A method for separating and extracting hafnia meliess by using penicillin and vancomycin

Abstract

This invention discloses a method for isolating and extracting *Havnia vesicularis* using penicillin and vancomycin. The method mainly includes the following steps: inclusion and screening of patients with bipolar disorder; collection and processing of feces from these patients; isolation and treatment of *Havnia vesicularis* by adding penicillin and vancomycin to the feces; and identification of *Havnia vesicularis*. This invention improves the isolation efficiency and success rate of *Havnia vesicularis*, solving the problems of low isolation efficiency and poor identification accuracy in existing technologies.

Description

Technical Field

[0001] This invention relates to a method for isolating and purifying Haffnia apiae, and more particularly to a method for isolating and extracting Haffnia apiae using penicillin and vancomycin. Background Technology

[0002] Bipolar disorder (BD) is a common and severe affective disorder characterized by recurrent episodes of depression or (hypo)mania, with a global prevalence of 2-3%. BD carries a significant disease burden, often impairing cognitive and social functioning, and is a leading cause of disability and death among adolescents. In recent years, increasing research has shown that gut microbiota is a crucial environmental factor influencing brain function, and the brain-gut-microbiota axis is involved in the pathogenesis of affective disorders. Gut microbiota refers to bacteria, fungi, viruses, and other microorganisms that colonize the gut. They synthesize a variety of metabolites, including hormones, cytokines, and neurotransmitters, and exert bidirectional regulatory effects on the brain through pathways via the nervous system (enteric nervous system, vagus nerve), endocrine system, and immune system, playing a vital physiological role in maintaining homeostasis.

[0003] *Hafnia alvei* is a facultative anaerobic, Gram-negative, flagellated bacillus belonging to the genus *Hafnia* in the family Enterobacteriaceae. It is both a bacterial food contaminant and an opportunistic pathogen. Recent studies have found that *Hafnia alvei* may be associated with intestinal diseases, food spoilage, and opportunistic infections. Some studies have found significant differences in the gut microbiota composition of patients with bipolar disorder compared to healthy individuals, including an increased abundance of certain opportunistic pathogens such as *Hafnia alvei*. Existing research suggests that *Hafnia alvei* may affect the host's neuroendocrine and immune systems by producing metabolites such as short-chain fatty acids and neuroactive substances. The relationship between *Hafnia alvei* and bipolar disorder is an emerging research area.

[0004] Existing evidence suggests that gut microbiota (including *Havnia vesicularis*) may influence the occurrence and development of bipolar disorder through the gut-brain axis. Therefore, to investigate the relationship between *Havnia vesicularis* and bipolar disorder, *Havnia vesicularis* was isolated from the stool of bipolar disorder patients. However, due to its similarity to other Enterobacteriaceae, isolating and identifying *Havnia vesicularis* presents significant technical challenges. Summary of the Invention

[0005] In view of the shortcomings of existing technologies and practical needs, the purpose of this invention is to provide a method for isolating Haffnia vesicae from the stool of bipolar disorder patients using penicillin and vancomycin, in order to improve the isolation efficiency and success rate of Haffnia vesicae and solve the problems of low isolation efficiency and poor identification accuracy in existing technologies.

[0006] This invention provides an efficient and reliable method for isolation and identification, offering technical support for the research and application of Hafnia apiae.

[0007] The technical solution adopted in this invention is:

[0008] The present invention mainly includes the following steps: collecting and processing feces from patients with bipolar disorder, isolating and purifying Haffnia vesicae from the feces, and identifying Haffnia vesicae.

[0009] I. Application of penicillin and vancomycin in the isolation and purification of Haffnia avesicae.

[0010] The Latin scientific name of the fungus *Havnia vesicae* is... Hafinia alvei .

[0011] II. A method for isolating and extracting *Havnia vesiclei* using penicillin and vancomycin, wherein the method comprises the following steps:

[0012] (1) Collect feces from patients with bipolar disorder and process them to obtain a separation culture medium;

[0013] (2) Add penicillin and vancomycin to the isolation medium to isolate Haffnia apiae;

[0014] (3) Further purification of Haffnia apiae.

