Novel bacterial culture method
By covering agar medium with a specific formula on the surface of ex vivo tissue, the problem of low positive rate of bacterial culture in patients with osteomyelitis is solved, the diagnostic accuracy and treatment effect are improved, and the production of drug-resistant bacteria is reduced.
Patent Information
- Application Number
- CN202510683520.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-15
AI Technical Summary
In the prior art, the positive rate of bacterial culture in patients with osteomyelitis is low, which affects diagnosis and treatment, and improper use of antibiotics leads to the production of drug-resistant bacteria.
The ex vivo tissue was covered with agar medium containing glycerol, coconut water, MgSO4, FeCl3, CaCl2 and lecithin, simulated the in vivo growth environment and improved the success rate of bacterial culture.
It significantly improves the positive rate of bacterial culture in patients with osteomyelitis, shortens the culture time, reduces the difficulty of clinical diagnosis and treatment, and reduces the abuse of antibiotics and the production of drug-resistant bacteria.
Smart Images
Figure CN120485060A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a novel bacterial culture method. Background Art
[0002] Chronic osteomyelitis is an inflammatory disease primarily characterized by bone destruction caused by bacterial infection. Although the discovery of antibiotics has cured some cases of osteomyelitis, a significant number of patients still experience recurrent attacks and persistent disease. Studies have shown that the recurrence rate of chronic osteomyelitis is as high as 20-30%. In recent years, with the development of society and the increase in life expectancy, the incidence of implant-related and diabetes-related osteomyelitis has increased annually. Numerous implants, such as plates, intramedullary nails, and prostheses, are used in orthopedic surgery each year. Literature indicates that the incidence of postoperative infection after implant surgery for closed fractures is 1-2%, while the incidence for open fractures is as high as 30%. The incidence rate among patients with diabetes is also increasing annually. Bone infection increases the disease's complications and mortality rate, and the high cost of treatment places an economic burden on families and society.
[0003] The vast majority of osteomyelitis cases are primarily caused by bacterial infections. If the causative bacteria can be identified, the diagnosis is established, and antibiotic treatment targeting that bacteria is theoretically curative. However, due to numerous factors, such as the use of antibiotics, the inability to obtain specimens containing bacteria, the bacterial death caused by prolonged tissue inactivation, and varying bacterial culture methods, the overall bacterial culture positivity rate is low, hindering both diagnosis and treatment. Currently, there are few methods to improve the bacterial culture positivity rate in osteomyelitis patients. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for improving the positive rate of bacterial culture, avoiding the abuse of antibiotics and reducing the generation of drug-resistant bacteria.
[0005] The present invention provides a method for improving the positive rate of bacterial culture, which comprises the following steps: The ex vivo tissue is spread on the surface of an agar culture medium, and then a layer of melted agar culture medium is poured on the surface of the ex vivo tissue to completely cover the ex vivo tissue for culturing; the agar culture medium is composed of a basal culture medium, 0.5wt% to 2wt% of glycerol, 5v / v% to 10v / v% of coconut water, 0.01 to 0.1mol / L of MgSO4, 0.01 to 0.1mol / L of FeCl3, 0.1 to 1mol / L of CaCl2, and 0.2 to 2.0g / L of lecithin.
[0006] Preferably, the agar medium consists of a basal medium, 0.5wt%~1wt% of glycerol, 5v / v%~6v / v% of coconut water, 0.02~0.05mol / L of MgSO4, 0.02~0.05mol / L of FeCl3, 0.5~1mol / L of CaCl2 and 0.5~1.0g / L of lecithin.
[0007] Preferably, the basal culture medium is any one of TSA trypticase soy agar medium, BHIA brain heart infusion agar medium, LB medium and MH Mueller-Hinton medium.
[0008] Preferably, the temperature of the melted agar medium is 40°C to 45°C.
[0009] Preferably, the culture condition is 32° C. to 37° C.; and the culture time is 24 to 48 hours.
[0010] Preferably, the ex vivo tissue is from a patient with osteomyelitis.
[0011] Preferably, the ex vivo tissue is infected bone marrow, infected cortical bone, pus at an abscess site, or abscess wall tissue.
[0012] Preferably, the bacteria include one or more of Staphylococcus aureus, coagulase-negative Staphylococcus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterococcus, Proteus and Mycobacterium.
