Co-culture method of methicillin-resistant staphylococcus aureus and pseudomonas aeruginosa

Abstract

The invention discloses a co-culture method of methicillin-resistant staphylococcus aureus and pseudomonas aeruginosa, and relates to the technical field of microbiology. Comprising the following steps: preparing materials: preparing methicillin-resistant staphylococcus aureus MRSA, pseudomonas aeruginosa PA and a culture medium; performing strain culture and activation, inoculating MRSA and PA, and performing subculture; preparing a bacterial suspension, and culturing MRSA and PA overnight; adding overnight culture bacterium liquid MRSA and PA into the culture medium, oscillating and uniformly mixing to prepare a bacterium suspension, and diluting for later use; the complete processes of strain preparation, bacterial suspension preparation, direct contact and indirect contact co-culture, drug sensitivity testing and the like are covered, and the interaction between MRSA and PA can be comprehensively researched; indirect contact co-culture is realized by adopting a Transwell insert, and a new method is provided for researching the interaction of two bacteria under the non-direct contact condition.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbiology, in particular to a co-culture method of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. BACKGROUND

[0002] Osteomyelitis is a destructive disease of bone tissue caused by pathogenic microorganisms. Due to the complexity and long-term abnormality, osteomyelitis is one of the refractory diseases in orthopedics, which poses a serious threat to the health and quality of life of patients. It can lead to permanent loss of function, even amputation and life-threatening. The treatment of osteomyelitis requires the comprehensive use of various measures, including appropriate antibiotic application, thorough debridement, elimination of dead space, and stability of fracture, etc. These measures are crucial for controlling infection and promoting the recovery of bone tissue. Most cases of osteomyelitis are caused by a single pathogen, however, polymicrobial infection plays an important role in some types of osteomyelitis. For example, about 1 / 3 of post-traumatic osteomyelitis cases, 6-21% of prosthetic joint infection cases and up to 83% of diabetic foot infection cases are caused by polymicrobial infection. Polymicrobial infection is more complex than single organism infection, with prolonged and poor treatment, bringing greater challenges to clinical treatment.

[0003] Among the polymicrobial infections of osteomyelitis, the mixed infection of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) is particularly common. MRSA is a pathogen with high drug resistance and virulence, its characteristics such as biofilm formation, osteocyte-lacunar canalicular network colonization and drug resistance make treatment extremely difficult. Pseudomonas aeruginosa (PA) is also known for its ability to form biofilms, and is also prone to antibiotic resistance, making infection difficult to treat and eradicate. Studies have shown that the mixed infection of MRSA and PA accounts for 16.7% of clinical cases, and the treatment of such mixed infection is more difficult, requiring more in-depth research to explore its mechanism and effective treatment strategies.

[0004] The existing technology often lacks a systematic method to evaluate the growth kinetics characteristics of MRSA and PA and their influence on drug sensitivity when studying the mixed infection of MRSA and PA. Therefore, the present application proposes a co-culture method for studying the interaction of MRSA and PA, aiming to in-depth explore the growth kinetics characteristics of the two bacteria when co-incubated through in vitro experiments, evaluate the minimum inhibitory concentration (MIC) difference of the two bacteria to commonly used antibacterial drugs, and determine whether the mixed infection will lead to the enhancement of bacterial drug resistance or the decrease of drug sensitivity, thereby providing a theoretical basis for the clinical treatment of mixed infection. SUMMARY

[0005] The object of the present application is to solve the problems existing in the prior art, and to provide a co-culture method of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

[0006] In order to achieve the above object, the present application adopts the following technical scheme:

[0007] The co-culture method of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa comprises the following steps:

[0008] S1: material preparation, preparing methicillin-resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa PA and culture medium;

[0009] S2: strain culture and activation, inoculating MRSA and PA, and subculturing;

[0010] S3: preparation of bacterial suspension, overnight culturing MRSA and PA; adding the overnight cultured bacterial solution MRSA and PA into the culture medium, shaking and mixing to prepare a bacterial suspension, and diluting for standby;

[0011] S4: verification of screening medium, using different culture media for MRSA bacterial solution and PA bacterial solution respectively, and equally dividing the culture medium plates; adding 10ul of bacterial suspension of different concentrations on the plates, repeating 3 times per zone, recording data after direct contact culture or indirect contact culture;

[0012] S5: drug sensitivity test.

