A method for inducing the formation of viable non-culturable state bacteria in the presence of host cells and its use

Abstract

The application belongs to the technical field of microorganisms, and discloses a method for inducing formation of living unculturable state bacteria in the presence of host cells and application thereof. The method is as follows: cells in the 3th to 20th generation are transferred to a well plate to obtain induced cells; bacteria in the logarithmic growth phase are washed by a buffer, and modified cell culture solution is resuspended to obtain a bacterial suspension. The bacterial suspension is added to the induced cells, and after incubation in a constant-temperature incubator for 1-5 hours, 10-500 mg / L gentamicin-containing modified cell culture solution is added. After cell treatment for 3-10 days, intracellular bacteria are collected by adding cell lysis solution, and then washed, centrifuged and resuspended to obtain living unculturable state bacteria. The application can induce bacteria to enter the living unculturable state in the presence of host cells, which not only expands the understanding of living unculturable state bacteria, but also provides theoretical and technical support for recurrent and chronic infectious diseases in clinical practice.

Description

A method for inducing the formation of live, unculturable bacteria in the presence of host cells and its application. Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a method for inducing the formation of live, unculturable bacteria in the presence of host cells and its application. Background Technology

[0002] Live unculturable bacteria are a survival strategy employed by bacteria to cope with adverse environmental pressures such as nutrient deficiency, low temperature, low pH, high salinity, and antibiotics. Their main characteristic is that they cannot form colonies on nutrient-rich, non-selective bacterial culture media, but they still maintain the structural integrity of the bacteria (proteins, cell wall, nucleic acids) and their metabolic activity. Live unculturable bacteria not only have increased tolerance to unsuitable environments and exhibit antibiotic resistance, but under suitable conditions, they may also revert to culturable bacteria and retain their harmfulness.

[0003] In most cases, the host immune system can eliminate invading bacteria. However, in some infections, bacteria may evade the host immune system and persist within the host. Furthermore, the intracellular environment is complex, with factors such as nutrient deficiency, low pH, nitric oxide, ROS, and antimicrobial peptides acting adversely on bacteria over time. Bacteria can gradually adapt (becoming a live, unculturable state) to survive within the intracellular host environment. These live, unculturable bacteria can evade immune recognition, extracellular host defense, and extracellular antibiotic action. Additionally, bacteria in this live, unculturable state develop increased tolerance, and with cell division, exocytosis releases them back into a suitable extracellular environment, causing them to revive and regain their original harmfulness. The presence of live, unculturable bacteria in the human body can lead to asymptomatic carriers of pathogens, prolong the incubation period of pathogenic bacteria, and allow them to evade antibiotic treatment, thus posing a potential threat to public health. Therefore, it is essential to delve into the evolutionary mechanisms of live, unculturable bacteria within host cells. Summary of the Invention

[0004] To address the shortcomings and drawbacks of existing technologies, the primary objective of this invention is to provide a method for inducing the formation of live, unculturable bacteria in the presence of host cells. By adjusting conditions such as different cell and bacterial species, cell numbers, bacterial concentrations, treatment time, and gentamicin concentration, live, unculturable bacteria can be stably obtained. This provides a model method for clinical research on the toxicity and drug resistance of live, unculturable bacteria, and offers theoretical and technical support for recurrent and chronic infectious diseases in clinical practice. The method of inducing bacteria into a live, unculturable state through cell induction allows for a better understanding of the long-term survival and evolution of bacteria within human cells. Using this model not only enables the rapid and stable induction of live, unculturable bacteria, but also expands our understanding of these bacteria, further elucidating the survival and evolutionary mechanisms of live, unculturable bacteria within cells.

[0005] Another object of the present invention is to provide live, unculturable bacteria induced by the above method, further elucidating their formation mechanism within host cells.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] S1. Discard the cell culture medium with a cell confluence of 70-90%, wash with buffer, digest with trypsin, collect the cells, determine the cell concentration by cell counting, and then transfer the obtained cells to cell culture dishes of different sizes and place them in an incubator for culture to obtain induced cells;

[0008] S2. Collect the bacteria cultured overnight, wash and centrifuge them with buffer, then resuspend them in modified cell culture medium, adjust the OD value of the bacteria to equal the concentration value using an ELISA reader, and obtain a bacterial suspension by serial dilution of the known concentration of bacteria.

