Method for inducing bacteria to enter viable and non-culturable state in host body and application thereof

Abstract

The invention discloses a method for inducing bacteria to enter a viable and non-culturable state in a host body and application of the method. According to the induction method, the respiratory tract environment of a rat is used as an induction condition to enable bacteria to enter a VBNC dormant state, and different from an induction mode of in-vitro environment pressure or an in-vitro cell system, the method successfully and effectively induces the bacteria to enter the VBNC state by using a complete biological individual, and is simple and convenient to operate and easy to prepare; the induction method can effectively realize induction of VBNC state bacteria in a host body, has very important significance for further research on induction of viable and non-culturable bacteria and subsequent health risk evaluation, and can be used for carrying out research on analysis of bacteria and host reactions under the condition of interaction of multiple innate immune pathways; the research on the drug resistance change and the like of the VBNC state bacteria induced in the host has important significance for understanding the drug resistance development mechanism of the bacteria in the environment in the host.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular, relates to a method for inducing bacteria to enter a live, uncultivable state in a host and an application thereof. Background Art

[0002] Many studies have pointed out that in harsh environments or when nutrition is scarce, bacteria enter a dormant state to reduce energy consumption and prolong survival time. Bacteria use dormancy to escape environmental threats. Viable but non-culturable state (VBNC) bacteria, as a type of dormant bacteria, can escape host attack and elimination. Whether bacteria that enter the respiratory tract will enter a dormant state due to the difference between the respiratory environment and the external environment or the host's defense has attracted special attention. As a deeper dormancy state, whether the viable but non-culturable state can cause exogenous bacteria to enter a viable but non-culturable state in the host's respiratory environment remains to be studied.

[0003] Bacteria can persist in a viable, unculturable state and are not susceptible to antibiotics, which may facilitate asymptomatic carriage of such pathogens, prolong the incubation period of pathogenic bacteria, lead to recurrent infections, and promote bacterial survival during antibiotic treatment, posing a risk to public health. Many studies have elucidated the mechanisms by which bacterial variation occurs in vitro. Currently, most studies use adverse environmental factors to induce bacteria to enter the VBNC state, and there is little information on bacterial phenotypic variation in vivo. Whether bacteria can become VBNC in intact animals rich in multiple immune pathways has not been studied. In order to further study the formation mechanism of VBNC bacteria in animals, studies should be conducted on intact animals that allow analysis of host responses under conditions where multiple innate immune pathways interact. Analyzing the host response of intact animals and whether various immune pathways can cause bacteria to enter the VBNC state is important for assessing human health risks by analyzing the changes in drug resistance of VBNC bacteria induced under this pathway. Therefore, there is an urgent need for an induction method that causes bacteria to become VBNC in the host without interference from other environmental factors. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a method for inducing bacteria to enter a live, uncultivable state in a host.

[0005] The second object of the present invention is to provide application of the method.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention first provides a method for inducing bacteria to enter a live and unculturable state, comprising exposing bacterial liquid to the respiratory tract of rats for 0 to 14 consecutive days to induce live and unculturable bacteria in the respiratory tract of the rats.

[0008] The present invention uses the respiratory environment of rats as an induction condition for the first time, successfully causing bacteria to enter a viable non-culturable state (VBNC); specifically, the bacterial solution is exposed to the inside of the rat respiratory tract, and by regulating the appropriate bacterial solution exposure concentration, exposure days, and each exposure dose, after a certain number of days of continuous exposure, the differences in the respiratory environment of the rats can be used to induce the bacteria to enter the VBNC state. The method can effectively achieve the induction of bacteria in the VBNC state, which is of great significance for studying the induction of viable non-culturable bacteria. Different from the induction method of in vitro environmental pressure or in vitro cell system, this method uses a complete biological individual to successfully induce bacteria to enter a dormant state, and the advantage is that it can carry out research on the analysis of bacteria and host responses under the conditions of the interaction of multiple innate immune pathways.

[0009] Furthermore, the bacterial solution concentration is 1×10 7 ~3×10 9 CFU / mL.

[0010] Preferably, the concentration of the bacterial solution is 2.0 to 3.0×10 9 CFU / mL.

[0011] Furthermore, the respiratory tract includes the nasal passages, pharynx, trachea and lungs.

[0012] Furthermore, the bacteria include but are not limited to one or more of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.

[0013] Furthermore, the bacteria are in a logarithmic growth phase.

