Method for culturing infectious bacteria in ascites

Abstract

The invention discloses a method for culturing infectious bacteria in ascites. Compared with a traditional culture method, the culture method is high in positive rate, and the ascites culture positive rate can be increased to 30% from 16%; meanwhile, the culture method disclosed by the invention is short in detection time and stable in detection time; according to the method, immediate bedside inoculation is not needed, and 1-2 hours of specimen transfer time can be reserved; and meanwhile, the economic cost is also reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for culturing bacteria that infect ascites. Background Technology

[0002] Spontaneous bacterial peritonitis (SBP) is one of the common sites of infection and complications in patients with end-stage liver diseases such as cirrhosis. It is also a major cause of death in patients with cirrhosis. Early diagnosis and timely and appropriate antibacterial treatment are crucial to improving the survival rate of patients with this disease.

[0003] Microbiological diagnosis of bacterial infection in ascites of patients with cirrhosis is based on ascites culture. Currently, multiple medical practice guidelines recommend that for all newly diagnosed ascites patients and those suspected of having abdominal infection, ascites fluid (at least 10 mL) should be aspirated at the bedside and directly injected into blood culture bottles (aerobic + anaerobic) before antibiotic administration and sent to an incubator for culture. However, domestic and international guidelines or consensus on ascites management have concluded that the positive rate of ascites culture is as low as 5%–20%, especially after antibiotic use, the positive rate further decreases. Even in some patients, the PMN in the ascites fluid is ≥250 cells / mm³. 3 However, culture results can still be negative. Furthermore, this method is time-consuming; after a positive culture alarm, agar plate subculturing is required, followed by pathogen identification and drug sensitivity testing, averaging 4-7 days. Additionally, this method has high requirements for specimen collection; after obtaining ascites specimens, bedside blood culture bottles must be inoculated immediately.

[0004] Metagenomic next-generation sequencing (mNGS) is a new technology applied in the diagnosis of infectious diseases in recent years. Compared with culture methods, mNGS has advantages such as broad pathogen coverage, high timeliness, and the ability to provide predictive analysis of antibiotic resistance, thus better guiding treatment and prognosis. Multiple studies on abdominal infections have shown that mNGS plays an important role in screening rare pathogens and improving detection efficiency. However, ascites fluid itself contains low levels of bacteria, and sequencing technology itself has extremely high sensitivity, capable of detecting very low concentrations of DNA. This easily leads to cross-contamination originating from non-sample DNA and between samples. Secondly, the DNA detected by sequencing is not necessarily from live bacteria; it may be generated by circulating immune cells during bacterial translocation, or bacteria may be killed by leukocyte attraction or regulatory mechanisms in the ascites fluid, while the DNA of dead bacteria can still be detected. Therefore, it is impossible to directly determine whether the sequence detected by sequencing comes from bacterial DNA or from the bacteria themselves. Furthermore, this technology is expensive, and not all patients can afford it. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for infecting ascites fluid with bacteria that is time-efficient, has a high positive culture rate, saves labor costs, and reduces economic costs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a method for culturing bacteria infected in ascites fluid.

[0008] Furthermore, the method includes the following steps:

[0009] (1) Centrifuge the ascites sample, discard the supernatant, and resuspend the precipitate in PBS;

[0010] (2) Centrifuge the above mixture, discard the supernatant, add Triton solution, and mix well;

[0011] (3) Centrifuge again, discard part of the supernatant, resuspend the precipitate using the remaining supernatant, and incubate the precipitate mixture on blood plates and chocolate plates.

[0012] Furthermore, the method also includes the step of adding triton solution, mixing well, and then allowing it to stand.

[0013] Furthermore, the settling time is 1 min to 10 min.

[0014] Preferably, the settling time is 1 minute.

[0015] Furthermore, the specific centrifugation conditions are 8000 rpm for 3 minutes.

[0016] Furthermore, the amount of PBS used is 1 mL.

[0017] Furthermore, the amount of the triton solution used is 1 mL.

[0018] Furthermore, the concentration of the triton solution is 0.05% to 1.0%.

