is 35 ℃; the rotation speed of the fermentation culture is 140-160 r/min; the culture time of the fermentation culture is 70-74 h. In one embodiment, the fermentation culture conditions are: shaking and culturing at 35 deg.C and 150r/min for 72 h.

Furthermore, in the step of adding the seed solution into the fermentation medium, the volume ratio of the seed solution added is 2-10 per mill (namely the volume ratio of the seed solution to the fermentation medium); for example, the seed solutions are added to the fermentation medium at a ratio (V/V) of 2%, 4%, 6%, 8%, 10%, respectively. Further, after obtaining the fermentation liquid, the method also comprises the step of centrifuging the fermentation liquid to obtain fermentation supernatant.

Finally, the embodiment of the invention also provides application of the human staphylococcus and/or the fermentation liquor obtained by the culture method of the human staphylococcus in preparation of a medicine for treating pertussis.

Specifically, the medicament for treating pertussis is a microecological preparation. The microecological preparation has been applied to the prevention and treatment of various diseases in clinic, including gastrointestinal diseases, iatrogenic infectious diseases, liver diseases, hypercholesterolemia, cancer and the like, and the application to respiratory diseases is less. According to the embodiment of the invention, a preparation which can effectively inhibit growth of bordetella pertussis, ensure no side effect and avoid adaptive variation is developed. The microecological preparation mutually permeates and cooperates with the field of pharmacy through the microecological field, and provides a new idea for treating and preventing the traditional pertussis diseases.

The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.

Example 1

Separation, purification, screening and identification of human Staphylococcus aureus hominis WHG2019031202

1) Separation and purification of human Staphylococcus aureus hominis WHG2019031202

Selecting 10 healthy infants, and wiping secretions on faucial arches, pharynx and tonsils on two sides by using a disposable sterile swab in a sensitive and soft action after the throat is exposed by opening the mouth with a sound;

smearing the swab carrying the throat secretion on a blood agar plate culture medium (hereinafter referred to as a blood plate) in a Z-shaped line drawing mode, inversely putting the blood agar plate culture medium into a constant-temperature incubator at 35 ℃, and culturing for 24 hours;

thirdly, observing and recording the growth condition of the bacterial colony after 24 hours;

picking single bacterial colonies with different forms on the blood plate by using a disposable inoculating loop, carrying out inoculating pure culture by a three-region marking method one by one, inversely buckling, putting into a constant-temperature incubator with the temperature of 35 ℃, and culturing for 24 hours;

taking out the blood plate after 24h, and storing the blood plate in a refrigerator at 4 ℃ for subsequent experiments.

As a result: 4 strains of single bacteria are obtained by separation.

The blood agar plate culture medium comprises: 10.0g of peptone, 3.0g of beef extract, 5.0g of sodium chloride, 50mL of defibered sheep blood, 15.0g of agar, 1L of distilled water, 1M NaOH and 1M HCl.

2) Screening of human Staphylococcus aureus hominis WHG2019031202

Firstly, using a disposable inoculating loop to pick Bordetella pertussis and smearing the Bordetella pertussis on a whole charcoal agar plate culture medium (hereinafter referred to as a charcoal plate) in a Z-shaped line drawing mode;

selecting 4 single bacteria separated and purified in the previous step in physiological saline to prepare corresponding bacterial suspension;

cutting a plurality of sterile filter paper sheets with the diameter of 0.5cm, sucking 0.5 mu L of bacterial suspension liquid into the center of the paper sheets, placing the paper sheets into a charcoal flat plate with bordetella pertussis after air drying, wherein the interval of each filter paper sheet is 2cm, then placing the culture medium into a carbon dioxide constant-temperature incubator, culturing at 35 ℃ for 72h, and observing and recording.

As a result: only the WHG2019031202 strain exhibited a significant zone of inhibition.

The charcoal agar culture medium comprises: 10.0g of Lad-Lemco powder, 10.0g of peptone powder, 10.0g of starch, 4.0g of charcoal bacterial powder, 5.0g of sodium chloride, 0.001g of nicotinic acid, 12.0g of agar, 1.0L of distilled water and 100.0mL of degreased sheep blood.

3) Routine identification of strain species

The morphological and physiological and biochemical characteristics of the WHG2019031202 strain were measured by referring to the method described in "Manual of identification of common bacterial systems", published by Dongxu bead et al (Beijing: scientific Press, 2001:349-398), and the measurement results were as follows:

the bacterial colony is gray white, round and convex, the surface is wet and smooth, the bacterial colony is not transparent, and the edge is neat after the WHG2019031202 bacterial strain grows for 24 hours on a blood agar culture medium, as shown in figure 2.

