Construction method of in-vitro staphylococcus aureus abscess community three-dimensional model

Abstract

The invention provides a construction method of an in-vitro staphylococcus aureus abscess community three-dimensional model. The construction method comprises the steps of strain activation, bacterial suspension preparation, collagen gel matrix preparation, model assembly and plasma treatment. The model constructed by the method disclosed by the invention provides a standardized experimental platform for researching a staphylococcus aureus chronic infection mechanism and evaluating an antibiotic curative effect and host immune clearance efficiency through in-vitro reproduction of a fibrous protein false capsule structure, bacterial antibiotic tolerance and immune escape characteristics; the method can be applied to development of novel anti-infection drugs, optimization of personalized treatment schemes and research and development of vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a Staphylococcus aureus abscess colony model, in particular to a method for constructing a three-dimensional model of a Staphylococcus aureus abscess colony in vitro. BACKGROUND

[0002] Staphylococcus aureus is a gram-positive commensal bacterium that mainly colonizes the nostrils and skin surface of humans. As an opportunistic pathogen, it can cause skin and soft tissue infections (SSTI), osteomyelitis, orthopedic implant-related infections (ODRI), and other diseases, and in severe cases, it can even endanger life due to bacteremia, metastatic infection, or sepsis. Due to the increasing antibiotic resistance, the treatment failure rate of Staphylococcus aureus infection remains high. For example, up to 20% of patients with skin and soft tissue infections and chronic osteomyelitis patients will have recurrent infections, and there is an urgent need for more effective treatment strategies.

[0003] A key feature of Staphylococcus aureus infection is the induction of tissue abscess formation, which is commonly seen in skin and soft tissue infections, osteomyelitis, and endocarditis. The core of these abscesses contains a Staphylococcus aureus abscess colony (SAC) composed of dense Staphylococcus aureus aggregates, surrounded by a layer of pseudocapsule formed mainly by fibrin deposits. The Staphylococcus aureus abscess colony structure is surrounded by immune cells.

[0004] It is generally believed that Staphylococcus aureus escapes host immune attack by forming a Staphylococcus aureus abscess colony, and the fibrin pseudocapsule on its periphery can act as a physical barrier to hinder the penetration of immune cells. The host attempts to control the infection by encapsulating the Staphylococcus aureus abscess colony and immune cells with fibrous tissue to form an abscess structure, but this encapsulation ultimately fails to eliminate the pathogen, as the Staphylococcus aureus abscess colony can survive in the body for a long time. This is likely one of the reasons for the high recurrence rate of infection in patients. In addition, the potential antibiotic resistance within the Staphylococcus aureus abscess colony may also contribute to the recurrence.

[0005] Due to the long-term co-evolution of Staphylococcus aureus with the human host, its virulence factors exhibit significant human host specificity. This specificity makes it difficult for traditional animal models to fully simulate the key host-pathogen interactions in human infections, which is manifested in the following two aspects:

[0006] 1. Species differences in thrombin activity: Staphylocoagulase produced by Staphylococcus aureus can activate prothrombin in mouse plasma, but its activation efficiency on human prothrombin is significantly higher than that on mouse prothrombin. This difference directly affects the formation of the fibrin pseudocapsule, the core structure of the infection, which is a key barrier for Staphylococcus aureus to achieve immune escape and antibiotic resistance.

[0007] 2. Human specificity of plasmin activation: Staphylococcal kinase secreted by S. aureus can specifically activate human plasminogen to generate plasmin. Plasmin promotes bacterial dissemination by degrading fibrin. However, this process cannot be effectively reproduced in animal models such as mice due to species specificity.

[0008] Therefore, it is of great significance to develop a suitable three-dimensional model of S. aureus abscess colony in vitro to study the physical properties of S. aureus abscess in vivo and the resistance to antibiotic treatment. SUMMARY

[0009] To solve the problems in the prior art, the present application provides a method for constructing a three-dimensional model of S. aureus abscess colony in vitro. The model constructed by the method of the present application reproduces the fibrin pseudo-capsule structure, bacterial antibiotic resistance and immune escape characteristics in vitro, providing a standardized experimental platform for studying the mechanism of chronic S. aureus infection, evaluating the efficacy of antibiotic treatment and the efficiency of host immune clearance. It can be applied to the development of new anti-infective drugs, optimization of personalized treatment plans and vaccine research.