[0015] The specific steps (1) are as follows:

[0016] Fecal samples from bipolar disorder patients were placed in a 30% (w / v) glycerol aqueous solution and irradiated in a clean bench for 30 minutes. The fecal samples were then removed from the glycerol solution and placed in sterile saline solution and mixed with a vortex mixer.

[0017] Then, a portion was extracted and added to a test tube containing 9 mL of separation culture medium, and then subjected to gradient dilution to obtain a fecal suspension.

[0018] The gradient dilution specifically refers to using the culture medium added to the liquid culture medium as a 10-fold gradient dilution of the original concentration of the stock solution, i.e., 10... -1 Then continue with multiple 10-fold serial dilutions until no turbidity is visible in the dilution tube, i.e., 10-fold. -6Starting from the point where the turbidity of the dilution tube is no longer visible, continue to dilute down by 4 10-fold gradients, and take the culture medium from all 10-fold dilution gradients starting from the point where the turbidity of the dilution tube is no longer visible as the result for further processing in step (2).

[0019] The separation medium is prepared as follows: 29.6g of LB liquid medium is dissolved in 800mL of purified water, autoclaved for 15min, and cooled to 60℃. Using a 0.22μm sterile filter tip, 200μL of 30μg / mL penicillin and 20μg / mL vancomycin are added, shaken well, and poured into a 15mL centrifuge tube to form the separation medium.

[0020] Step (2) specifically involves:

[0021] (21) Add each of the culture media obtained in step (1) to their respective isolation media containing Haffnia hymeni, penicillin and vancomycin and incubate for 24 h.

[0022] (22) After culturing for 24 hours, the culture medium was extracted and diluted 10 times again to multiple dilution gradients. Multiple separation tubes were transferred to each dilution gradient for culturing. Each separation tube was then placed in a 37°C constant temperature incubator for 24 hours.

[0023] (23) After 24 hours of incubation, remove the separation tubes in a sterile gas environment and process and select the turbid tubes according to the following principles:

[0024] ① Select a turbid separation tube;

[0025] ② Select the most uniform, suspended, and free of precipitate, clumps, or filaments from the turbid separation tubes as the final turbid tubes;

[0026] (24) The selected turbid tubes were passaged and cultured in a new isolation medium containing penicillin and vancomycin, and then placed in a constant temperature incubator at 37°C for 24 hours.

[0027] (25) Repeat the above steps (22) to (24) multiple times to carry out subculture until no contaminants are present, and obtain the final screening culture medium as the final isolation medium;

[0028] (26) Finally, the final culture medium was extracted from the final separation medium and subjected to multiple 10-fold serial dilutions. The final culture medium and the culture medium of all the 10-fold serial dilutions were spread on the MHA purified solid medium which is conducive to the growth of Haffnia apiae and placed in a constant temperature incubator at 37°C for 24 hours.

[0029] The separation medium was prepared as follows: 29.6 g of LB liquid medium was dissolved in 800 mL of purified water, autoclaved for 15 min, and cooled to 60 °C. Using a 0.22 μm sterile filter tip, 200 μL of 30 μg / mL penicillin and 20 μg / mL vancomycin were added, shaken well, and poured into a 15 mL centrifuge tube to form the separation medium.

[0030] The MHA purification solid culture medium was prepared as follows: 30.4g of MHA culture medium was taken and placed in 800mL of purified water, autoclaved for 15min, and cooled to 60℃. Using a 0.22μm sterile filter head, 200μL of 30μg / mL penicillin and 20μg / mL vancomycin were added, shaken well, and poured into 15mL plates per petri dish. The plates were left overnight, and after solidification, they were inverted and stored at 4℃.