[0014] Furthermore, the method for preparing the bacterial culture liquid is to culture the bacteria to the logarithmic growth phase, collect the bacterial cells, wash with a buffer solution, centrifuge and resuspend, and adjust to a suitable concentration to obtain the bacterial culture liquid.

[0015] Preferably, the buffer is a sterile phosphate buffer, and the centrifugation is performed at 500 to 8000 rpm for 1 to 30 min.

[0016] Furthermore, the exposure days are 3 to 14 days.

[0017] Preferably, the continuous exposure days are 7 to 14 days.

[0018] Preferably, the continuous exposure days are 7 days.

[0019] Furthermore, the method of exposing the bacterial liquid to the respiratory tract of rats is to expose the rats in a coma state by nasal drops.

[0020] Preferably, the rat in coma is anesthetized with ether.

[0021] Furthermore, the nasal drop dosage of the nasal drop method is 100 to 300 μL.

[0022] Preferably, the nasal drop dosage is 100 to 200 μL, more preferably 200 μL.

[0023] Preferably, the nasal drops are administered 1 to 5 times a day.

[0024] Preferably, the dibi method is nasal drops 1 to 4 times a day.

[0025] More preferably, the nasal drops are administered once a day.

[0026] Furthermore, the rats are SD rats that have been raised for 2 to 14 days under the environmental conditions of 18 to 26° C. and a relative humidity of 40% to 70% and have a body weight of 100 to 400 g. The SD rats are raised under the above-mentioned appropriate environmental conditions for a period of time to adapt to the surrounding environment to avoid stress reactions during the experiment.

[0027] Furthermore, the method for collecting the live unculturable bacteria is to kill the rats and collect the bacterial samples in the rats' respiratory tract using a sterile buffer solution; the respiratory tract includes the nasal passages, throat, trachea, and lungs.

[0028] The present invention provides living unculturable bacteria obtained by any of the above methods.

[0029] The present invention also provides the application of any of the above methods in human health risk assessment. Specifically, the induction method can be used to obtain live, unculturable bacteria induced in intact animals, explore the changes in the number of VBNC bacteria in different parts of the respiratory tract, the growth curve, and the changes in drug resistance, and pay attention to the changes in rat weight, pulmonary edema, and pulmonary inflammation, which can be used to assess the health risks of bacteria to humans.

[0030] The present invention provides use of any of the above methods in evaluating changes in drug resistance of live, unculturable bacteria in the respiratory tract.

[0031] The present invention provides the use of live unculturable bacteria obtained by any of the above methods in preparing a product for evaluating the antibiotic resistance of live unculturable bacteria in a host.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a method for inducing bacteria in a live, unculturable state in the respiratory tract of a host, using the respiratory environment of rats as an induction condition to cause the bacteria to enter a VBNC dormant state. Different from the induction method of in vitro environmental pressure or in vitro cell system, the present method uses a complete biological individual to successfully and effectively induce bacteria to enter a VBNC state, is simple to operate, and is easy to prepare; the method can effectively achieve the induction of bacteria in the VBNC state in the host, which is of great significance for further research on the induction of live, unculturable bacteria. The advantage of using the induction method of the present invention to obtain bacteria in the VBNC state is that it is possible to carry out research on analyzing bacteria and host responses under the conditions of interaction of multiple innate immune pathways, explore the changes in drug resistance of bacteria in the VBNC state induced in the complete host, etc., which is of great significance for understanding the development mechanism of drug resistance of bacteria in the in vivo environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Flow chart of the method for inducing live, unculturable bacteria in the host's respiratory tract.

[0035] Figure 2 The growth of culturable bacteria isolated from the nasal cavity (A), pharynx (B), trachea (C) and lungs (D) of SD rats in Example 1 on cetyl trimethylammonium bromide agar medium.

[0036] Figure 3 This is a graph showing the number of live and culturable Pseudomonas aeruginosa bacteria in samples collected from various parts of the respiratory tract of SD rats in Example 1.

[0037] Figure 4 This is a scanning electron micrograph of wild-type Pseudomonas aeruginosa.

[0038] Figure 5 These are scanning electron micrographs of bacteria collected from the nasal cavity (A), pharynx (B), trachea (C) and lungs (D) of SD rats in Example 2.