[0013] Beneficial effects of the present invention: The agar culture medium of the present invention is compounded with a variety of nutrients and growth factors. The addition of glycerol can stabilize the properties of the culture medium, has a certain moisturizing effect, and can also provide a certain carbon source, which is conducive to the growth and metabolism of bacteria. Coconut water is rich in various nutrients, such as sugars, amino acids, minerals, etc., which provides abundant natural nutrition for bacteria and is conducive to promoting the growth and reproduction of bacteria. The addition of MgSO4, FeCl3, and CaCl2 can meet the demand for trace elements in the bacterial growth process, regulate the ionic strength and acid-base balance of the culture medium, and create a good chemical environment for bacterial growth. The addition of lecithin may help simulate the bacterial growth environment in the body, promote bacterial growth and metabolism, and improve the success rate and positive rate of culture.
[0014] The present invention is compatible with a variety of commonly used basal culture media, and suitable basal culture media can be selected according to different bacterial species and culture requirements, thereby enhancing the applicability and flexibility of the method and being widely applicable to the culture of a variety of bacteria.
[0015] The present invention cultures in vitro tissues such as infected bone marrow, infected cortical bone, pus at the abscess site, or abscess wall tissue, and can more directly obtain information about pathogenic bacteria at the infection site. This is of great significance for the etiological diagnosis and formulation of treatment plans for osteomyelitis, and helps to improve the treatment effect and prognosis of osteomyelitis.
[0016] The method provided by the present invention can improve the positive rate of bacterial culture in patients with osteomyelitis, and to a certain extent reduce the difficulty of clinical diagnosis and treatment of osteomyelitis. After screening the bacteria infected by patients with osteomyelitis, drug sensitivity tests can be performed on pathogenic bacteria, thereby improving the cure rate, avoiding the abuse of antibiotics, and reducing the generation of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0018] Figure 1 This is a flow chart of a method for culturing a novel bacterium in Example 1 of the present invention.
[0019] Figure 2 Schematic diagram of in vitro tissue culture from patients with osteomyelitis. DETAILED DESCRIPTION
[0020] In order to further illustrate the present invention, a novel bacterial culture method provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0021] Unless otherwise specified, the substances used in the examples of the present invention are conventional commercially available products.
[0022] Example 1 according to Figure 1 The process shown is for culturing tissue isolated from patients with osteomyelitis.
[0023] TSA trypticase soy peptone agar medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.6wt%, coconut water 5v / v%, MgSO40.03mol / L, FeCl30.03mol / L, CaCl20.5mol / L, and lecithin 0.8g / L.
[0024] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 35°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0025] Example 2 LB medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.8wt%, coconut water 5v / v%, MgSO4 0.04mol / L, FeCl3 0.02mol / L, CaCl2 0.5mol / L, and lecithin 0.8g / L.
[0026] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 37°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0027] Example 3 BHIA brain heart infusion agar medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.5wt%, coconut water 6v / v%, MgSO4 0.02mol / L, FeCl3 0.05mol / L, CaCl2 0.6mol / L, and lecithin 1.0g / L.
[0028] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 35°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0029] Example 4 MH Mueller-Hinton medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.8wt%, coconut water 6v / v%, MgSO40.05mol / L, FeCl30.05mol / L, CaCl21mol / L, and lecithin 0.5g / L.
[0030] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 35°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0031] Comparative Example 1 The difference from Example 1 is that agar culture medium is not poured on the surface of the isolated tissue, specifically as follows: TSA trypticase soy peptone agar medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.6wt%, coconut water 5v / v%, MgSO40.03mol / L, FeCl30.03mol / L, CaCl20.5mol / L, and lecithin 0.8g / L.
[0032] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. The culture medium is then covered and incubated at 35°C in a bacterial incubator. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0033] Comparative Example 2 The difference from Example 1 is that only basal culture medium is used for culture, which is as follows: Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on TSA trypticase soy agar. A layer of TSA medium melted to 45°C is then poured over the ex vivo tissue, completely covering the tissue. The culture is then incubated at 35°C. The medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0034] Comparative Example 3 The difference from Example 1 is that glycerol is not used in the culture medium, specifically as follows: TSA trypticase soy peptone agar medium was used as the basal culture medium, and the final concentrations of each substance in the prepared agar medium were: coconut water 5v / v%, MgSO4 0.03mol / L, FeCl3 0.03mol / L, CaCl2 0.5mol / L, and lecithin 0.8g / L.