[0031] The specific steps (3) are as follows: pick single colonies from the MHA purified solid medium obtained in step (2) by streaking the medium, and inoculate the colonies with a sterile pipette tip under strong light and next to an alcohol lamp into LB liquid medium for enrichment of Haffnia apis. Place it in a 37°C constant temperature incubator for 24 hours to obtain purified Haffnia apis single bacteria.

[0032] The specific method for enriching Haffnia apiae with LB liquid culture medium is as follows: Dissolve 29.6g of LB liquid culture medium in 800mL of purified water, autoclave for 15min, cool to 60℃, shake well and pour 9mL into a 15mL centrifuge tube to form the enrichment LB liquid culture medium.

[0033] In a first aspect, the present invention provides the use of penicillin and vancomycin in the preparation of culture media for the isolation and purification of Haffnia auriculata.

[0034] This invention, through selective culture media and gradient dilution methods, unexpectedly discovered in studies on isolating *Havnia vesicae* that by adding penicillin and vancomycin during the isolation and screening process, penicillin specifically targets bacterial peptidoglycan synthesis (a major component of the cell wall) and blocks transpeptidase activity by binding to penicillin-binding proteins (PBPs). This leads to the inhibition of peptidoglycan cross-linking, preventing the bacteria from forming a complete cell wall.

[0035] Vancomycin binds with a high affinity to the cell wall precursor of Gram-positive bacteria (D-alanyl-D-alanine terminus), blocking the activity of transpeptidase and transglycosylase. This inhibits Gram-positive bacteria but has no effect on Gram-negative bacteria.

[0036] The combined use of two antibiotics can effectively inhibit the growth of other microorganisms in samples (such as feces) without inhibiting the growth of Havnia apis, thus significantly improving the isolation efficiency and success rate of Havnia apis, and shortening the screening cycle, providing a new approach for the isolation, purification and enrichment of Havnia apis.

[0037] Secondly, this invention proposes a method for purity identification of *Havnia vesicae* by combining morphological identification and mass spectrometry.

[0038] This invention combines morphological identification and mass spectrometry in the purification process of *Havnia solani*, enabling strain identification to be completed within minutes, significantly shortening the identification time and achieving rapid identification. Furthermore, the mass spectrometry results are accurate and reliable, avoiding errors inherent in traditional biochemical identification and greatly improving the accuracy of strain identification.

[0039] Morphological identification involves picking single colonies that are free of surrounding bacteria, less than 2 mm in diameter, milky white, and viscous from the prepared solid culture medium.

[0040] Mass spectrometry involves taking a small amount of purified bacterial colonies, washing them with sterile water, spreading them evenly on a mass spectrometry plate, and then extracting them using the formic acid method, which involves adding a 1:1 mixture of 70% formic acid and acetonitrile matrix powder.

[0041] The analysis was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass spectra were compared with strain databases (such as the Bruker MALDI Biotyper database) to confirm the strain species. The database for this strain was built in advance.

[0042] The single colony was streaked in a new solid culture medium under aseptic conditions, and cultured for a period of time. Single colonies were then picked to obtain Haffnia camara. The purity of the strains screened by this method can reach the species level.

[0043] The purity of Haffnia apiacea obtained by the method of this invention can reach over 99.9%.

[0044] The beneficial effects of this invention are:

[0045] (1) The stool used in this invention comes from outpatients with bipolar disorder, which is easy to obtain and has low cost.

[0046] (2) When isolating this bacterium from feces, the combined use of penicillin and vancomycin strictly inhibits the growth of other bacteria, while having no effect on Haffnia apis, which significantly improves the isolation efficiency and success rate, and shortens the screening cycle, providing a new approach for the isolation, purification and enrichment of Haffnia apis.

[0047] (3) Combining morphological identification and mass spectrometry identification methods improves identification efficiency, and the purity of the screened strains can reach the species level.

