Construction method of resuscitation model of salmonella in holding state

Abstract

The invention provides a resuscitation model of salmonella in a holding state, which is shown as a formula (2), and provides an establishment method of the resuscitation model. According to the model, the resuscitation condition of the salmonella in the holding state can be accurately, objectively and quantitatively reflected, a data basis is provided for research on the resuscitation model of the bacteria in the holding state, and resuscitation of the bacteria in the holding state can be known. Y = c + A1e-at + A2e-bt = 0.21787 to 0.08087 * e <-0.02116 > t to 0.004 * e <-98.57626 > t (2), and the formula is shown in the description.

Description

Technical Field

[0001] The invention relates to a method for constructing a persistent-state Salmonella resuscitation model. Background Art

[0002] Salmonella, as a widely distributed Gram-negative foodborne pathogen, poses a serious challenge to food safety and public health. This bacterium can be transmitted to humans through contamination of foods such as meat, eggs and dairy products, causing a series of serious diseases, including typhoid fever, acute gastroenteritis and sepsis. In particular, when Salmonella invades blood tissue, the systemic inflammation it triggers may lead to fatal consequences.

[0003] During food processing and storage, Salmonella may encounter adverse environments such as extreme pH, limited nutrients, low temperature and high osmotic pressure. These conditions can cause Salmonella to enter a persister state. The persister state is an adaptive survival strategy adopted by Salmonella when faced with environmental conditions that are unfavorable for its own survival. In this state, Salmonella grows slowly or enters a dormant state, and this phenotype is not heritable. When the adverse environment disappears, Salmonella in the persister state can recover and once again pose a threat to the surrounding environment and human safety. At present, there are few studies on the recovery of persister bacteria.

[0004] The persistence bacteria recovery model is an important data basis for understanding and preventing the recovery of persistence bacteria. 600 The model was fitted to the data according to the change of the value. This model provides data support for the research on the recovery model of bacteria in the retention state. Summary of the invention

[0005] The present invention provides a method for constructing a persistent-state Salmonella resuscitation model, and the resuscitation of the persistent-state Salmonella can be understood through the model. The deviation between the model value and the experimental data is small, and the relationship between the resuscitation of the persistent-state bacteria and time can be well fitted.

[0006] The technical solution adopted by the present invention is:

[0007] A method for constructing a persistent Salmonella resuscitation model, the method comprising the following steps:.

[0008] (1) adding a Salmonella bacterial solution to a low water activity LB broth medium and culturing;

[0009] (2) adding ciprofloxacin stock solution to the Salmonella cultured in step (1), and culturing;

[0010] (3) centrifuging the bacterial solution obtained by culturing in step (2), and the obtained precipitate is a substance containing persister Salmonella, and the live bacteria in the precipitate are persister Salmonella;

[0011] (4) adding the persister Salmonella obtained by centrifugation in step (3) to LB broth medium for culturing and resuscitation;

[0012] (5) During the recovery process in step (5), starting from 0 minute, measure the OD with an ELISA reader every 5 minutes. 600 value.

[0013] (6) Fitting the Salmonella persister recovery curve using the nonlinear model of formula (1)

[0014] y=c+A1e -at +A2e -bt (1)

[0015] Where t is the culture recovery time, in minutes; a is the growth rate of cells in the normal state; b is the conversion rate from the retention state to the normal state; c is the OD value of the bacterial solution when reaching stability. 600 Value; A1 is the potential factor affecting the growth of normal cells; A2 is the potential factor affecting the recovery of persister cells; y is the OD of the bacterial solution at time t 600 Value, a, b are the parameters to be fitted of the nonlinear equation;

[0016] Use nonlinear fitting to find a and b.