[0019] Preferably, the concentration of the triton solution is 0.05% to 0.5%.

[0020] More preferably, the concentration of the triton solution is 0.05%.

[0021] Furthermore, the amount of the remaining supernatant in step (3) is 200 μL.

[0022] Furthermore, the amount of the precipitate mixture aspirated onto the blood plate and chocolate plate is 100 μL, respectively;

[0023] Furthermore, the culture temperature is 37°C.

[0024] Furthermore, the culture time is 18h~24h.

[0025] The Triton solution described in this invention is Triton X-100, which is a nonionic surfactant. The amount of Triton solution used in this invention is not limited; its amount can be easily determined by those skilled in the art for the purposes of this invention.

[0026] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0027] Furthermore, the ascites samples were obtained from patients with cirrhosis or liver cancer.

[0028] Preferably, the ascites sample is derived from a patient with cirrhosis.

[0029] This invention addresses situations where antibiotics have been administered before culture. After centrifuging 5-10 mL of ascites fluid, the fluid is washed with PBS to remove the antibiotics. To address the presence of leukocytes phagocytosing bacteria in the ascites fluid, this study uses 0.5% Triton solution to lyse the cells and release the phagocytosed bacteria. To address the issue that some peritoneal infection pathogens in ascites fluid cannot grow in vitro on general culture media, this study uses nutrient-rich chocolate agar plates and blood agar plates for ascites fluid culture. This invention innovatively discovers that applying the above methods can significantly improve the positive rate of ascites fluid culture. Furthermore, this study uses an overnight agar plate culture method, which shortens the detection time to 2 days and ensures stable detection.

[0030] A second aspect of the present invention provides a method for identifying the types of bacteria infecting ascites.

[0031] Furthermore, the method includes culturing infectious bacteria in ascites fluid using the method described in the first aspect of the present invention; and identifying the bacterial colonies cultured.

[0032] Furthermore, the methods for identifying the bacterial strains include morphological identification, biochemical identification, serological identification, molecular biological identification, and / or BIOLOG automated identification system.

[0033] Furthermore, the molecular biological identification includes PCR technology and nucleic acid hybridization.

[0034] Furthermore, the PCR technique includes sequence alignment using 16S rRNA.

[0035] In this invention, the morphological identification includes observing the morphology (cocci, bacilli, etc.), size, and arrangement of bacteria through staining (such as Gram staining) and microscopy; it also includes observing the phenotypic characteristics of colonies on solid culture media, such as shape, color, and edge.

[0036] In this invention, the biochemical identification refers to detecting the ability of bacteria to utilize substrates through metabolic reactions (such as sugar fermentation test, oxidase test), and the metabolites of different bacterial species have specificity.

[0037] In this invention, the serological identification refers to the identification of bacterial species using antigen-antibody reactions (such as agglutination tests), which has high specificity and is often used for rapid screening of pathogens.

[0038] In this invention, the molecular biological identification includes PCR technology and nucleic acid hybridization; the PCR technology refers to amplifying a specific gene (such as 16S rRNA) for sequence alignment, and is suitable for difficult-to-culture bacterial species. The nucleic acid hybridization refers to confirming the bacterial species by the binding of a probe to the target DNA.

[0039] In this invention, the BIOLOG system is an automated identification system that generates fingerprint profiles based on the metabolic patterns of microorganisms to different carbon sources, thereby enabling rapid identification.

[0040] In this invention, the method further includes a separation and purification step before strain identification, wherein the separation and purification includes streak plating and dilution plating.

[0041] In this invention, the above methods for identifying microbial strains can be flexibly combined and used according to the characteristics of the microbial strains and experimental requirements.

[0042] The third aspect of the present invention provides the application of the method described in the first aspect of the present invention in identifying the types of bacteria infecting ascites.

[0043] The fourth aspect of the present invention provides the application of the method described in the first aspect of the present invention or the method described in the second aspect of the present invention in diagnosing the types of bacterial infections in ascites of patients.

[0044] Preferably, the patients include patients with cirrhosis or liver cancer.

