Induction method of staphylococcus aureus in VBNC state, resuscitation culture medium and application

Abstract

The invention relates to the technical field of microorganisms, and discloses a VBNC state staphylococcus aureus induction method, a resuscitation medium and application, the resuscitation medium comprises Rpf recombinant protein, MgSO4, peptone, beef extract and sodium chloride, and the mass concentration ratio of the Rpf recombinant protein to the MgSO4 to the peptone to the beef extract to the sodium chloride is (90 ng / L to 110 ng / L): (0.5 g / L to 0.7 g / L): (9 g / L to 11 g / L): (4 g / L to 6 g / L): (70 g / L to 80 g / L); the nucleotide sequence of the Rpf recombinant protein is as shown in SEQ ID NO. 1. The resuscitation medium provided by the invention can successfully resuscitate staphylococcus aureus in a VBNC state.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for inducing Staphylococcus aureus in the VBNC state, a resuscitation medium, and its application. Background Technology

[0002] Staphylococcus aureus (S. aureus), also known as "Staphylococcus aureus," belongs to the genus Staphylococcus and is a representative Gram-positive bacterium, a common foodborne pathogen. Its optimal growth temperature is 37°C, and its optimal pH is 7.4. It is tolerant of high salt concentrations, growing in environments with salt concentrations approaching 10%. Staphylococcus aureus commonly resides on the skin, nasal cavity, pharynx, gastrointestinal tract, boils, and abscesses of humans and animals, and is also present in air and sewage. When testing samples of food, pharmaceuticals, and process water, the low bacterial contamination, low bacterial count, and the presence of difficult-to-culture VBNC bacteria make the detection sensitivity insufficient to meet the requirements for pathogen control, creating a detection blind spot and seriously threatening the microbial control of food and pharmaceuticals.

[0003] In existing technologies, the detection of Staphylococcus aureus in food and pharmaceutical samples mainly relies on culture methods. However, because Staphylococcus aureus can exist in a VBNC (virtual bacterial count), conventional microbial culture methods cannot detect it. US patent application US20200340040A1 uses PCR amplification technology to detect microorganisms in the VBNC state. However, PCR detection is costly, and its sensitivity is insufficient for most detection requirements; the sensitivity of PCR methods generally requires a viable count of 10⁻⁶ in the sample. 4 VBNC bacteria counts are above 10 cells / mL, while in food and pharmaceutical samples they are typically between 0-10. 4 Therefore, there is an urgent need for a culture medium that can successfully detect Staphylococcus aureus in the VBNC state. Summary of the Invention

[0004] This invention provides a method for inducing Staphylococcus aureus in the VBNC state, a resuscitation culture medium, and its application.

[0005] The technical solution adopted in this invention is as follows.

[0006] This invention provides a resuscitation medium comprising Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride, wherein the mass concentration ratio of Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L). The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the resuscitation medium also includes sodium pyruvate and glutamine, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (0.27 g / L-2.2 g / L): (1 g / L-3 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L).

[0008] Furthermore, the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

[0009] Furthermore, the pH value of the resuscitation medium is 7.2-7.6.

[0010] The present invention also provides the application of the resuscitation medium in culturing Staphylococcus aureus in VBNC state: Staphylococcus aureus in VBNC state was cultured in resuscitation medium at a temperature of 36℃-38℃ for 18h-48h.

[0011] The present invention also provides a method for inducing Staphylococcus aureus in the VBNC state, the induction method comprising: adjusting the pH value of a solution containing vancomycin and sodium chloride with glacial acetic acid, mixing them evenly, transferring Staphylococcus aureus not in the VBNC state into the evenly mixed solution, and incubating it at a temperature of 2℃-8℃ for 22h-24h.

[0012] Furthermore, the pH of the solution containing vancomycin and sodium chloride was adjusted to 2.6-3 using glacial acetic acid.

[0013] Furthermore, the vancomycin concentration in the solution containing vancomycin and sodium chloride is 60 mg / L-68 mg / L.