[0017] After a and b are obtained by nonlinear fitting, the OD of the persister recovery can be obtained. 600 The relationship between value change and time is as follows (2):

[0018] y=c+A1e -at +A2e -bt =0.21787-0.08087×e -0.02116t -0.004×e -98.57626t (2)

[0019] Furthermore, the present invention uses Salmonella Typhimurium, numbered ATCC14028, provided by the National Light Industry Food Quality Supervision and Inspection Nanjing Station.

[0020] In step (1), the preparation method of the Salmonella liquid is to activate the Salmonella overnight to obtain an activated liquid, and then add the Salmonella activated liquid to a low water activity LB broth medium and culture it at 37°C until the bacterial count reaches 10 8 CFU / mL.

[0021] In step (1), the method for preparing the low water activity LB broth culture medium is to adjust the mass fraction of NaCl in the LB broth culture medium to 2%.

[0022] In step (2), the concentration of ciprofloxacin in the ciprofloxacin mother solution is above 1000 μg / mL, preferably 1280 μg / mL, and the solvent is 0.7 vt% phosphoric acid aqueous solution.

[0023] In step (2), the amount of ciprofloxacin mother solution added is controlled so that its final concentration is 25-100 times, preferably 50 times, the minimum inhibitory concentration of ciprofloxacin against Salmonella.

[0024] In step (3), the method for isolating the Salmonella persisters is to add a 50-fold minimum inhibitory concentration of ciprofloxacin solution to a Salmonella solution, culture the mixture at 37° C. for 4 hours, and then centrifuge to obtain the Salmonella persisters.

[0025] In step (4), the culture temperature for resuscitation of Salmonella persisters is 37°C.

[0026] In step (5), the recovery of the persisting Salmonella at different times was measured. 100 μL of the bacterial solution was taken out every 5 minutes, and the OD was measured using an ELISA reader with the absorbance set at 600 nm. 600 value.

[0027] Generally, the experiment was repeated three times at each time point, and the average value of the three experiments was taken.

[0028] The OD of bacteria in this study 600 The value changes were expressed as mean and standard deviation, and data processing was performed using Microsoft Excel 2021. In this study, the nonlinear fitting function in Origin 2021 software was used to fit the data.

[0029] The present invention also provides a persistent Salmonella resuscitation model, as shown in the following formula (2):

[0030] y=c+A1e -at +A2e -bt =0.21787-0.08087×e -0.02116t -0.004×e -98.57626t (2)

[0031] Where t is the culture recovery time, in minutes; a is the growth rate of cells in the normal state; b is the conversion rate from the retention state to the normal state; c is the OD value of the bacterial solution when reaching stability. 600 Value; A1 is the potential factor affecting the growth of normal cells; A2 is the potential factor affecting the recovery of persister cells; y is the OD of the bacterial solution at time t 600 value.

[0032] When establishing the model, the present invention found that during the recovery process of persister Salmonella, as time goes on, the recovery of persister Salmonella shows a nonlinear law, and the recovery trend is very slow. The present invention uses a nonlinear equation to fit the bacterial OD 600 The relationship between value and time shows a significant nonlinear relationship. The corrected correlation coefficient R 2 The residual sum of squares is 0.99142, which indicates that the model prediction value has little deviation from the experimental data and can better fit the bacterial OD 600 Value and time relationship.

[0033] The beneficial effects of the present invention are as follows: the present invention provides a method for constructing a model for the resuscitation of persisting Salmonella, and the model can accurately, objectively and quantitatively reflect the resuscitation of persisting Salmonella, provide a data basis for relevant research on the resuscitation of persisting Salmonella, and be beneficial to the control, risk warning and assessment of persisting Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and other advantages of the present invention will become more clear.

[0035] Figure 1 Flowchart for the resuscitation of persister Salmonella.

[0036] Figure 2 Recovery trend of persister Salmonella.