[0039] Figure 6 The bacterial concentration is 10 9 CFU / mL, under the condition of daily nasal drops dose of 200 μL, the percentage of live unculturable Pseudomonas aeruginosa in samples collected from various parts of the respiratory tract of SD rats changes with the number of days of nasal drops exposure.

[0040] Figure 7 The figure shows the change in the percentage of live, uncultivable Pseudomonas aeruginosa in samples collected from various parts of the respiratory tract of SD rats with the concentration of nasal drops under the condition of nasal drops of 200 μL per day for 7 days.

[0041] Figure 8 The concentration of the bacterial solution is 10 9 CFU / mL, under the condition of nasal drops once a day, the percentage of live uncultivable Pseudomonas aeruginosa in samples collected from various parts of the respiratory tract of SD rats changes with exposure dose. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1

[0045] A flow chart of an induction method for converting bacteria into a viable, unculturable state in a host Figure 1 As shown, the specific method is as follows:

[0046] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0047] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL.

[0048] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0049] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of a bacterial solution of Pseudomonas aeruginosa at a concentration of CFU / mL (the rats in the control group were not subjected to this treatment and were immediately killed after the adaptation period in step 3 to obtain respiratory bacteria samples).

[0050] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 7 consecutive days. On the 8th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0051] 6. Count the live bacteria (PMA combined with qPCR) and culturable bacteria in bacterial samples from various parts of the respiratory tract. The growth of culturable bacteria in bacterial samples collected from the nasal passages, throat, trachea, and lungs is as follows: Figure 2 As shown, Figure 3 This is a graph showing the number of live and culturable Pseudomonas aeruginosa bacteria in samples collected from various parts of the respiratory tract of SD rats. It can be seen that the number of live bacteria in various parts of the respiratory tract is greater than the number of culturable bacteria, indicating that there are live unculturable bacteria in various parts of the respiratory tract. The bacterial samples collected from the respiratory tract of SD rats were observed using a scanning electron microscope to observe their morphology. It was found that the morphology of the bacteria extracted from the respiratory tract was significantly shortened compared with the wild-type bacteria, which proves that the induction of live unculturable bacteria was successful.

[0052] Example 2

[0053] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0054] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 5000 rpm for 5 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL.

[0055] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0056] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of a bacterial solution of Pseudomonas aeruginosa at a concentration of CFU / mL (the rats in the control group were not subjected to this treatment and were immediately killed after the adaptation period in step 3 to obtain respiratory bacteria samples).

[0057] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 14 consecutive days. On the 15th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0058] 6. The number of live bacteria and culturable bacteria in the bacterial samples from various parts of the respiratory tract was counted, and the number of live bacteria was greater than the number of culturable bacteria. The bacterial samples collected from the respiratory tract of SD rats were observed using a scanning electron microscope to observe their morphology and found that they were different from wild-type bacteria ( Figure 4 ) compared with the VBNC bacteria extracted from various parts of the respiratory tract (nasal passages, throat, trachea, lungs) were significantly shorter ( Figure 5 ), which can prove that the living unculturable bacteria are successfully induced.

[0059] Example 3

[0060] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0061] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 5000 rpm for 5 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL.

[0062] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0063] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of a bacterial solution of Pseudomonas aeruginosa at a concentration of CFU / mL (the rats in the control group were not subjected to this treatment and were immediately killed after the adaptation period in step 3 to obtain respiratory bacteria samples).

[0064] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 3 consecutive days. On the 4th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0065] 6. Count the live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract, and the number of live bacteria is greater than the number of culturable bacteria. Use a scanning electron microscope to observe the morphology of bacterial samples collected from the respiratory tract of SD rats, and find that the morphology of bacteria extracted from the respiratory tract is significantly shortened compared with wild-type bacteria, which proves that the induction of live unculturable bacteria is successful.

[0066] Combining Examples 1, 2, and 3, the effects of different exposure days on the number of induced VBNC bacteria can be statistically analyzed. The results are as follows: Figure 6 As shown in the figure, when the exposure days were 3, 7, and 14 days, the proportion of VBNC bacteria in the nasal cavity, trachea, and lungs was close to 100%, while the proportion of VBNC bacteria in the throat was greatly affected by the exposure time, and the proportion was close to 100% when the exposure day was 7 days. When the exposure day was extended to 14 days, the proportion of VBNC bacteria decreased.

[0067] Example 4

[0068] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0069] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 0.1, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 8 CFU / mL.