[0010] The present application adopts the following technical solutions:

[0011] A method for constructing a three-dimensional model of S. aureus abscess colony in vitro, comprising the steps of activating the bacteria, preparing a bacterial suspension, preparing a collagen gel matrix, assembling the model and treating the plasma.

[0012] According to an embodiment of the present application, the bacterial suspension is prepared by suspending the activated single colony in RPMI1640 medium, and the absorbance is measured at a wavelength of 620 nm using a spectrophotometer. The absorbance is adjusted to 0.6-0.8 at OD620. The bacterial suspension is diluted to obtain an inoculum with a final concentration of about 13-15 CFU / 25 μl.

[0013] According to an embodiment of the present application, the collagen gel matrix is prepared by diluting the rat type I collagen solution with 1×RPMI 1640 medium to a final concentration of 1.7-1.9 mg / ml.

[0014] According to an embodiment of the present application, the model assembly is performed by adding the collagen gel solution in a 24-well Transwell system, polymerizing for 1-2 hours in a 36-38℃ humidified incubator, and adding the bacterial suspension on the surface of the gel; at the same time, fresh collagen gel is covered.

[0015] According to one embodiment of the present application, the model is assembled by adding 80 μl of collagen gel solution to each well of a 24-well Transwell system, polymerizing for 1 hour in a 37°C humidified incubator, and adding 50 μl of bacterial suspension to the surface of the gel; meanwhile, 40 μl of fresh collagen gel is overlaid.

[0016] According to one embodiment of the present application, the collagen gel matrix is prepared by diluting a rat type I collagen solution with 1x RPMI 1640 medium (pH 7.4) to a final concentration of 1.78 mg / ml.

[0017] According to one embodiment of the present application, the bacterial suspension is prepared by suspending a single colony after activation in RPMI 1640 medium, using a spectrophotometer to measure the absorbance at a wavelength of 620 nm, and adjusting the OD620 to 0.7±0.05; the bacterial suspension is diluted at a ratio of 1:200000 to obtain an inoculum with a final concentration of about 14 CFU / 25 μl.

[0018] According to one embodiment of the present application, the bacterial strain is activated by inoculating a frozen-preserved Staphylococcus aureus strain on an LB agar plate; the plate is incubated in a 37°C constant-temperature incubator for 12-18 hours for activation.

[0019] According to one embodiment of the present application, the plasma treatment is performed by adding 200 μl of mixed human plasma to each well after the model is constructed; 200 μl of the same plasma is added after 5 hours, and the model is observed using a fluorescence microscope after 24 hours to determine whether the model is successfully constructed.

[0020] A method for constructing a three-dimensional model of a Staphylococcus aureus abscess colony in vitro, comprising the following steps:

[0021] (1). Bacterial strain activation step:

[0022] A frozen-preserved Staphylococcus aureus strain is inoculated on an LB agar plate; the plate is incubated in a 37°C constant-temperature incubator for 12-18 hours for activation;

[0023] (2). Bacterial suspension preparation:

[0024] A single colony after activation is suspended in RPMI 1640 medium, and the absorbance is measured at a wavelength of 620 nm using a spectrophotometer; the OD620 is adjusted to 0.7±0.05; the bacterial suspension is diluted at a ratio of 1:200000 to obtain an inoculum with a final concentration of about 14 CFU / 25 μl;

[0025] (3). Collagen gel matrix preparation:

[0026] Rat collagen type I solution was taken according to the manufacturer's instructions; diluted with 1x RPMI 1640 medium (pH 7.4) to a final concentration of 1.78 mg / ml;

[0027] (4). Model assembly:

[0028] For the 24-well Transwell system, 80 μl of collagen gel solution was added to each well; polymerized in a 37℃ humidified incubator for 1 hour; 50 μl of bacterial suspension was added to the surface of the gel; at the same time, 40 μl of fresh collagen gel was covered;

[0029] (5). Plasma treatment:

[0030] After the model was completed, 200 μl of mixed human plasma was added to each well; 200 μl of the same plasma was added after 5 hours, and the model was observed for success or failure using a fluorescence microscope after 24 hours.