[0037] Figure 3 OD of the culture solution when the persister Salmonella was revived 600 A plot of the values ​​of a nonlinear equation fit over time. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to the accompanying drawings:

[0039] 1. Materials and methods

[0040] (1) Experimental consumables, strains and equipment

[0041] Consumables: several 96-well plates, several test tubes, 6 150mL conical flasks, several disposable culture dishes, a 50mL beaker, several 10μL, 200μL, 1000μL pipette tips, several 1.5mL centrifuge tubes, several 50mL centrifuge tubes, several 10mL centrifuge tubes, LB broth medium, agar powder, 0.85% saline, UP water.

[0042] Equipment: one 10μL, one 200μL, one 1000μL pipette, clean bench, autoclave, biochemical incubator, and microplate reader.

[0043] Bacteria: This experiment used Salmonella Typhimurium, number ATCC14028, provided by the National Light Industry Food Quality Supervision and Inspection Nanjing Station.

[0044] Antibiotic: Ciprofloxacin.

[0045] 2. Experimental preparation:

[0046] (1) Prepare 150 mL LB broth medium (1 10 mL LB (test tube) + 3 40 mL LB (conical flasks)) and 1 L LB agar medium.

[0047] (2) Adjust the mass fraction of NaCl in 40 mL LB broth to 2%.

[0048] (3) Prepare 2000 mL of normal saline, 1000 mL of sterile water, and 1280 μg / mL of ciprofloxacin stock solution in 0.7 vt% phosphoric acid aqueous solution and store in a 4°C refrigerator for no more than 6 months.

[0049] (4) Sterilize the culture medium, physiological saline, beakers, pipette tips, centrifuge tubes, test tubes, conical flasks, etc. prepared above at 121°C for 15 min.

[0050] 3. Cultivation of Salmonella

[0051] (1) Activate Salmonella and pick a single colony in 10 mL LB broth medium and culture it overnight at 37°C and 160 rpm, which is called activation solution.

[0052] (2) Take 100 μL of the activated bacterial solution and culture it in 40 mL of LB broth medium with a mass fraction of 2% NaCl at 37°C for 5 hours until the culture temperature reaches 10 8 CFU / mL.

[0053] (3) This step is to verify the initial concentration: take 1 mL of the bacterial solution in (2) and dilute it 10 times to 10 -4 10 5 10 6 Then, 100 μL was spread on LB agar medium and cultured at 37°C for 24 h to count the initial bacterial solution concentration of about 10 8 CFU / mL or so.

[0054] 4. Isolation of Salmonella persisters

[0055] (1) Add 50 μL of ciprofloxacin stock solution to the remaining bacterial solution in step 3 (2) to make the final ciprofloxacin concentration 50 times the MIC, and culture in a shaker at 37°C and 160 rpm for 4 hours.

[0056] (2) This step is to verify the initial concentration: take 1 mL of the bacterial solution in (1), centrifuge at 8000 rpm for 5 min, remove the supernatant, resuspend the precipitate in 1 mL of sterile saline and dilute it 10-fold to 10 2 10 3 10 4 After that, 100 μL was spread on LB agar medium and cultured at 37°C for 24 h to count the initial Salmonella persister concentration of about 10 5 CFU / mL or so.

[0057] 5. Salmonella persisters resuscitation, such as Figure 1 Shown

[0058] (1) Centrifuge the remaining bacterial solution in step 4 (1), mix with an equal amount of LB broth medium, and culture and revive in a shaking incubator at 37°C and 160 rpm.

[0059] (2) The recovery process starts at 0 minute. Every five minutes, 100 μL of bacterial solution is taken out and added to a 96-well plate. The OD is measured using an ELISA reader. 600 value.

[0060] 6. Model building

[0061] The simulated recovery conditions for persister Salmonella in this study were simple, considering only the bacterial OD at a fixed temperature. 600 Therefore, the change in OD600 value (y) during the recovery of persisting Salmonella was calculated according to the nonlinear equation (1):

[0062] y=c+A1e -at +A2e -bt (1)

[0063] Where t is the culture recovery time, in minutes; a is the growth rate of cells in the normal state; b is the conversion rate from the retention state to the normal state; c is the OD value of the bacterial solution when reaching stability. 600 Value; A1 is the potential factor affecting the growth of normal cells; A2 is the potential factor affecting the recovery of persister cells; y is the OD of the bacterial solution at time t 600 value.