[0009] S3. In a clean bench, add the bacterial suspension from step S2 into the induced cells, centrifuge to allow the bacteria to fully enter the cells, and then transfer to a constant temperature incubator at 10-60℃ and 0.5-30% CO2 for 1-5 hours.

[0010] S4. In a clean bench, remove the bacterial suspension from the cell culture dish in step S3, wash with buffer, then add cell culture medium containing 50-500 mg / L gentamicin, incubate in an incubator, then aspirate the culture medium, and then add cell culture medium containing 10-100 mg / L gentamicin to prevent reinfection by bacteria released from infected dead cells. Monitor the cell condition using an optical microscope and replace the cell culture medium as needed.

[0011] S5. After 3 to 10 days of cell treatment, collect the bacteria in the induced cells. First, remove the bacterial suspension and wash with buffer. Then, add 0.1 to 10 wt% SDS cell lysis buffer and incubate at room temperature to release the intracellular bacteria. Add buffer to wash, centrifuge, wash, and resuspend in buffer to obtain live, unculturable bacteria.

[0012] Preferably, the cells in step S1 are cell lines of generation 3 to 20, and the bacteria are in the logarithmic growth phase.

[0013] Preferably, the cells in step S1 are 16HBE, Base-2B, or HaCaT human cells, and the bacteria are one or more of Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa.

[0014] Preferably, the cell culture medium in steps S1 and S4 is a basic cell culture medium, FBS, and penicillin-streptomycin. The content of the basic cell culture medium is 30-99.9 wt%, the content of FBS in the basic cell culture medium is 0.05-50 wt%, and the content of penicillin-streptomycin in the basic cell culture medium is 0.05-5 wt%. The basic cell culture medium is DMEM, RPMI1640, MEM, or F-12K.

[0015] Preferably, the modified cell culture medium in step S2 is a basic cell culture medium and FBS; the content of the basic cell culture medium is 30-99.95 wt%, and the content of FBS in the basic cell culture medium is 0.05-50 wt%; the basic cell culture medium is DMEM, RPMI 1640, MEM, or F-12K.

[0016] Preferably, the concentration of cells in step S1 is 10. 4 ~10 7 The bacterial suspension concentration described in step S2 for cell / well is 10. 4 ~10 8 CFU / mL.

[0017] Preferably, the centrifugation speed in step S2 is 500-2000 rpm, and the centrifugation time is 5-30 min.

[0018] Preferably, the buffer solution described in steps S1 to S5 is a sterile phosphate buffer solution.

[0019] The live, unculturable bacteria are prepared by the method described above.

[0020] The application of the described method for inducing the formation of live, unculturable bacteria in clinical settings for evaluating recurrent and chronic infectious diseases.

[0021] This invention provides a method for inducing the formation of live, unculturable bacteria in the presence of host cells, and experiments have demonstrated that live, unculturable bacteria can be induced to form within host cells, posing a significant threat to public health and safety.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention is the first to use human host cells to induce bacteria into a live, unculturable state. By selecting cell culture dishes of different sizes, designing different cell numbers, adjusting different bacterial concentrations, and varying the interaction time between cells and bacteria, the operation is simple, quick, and has a high probability of stably obtaining live, unculturable bacteria.

[0024] 2. In this invention, the addition of cell culture medium containing gentamicin during the induction process can not only remove extracellular bacteria and prevent secondary bacterial infection during the induction process, but also maintain the normal activity of cells.

[0025] 3. This invention can expose pathogenic bacteria capable of entering a live, unculturable state (as evidenced by existing literature) to human host cells. The health risks posed by pathogens entering human cells in a realistically simulated environment during antibiotic treatment—namely, the survival of these pathogens in a live, unculturable state within human cells during antibiotic therapy—may lead to antibiotic failure and recurrent infections.

[0026] 4. This invention can easily separate host cell components from live unculturable bacteria, characterize the shape of live unculturable bacteria independently, and elucidate the formation mechanism of the live unculturable state from a bacterial perspective.

[0027] 5. This invention can provide an efficient cell induction model for the study, prevention and control of the spread, proliferation and blocking of live unculturable bacteria in clinical practice, and expands the new understanding of live unculturable bacteria. Bacteria can persist in host cells in this dormant state of live unculturable bacteria, and re-examine the potential harm of live unculturable bacteria in the human environment to public health. Attached Figure Description

[0028] Figure 1 is a flowchart of the cell-induced live, unculturable bacteria of the present invention.