[0031] Beneficial effects:

[0032] For the study of the mechanism of Staphylococcus aureus infection, especially the dynamic process involving fibrin pseudo-capsule formation and degradation, it must be carried out in a system containing human cells and host factors (such as human thrombin and plasminogen). The existing preclinical animal models cannot fully evaluate these key interactions due to their inherent species limitations.

[0033] The present application provides a method for constructing an in vitro Staphylococcus aureus abscess colony three-dimensional model, which comprises the steps of activating the bacteria, preparing the bacterial suspension, preparing the collagen gel matrix, assembling the model, and treating the plasma. The model constructed by the method of the present application reproduces the fibrin pseudo-capsule structure, bacterial antibiotic resistance, and immune escape characteristics in vitro, providing a standardized experimental platform for studying the mechanism of chronic Staphylococcus aureus infection, evaluating the efficacy of antibiotic therapy and the efficiency of host immune clearance. It can be applied to the development of new anti-infective drugs, optimization of personalized treatment plans, and vaccine research and development. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a flow chart of the method for constructing an in vitro three-dimensional Staphylococcus aureus abscess model;

[0035] Figure 2 is a morphological feature diagram of an in vitro SAC;

[0036] Figure 3 is a fluorescence diagram of MRSA-CHERRY SAC constructed using red fluorescence, A is a bright field diagram of MRSA-CHERRY SAC constructed using white light, and B is a fluorescence diagram of MRSA-CHERRY SAC constructed using red light. DETAILED DESCRIPTION

[0037] In order to further illustrate the present application and its advantages, the technical solutions of the present application are further illustrated by specific embodiments below, and it should be understood that these embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application. Unless otherwise specified, the parts described in the present application are parts by weight, and the percentages described are mass percentages.

[0038] Example 1

[0039] The process of constructing a three-dimensional model of Staphylococcus aureus abscess in vitro is shown in Figure 1

[0040] ①Add a collagen solution with a concentration of 1.75 mg / ml (80 μl) to a 24-well filter plate, and supplement 500 μl of PBS at the bottom of the filter plate, then incubate at 37°C for 1 hour;

[0041] ②After 1 hour, add 50 μl of bacterial suspension to the well, then add 40 μl of collagen solution above the bacterial solution, followed by 200 μl of rat plasma, then polymerize in a 37°C humidified incubator for 5 hours;

[0042] ③After 5 hours, continue to use 200 μl of rat plasma;

[0043] ④After 24 hours, SAC will form between the two layers of collagen.

[0044] Example 2

[0045] Method for constructing a three-dimensional in vitro Staphylococcus aureus abscess colony model

[0046] (1). Bacterial activation step:

[0047] Inoculate the frozen Staphylococcus aureus strain into an LB agar plate; incubate in a 37°C incubator for 12-18 hours for activation.

[0048] (2). Preparation of bacterial suspension:

[0049] Take the activated single colony and suspend it in RPMI 1640 medium; use a spectrophotometer to measure the absorbance at 620 nm wavelength, and adjust the OD620 to 0.7±0.05; dilute the bacterial suspension at a ratio of 1:200000 to obtain an inoculum with a final concentration of about 14 CFU / 25 μl.

[0050] (3). Preparation of collagen gel matrix:

[0051] ​Rat type I collagen solution was taken according to the manufacturer's instructions; diluted with 1x RPMI 1640 medium (pH 7.4) to a final concentration of 1.78 mg / ml.

[0052] (4). Model assembly:

[0053] For the 24-well Transwell system (polyester membrane pore size 0.4 μm, purchased from Corning Life Sciences Corporation), 80 μl of collagen gel solution was added to each well; polymerized for 1 hour in a 37°C humidified incubator; 50 μl of bacterial suspension prepared in step (2) was added to the surface of the gel; at the same time, 40 μl of fresh collagen gel was covered;

[0054] (5). Plasma treatment:

[0055] After the model was completed, 200 μl of mixed human plasma was added to each well; 200 μl of the same plasma was added after 5 hours.