[0035] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 35°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0036] Comparative Example 4 The difference from Example 1 is that coconut water is not added to the culture medium, specifically as follows: TSA trypticase soy peptone agar medium was used as the basal medium, and the final concentrations of each substance in the prepared agar medium were: glycerol 0.6wt%, MgSO4 0.03mol / L, FeCl3 0.03mol / L, CaCl2 0.5mol / L, and lecithin 0.8g / L.
[0037] Infected cortical bone from patients with osteomyelitis is used as ex vivo tissue and plated on an agar culture medium. A layer of agar culture medium melted to 45°C is then poured over the ex vivo tissue to completely cover the tissue. The culture medium is then incubated at 35°C. The culture medium is regularly observed for the appearance of colonies. If colonies appear, their morphology, size, color, and other characteristics are recorded. Typical colonies are selected from the culture medium for purification, and 16S rRNA sequencing is used to determine the bacterial species and whether they are pathogenic.
[0038] Test Example 1 The time for obtaining colonies in Examples 1 to 4 and Comparative Examples 1 to 4 was counted, and the positive rate of bacterial culture was calculated. The results are shown in Table 1.
[0039] Positive rate = number of positive samples / total number of samples × 100% Table 1 Bacterial culture positive rate results
[0040] As shown in Table 1, compared to the comparative example, the incubation time of Examples 1 to 4 is significantly reduced, indicating that the incubation time can be effectively shortened using the method provided by the present invention to cultivate, thereby improving bacterial culture efficiency. Compared to comparative example 1, the bacterial culture positive rate of embodiment has been significantly improved, indicating that the present invention can effectively improve bacterial culture positive rate by pouring agar culture medium on the surface of in vitro tissue after melting, and the present invention can cover in vitro tissue after pouring agar culture medium on the surface of in vitro tissue, thereby reducing the risk of in vitro tissue dehydration, and providing a good growth environment for bacteria. The bacterial culture positive rate of comparative example 3 and comparative example 4 is significantly higher than comparative example 2, but lower than embodiment 1, indicating that the addition of glycerol and coconut water is conducive to promoting bacterial growth, thereby improving positive rate, and the two are more effective after synergistic action.
[0041] Test Example 2 The pathogenic bacteria species cultured in the examples were counted, and the absolute abundance of each pathogenic bacteria in each example (the number of occurrences of each pathogenic bacteria species in all samples) was counted as shown in Table 2.
[0042] Table 2 Types of pathogenic bacteria
[0043] As shown in Table 2, the pathogenic bacteria that infect patients with osteomyelitis mainly include Staphylococcus aureus, coagulase-negative Staphylococci, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterococci, Proteus and Mycobacterium.
[0044] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A method for improving the positive rate of bacterial culture, characterized in that: The method comprises the following steps: The ex vivo tissue is spread on the surface of the agar culture medium, and then a layer of melted agar culture medium is poured on the surface of the ex vivo tissue to completely cover the ex vivo tissue for culture; The agar medium consists of a basic medium, 0.5wt%-2wt% of glycerol, 5v / v%-10v / v% of coconut water, 0.01-0.1mol / L of MgSO4, 0.01-0.1mol / L of FeCl3, 0.1-1mol / L of CaCl2, and 0.2-2.0g / L of lecithin.
2. The method according to claim 1, characterized in that The agar medium consists of a basic medium, 0.5wt%~1wt% of glycerol, 5v / v%~6v / v% of coconut water, 0.02~0.05mol / L of MgSO4, 0.02~0.05mol / L of FeCl3, 0.5~1mol / L of CaCl2 and 0.5~1.0g / L of lecithin.
3. The method according to claim 1, characterized in that The basic culture medium is any one of TSA trypticase soy peptone agar medium, BHIA brain heart infusion agar medium, LB medium and MH Mueller-Hinton medium.
4. The method according to claim 1, wherein The temperature of the melted agar medium is 40°C to 45°C.
5. The method according to claim 1, wherein The culture condition is 32° C. to 37° C.; and the culture time is 24 to 48 hours.
6. The method according to claim 1, characterized in that The ex vivo tissue is from a patient with osteomyelitis.
7. The method according to claim 1, characterized in that The ex vivo tissue is infected bone marrow, infected bone cortex, pus at the abscess site or abscess wall tissue.
8. The method according to any one of claims 1 to 7, characterized in that The bacteria include one or more of Staphylococcus aureus, coagulase-negative Staphylococcus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterococcus, Proteus and Mycobacterium.