[0029] Figure 2 shows the changes in culturable bacteria within the host cell in Example 1.

[0030] Figure 3 is an electron micrograph of the live, unculturable Escherichia coli obtained in Example 2. Detailed Implementation

[0031] The following specific embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0032] The cell culture medium in this embodiment of the invention includes 30-99.9 wt% of one of DMEM (Dulbecco's Modified Eagle), MEM (Minimum Essential Medium), F-12K (Ham's F-12), or RPMI (Roswell Park Memorial Institute), 0.05-50 wt% of FBS (Fetal Bovines Serum), and 0.05-5 wt% of penicillin-streptomycin.

[0033] The modified cell culture medium includes one of 30-99.95% DMEM (Dulbecco's Modified Eagle), MEM (Minimum Essential Medium), F-12K (Ham's F-12), or RPMI (Roswell Park Memorial Institute), and 0.05-50 wt% FBS (Fetal Bovines Serum).

[0034] Example 1

[0035] Figure 1 is a flowchart of the host cell induction of live unculturable bacteria in an embodiment of the present invention, and the specific steps are as follows:

[0036] 1. 16HBE cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, followed by digestion, centrifugation, and resuspending to obtain a cell suspension. This suspension was then seeded into 12-well plates at a cell concentration of 1 × 10⁻⁶ cells / well. 5 The cells / wells are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0037] 2. The *E. coli* bacteria cultured overnight to the logarithmic growth phase were centrifuged at 8000 rpm for 2 min at room temperature. The suspension was washed three times with sterile phosphate-buffered saline (PBS) and then resuspended in modified cell culture medium to obtain an *E. coli* suspension. The OD value of the *E. coli* suspension was then adjusted to 0.1 using a microplate reader, at which point the concentration of the *E. coli* suspension was 10-1. 8 CFU / mL, by serially diluting a known concentration of E. coli suspension to obtain the desired concentration of infected E. coli suspension. 6 CFU / mL.

[0038] 3. In a clean bench, remove the cell culture medium from the 12-well plate and wash three times with phosphate buffer to remove residual antibiotics. Add 2 mL of the infected E. coli suspension to the induced cells, place the 12-well plate in a plate centrifuge, centrifuge at 1000 rpm for 10 min, and then incubate the centrifuged plate in a cell culture incubator for 2 h.

[0039] 4. Discard the infected E. coli suspension in the 12-well plate, then add 2 mL of cell culture medium containing 100 mg / L gentamicin and incubate for 20 min. After removing the E. coli adhering to the surface of the induced cells, add 2 mL of cell culture medium containing 50 mg / L gentamicin. This step prevents reinfection by E. coli released from infected dead cells and introduces the cells into a live, unculturable state. During induction, monitor the cells using an optical microscope and change the cell culture medium containing 50 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0040] 5. Remove the cell culture medium containing gentamicin, wash three times with sterile phosphate buffer, then add 500 μL of 0.1 wt% SDS solution to lyse the cells and release the intracellular E. coli. Then add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live, unculturable E. coli.

[0041] Figure 2 shows the changes in live culturable Escherichia coli in the host cell in Example 1. As can be seen from Figure 2, the number of culturable Escherichia coli in the host cell decreases with the increase of culture time. By the 36th hour, all bacteria have entered the live unculturable state, indicating that the cell can induce bacteria to enter the live unculturable state.

[0042] Example 2

[0043] 1. 16HBE cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, and the cells were digested, centrifuged, and resuspended to obtain a cell suspension. This suspension was then seeded into 6-well plates at a cell concentration of 1 × 10⁻⁶ cells / well. 6 The cells / wells are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0044] 2. The *E. coli* bacteria cultured overnight to the logarithmic growth phase were centrifuged at 8000 rpm for 2 min at room temperature. The suspension was washed three times with sterile phosphate-buffered saline (PBS) and then resuspended in modified cell culture medium to obtain an *E. coli* suspension. The OD value of the *E. coli* suspension was then adjusted to 0.1 using a microplate reader, at which point the concentration of the *E. coli* suspension was 10-1. 8 CFU / mL, by serially diluting a known concentration of E. coli suspension to obtain the desired concentration of infected E. coli suspension. 7CFU / mL.