[0013] Preferably, in S1, the culture medium comprises LB broth, LB nutrient agar, MH medium, mannitol high salt agar, pseudomonas agar base medium and sterile PBS.

[0014] Preferably, in S2, the specific method of strain culture and activation is:

[0015] Inoculating MRSA and PA in LB broth liquid medium and shaking culture; subculturing to the 2nd generation, recovering after culturing the strain to the 3rd generation; when culturing the 3rd generation strain, first diluting the bacterial suspension of the 2nd generation strain and inoculating it on the solid plate culture medium.

[0016] Preferably, in S2, the environmental temperature of shaking culture is controlled at 37℃, and the rotation speed of shaking culture is controlled at 200r·min －1 .

[0017] Preferably, in S3, the preparation of bacterial suspension is as follows:

[0018] After overnight culturing of MRSA and PA, the overnight cultured bacterial cultures of MRSA and PA were added to LB broth, thoroughly shaken and mixed to prepare bacterial suspensions, and then serially diluted 10-fold to prepare bacterial suspensions for later use.

[0019] Preferably, in step S3, the overnight culture method is as follows:

[0020] Bacteria from MRSA single-colony plates were picked and inoculated into LB broth medium, shaken well, and incubated at 37°C and 200 rpm. －1 Incubate overnight; pick bacteria from PA single-colony plates and inoculate them into LB broth medium, shake well, and incubate at 37°C and 200 rpm. －1 Overnight incubation.

[0021] Preferably, in step S4, the direct contact culture method is as follows:

[0022] Mixed bacterial suspensions were prepared by directly mixing MRSA and PA bacterial suspensions at three ratios: 1:1, 1:100, and 100:1; and then incubated at 37°C and 200 rpm. －1 Under the conditions of shaking culture, 100 μL of bacterial suspension was added to 900 μL of sterile PBS at time points of 0, 1, 3, 6, 12, 24, 36, and 48 hours, and then serially diluted 10-fold to obtain a series of bacterial suspensions with 8 concentrations. Two plates of each of the two selection media were used at each time point, and the media plates were divided into 4 equal zones. 10 μL of the 4 concentrations of bacterial suspension was added to one plate, and each zone was repeated 3 times. The bacterial concentration was recorded after the culture was completed.

[0023] Preferably, in step S4, the indirect contact culture method is as follows:

[0024] Experiments were conducted using Transwell inserts, in which bacteria shared a liquid culture medium while avoiding direct contact. Different concentrations of bacteria were added to the upper and lower chambers at ratios of 1:1, 1:100, and 100:1. 100 μL of bacterial culture was collected into 96-well plates at time points of 0, 1, 3, 6, 12, 24, 36, and 48 hours. LB broth was used as a blank control. The absorbance was measured at a wavelength of 600 nm, and the data were recorded.

[0025] Preferably, in step S5, the MIC values ​​of bacteria isolated from methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa are determined by co-culturing them at a ratio of 1:1 in broth for 24 h and 48 h using a micro-broth dilution method.

[0026] Preferably, in step S5, antibiotics are diluted to different concentrations, and 100 μL of antibiotic is added to each well of a 96-well plate. 100 μL of cultured bacterial solution is added to the well containing antibiotic. Growth control well MH + bacterial solution and blank control well MH are set up. Each group is repeated six times and cultured at 37°C for 24 hours. The lowest drug concentration at which no bacteria grow is the minimum inhibitory concentration of the drug for that bacterium.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This invention covers a complete process including strain preparation, bacterial suspension preparation, direct and indirect co-culture, and drug sensitivity testing, enabling a comprehensive study of the interaction between MRSA and PA.