[0048] This invention can improve the isolation efficiency and success rate of Haffnia apiacea, and solve the problems of low isolation efficiency and poor identification accuracy in the prior art. Attached Figure Description

[0049] Figure 1 This is a flowchart of the method of the present invention;

[0050] Figure 2 This is a schematic diagram of a cultured single colony according to an embodiment of the present invention;

[0051] Figure 3 This is a schematic diagram of a single colony identified by mass spectrometry in an embodiment of the present invention;

[0052] Figure 4 This is a diagram showing the identification results of Haffnia apiacea isolated in an embodiment of the present invention;

[0053] Figure 5 This is a diagram showing the identification results of Haffnia apiacea isolated in the comparative proportion of this invention. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0055] The embodiments of the present invention are as follows:

[0056] Example 1: The process is as follows Figure 1 As shown.

[0057] (1) Inclusion and screening of patients with bipolar disorder.

[0058] The DSM-IV-TR structured clinical interview (SCID) was used to include patients with bipolar disorder. All included patients had a Hamilton Depression Rating Scale (HAMD-24) score ≥20 and a Young Mania Rating Scale (YMRS) score <6.

[0059] All enrolled patients had not consumed probiotic preparations such as yogurt within one week prior to enrollment, nor had they taken antibiotic preparations within one week prior to enrollment.

[0060] (2) Collect and process feces from patients with bipolar disorder.

[0061] One gram of stool samples from each of the three previously collected patients was sent to the company for sequencing, and the samples were found to contain Haffnia vesicae.

[0062] Fresh stool samples were collected from patients and preserved in a 30% (w / v) glycerol aqueous solution. When needed, 1g of stool was weighed from the glycerol aqueous solution and placed in Green's solution containing cysteine. The sample was mixed using a vortex mixer and used as the stock solution. 1mL of the stock solution was aseptically added to a test tube containing 9mL of liquid culture medium, thus creating a 10-fold serial dilution of the original concentration. -1 Then continue with five more 10-fold serial dilutions until the dilution tubes show no turbidity (i.e., 10-fold dilution). -6 Continue diluting down by 3 gradients to 10. -9 The last four dilution gradients with 10x intervals were then applied. -6 ~ 10 -9 That is, 10 each -6 10 -7 10 -8 10 -9 The dilution gradient was used to add 1 mL of each tube to an isolation medium containing 9 mL of Haffnia aeruginosa, penicillin, and vancomycin, and incubate for 24 h.

[0063] The above-mentioned separation medium is prepared as follows: 29.6g of LB liquid medium is dissolved in 800mL of purified water, autoclaved for 15min, and cooled to 60℃. Using a 0.22μm sterile filter tip, 200μL of 30μg / mL penicillin and 20μg / mL vancomycin are added, shaken well, and poured into a 15mL centrifuge tube to form the separation medium.

[0064] (3) Isolation of Haffnia apiae from feces

[0065] For the results from each dilution tube after 24 hours of culture in step (2), extract them and perform multiple 10-fold serial dilutions until the selected tube is diluted to 10. -3 ~ 10 -8 These six dilution gradients, namely 10... -3 10 -4 10 -5 10 -6 10 -7 10 -8 The dilution gradients are such that three separation tubes are connected simultaneously under each dilution gradient.

[0066] A total of 4*6*3=72 isolation tubes were obtained. Each isolation tube was placed in a 37℃ constant temperature incubator for 24 hours.

[0067] After 24 hours of incubation, remove the separation tubes in a sterile gas environment and process and select the turbid tubes according to the following principles:

[0068] ① Select a turbid separation tube;

[0069] ② Select the most uniform, suspended, and free of precipitates, clumps, or filaments from the turbid separation tubes as the final turbid tubes.

[0070] The above process ensures that the selected turbid tubes include separation tubes with a lower dilution gradient, such as 10. -3 and 10 -4 All of the above requirements were met, and the turbid tubes from the two dilutions were selected. Furthermore, turbid tubes with different dilutions can be included, provided that the above requirements are met.

[0071] The selected turbid tubes from the previous step were passaged into isolation medium containing penicillin and vancomycin. 1 mL of the medium was added to each tube containing 9 mL of isolation medium, and the tubes were placed in a 37°C incubator for 24 hours.