[0045] 3. In a clean bench, remove the cell culture medium from the well plate and wash three times with phosphate buffer to remove residual antibiotics. Add 3 mL of the infected E. coli suspension to the induced cells, place the 6-well plate in a centrifuge, centrifuge at 1000 rpm for 10 min, and then incubate the centrifuged plate in a cell culture incubator for 2 h.

[0046] 4. Discard the infected E. coli suspension in the 6-well plate, then add 1 mL of cell culture medium containing 100 mg / L gentamicin and incubate for 20 min. After removing the E. coli adhering to the surface of the induced cells, add 3 mL of cell culture medium containing 50 mg / L gentamicin. This step prevents reinfection by E. coli released from infected dead cells and introduces the cells into a live, unculturable state. During induction, monitor the cells using an optical microscope and change the cell culture medium containing 50 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0047] 5. Remove the cell culture medium containing gentamicin, wash three times with sterile phosphate buffer, then add 500 μL of 0.1 wt% SDS solution to lyse the cells and release the intracellular E. coli. Then add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live, unculturable E. coli.

[0048] Figure 3 is an electron micrograph of the live, unculturable Escherichia coli obtained in Example 2. As can be seen from Figure 3, after entering the live, unculturable state, some Escherichia coli bacteria changed from rod-shaped to spherical in appearance.

[0049] Example 3

[0050] 1. Base-2B cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, and the cells were digested, centrifuged, and resuspended to obtain a cell suspension. This suspension was then seeded into 6-well plates at a cell concentration of 1 × 10⁶ cells / well. 6 The cells / wells are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0051] 2. The *E. coli* bacteria cultured overnight to the logarithmic growth phase were centrifuged at 8000 rpm for 2 min at room temperature. The suspension was washed three times with sterile phosphate-buffered saline (PBS) and then resuspended in modified cell culture medium to obtain an *E. coli* suspension. The OD value of the *E. coli* suspension was then adjusted to 0.1 using a microplate reader, at which point the concentration of the *E. coli* suspension was 10-1. 8 CFU / mL, by serially diluting a known concentration of E. coli suspension to obtain the desired concentration of infected E. coli suspension. 7 CFU / mL.

[0052] 3. In a clean bench, remove the cell culture medium from the well plate and wash three times with phosphate buffer to remove residual antibiotics. Add 3 mL of the infected E. coli suspension to the induced cells, place the 6-well plate in a centrifuge, centrifuge at 1000 rpm for 10 min, and then incubate the centrifuged plate in a cell culture incubator for 2 h.

[0053] 4. Discard the infected E. coli suspension in the 6-well plate, then add 3 mL of cell culture medium containing 100 mg / L gentamicin and incubate for 20 min. After removing the E. coli adhering to the surface of the induced cells, add 3 mL of cell culture medium containing 50 mg / L gentamicin. This step prevents reinfection by E. coli released from infected dead cells and introduces the cells into a live, unculturable state. During induction, monitor the cells using an optical microscope and change the cell culture medium containing 50 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0054] 5. Remove the cell culture medium containing gentamicin, wash three times with sterile phosphate buffer, then add 500 μL of 0.1 wt% SDS solution to lyse the cells and release the intracellular E. coli. Then add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live, unculturable E. coli.

[0055] Example 4

[0056] 1. 16HBE cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, and the cells were digested, centrifuged, and resuspended to obtain a cell suspension. This suspension was then seeded into 6-well plates at a cell concentration of 1 × 10⁻⁶ cells / well. 6 The cells / wells are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0057] 2. The *E. coli* bacteria cultured overnight to the logarithmic growth phase were centrifuged at 8000 rpm for 2 min at room temperature. The suspension was washed three times with sterile phosphate-buffered saline (PBS) and then resuspended in modified cell culture medium to obtain an *E. coli* suspension. The OD value of the *E. coli* suspension was then adjusted to 0.1 using a microplate reader, at which point the concentration of the *E. coli* suspension was 10-1. 8 CFU / mL, the desired concentration of E. coli suspension was obtained by serial dilution of known concentrations of CFU / mL. 7 A suspension of E. coli containing CFU / mL.

[0058] 3. In a clean bench, remove the cell culture medium from the well plate and wash three times with phosphate buffer to remove residual antibiotics. Add 3 mL of the infected E. coli suspension to the induced cells, place the 6-well plate in a centrifuge, centrifuge at 1000 rpm for 10 min, and then incubate the centrifuged plate in a cell culture incubator for 2 h.