Observation by a microscope to obtain: the bacterial body of the WHG2019031202 has a diameter of about 0.8 μm, is round or oval, is arranged like a grape, is not capsulated, is not sporulated, is not flagellated, and cannot move, as shown in figure 1.

The physiological and biochemical characteristics of the WHG2019031202 strain are shown in Table 1.

TABLE 1

Note: "+" indicates positive, and "-" indicates negative.

Fourthly 16S rDNA identification of WHG2019031202 strain

Extracting DNA from a material of a strain WHG2019031202 by using an Omega D3350 Bacterial DNA Kit, and taking a product as an amplification template by adopting the following primer pairs:

forward primer (Forward primer) 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);

reverse primer (Reverse primer) 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

Performing PCR amplification reaction, wherein the PCR amplification reaction system is as follows: mu.L of 2 XPCR Mix 25. mu.L, 3.0. mu.L of each 2.0. mu. L, DNA of forward and reverse primers (10mM), and 18.0. mu.L of deionized and sterilized ultrapure water, in a total volume of 50.0. mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 45 s; total extension at 72 ℃ for 5min (30 cycles total).

The PCR amplification products were sequenced by ABI-3730xl sequencer. The measured read1, read2 sequences were edited with the software ChromasPro (version2.1.8) to yield a complete sequence. The sequences were subjected to homology alignment by registering with NCBI GenBank, and phylogenetic trees were constructed using the Maximum reduction method of MEGA7.0 (Maximum-parsimony; MP) together with the 24 sequences having homology. Based on phylogenetic tree analysis, the strain human Staphylococcus hominis WHG2019031202 is clustered with Staphylococcus hominis (NR036956.1, human Staphylococcus), Staphylococcus hominis subsp. novobiosepticus (NR041323.1, human Staphylococcus) in a large branch, and the self-development value reaches more than 90, so that the strain can be proved to be human Staphylococcus.

The strain is confirmed to be human Staphylococcus aureus hominis WHG2019031202 by combining common bacteria identification manual and comparing morphological characteristics, physiological and biochemical characteristics and phylogenetic analysis of the strain WHG 2019031202.

The strain Staphylococcus aureus hominis WHG2019031202 has been deposited in Guangdong province microbial culture collection center (GDMCC) in 13.3.2019, is located in Michelia intermedia 100 No. 5 building of Dazhou college No. 59, Guangdong province microbial research institute, and has a deposition number: GDMCC No. 60608.

The preservation culture medium of the Staphylococcus aureus hominis WHG2019031202 strain is a culture medium of a ferric saccharate slant. The culture medium of the Ke's disaccharide iron slant is as follows: 20.0g of peptone, 3.0g of beef extract powder, 3.0g of yeast extract powder, 10.0g of lactose, 1.0g of glucose, 5.0g of sodium chloride, 0.5g of ferric ammonium citrate, 0.5g of sodium thiosulfate, 12.0g of agar, 0.025g of phenol red, and the pH value: 7.4 +/-0.2, and the culture temperature is 35 ℃.

Example 2

Determination of bacteriostatic activity of fermentation liquor of human Staphylococcus aureus hominis WHG2019031202 strain

1) Preparation of sterile filtrate of fermentation broth

Firstly, selecting a Staphylococcus aureus hominis WHG2019031202 lawn after one-fungus-loop purification, inoculating into a test tube filled with 10mL of broth culture medium, putting into a constant-temperature incubator at 35 ℃, and culturing for 24 hours to obtain a bacterial suspension as a seed solution.

Broth culture medium: 10.0g of peptone, 3.0g of beef extract, 15.0g of NaCl15, and 1L of distilled water, wherein the pH value is 7.0-7.2.

② adding the seed liquid into 5 100mL triangular flasks containing 50mL basic fermentation medium according to the proportion (V/V) of 2 per mill, 4 per mill, 6 per mill, 8 per mill and 10 per mill, marking correspondingly, culturing at 35 ℃ for 150r/min under shaking for 72 h.

Basic fermentation medium: glucose 20.0g, tryptone 20.0g, NaCl5.0g, KH₂PO₄ 1.0g，K₂HPO₄2.0g, 1L distilled water, pH 7.0-7.2.

③ respectively sucking 1mL of fermentation liquor with the concentration of 2 per thousand, 4 per thousand, 6 per thousand, 8 per thousand and 10 per thousand into a 1.5mL centrifugal tube, and carrying out centrifugal treatment (13000r/min, 10min) to obtain fermentation supernatant; 500. mu.L of the fermentation supernatant was aspirated into each tube, and the resulting solution was filtered through a 0.45 μm sterile filter to obtain a sterile filtrate.