[0014] Furthermore, the mass concentration of sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

[0015] The present invention has the following technical effects: 1. The Rpf recombinant protein in the resuscitation medium of this invention has good peptidoglycan hydrolase activity, which can degrade the peptidoglycan in the cell wall of Staphylococcus aureus, making the cell wall loose. The degraded peptidoglycan can enter the cell through the loose cell wall, thereby reviving the cell. Magnesium ions in magnesium sulfate provide divalent metal ions for the activity of Rpf recombinant protein. Sodium pyruvate can not only act as a scavenger of reactive oxygen free radicals, which can remove reactive oxygen free radicals that damage DNA in the cell and put the cell in a recoverable state, but also act as an energy substance, playing an important role in glycolysis. When Rpf recombinant protein and sodium pyruvate are present in the culture medium, glutamine can be added to enhance the synergistic effect of Rpf recombinant protein and sodium pyruvate, which can relieve the VBNC state of Staphylococcus aureus. In addition, glutamine can also provide sufficient nutrition and energy for the resuscitation process, promoting the transformation of bacteria from the VBNC state to the culturable state.

[0016] 2. This invention also successfully provides a method for inducing Staphylococcus aureus to be in a VBNC state, which can successfully prepare bacteria with a total viable count of not less than 10. 4 Staphylococcus aureus suspension in VBNC state with a cell / mL and a culturable number of 0 cells / mL. Attached Figure Description

[0017] Figure 1 These are the stress experiment results of the embodiments and comparative examples of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] In a first aspect, embodiments of the present invention provide a resuscitation culture medium comprising Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride, wherein the mass concentration ratio of the Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L). The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0020] In this embodiment, the resuscitation medium further includes sodium pyruvate and glutamine, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (0.27 g / L-2.2 g / L): (1 g / L-3 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L).

[0021] In this embodiment, the resuscitation medium also includes water.

[0022] In this embodiment, the resuscitation medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride, with a mass concentration ratio of 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

[0023] In this implementation method, the pH value of the resuscitation medium is 7.2-7.6.

[0024] Secondly, the present invention also provides an application of a resuscitation medium in culturing Staphylococcus aureus in the VBNC state.

[0025] In this embodiment, Staphylococcus aureus in VBNC state is cultured in resuscitation medium at a temperature of 36-38°C for 18-24 hours.

[0026] Thirdly, the present invention also provides a method for inducing Staphylococcus aureus in the VBNC state, the induction method comprising: The pH of the solution containing vancomycin and sodium chloride was adjusted with glacial acetic acid, mixed thoroughly, and Staphylococcus aureus not in the VBNC state was transferred into the well-mixed solution and incubated at 2℃-8℃ for 22h-24h.

[0027] In this implementation method, the well-mixed solution is sterilized before transferring Staphylococcus aureus that is not in the VBNC state.

[0028] In this embodiment, the solution containing vancomycin and sodium chloride is prepared by dispersing vancomycin and sodium chloride in water.

[0029] In this implementation method, glacial acetic acid is used to adjust the pH of the solution containing vancomycin and sodium chloride to 2.6-3.0.

[0030] In this embodiment, the vancomycin has a mass concentration of 60 mg / L to 68 mg / L in a solution containing vancomycin and sodium chloride.

[0031] In some embodiments, the sodium chloride concentration in the solution containing vancomycin and sodium chloride is 140 g / L to 160 g / L.

[0032] The following detailed explanation is provided with reference to specific embodiments and comparative examples: Example 1: 1. Preparation of Staphylococcus aureus in VBNC state: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0033] (2) Bacterial cell count: Take 1 mL of fresh cultured bacterial solution and perform a 10-fold serial dilution with sterile PBS buffer solution (10 mmol, pH=7.2-7.4). Take 100 μL of each dilution and spread it onto tryptic soy agar medium, including 10... 3 times, 10 4 times, 10 5 times, 10 6 Double the volume and incubate overnight in a 37°C biochemical incubator. Observe and count the colonies the next day.

[0034] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 rpm for 10 minutes, and the supernatant was removed. The tubes were then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution contained 64 mg / L vancomycin and 150 g / L NaCl. The pH of the stress solution was adjusted to 2.8 with glacial acetic acid. After resuspending thoroughly, the tubes were incubated at 2℃-8℃. Samples were taken to determine the culturable count and total viable count.