[0059] 4. Discard the infected E. coli suspension in the 6-well plate, then add 1 mL of cell culture medium containing 100 mg / L gentamicin and incubate for 20 min. After removing the E. coli adhering to the surface of the induced cells, add 3 mL of cell culture medium containing 25 mg / L gentamicin. This step prevents reinfection by E. coli released from infected dead cells and introduces the cells into a live, unculturable state. During induction, monitor the cells using an optical microscope and change the cell culture medium containing 25 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0060] 5. Remove the cell culture medium containing gentamicin, wash three times with sterile phosphate buffer, then add 500 μL of 0.1 wt% SDS solution to lyse the cells and release the intracellular E. coli. Then add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live, unculturable E. coli.

[0061] Example 5

[0062] 1. 16HBE cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, and the cells were digested, centrifuged, and resuspended to obtain a cell suspension. This suspension was then seeded into 6-well plates at a concentration of 1 × 10⁶ cells / well. 6 The cells / wells are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0063] 2. Centrifuge the overnight-cultured Staphylococcus aureus in the logarithmic growth phase at 8000 rpm for 2 min at room temperature. Wash three times with sterile phosphate buffer, then resuspend in modified cell culture medium to obtain a Staphylococcus aureus suspension. Adjust the OD value of the Staphylococcus aureus suspension to 0.1 using a microplate reader. At this point, the concentration of the Staphylococcus aureus suspension is 10⁻⁶. 8 CFU / mL, to obtain the desired concentration of infected Staphylococcus aureus suspension by serial dilution of known concentrations of Staphylococcus aureus suspension. 7 CFU / mL.

[0064] 3. In a clean bench, remove the cell culture medium from the well plate and wash three times with sterile phosphate buffer to remove residual antibiotics. Add 3 mL of Staphylococcus aureus suspension containing the antibiotic to the induced cells. Place the 6-well plate in a centrifuge and centrifuge at 1000 rpm for 10 min. Then incubate the centrifuged plate in a 37°C, 5% CO2 incubator for 2 h.

[0065] 4. Discard the Staphylococcus aureus suspension in the 6-well plate, then incubate with cell culture medium containing 100 mg / L gentamicin for 20 min to remove Staphylococcus aureus adhering to the cell surface. Afterward, incubate with cell culture medium containing 50 mg / L gentamicin for 5 days. This procedure prevents reinfection by Staphylococcus aureus released from infected dead cells and introduces the bacteria into a viable, unculturable state. During induction, monitor cell condition using an optical microscope and change the cell culture medium containing 50 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0066] 5. Remove the cell culture medium containing gentamicin, wash three times with sterile phosphate buffer, then add 1 mL of 0.1 wt% SDS solution to lyse the cells and release intracellular Staphylococcus aureus. Then add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live but unculturable Staphylococcus aureus.

[0067] Example 6

[0068] 1. 16HBE cells cultured to passage 5 were used for plating. First, the cell culture medium was removed, and the cells were digested, centrifuged, and resuspended to obtain a cell suspension. This suspension was then seeded into T25 flasks at a concentration of 1×10⁶ cells / mL. 6 The cells / flasks are placed in an incubator until they completely adhere to the incubator, thus obtaining induced cells.

[0069] 2. The *Pseudomonas aeruginosa* strain cultured overnight to the logarithmic growth phase was centrifuged at 8000 rpm for 2 min at room temperature. It was washed three times with sterile phosphate-buffered saline (PBS) and then resuspended in modified cell culture medium to obtain a *P. aeruginosa* suspension. The OD value of the *P. aeruginosa* suspension was then adjusted to 0.1 using a microplate reader. At this point, the concentration of the *P. aeruginosa* suspension was 10⁻⁶. 8 CFU / mL, the known concentration of Pseudomonas aeruginosa suspension was serially diluted to obtain the desired concentration of 10. 7 CFU / mL of Pseudomonas aeruginosa suspension.

[0070] 3. In a clean bench, remove the cell culture medium from the 6-well plate and wash three times with sterile phosphate buffer to remove residual antibiotics. Add 5 mL of the infected Pseudomonas aeruginosa suspension to the induced cells. Centrifuge the 6-well plate at 1000 rpm for 10 min, and then incubate the centrifuged plate in a 37°C, 5% CO2 incubator for 2 h.