[0029] 2. This invention uses Transwell inserts to achieve indirect contact co-culture, providing a new method for studying the interaction between two bacteria under non-direct contact conditions.

[0030] 3. This invention evaluates the impact of co-culture on bacterial drug resistance through drug susceptibility testing, providing a theoretical basis for clinical treatment and having practical application value.

[0031] 4. The experiments in this invention not only reveal the interaction mechanism between MRSA and PA, but also provide specific guidance for the development of clinical treatment plans; by evaluating the impact of co-culture on bacterial resistance, it helps to develop more effective treatment strategies to reduce the emergence of resistance. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the verification screening medium of the present invention;

[0033] Figure 2 This is a growth curve of the present invention cultured alone in vitro for 48 hours;

[0034] Figure 3 This is a growth curve of the present invention after 48 hours of in vitro direct contact mixed culture;

[0035] Figure 4 This is a growth curve of the in vitro indirect contact mixed culture of the present invention after 48 hours;

[0036] Figure 5 This is a comparative analysis diagram of in vitro indirect contact mixed culture according to the present invention;

[0037] Figure 6 This is a graph showing the MRSA drug susceptibility test results of the present invention;

[0038] Figure 7 This is a graph showing the PA drug susceptibility test results of the present invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0040] Example 1:

[0041] Methods for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, such as... Figures 1-7 As shown, it includes:

[0042] S1: Material Preparation

[0043] bacterial strains:

[0044] Methicillin-resistant Staphylococcus aureus (MRSA) [ATCCBAA-1717] and Pseudomonas aeruginosa (PA) [ATCC15692] were purchased from Baosai Biotechnology.

[0045] Instruments and reagents:

[0046] HCB-1300V clean bench, HDPF-150 electric thermostatic incubator, thermostatic shaker, LDZM-60KCS-Ⅱ vertical pressure steam sterilizer, full-function microplate reader, UPU ultrapure water production system, etc.; LB broth, LB nutrient agar, MH medium, mannitol high-salt agar, Pseudomonas agar basal medium, sterile PBS, etc.

[0047] S2: Microbial culture and activation

[0048] MRSA and PA were inoculated into LB broth liquid medium and incubated at 37°C and 200 rpm. －1 Shaking culture; subculture to the second generation, and the strain is revived after being cultured to the third generation; when culturing the third generation strain, the bacterial suspension of the second generation strain is first diluted and inoculated onto a solid agar plate to facilitate the picking of individual colonies;

[0049] S3: Preparation of bacterial suspension

[0050] Bacteria from MRSA single-colony plates were picked and inoculated into LB broth medium, shaken well, and incubated at 37°C and 200 rpm. －1 Incubate overnight; pick bacteria from PA single-colony plates and inoculate them into LB broth medium, shake well, and incubate at 37°C and 200 rpm. －1Overnight culture; add the overnight cultured bacterial suspensions MRSA and PA to an appropriate amount of LB broth, shake thoroughly to mix and prepare a bacterial suspension of a certain concentration (approximately 107 CFU / ml, OD600 = 0.06), and then serially dilute 10 times to prepare a bacterial suspension of approximately 105 CFU / ml for later use.

[0051] S4: Validation of the screening medium

[0052] MRSA and PA bacterial suspensions were prepared using one plate of each of the three culture media. The culture media plates were divided into four equal sections. 10 μL of bacterial suspensions of four different concentrations were added to one plate, and each section was repeated three times. All inoculated plates were incubated under suitable conditions.

[0053] direct contact culture

[0054] Mixed bacterial suspensions were prepared by directly mixing MRSA and PA bacterial suspensions at three ratios: 1:1, 1:100, and 100:1; incubation was carried out at 37℃ for 200 rpm. －1 Shaking culture was performed, and 100 μL of the bacterial suspension was added to 900 μL of sterile PBS at time points of 0, 1, 3, 6, 12, 24, 36, and 48 hours. The suspension was serially diluted 10-fold to obtain a series of bacterial suspensions with 8 concentrations. Two plates of each of the two selection media were used at each time point, and the media plates were divided into 4 equal zones. 10 μL of the 4 concentrations of bacterial suspension was added to one plate, and each zone was repeated 3 times. All inoculated plates were cultured under appropriate conditions, and the bacterial concentration was recorded after the culture was completed.