Note: the concentrations of the fermentation liquor, the fermentation supernatant and the sterile filtrate are based on the inoculation of seed liquid with different volumes, namely 2 per thousand, 4 per thousand, 6 per thousand, 8 per thousand and 10 per thousand.

2) Inhibition of Bordetella pertussis activity

Picking up bordetella pertussis by utilizing a disposable sterile inoculating loop, preparing bacterial suspension with the concentration of 0.3MCF (Mycoleptodonoides aitchisonii), 0.6MCF and 0.9MCF by using sterile normal saline, respectively sucking 100uL of the bacterial suspension, adding the bacterial suspension into 3 same charcoal flat plates, uniformly coating the whole flat plate by using a coating rod, and storing the flat plate in a refrigerator at 4 ℃;

preparing a plurality of sterile filter paper sheets with the diameter of 0.5cm, respectively sucking the fermentation liquid, the fermentation supernatant and the sterile filtrate of the fermentation supernatant with the concentration of 2 per thousand, 4 per thousand, 6 per thousand, 8 per thousand and 10 per thousand into the center of a sterile filter paper disc with the diameter of 0.5cm, and placing the sterile filter paper disc at room temperature for airing.

Thirdly, placing filter paper sheets of fermentation liquor, fermentation supernatant and sterile filtrate with the same concentration and a blank control into charcoal flat plates of 0.3MCF, 0.6MCF and 0.9MCF bordetella pertussis respectively, wherein the interval of each paper sheet is 2cm, 15 groups are formed, and 3 repeats are arranged in each group. The medium was then placed in a carbon dioxide incubator, incubated at 35 ℃ for 72h, and the results observed and recorded (FIG. 4, Table 2). Table 2 shows the results of the plate inhibition experiment of the WHG2019031202 strain on fermentation broth, fermentation supernatant and sterile filtrate at different concentrations.

TABLE 2

From table 2, it can be seen that: the thallus with different concentrations has certain inhibiting effect on the Bordetella pertussis with the same concentration, and the inhibiting effect is enhanced along with the increase of the thallus concentration in a certain range.

The experiments prove that the preparation (such as a microecological preparation) taking the staphylococcus hominis WHG2019031202 thalli as the main component has higher safety for treating the pertussis diseases, and can improve respiratory flora and avoid other intestinal diseases.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

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Claims

1. The staphylococcus hominis is deposited in the Guangdong provincial culture collection center, and the deposit number of the staphylococcus hominis is GDMCC No. 60608.

2. The staphylococcus hominis according to claim 1, wherein the primers for identifying staphylococcus hominis are shown as SEQ ID No.1 and SEQ ID No. 2.

3. A method for culturing human staphylococcus, which is characterized by comprising the following steps:

inoculating the staphylococcus hominis of claim 1 or 2 into a broth culture medium, and performing scale-up culture to obtain a seed solution;

4. The method for culturing Staphylococcus hominis according to claim 3, wherein the culture broth comprises peptone, beef extract and NaCl; and/or the presence of a gas in the gas,

the broth medium has a pH of 7.0 to 7.2.

5. The method for culturing Staphylococcus hominis according to claim 3, wherein the temperature of the scale-up culture is 35 ℃; and/or the presence of a gas in the gas,

the culture time of the amplification culture is 22-26 h.

6. The method for culturing Staphylococcus hominis according to claim 3, wherein the fermentation medium comprises glucose, tryptone, NaCl, KH₂PO₄And K₂HPO₄(ii) a And/or the presence of a gas in the atmosphere,

the pH of the fermentation medium is 7.0-7.2.

7. The method for culturing Staphylococcus hominis according to claim 3, wherein the temperature of the fermentation culture is 35 ℃; and/or the presence of a gas in the gas,

the rotation speed of the fermentation culture is 140-160 r/min; and/or the presence of a gas in the gas,

the culture time of the fermentation culture is 70-74 h.

8. The method for culturing Staphylococcus hominis according to any one of claims 3 to 7, wherein the seed solution is added to the fermentation medium at a volume ratio of 2 to 10 ‰; and/or the presence of a gas in the gas,

after the fermentation liquor is obtained, the method also comprises the step of centrifuging the fermentation liquor to obtain fermentation supernatant.

9. Use of the human staphylococci according to claim 1 or 2 and/or the fermentation broth obtained from the method for culturing human staphylococci according to any one of claims 3-8 for the manufacture of a medicament for the treatment of pertussis.

10. The use of claim 9, wherein the medicament for the treatment of pertussis is a probiotic.