[0070] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0071] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×10 8 SD rats were exposed to a single nasal drop of a bacterial solution of Pseudomonas aeruginosa at a concentration of CFU / mL (the rats in the control group were not subjected to this treatment and were immediately killed after the adaptation period in step 3 to obtain respiratory bacteria samples).

[0072] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 7 consecutive days. On the 8th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0073] 6. Count the live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract, and the number of live bacteria is greater than the number of culturable bacteria. Use a scanning electron microscope to observe the morphology of bacterial samples collected from the respiratory tract of SD rats, and find that the morphology of bacteria extracted from the respiratory tract is significantly shortened compared with wild-type bacteria, which proves that the induction of live unculturable bacteria is successful.

[0074] Example 5

[0075] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0076] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 0.1, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 8 CFU / mL, and then dilute the bacterial solution with sterile phosphate buffer to a concentration of 2.0-3.0×10 7 CFU / mL.

[0077] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0078] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×10 7 SD rats were exposed to a single nasal drop of a bacterial solution of Pseudomonas aeruginosa at a concentration of CFU / mL (the rats in the control group were not subjected to this treatment and were immediately killed after the adaptation period in step 3 to obtain respiratory bacteria samples).

[0079] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 7 consecutive days. On the 8th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0080] 6. Count the live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract, and the number of live bacteria is greater than the number of culturable bacteria. Use a scanning electron microscope to observe the morphology of bacterial samples collected from the respiratory tract of SD rats, and find that the morphology of bacteria extracted from the respiratory tract is significantly shortened compared with wild-type bacteria, which proves that the induction of live unculturable bacteria is successful.

[0081] Combined with Examples 3, 4, and 5, the effects of different nasal drops concentrations on the number of induced VBNC bacteria can be statistically obtained. The results are as follows: Figure 7 As shown in the figure, with the increase of the concentration of nasal drops, the proportion of induced VBNC bacteria in various parts of the respiratory tract increased, ranging from 2.0 to 3.0×10 9 CFU / mL, the proportion of VBNC bacteria in various parts of the respiratory tract was close to 100%

[0082] Example 6

[0083] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0084] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL.

[0085] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0086] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 100 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of Pseudomonas aeruginosa liquid containing CFU / mL.

[0087] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 7 consecutive days. On the 8th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0088] 6. Count the live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract, and the number of live bacteria is greater than the number of culturable bacteria. Use a scanning electron microscope to observe the morphology of bacterial samples collected from the respiratory tract of SD rats, and find that the morphology of bacteria extracted from the respiratory tract is significantly shortened compared with wild-type bacteria, which proves that the induction of live unculturable bacteria is successful.

[0089] Example 7

[0090] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0091] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL.

[0092] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0093] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 300 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of Pseudomonas aeruginosa liquid containing CFU / mL.

[0094] 5. According to the above-mentioned nasal drops dosage, SD rats were exposed once a day for 7 consecutive days. On the 8th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0095] 6. Count the live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract, and the number of live bacteria is greater than the number of culturable bacteria. Use a scanning electron microscope to observe the morphology of bacterial samples collected from the respiratory tract of SD rats, and find that the morphology of bacteria extracted from the respiratory tract is significantly shortened compared with wild-type bacteria, which proves that the induction of live unculturable bacteria is successful.

[0096] Combined with Examples 1, 6, and 7, the effects of different exposure doses on the number of induced VBNC bacteria can be statistically obtained. The results are as follows: Figure 8 As shown, when the nasal drop exposure dose was 200 μL each time, the proportion of VBNC bacteria induced in various parts of the respiratory tract was the highest. When the nasal drop exposure dose was increased to 300 μL each time, the VBNC bacteria induced in various parts of the respiratory tract decreased slightly.

[0097] Example 8

[0098] 1. Cultivate Staphylococcus aureus overnight in a shaker at 37°C and 140 rpm until OD600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0099] 2. Centrifuge the Staphylococcus aureus cultured overnight to the logarithmic growth phase at 4°C, 2000 rpm for 10 min, wash three times with sterile phosphate buffer, and resuspend to obtain a Staphylococcus aureus suspension. Then use an ELISA instrument to adjust the OD value of the Staphylococcus aureus suspension to 1.0. At this time, the concentration of the Staphylococcus aureus suspension is 2.0-3.0×10 9 CFU / mL.

[0100] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0101] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 100 μL of the above-mentioned concentration of 2.0-3.0×10 9 SD rats were exposed to a single nasal drop of Staphylococcus aureus liquid containing CFU / mL.