[0045] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0046] In the description of this invention, the term "and / or" includes all and any combination of one or more of the associated listed items.

[0047] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0048] Advantages and beneficial effects of the present invention:

[0049] 1) High positive rate: Compared with traditional culture methods, the method of the present invention can increase the positive rate of ascites culture from 16% to 30%;

[0050] 2) Detection time is shortened and stabilized: The average detection time of traditional culture methods is 4-7 days, while the method of this invention can be shortened to 48 hours;

[0051] 3) Saves labor costs: The method of this invention does not require immediate bedside inoculation, allowing 1-2 hours for specimen transport.

[0052] 4) Reduced economic costs: A set of blood culture bottles costs about 270 yuan using traditional culture methods. The method of this invention uses commonly used laboratory reagents, which significantly reduces costs. Attached Figure Description

[0053] Figure 1 The figure shows the effect of different membrane breaking concentrations and breaking times on the culture positivity rate. Detailed Implementation

[0054] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the following embodiments. The invention provides a general and / or specific description of the materials and methods used in the experiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, the reagents, biological materials, etc., used in the following embodiments are commercially available.

[0055] Example

[0056] 1. Experimental materials

[0057] PBS, Triton X100, blood agar plates, chocolate agar plates;

[0058] Sample source: Patients with cirrhosis and ascites who were hospitalized in the Department of Hepatology, Department of Integrated Traditional Chinese and Western Medicine, and Intensive Care Unit of Beijing Ditan Hospital, affiliated with Capital Medical University, from December 2024 to March 2025.

[0059] Inclusion criteria: (1) Age greater than 18 years; (2) Liver cirrhosis; (3) Moderate to large amount of ascites (ascites depth > 3cm); (4) Planned abdominal catheterization after assessment by the attending physician; (5) Signed informed consent form;

[0060] Exclusion criteria: (1) Infection, perforation, trauma, etc. of intra-abdominal organs; (2) Peritoneal dialysis patients; (3) Ascites caused by other reasons, such as renal ascites, cardiac ascites, pancreatic ascites; (4) Uncontrolled tumors; (5) Contraindications to abdominal paracentesis; (6) Pregnancy.

[0061] 2. Experimental Methods

[0062] 1) Collect 5-10 mL of ascites fluid from patients with cirrhosis, centrifuge at 8000 rpm for 3 min; discard the supernatant, add 1 mL of PBS, and resuspend the precipitate;

[0063] 2) Centrifuge at 8000 rpm for 3 min, discard the supernatant, add 1 mL of 0.5% Triton solution, mix by pipetting, and let stand for 1 min;

[0064] 3) Centrifuge again at 8000 rpm for 3 min, discard 800 μL of supernatant, resuspend the precipitate in the remaining 200 μL of supernatant, and aspirate 100 μL of each onto blood agar plates and chocolate agar plates for plating. Then incubate the plates overnight in a 37°C incubator.

[0065] 4) On the second day, if colony growth is observed on the plate, observe the colony morphology and pick 4-6 single bacteria for incubation in BHI liquid medium. After 4-6 hours, aspirate the bacterial solution for PCR and send it to a 16S primer for species identification. PCR steps: Aspirate 200 μL of bacterial solution, centrifuge at 10,000 rpm for 2 min, and discard the supernatant; resuspend in 40 μL of sterile, enzyme-free water; lyse at 99℃ for 10 min, centrifuge at 10,000 rpm for 1 min, and use the supernatant as a DNA template; perform PCR using 16S primers, and send the product to a 16S primer for species identification. Store the remaining bacterial solution in glycerol at -80℃.

[0066] 3. Effects of different concentrations of Triton solution and membrane breaking time on the results

[0067] To explore whether different concentrations of Triton solution and the incubation time after membrane disruption would affect the positive rate of culture, we evaluated the effects of 0.05%, 0.1%, 0.5%, and 1.0% concentrations of Triton solution and the incubation time on the positive rate.