[0056] After 24 hours, the fluorescence microscope was used to observe as Figure 2 and 3 It is shown that the results indicate that the fibrous capsule is wrapped around MRSA (Staphylococcus aureus), and it can be seen that the in vitro model is successfully constructed.

[0057] Although the present application has been disclosed in the above with preferred embodiments, it is not intended to limit the present application. Those skilled in the art without departing from the spirit and scope of the present application can make various modifications and improvements. Therefore, the scope of protection of the present application shall be subject to the scope defined by the claims.

Claims

1. A method for constructing a three-dimensional model of Staphylococcus aureus abscess community in vitro, comprising the steps of strain activation, bacterial suspension preparation, collagen gel matrix preparation, model assembly and plasma treatment; The bacterial suspension preparation is to suspend the activated single colony in RPMI 1640 medium, and adjust the absorbance to 0.6-0.8 at 620 nm wavelength by using a spectrophotometer;The bacterial suspension is diluted to obtain an inoculum with a final concentration of about 13-15 CFU / 25 μl; The collagen gel matrix preparation is to dilute the rat type I collagen solution with 1×RPMI 1640 medium to a final concentration of 1.7-1.9 mg / ml; The model assembly is to add collagen gel solution in a 24-well Transwell system, polymerize for 1-2 hours in a 36-38℃ humidified incubator, and add bacterial suspension on the surface of the gel;Meanwhile, cover fresh collagen gel.

2. The method of claim 1, wherein, The model assembly is to add 80 μl of collagen gel solution in each well of a 24-well Transwell system, polymerize for 1 hour in a 37℃ humidified incubator, and add 50 μl of bacterial suspension on the surface of the gel;Meanwhile, cover 40 μl of collagen gel.

3. The method of claim 1, wherein, The collagen gel matrix preparation is to dilute the rat type I collagen solution with 1×RPMI 1640 medium to a final concentration of 1.78 mg / ml.

4. The method of claim 1, wherein, The bacterial suspension preparation is to suspend the activated single colony in RPMI 1640 medium, and adjust the absorbance to 0.7±0.05 at 620 nm wavelength by using a spectrophotometer;The bacterial suspension is diluted at a ratio of 1:200000 to obtain an inoculum with a final concentration of about 14 CFU / 25 μl.

5. The method according to any one of claims 1 to 4, characterized in that, The strain activation is to inoculate the frozen Staphylococcus aureus strain on an LB agar plate;Incubate in a 37℃ constant temperature incubator for 12-18 hours for activation.

6. The method according to any one of claims 1 to 4, wherein The plasma treatment is to add 200 μl of mixed human plasma to each well after the model is constructed;After 5 hours, add another 200 μl of the same plasma, and observe and determine whether the model is successfully constructed using a fluorescence microscope after 24 hours. 7.A method for constructing a three-dimensional model of Staphylococcus aureus abscess community in vitro, comprising the following steps: (1). Strain activation step: Inoculate the frozen Staphylococcus aureus strain on an LB agar plate;Incubate in a 37℃ constant temperature incubator for 12-18 hours for activation; (2). Bacterial suspension preparation: Suspend the activated single colony in RPMI 1640 medium, and adjust the absorbance to 0.7±0.05 at 620 nm wavelength by using a spectrophotometer;Dilute the bacterial suspension at a ratio of 1:200000 to obtain an inoculum with a final concentration of about 14 CFU / 25 μl; (3). Collagen gel matrix preparation: Take the rat type I collagen solution according to the manufacturer's instructions;Dilute with 1×RPMI 1640 medium to a final concentration of 1.78 mg / ml; (4). Model assembly: For a 24-well Transwell system, add 80 μl of collagen gel solution to each well; polymerize in a 37°C humidified incubator for 1 hour; add 50 μl of bacterial suspension to the gel surface; and cover with 40 μl of fresh collagen gel. (5) Plasma treatment: After the model was constructed, 200 μl of mixed human plasma was added to each well; 200 μl of the same plasma was added 5 hours later, and the success of the model construction was determined by fluorescence microscopy after 24 hours.

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