[0071] 4. Remove the *Pseudomonas aeruginosa* suspension from the 6-well plate and aspirate it. Then, incubate the plate for 30 minutes with cell culture medium containing 100 mg / L gentamicin to remove *P. aeruginosa* adhering to the surface of the induced cells. Next, incubate with cell culture medium containing 50 mg / L gentamicin. This step prevents reinfection by *P. aeruginosa* released from infected dead cells and introduces the bacteria into a viable, unculturable state. During induction, monitor cell condition using an optical microscope and change the cell culture medium containing 50 mg / L gentamicin daily to ensure the cells are in a normal physiological state.

[0072] 5. First, remove the cell culture medium containing gentamicin, then wash three times with sterile phosphate buffer, add 1 mL of 0.1% SDS solution to lyse the cells and release the intracellular Pseudomonas aeruginosa. Then, add sterile phosphate buffer to wash and transfer to a centrifuge tube, centrifuge, wash, and finally resuspend with sterile phosphate buffer to obtain a suspension of live but unculturable Pseudomonas aeruginosa.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for inducing the formation of live, unculturable bacteria in the presence of host cells, characterized in that, Includes the following steps: S1. Discard the cell culture medium with a cell confluence of 70-90%, wash with buffer, digest with trypsin, collect the cells, determine the cell concentration by cell counting, and then transfer the obtained cells to cell culture dishes of different sizes and incubate them in an incubator to obtain induced cells; the cells are 16HBE, Base-2B, or HaCaT human cells, and the bacteria are one or more of Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa; the cell number is 10. 4 ~10 7 cell / well, bacterial suspension concentration of 10 4 ~10 8 S2. Collect the bacteria cultured overnight, wash and centrifuge with buffer, then resuspend in modified cell culture medium. Adjust the OD value of the bacteria to equal the bacterial concentration using an ELISA reader. Dilute the bacteria of known concentrations serially to obtain a bacterial suspension. The modified cell culture medium consists of basal cell culture medium and FBS. The content of the basal cell culture medium is 30-99.95 wt%, and the content of FBS in the basal cell culture medium is 0.05-50 wt%. The basal cell culture medium is DMEM, RPMI 1640, MEM, or F-12K. S3. In a clean bench, add the bacterial suspension from step S2 to the induced cells, centrifuge to ensure the bacteria fully enter the cells, and then transfer to a constant temperature incubator at 10-60℃ and 0.5-30 vol% CO2 for 1-5 h. S4. In a clean bench, remove the bacterial suspension from the cell culture dish from step S3, wash with buffer, and add a solution containing 10-500 CFU / mL. Cell culture medium containing 10-500 mg / L gentamicin was placed in an incubator for incubation. The culture medium was then aspirated, and cell culture medium containing 10-500 mg / L gentamicin was added to prevent reinfection by bacteria released from infected dead cells. Cell condition was monitored using an optical microscope, and cell culture medium was replaced promptly. S5. After 3-10 days of cell treatment, collect bacteria from the host cells. First, remove the bacterial suspension and wash with buffer. Then, add 0.1-10 wt% SDS cell lysis buffer and incubate at room temperature to release intracellular bacteria. Wash with buffer, centrifuge, wash, and resuspend in buffer to obtain live, unculturable bacteria. The cell culture medium consists of basal cell culture medium, FBS, and penicillin-streptomycin. The content of basal cell culture medium is 30-99.9 wt%, the content of FBS in basal cell culture medium is 0.05-50 wt%, and the content of penicillin-streptomycin in basal cell culture medium is 0.05-5 wt%. The basal cell culture medium is DMEM, RPMI 1640, MEM, or F-12K.

2. The method for inducing the formation of live, unculturable bacteria in the presence of host cells according to claim 1, characterized in that, In step S1, the cells are cell lines from generation 3 to 20, and the bacteria are in the logarithmic growth phase.

3. The method for inducing the formation of live, unculturable bacteria in the presence of host cells according to claim 1, characterized in that, In step S2, the centrifugation speed is 500~2000 rpm and the centrifugation time is 5~30 min.

4. The method for inducing the formation of live, unculturable bacteria in the presence of host cells according to claim 1, characterized in that, The buffer solutions used in steps S1 to S5 are all sterile phosphate buffer solutions.

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