[0102] 5. The above-mentioned nasal drops were administered to SD rats twice a day for 14 consecutive days. The rats were bled to death on the 15th day, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0103] 6. The live bacteria and culturable bacteria in bacterial samples from various parts of the respiratory tract were counted, and the result showed that the number of live bacteria was much greater than the number of culturable bacteria. The bacterial samples collected from the respiratory tract of SD rats were then observed under a scanning electron microscope to observe their morphology. It was found that compared with wild-type bacteria, the diameter of the cocci extracted from the respiratory tract was shortened, which proved that the induction of live, unculturable bacteria was successful.

[0104] Example 9

[0105] 1. Grow E. coli overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0106] 2. Centrifuge the E. coli cultured overnight to the logarithmic growth phase at 4°C, 6000 rpm for 5 min, wash three times with sterile phosphate buffer, and resuspend to obtain an E. coli suspension. Then use an ELISA reader to adjust the OD value of the E. coli suspension to 0.1. At this time, the concentration of the E. coli suspension is 2.0-3.0×10 8 CFU / mL.

[0107] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 65% for 14 days to allow them to adapt to the environment to avoid stress reactions during the experiment.

[0108] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 100 μL of the above-mentioned concentration of 2.0-3.0×10 8 SD rats were exposed to CFU / mL of Escherichia coli solution by nasal drops once.

[0109] 5. The above-mentioned nasal drops were administered to SD rats 4 times a day for 14 consecutive days. The rats were bled to death on the 15th day, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer.

[0110] 6. Count the live bacteria and culturable bacteria in the bacterial samples from various parts of the respiratory tract. The live bacteria count is greater than the culturable bacteria count. Then use a scanning electron microscope to observe the bacterial samples collected from the respiratory tract of SD rats and observe their morphology. It is found that the morphology of the bacteria extracted from the respiratory tract is significantly shorter than that of the wild-type bacteria, which proves that the live unculturable bacteria have been successfully induced.

[0111] Comparative Example

[0112] 1. Grow Pseudomonas aeruginosa overnight in a shaker at 37°C and 140 rpm until OD 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0113] 2. The Pseudomonas aeruginosa cultured overnight to the logarithmic growth phase was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD of the Pseudomonas aeruginosa suspension was adjusted using an ELISA instrument. 600nm The value is equal to 1.0, at which time the concentration of Pseudomonas aeruginosa suspension is 2.0-3.0×10 9 CFU / mL, and the bacterial solution concentration was adjusted to 2.0-3.0×10 6 CFU / mL.

[0114] 3. SD rats weighing about 200 g were selected and raised in an environment with a temperature of 25°C and a humidity of 55% for 7 days to allow them to adapt to the environment to avoid stress response during the experiment.

[0115] 4. Place the SD rats that have been adapted into an airtight anesthesia box, put 3-4 ether cotton balls into the box, wait for about 10 minutes until the rats enter a coma, and immediately take 200 μL of the above-mentioned concentration of 2.0-3.0×106 SD rats were exposed to a single nasal drop of Pseudomonas aeruginosa liquid containing CFU / mL.

[0116] 5. According to the above nasal drop dosage, administer SD rats 1 time per day.

[0117] The results showed that when the concentration of nasal drops was 2.0-3.0×10 6 CFU / mL, the exposure days need to be greatly extended to successfully induce live unculturable bacteria in the respiratory tract. It requires at least two months of continuous exposure, which greatly prolongs the experimental time and easily affects the progress of subsequent research.

[0118] Application Example 1 Evaluation of drug resistance changes of live, unculturable bacteria in the respiratory tract

[0119] 1. Bacterial culture:

[0120] E. coli was cultured overnight in a shaker at 37°C and 140 rpm to an OD of 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0121] 2. Bacteria collection and concentration adjustment:

[0122] The overnight cultured Pseudomonas aeruginosa was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain an E. coli suspension. Then, the OD value of the E. coli suspension was adjusted to 1.0 using an ELISA reader for later use. At this time, the concentration of the Pseudomonas aeruginosa suspension was 1.0-3.0×10 9 CFU / mL.

[0123] 3. Rats exposed to nasal drops:

[0124] SD rats weighing about 200 g were selected and the concentration of 1.0-3.0×10 9 Rats were exposed to bacterial solution with a daily nasal drop dose of 200 μL for 14 consecutive days.