[0068] 4. Experimental results compared with traditional culture methods

[0069] This study included 124 patients with cirrhosis. The positive bacterial cultures from their ascites are shown in Table 1. Using the improved culture method of this invention, 38 bacteria were cultured, with a positive rate of 38 / 124 = 30.6%. Using the traditional culture method, 20 bacteria were cultured, with a positive rate of 20 / 124 = 16.1%. It is evident that the culture method of this invention can improve the positive rate and shorten the detection time (the average detection time for the traditional culture method is 4-7 days, while in this study it can be shortened to 48 hours).

[0070] Table 1 Experimental Results

[0071] 5. Results of the effect of different concentrations of Triton solution and membrane rupture standing time on the positive rate

[0072] The effects of different concentrations of Triton solution and membrane permeation time on the positive rate are shown in Table 2 and... Figure 1 As shown, Group 1 (Triton concentration 0.05%, membrane rupture time 1 min) had the highest positive rate, which was statistically different from Group 4 (Triton concentration 1.0%, membrane rupture time 1 min) (84.6% vs 38.5%, P=0.016), suggesting that lower Triton concentrations result in higher positive rates. Furthermore, the positive rate of Group 1 was statistically different from that of Group 5 (Triton concentration 0.05%, membrane rupture time 10 min), suggesting that shorter membrane rupture times result in higher positive rates.

[0073] Table 2. Effects of different concentrations of Triton solution and membrane breakage time on the positive rate.

[0074] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the technical methods of the present invention. Any technical method that can be implemented based on the above embodiments without creative effort should be considered to fall within the scope of protection of the present invention. Furthermore, various different embodiments of the present invention can be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed by the present invention.

Claims

1. A method for culturing bacteria in ascites fluid, characterized in that, The method includes the following steps: (1) Centrifuge the ascites sample, discard the supernatant, and resuspend the precipitate in PBS; (2) Centrifuge the above mixture, discard the supernatant, add Triton solution, and mix well; (3) Centrifuge again, discard part of the supernatant, resuspend the precipitate using the remaining supernatant, and incubate the precipitate mixture on blood plates and chocolate plates.

2. The method according to claim 1, characterized in that, The method also includes the step of adding Triton solution, mixing well, and then letting it stand. Preferably, the settling time is 1 min to 10 min; More preferably, the settling time is 1 minute.

3. The method according to claim 1, characterized in that, The specific centrifugation conditions were 8000 rpm for 3 minutes. Preferably, the amount of PBS used is 1 mL; Preferably, the amount of the triton solution used is 1 mL; Preferably, the concentration of the triton solution is 0.05% to 1.0%; Preferably, the concentration of the triton solution is 0.05% to 0.5%; More preferably, the concentration of the triton solution is 0.05%.

4. The method according to any one of claims 1-3, characterized in that, The amount of supernatant remaining in step (3) is 200 μL; Preferably, the amount of the precipitate mixture aspirated onto the blood plate and chocolate plate is 100 μL, respectively; Preferably, the culture temperature is 37°C; Preferably, the culture time is 18h to 24h.

5. The method according to claim 1, characterized in that, The ascites samples were obtained from patients with cirrhosis or liver cancer. Preferably, the ascites sample is derived from a patient with cirrhosis.

6. A method for identifying the types of bacteria causing infection in ascites, characterized in that, The method includes culturing infectious bacteria in ascites fluid using the method described in any one of claims 1-5; and identifying the bacterial colonies cultured.

7. The method according to claim 6, characterized in that, The methods for identifying the strains include morphological identification, biochemical identification, serological identification, molecular biological identification, and / or BIOLOG automated identification system; Preferably, the molecular biological identification includes PCR technology and nucleic acid hybridization.

8. The method according to claim 7, characterized in that, The PCR technique includes sequence alignment using 16S rRNA.

9. The application of the method according to any one of claims 1-5 in identifying the types of bacteria causing infection in ascites.

10. The use of the method according to any one of claims 1-5 or the method according to any one of claims 6-8 in diagnosing the type of bacterial infection in ascites of patients; Preferably, the patients include patients with cirrhosis or liver cancer.

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