[0072] (4) Repeat step (3) three times, and subculture three times until no other bacteria are present. Then, perform multiple 10-fold serial dilutions on the culture medium obtained after final screening for each culture. -1 ~ 10 -9 Choose 10 -3 ~ 10 -9 Seven dilution gradients were plated onto MHA purified solid medium that promotes the growth of Hafnia solani.

[0073] Preparation of purified solid culture medium for MHA: Take 30.4g of MHA culture medium in 800mL of purified water, autoclave for 15min, and let it cool to 60℃. Use a 0.22μm sterile filter tip to add 200μL of 30μg / mL penicillin and 20μg / mL vancomycin, shake well, pour 15mL into each petri dish, let stand overnight, and after solidification, invert and store at 4℃.

[0074] For each of the seven dilution gradients, 100 μL of the diluted solution was pipetted into each petri dish and spread using an L-shaped spreader, with three dishes spread for each dilution. The dishes were then placed in a 37°C incubator and incubated for 24 hours.

[0075] In specific implementation, the plate mentioned above is a solid plate.

[0076] (5) Identification of Haffnia apiosa

[0077] Take out the cultured petri dishes as follows: Figure 2As shown, under strong light, 5-10 single colonies with no surrounding bacteria, a diameter of less than 2 mm, a milky white, viscous appearance, were selected from each petri dish. A small amount of each colony was taken, washed with sterile water, and then evenly spread onto a MALDI target plate (mass spectrometry plate). The bacterial protein was extracted using the formic acid method, i.e., 2 μL of 70% formic acid was added. After drying, 2 μL of matrix solution such as α-cyano-4-hydroxycinnamic acid (HCCA) was added to co-crystallize with the bacterial protein. After drying to a milky white color, the sample was placed in a mass spectrometer. Analyzing was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to generate a mass spectrum (recording the m / z values ​​and intensities of each protein peak to form a fingerprint spectrum).

[0078] The experimental spectra were compared with a built-in bacterial strain database (such as the Bruker MALDI Biotyper database), and the matching degree was calculated using algorithms (such as principal component analysis and cluster analysis). Matching degree ≥ 2.0: High confidence, purity usually > 90% (target strain is dominant), identification to the species level; 1.7-2.0: Medium confidence, requires verification, purity may be between 70%-90%, possibly identification to the genus level; < 1.7: Results are unreliable, may indicate significant contamination or mixed flora, requires verification.

[0079] Select single colonies of *Havnia vesicae* that have reached the inoculum level. Using a sterilized inoculation loop, pick up a small number of single colonies near an alcohol lamp and streak them in a zigzag pattern on the purified medium. After streaking, invert the petri dish and incubate it in a 37°C incubator for 24 hours.

[0080] (6) Purification of Haffnia apiae

[0081] Following the steps described above, select single colonies from the primary culture after streaking on the isolation medium. Under strong light, select 10 single colonies from each dish and identify them by mass spectrometry. After identification, they are confirmed to be *Havnia vesicae* at the species level. Inoculate these colonies into 15 mL of *Havnia vesicae* enrichment LB liquid medium near an alcohol lamp and incubate at 37°C for 24 hours to obtain purified *Havnia vesicae*.

[0082] In a specific embodiment, a single colony, less than 2 mm in diameter, milky white, and viscous in color, is picked from the cultured solid culture medium, free of surrounding bacteria. Figure 3 As shown.

[0083] A small amount of purified colonies were taken, washed with sterile water, and evenly spread on a mass spectrometry plate. Extraction was performed using the formic acid method, which involves adding a mixture of 70% formic acid and acetonitrile matrix powder (1:1). The analysis was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass spectrum was compared with a strain database (such as the Bruker MALDI Biotyper database) to confirm that the strain was *Havonia betel nut*.