[0055] Indirect contact culture

[0056] Experiments were conducted using Transwell inserts, in which bacteria shared a liquid culture medium while avoiding direct contact. Different concentrations of bacteria were added to the upper and lower chambers at ratios of 1:1, 1:100, and 100:1. 100 μL of bacterial culture was collected into 96-well plates at time points of 0, 1, 3, 6, 12, 24, 36, and 48 hours. LB broth was used as a blank control. The absorbance was measured at 600 nm and the data were recorded.

[0057] S5: Drug sensitivity testing

[0058] According to the operational requirements and judgment criteria of the Clinical and Laboratory Standards Institute (CLSI, 2021), the microbroth dilution method was used to detect the MIC values ​​of bacteria isolated from a 1:1 co-culture of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa for 24 and 48 hours. The antibiotics corresponding to MRSA were: cefoxitin (screening), benzylpenicillin, ampicillin, oxacillin, cefuroxime, gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, clindamycin (inducing clindamycin resistance), azithromycin, clarithromycin, erythromycin, clindamycin, linezolid, dapoxetine, teicoplanin, vancomycin, tigecycline, rifampin, and trimethoprim / sulfamethoxazole. The antibiotics corresponding to Pseudomonas aeruginosa were: ticarcillin / clavulanic acid. Piperacillin / tazobactam, ceftazidime, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, amikacin, tobramycin, ciprofloxacin, levofloxacin, doxycycline, minocycline, tigecycline, colistin, trimethoprim / sulfamethoxazole; antibiotics were diluted to 128, 64, 32, 16…0.125 μg / mL, and 100 μL of antibiotic was added to each well of a 96-well plate. 100 μL of the cultured bacterial solution was added to the well containing the antibiotic. Growth control wells (MH + bacterial solution) and blank control wells (MH) were also set up. Six replicates were performed for each group, and the plates were incubated at 37°C for 24 h. MIC interpretation: The lowest drug concentration at which no bacterial growth occurs is the minimum inhibitory concentration (MIC) of the drug for that bacterium.

[0059] The results are as follows:

[0060] By constructing direct and indirect co-culture systems of MRSA and PA, the growth dynamics and interactions of the two bacteria over 48 hours were systematically evaluated. Under direct contact conditions, when the two bacteria were co-cultured at a 1:1 ratio, PA exhibited a stronger growth advantage, suggesting that PA may dominate in resource competition. Under indirect contact conditions, using Transwell chambers to isolate and culture the two bacteria, it was found that PA secretions significantly affected the growth of MRSA, indicating that PA may inhibit MRSA proliferation by secreting certain metabolites or toxins. Analyzing the changes in the antibiotic sensitivity of MRSA and PA at different time points can provide important reference information for clinical treatment, helping doctors to develop more rational treatment plans, improve treatment efficacy, and reduce the development of drug resistance. These results provide experimental evidence for further elucidating the interaction mechanism between the two bacteria.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, characterized by, It comprises the following steps: S1: material preparation, prepare methicillin-resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa PA and culture medium; S2: strain culture and activation, inoculate MRSA and PA, and subculture; S3: bacterial suspension preparation, overnight culture MRSA and PA; add the overnight cultured MRSA and PA liquid to the culture medium, shake and mix to prepare a bacterial suspension, and dilute for standby; S4: verification of screening medium, use different culture media for MRSA and PA liquid respectively, and divide the medium plates into equal zones; add 10ul of bacterial suspension of different concentrations to the plates, repeat 3 times per zone, record the data after direct contact culture or indirect contact culture; S5: drug sensitivity test.

2. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 1, characterized by, In S1, the culture medium includes LB broth, LB nutrient agar, MH medium, mannitol high salt agar, pseudomonas agar base medium, and sterile PBS.

3. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 2, characterized by, In S2, the specific method of strain culture and activation is as follows: Inoculate MRSA and PA in LB broth liquid medium and shake culture; subculture to the 2nd generation, and recover the strain culture to the 3rd generation; when culturing the 3rd generation strain, dilute the bacterial suspension of the 2nd generation strain and inoculate it into the solid plate culture medium.

4. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 3, characterized by, In the S2, the environmental temperature of the oscillation culture is controlled at 37℃, and the rotation speed of the oscillation culture is controlled at 200 r·min －1 .

5. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 3, characterized by, In S3, the bacterial suspension preparation is as follows: After overnight culture of MRSA and PA, add the overnight cultured MRSA and PA liquid to LB broth, shake and mix thoroughly to prepare a bacterial suspension, and then prepare a 10-fold gradient dilution of the bacterial suspension for standby.

6. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 5, characterized by, In S3, the overnight culture method is as follows: Bacteria from MRSA single-colony plates were picked and inoculated into LB broth medium, shaken well, and incubated at 37°C and 200 rpm. －1 Incubate overnight; pick bacteria from PA single-colony plates and inoculate them into LB broth medium, shake well, and incubate at 37°C and 200 rpm. －1 Overnight incubation.

7. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 1, characterized by, In S4, the direct contact culture method is as follows: The mixed bacterial suspension was prepared by directly mixing MRSA and PA bacterial suspension at a ratio of 1:1, 1:100 and 100:

1. The bacterial suspension was cultured at 37°C and 200 r·min-1, and 100 ul of the bacterial suspension was taken at 0, 1, 3, 6, 12, 24, 36 and 48 hours and added to 900 ul of sterile PBS. The bacterial suspension was diluted by 10 times in sequence to obtain 8 concentrations of bacterial solution. Two pieces of each of the two kinds of screening medium were used at each time point, and the medium plates were equally divided into 4 regions. 10 ul of the bacterial suspension at 4 concentrations was added to one plate, and each region was repeated 3 times. The bacterial concentration was recorded after the culture ended. －1 The mixed bacterial suspension was prepared by directly mixing MRSA and PA bacterial suspension at a ratio of 1:1, 1:100 and 100:

1. The bacterial suspension was cultured at 37°C and 200 r·min-1, and 100 ul of the bacterial suspension was taken at 0, 1, 3, 6, 12, 24, 36 and 48 hours and added to 900 ul of sterile PBS. The bacterial suspension was diluted by 10 times in sequence to obtain 8 concentrations of bacterial solution. Two pieces of each of the two kinds of screening medium were used at each time point, and the medium plates were equally divided into 4 regions. 10 ul of the bacterial suspension at 4 concentrations 8. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 1, characterized by, In S4, the indirect contact culture method is as follows: Use Transwell inserts for the experiment, which share liquid culture medium while avoiding direct contact; add different concentrations of bacteria in a ratio of 1:1, 1:100, and 100:1 to the upper and lower chambers, take 100ul of bacterial liquid to a 96-well plate at time points of 0, 1, 3, 6, 12, 24, 36, and 48 hours, use LB broth as a blank control liquid, measure the absorbance at 600nm wavelength, and record the data.

9. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 1, characterized by, In S5, use the micro-broth dilution method to detect the MIC value of antibiotics for methicillin-resistant Staphylococcus aureus: Pseudomonas aeruginosa = 1:1 co-culture for 24h and 48h.

10. The method for co-culturing methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa according to claim 9, characterized by, In S5, dilute the antibiotics to different concentrations, add 100ul of antibiotics to each well of the 96-well plate, add 100ul of cultured bacterial liquid to the wells containing antibiotics, set growth control wells MH+ bacterial liquid and blank control wells MH, do six repeats for each group, and culture at 37℃ for 24h; the lowest drug concentration without bacterial growth is the minimum inhibitory concentration of the bacteria to the drug.

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