[0064] 7. Data Analysis

[0065] The OD of bacteria in this study 600The value changes are expressed as mean and standard deviation, and data processing is performed using Microsoft Excel 2021. In this study, the nonlinear fitting function in Origin 2021 software was used to fit the data (e.g. Figure 3 shown).

[0066] 8. Results and Discussion

[0067] (1) The resurgence of persistent Salmonella

[0068] The test showed that the OD600 value of the initial persisting Salmonella was 0.133. Figure 2 This is the recovery curve of the persisting Salmonella, such as Figure 2 As shown, within 165 minutes, the OD600 value of the bacterial solution increased by 0.084. As time went on, the recovery of the persisting Salmonella generally showed a nonlinear growth pattern.

[0069] The present invention provides a method for constructing a Salmonella resuscitation model in a persistent state. The methods and approaches for implementing the technical solution are very specific. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a persistent Salmonella resuscitation model, characterized in that: The steps include: (1) inoculating Salmonella into a low water activity LB broth medium, culturing, and obtaining a bacterial liquid; (2) mixing the bacterial solution obtained by culturing in step (1) with a ciprofloxacin mother solution, and culturing to obtain a bacterial solution; (3) centrifuging the bacterial solution obtained by culturing in step (2), and the resulting precipitate is a substance containing persister bacteria; (4) The persister bacteria obtained in step (3) were inoculated into LB broth medium for culture and recovery, and the OD of the bacterial solution at different culture and recovery times was measured. 600 value. (5) Fitting the growth curve using the model of formula (1) y=c+A1e -at +A2e -bt (1) Where t is the culture recovery time, in minutes; a is the growth rate of cells in the normal state; b is the conversion rate from the retention state to the normal state; c is the OD value of the bacterial solution when reaching stability. 600 Value; A1 is the potential factor affecting the growth of normal cells; A2 is the potential factor affecting the recovery of persister cells; y is the OD of the bacterial solution at time t 600 Value, a, b are the parameters of the nonlinear equation to be fitted. Calculate a and b by nonlinear fitting.

2. The method according to claim 1, characterized in that In step (1), the method for preparing the low water activity LB broth is to adjust the mass fraction of NaCl in the LB broth culture medium to 2%.

3. The method according to claim 1, characterized in that In step (2), the preparation method of the Salmonella liquid is to activate the Salmonella overnight to obtain an activated liquid, and then add the activated liquid to a low water activity LB broth medium and culture until the bacterial count is 10 8 CFU / mL.

4. The method according to claim 1, characterized in that In step (2), the concentration of ciprofloxacin in the ciprofloxacin mother solution is above 1280 μg / mL, and the solvent is 0.7 vt% phosphoric acid aqueous solution.

5. The method according to claim 1, characterized in that In step (2), the amount of ciprofloxacin mother solution added is controlled so that its final concentration is 50 times the minimum inhibitory concentration of ciprofloxacin.

6. The method according to claim 1, characterized in that The resuscitation temperature of the persisting Salmonella in step (4) is 37°C.

7. A persister Salmonella recovery model established according to the method of claim 1, characterized in that As shown in formula (2) y=c+A1e -at +A2e -bt =0.21787-0.08087*e -0.02116t −0.004*e -98.57626t (2) Where t is the culture recovery time, in minutes; a is the growth rate of cells in the normal state; b is the conversion rate from the retention state to the normal state; c is the OD value of the bacterial solution when reaching stability. 600 Value; A1 is the potential factor affecting the growth of normal cells; A2 is the potential factor affecting the recovery of persister cells; y is the OD of the bacterial solution at time t 600 value.

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