[0084] After mass spectrometry analysis, the experimental chromatograms were compared with the built-in strain database, showing that the bacterium was *Havnia vesicae*, and the identification score for *Havnia vesicae* was 2.21 (<1.7 indicates unreliable identification, 1.7-2.0 indicates possible genus-level identification, ≥2.0 indicates possible species-level identification), indicating that the identification reached the species level and the purity reached 98% (e.g., ...). Figure 4 (As shown).

[0085] (7) Preservation of Haffnia in beehives

[0086] Mix sterile water and glycerol in a 7:3 ratio, autoclave, and dispense 1.5 mL into each cryovial. Centrifuge the liquid culture medium containing Haffnia apiae (4000 rpm, 4℃, 10 min), discard the supernatant, and add the lower layer of bacterial sludge to the glycerol mixture. Label the mixture with the date, strain name, and stool number, and store it in a -80℃ freezer.

[0087] Comparative Example 1:

[0088] The only difference from the example is that penicillin and vancomycin were not added.

[0089] The isolation, purification, and mass spectrometry detection of *Havnia solani* were performed according to the example procedure. The mass spectrometry results showed the presence of other bacteria, making it impossible to isolate *Havnia solani*. Figure 5 As shown.

[0090] The isolation medium is prepared by dissolving 29.6g of LB liquid medium in 800mL of purified water, autoclaving for 15min, cooling to 60℃, shaking well, and pouring 9mL into a 15mL centrifuge tube.

[0091] (1) 1g of stool samples from the three patients were sent to the company for sequencing, and Haffnia auriculata was detected in the three stool samples.

[0092] Fresh stool samples were collected from patients and preserved in a 30% (w / v) glycerol aqueous solution. When needed, 1g of stool was weighed from the glycerol aqueous solution and placed in Green's solution containing cysteine. The sample was mixed using a vortex mixer and used as the stock solution. 1mL of the stock solution was aseptically added to a test tube containing 9mL of liquid culture medium, thus creating a 10-fold serial dilution of the original concentration. -1 Then continue with five more 10-fold serial dilutions until the dilution tubes show no turbidity (i.e., 10-fold dilution). -6 Continue diluting down by 3 gradients to 10. -9 The last four dilution gradients with 10x intervals were then applied. -6 ~ 10 -9 That is, 10 each -6 10 -7 10 -8 10 -9 The dilution gradient was used to add 1 mL of each tube to an isolation medium containing 9 mL of isolating Haffnia aeruginosa and incubate for 24 h.

[0093] (2) Isolation of Haffnia apiae from feces

[0094] For the results from each dilution tube after 24 hours of culture in step (1), extract them and perform multiple 10-fold serial dilutions until the selected tube is dilute to 10. -3 ~ 10 -8 These six dilution gradients, namely 10... -3 10 -4 10 -5 10 -6 10 -7 10 -8 The dilution gradients are such that three separation tubes are connected simultaneously under each dilution gradient.

[0095] A total of 4*6*3=72 isolation tubes were obtained. Each isolation tube was placed in a 37℃ constant temperature incubator for 24 hours.

[0096] After 24 hours of incubation, remove the separation tubes in a sterile gas environment and process and select the turbid tubes according to the following principles:

[0097] ① Select a turbid separation tube;

[0098] ② Select the most uniform, suspended, and free of precipitates, clumps, or filaments from the turbid separation tubes as the final turbid tubes.

[0099] The above process ensures that the selected turbid tubes include separation tubes with a lower dilution gradient, such as 10. -3 and 10 -4All of the above requirements were met, and the turbid tubes from the two dilutions were selected. Furthermore, turbid tubes with different dilutions can be included, provided that the above requirements are met.

[0100] Subculture the selected turbid tubes in the previous step into the same separation medium, adding 1 mL of the medium to each tube containing 9 mL of separation medium, and incubate in a 37°C incubator for 24 hours.

[0101] (3) Repeat step (3) three times, and subculture three times until no other bacteria are present. Then, perform multiple 10-fold serial dilutions on the culture medium obtained after final screening for each culture. -1 ~ 10 -9 Choose 10 -3 ~10 -9 Seven dilution gradients were plated onto MHA purified solid medium that promotes the growth of Hafnia solani.