[0125] 4. Respiratory sampling:

[0126] On the 15th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer to obtain live, unculturable Pseudomonas aeruginosa.

[0127] 6. Drug resistance assessment:

[0128] The drug resistance of Pseudomonas aeruginosa samples collected from the respiratory tract was evaluated by measuring the changes in its sensitivity to different antibiotics and evaluating the development of bacterial resistance. This step is of great significance for understanding the mechanism of drug resistance development of Pseudomonas aeruginosa in the respiratory environment.

[0129] Application Example 2 Risk Assessment of Pseudomonas aeruginosa to Human Health

[0130] 1. Bacterial culture:

[0131] Pseudomonas aeruginosa was cultured overnight in a shaker at 37°C and 140 rpm to an OD of 600nm =0.8~1.2, at this time the bacteria are in the logarithmic growth phase.

[0132] 2. Bacteria collection and concentration adjustment:

[0133] The overnight cultured Pseudomonas aeruginosa was centrifuged at 4°C, 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and resuspended to obtain a Pseudomonas aeruginosa suspension. Then, the OD value of the Pseudomonas aeruginosa suspension was adjusted to 1.0 using an ELISA instrument. At this time, the concentration of the Pseudomonas aeruginosa suspension was 1.0-3.0×10 9 CFU / mL, and the above concentration of bacterial solution was adjusted to 10 using sterile phosphate buffer. 7 CFU / mL is reserved.

[0134] 3. Establish animal model:

[0135] SD rats weighing about 200 g were selected and the concentration of 1.0-3.0×10 7 Rats were exposed to nasal drops of bacterial liquid with a daily nasal drop dose of 200 μL for 14 consecutive days to simulate human infection.

[0136] 4. Respiratory sampling:

[0137] On the 15th day, the rats were bled to death, and bacterial samples were collected from the nasal passages, throat, trachea, and lungs in sterile phosphate buffer to obtain live, uncultivable Escherichia coli.

[0138] 6. Observe and record:

[0139] Record the changes of bacteria in rats, including changes in the number of bacteria entering the viable non-culturable state (VBNC) in various parts of the respiratory tract, changes in growth curves, and changes in drug resistance. Record the physiological changes of rats, including changes in body weight, pulmonary edema, and changes in pulmonary inflammatory cells.

[0140] 7. Health risk assessment:

[0141] Severity and progression of infection: By observing the physiological responses of the rats and the proliferation of bacteria, we can assess the severity and progression of the infection.

[0142] Bacterial resistance: Through long-term exposure, the development of bacterial resistance to antibiotics can be assessed, which is crucial for developing effective treatment strategies.

[0143] Long-term effects of infection: Long-term observations can provide insights into the long-term effects of these bacteria on host health, including the potential for chronic infection and the host’s immune response.

[0144] This is of great significance for assessing the health risks of Pseudomonas aeruginosa to humans.

Claims

1. A method for inducing bacteria to enter a live, uncultivable state, characterized in that: The method comprises exposing bacterial liquid to the respiratory tract of rats for 0 to 14 consecutive days, thereby inducing live unculturable bacteria in the respiratory tract of rats.

2. The method according to claim 1, characterized in that: The bacteria are one or more of Pseudomonas aeruginosa, Escherichia coli or Staphylococcus aureus.

3. The method according to claim 1, characterized in that: The bacterial solution concentration is 1×10 7 ~3×10 9 CFU / mL.

4. The method according to claim 1, characterized in that: The method for exposing the bacterial liquid to the respiratory tract of rats is to expose the rats in a coma state by nasal drops.

5. The method according to claim 4, characterized in that: The nasal drops are performed 1 to 5 times a day.

6. The method according to claim 4, characterized in that: The dosage of the nasal drops is 100 to 300 μL per time.

7. The method according to claim 1, characterized in that: The bacteria in the bacterial liquid are in a logarithmic growth phase.

8. The method according to claim 1, characterized in that: The rats are SD rats that have been raised for 2 to 14 days under environmental conditions of 18 to 26° C. and a relative humidity of 40% to 70% and have a body weight of 100 to 400 g.

9. Use of the method according to any one of claims 1 to 8 in human health risk assessment.

10. Use of the live unculturable bacteria obtained by the method according to any one of claims 1 to 8 in the preparation of a product for evaluating the antibiotic resistance of live unculturable bacteria in a host.

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