[0102] Preparation of MHA purified solid culture medium: Take 30.4g of MHA culture medium in 800mL of purified water, autoclave for 15min, let it cool to 60℃, pour 15mL into each petri dish into a plate, let it stand overnight, and after solidification, invert it and store at 4℃.

[0103] For each of the seven dilution gradients, 100 μL of the diluted solution was pipetted into each petri dish and spread using an L-shaped spreader, with three dishes spread for each dilution. The dishes were then placed in a 37°C incubator and incubated for 24 hours.

[0104] (4) Identification of Haffnia apiosa

[0105] Remove the cultured petri dishes and, under strong light, select 5-10 single colonies from each dish that are free of surrounding bacteria, less than 2 mm in diameter, milky white, and viscous. Take a small amount of each colony, wash with sterile water, and evenly spread it onto a MALDI target plate (mass spectrometry plate). Extract bacterial proteins using the formic acid method (add 2 μL of 70% formic acid). After drying, add 2 μL of a matrix solution containing α-cyano-4-hydroxycinnamic acid (HCCA) and co-crystallize with the bacterial proteins. Allow it to dry to a milky white color before placing it in a mass spectrometer. Analyze using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to generate a mass spectrum (recording the m / z values ​​and intensities of each protein peak to form a fingerprint).

[0106] The experimental spectra were compared with the built-in strain database (such as the Bruker MALDI Biotyper database), and the matching degree was calculated using algorithms (such as principal component analysis and cluster analysis). Matching degree ≥ 2.0: identification to the species level; 1.7-2.0: possible identification to the genus level; < 1.7: results are unreliable and need to be verified.

[0107] A single colony of *Havnia vesicae* was identified at the genus level and was found to be contaminated with other bacteria. A small number of colonies were picked up with a sterilized inoculation loop near an alcohol lamp and streaked in a zigzag pattern on a purification medium. After streaking, the plate was inverted and incubated at 37°C for 24 hours. Mass spectrometry analysis and comparison of the experimental chromatogram with the built-in strain database confirmed that the bacterium was *Havnia vesicae*, but the matching score was only 1.73, and the purity reached 75%, indicating that the identification could only reach the genus level (e.g., *Havnia vesicae*). Figure 5 (As shown).

[0108] In summary, this invention addresses the isolation of Haffnia vesicae and discovers that the combined use of penicillin and vancomycin can effectively inhibit the growth of other microorganisms in samples (feces) without inhibiting the growth of Haffnia vesicae. This provides a new approach for the isolation, purification, and enrichment of Haffnia vesicae, further improving isolation efficiency and purity, shortening the screening cycle, achieving a purity of up to 98% for the screened strains, and employing a simple and low-cost culture medium with a straightforward isolation method.

[0109] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0110] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. An application of a combination of penicillin and vancomycin, characterized in that: In the application of isolating and purifying Hafnia alvei, the Latin name of the Hafnia alvei is Hafnia alvei .

2. A method for isolation of Hafnia alvei by using a combination of penicillin and vancomycin, characterized by: The method is performed in the following manner: (1) Collect feces from patients with bipolar disorder and process them to obtain a separation culture medium; (2) Isolate Haffnia apiae by adding penicillin and vancomycin to the isolation medium simultaneously; (3) Further purification of Haffnia apiosa; The separation medium was prepared as follows: 29.6 g of LB liquid medium was dissolved in 800 mL of purified water, autoclaved for 15 min, and cooled to 60 °C. Using a 0.22 μm sterile filter head, 200 μL of 30 μg / mL penicillin and 20 μg / mL vancomycin were added, shaken well, and poured into a 15 mL centrifuge tube.

3. The method for isolating and extracting Haffnia apiae using a combination of penicillin and vancomycin according to claim 2, characterized in that: The specific steps (1) are as follows: Fecal samples from bipolar disorder patients were placed in a 30% (w / v) glycerol aqueous solution and irradiated in a clean bench for 30 minutes. The fecal samples were then removed from the glycerol solution and placed in sterile saline solution and mixed with a vortex mixer. Then add it to a test tube containing 9 mL of separation culture medium and perform gradient dilution to obtain a fecal suspension.

4. A method for isolation and extraction of Hafnia cellulans by using combination of penicillin and vancomycin as claimed in claim 3, wherein the said method comprises the steps of: The gradient dilution is specifically 10-fold gradient dilution of the culture solution added to the liquid medium as the original concentration of the original solution, and then continuing 10-fold gradient dilution multiple times until no turbidity is observed in the dilution tube, i.e. 10 -6 From the start of no turbidity observed in the dilution tube, continue to dilute down 4 10-fold gradients, and take the culture solution from all of the 10-fold dilution gradients diluted down from the start of no turbidity observed in the dilution tube.

5. A method for isolation and extraction of Hafnia alvei by combination of penicillin and vancomycin as claimed in claim 3, wherein the said method comprises the steps of: Step (2) specifically involves: (21) Each culture medium obtained in step (1) is added to its respective isolation medium containing Haffnia apiae and penicillin and vancomycin and cultured for 24 h. (22) After culturing for 24 hours, the culture medium was extracted and diluted 10 times again to multiple dilution gradients. Multiple separation tubes were transferred to each dilution gradient for culturing. Each separation tube was then placed in a constant temperature incubator for 24 hours. (23) After 24 hours of incubation, remove the separation tubes in a sterile gas environment and process and select the turbid tubes according to the following principles: ① Select a turbid separation tube; ② Select the most uniform, suspended, and free of precipitate, clumps, or filaments from the turbid separation tubes as the final turbid tubes; (24) The selected turbid tubes were passaged and cultured in the same isolation medium containing penicillin and vancomycin, and then placed in a constant temperature incubator for 24 hours. (25) Repeat the above steps (22) to (24) multiple times to carry out subculture until no contaminants are present, and obtain the final screening culture medium as the final isolation medium; (26) Finally, the final culture medium was extracted from the final separation medium and subjected to multiple 10-fold serial dilutions. The final culture medium and the culture medium of all the 10-fold serial dilutions were spread on the MHA purified solid medium which is conducive to the growth of Haffnia apiae and placed in a constant temperature incubator at 37°C for 24 hours.

6. The method for isolating and extracting Haffnia apiae using a combination of penicillin and vancomycin according to claim 5, characterized in that: The separation medium was prepared as follows: 29.6 g of LB liquid medium was dissolved in 800 mL of purified water, autoclaved for 15 min, and cooled to 60 °C. Using a 0.22 μm sterile filter head, 200 μL of 30 μg / mL penicillin and 20 μg / mL vancomycin were added, shaken well, and poured into a 15 mL centrifuge tube.

7. The method for isolating and extracting Haffnia apiae using a combination of penicillin and vancomycin according to claim 5, characterized in that: The MHA purification solid culture medium was prepared as follows: 30.4g of MHA culture medium was taken and placed in 800mL of purified water, autoclaved for 15min, and cooled to 60℃. Using a 0.22μm sterile filter head, 200μL of 30μg / mL penicillin and 20μg / mL vancomycin were added, shaken well, and poured into 15mL plates per petri dish. The plates were left overnight, and after solidification, they were inverted and stored at 4℃.

8. A method for isolation and extraction of Hafnia alvei by combination of penicillin and vancomycin as claimed in claim 5, wherein the said method comprises the steps of: The specific steps (3) are as follows: pick a single colony from the MHA purified solid medium obtained in step (2), and inoculate the colony with a sterile pipette tip under the light and next to the alcohol lamp into the LB liquid medium for enrichment of Haffnia apis, and place it in a constant temperature incubator for 24 hours to obtain the purified Haffnia apis single bacteria.

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