[0035] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution are detected. The incubation time is determined based on the culturable bacterial count being 0 CFU / mL. Take 100 μL of bacterial solution and count the bacteria according to the method in step (2) above. Detect the culturable bacterial count in the bacterial solution. Detect once every 12 hours until the detected culturable bacterial count is 0 CFU / mL.

[0036] (5) Number of culturable bacteria: The plate count method was used for detection. When the detection result was 0 CFU / mL, the incubation time was 24h.

[0037] (6) Total viable count determination: 24 h after stress induction, 1 mL of bacterial suspension was taken. During the induction process, a large number of bacterial strains died. After induction, the bacterial suspension contained bacteria in three states: dead, culturable, and VBNC. Cells with respiratory activity were stained using the CTC bacterial viability assay kit (Tongren Chemical Research Institute), and the total viable count was determined by flow cytometry. The results are as follows: Figure 1 As shown.

[0038] (7) The content of VBNC bacteria is calculated according to the following formula: VBNC status bacteria = total number of viable bacteria - number of culturable bacteria.

[0039] 2. Culture Staphylococcus aureus in the VBNC state: Preparation of resuscitation medium: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0040] The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1, which is:

[0041] The source of the Rpf recombinant protein: It was obtained by mutating the amino acid sequence of Micrococcus luteus Rpf protein, and the mutated nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, thus obtaining the Rpf recombinant protein of the present invention.

[0042] The nucleotide sequence of the premutated Micrococcus luteus Rpf protein is shown in SEQ ID NO.2. SEQ ID NO.2 is:

[0043] Enzymatic activity assays were performed on the recombinant Rpf protein of SEQ ID NO.1 and the Rpf protein of SEQ ID NO.2. The results showed that the average amount of 4-methylumbelliferone catalyzed by the recombinant Rpf protein of SEQ ID NO.1 to generate 4-methylumbelliferone was 319.7 μmol / L, while the unmutated Rpf protein of SEQ ID NO.2... WT The average amount of 4-methylumbelliferone produced from the protein-catalyzed substrate (4-MUF-3-NAG) was 84.3 μmol / L. This indicates that the mutant protease activity was increased by more than twofold.

[0044] Example 2: The resuscitation medium for Example 2 consisted of: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.55 g / L), glutamine (1 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0045] Example 3: The resuscitation medium for Example 3 consisted of: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.275 g / L), glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0046] Example 4: The resuscitation medium for Example 4 consisted of Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. The Rpf recombinant protein was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein was added.

[0047] Comparative Example 1: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0048] (2) Counting of bacterial cells: Take 1 mL of fresh cultured bacterial solution and make a 10-fold serial dilution with sterile PBS buffer solution (10 mmol, pH=7.2-7.4). Take 100 μL of the appropriately diluted bacterial solution and spread it on tryptic soy agar medium. Incubate overnight in a biochemical incubator at 37℃. Observe and count the colonies the next day.

[0049] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 rpm for 10 minutes, and then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution was an aqueous solution containing 150 g / L NaCl, with the pH adjusted to 2.8 ± 0.2 using glacial acetic acid. The solution was incubated at 2℃–8℃, and samples were taken to determine the culturable count and total viable count.

[0050] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution are detected. The incubation time is determined based on the culturable bacterial count being 0 CFU / mL. Take 100 μL of bacterial solution and count the bacteria according to the method in step (2) above. Detect the culturable bacterial count in the bacterial solution. Detect once every 12 hours until the detected culturable bacterial count is 0 CFU / mL.

[0051] (5) Number of culturable bacteria: The plate count method was used for detection. When the detection result was 0 CFU / mL, the incubation time was 7 days.

[0052] (6) Total viable count determination: Seven days after stress induction, 1 mL of bacterial culture was taken. Since a large number of bacterial strains die during the induction process, the total viable count was determined by flow cytometry after staining with a CTC bacterial viability assay kit (Tongren Chemical Research Institute). The results are as follows: Figure 1 As shown.

[0053] (7) The content of VBNC bacteria is calculated according to the following formula: VBNC status bacteria = total number of viable bacteria - number of culturable bacteria.

[0054] Comparative Example 2: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0055] (2) Counting of bacterial cells: Take 1 mL of fresh cultured bacterial solution and make a 10-fold serial dilution with sterile PBS buffer solution (10 mmol, pH=7.2-7.4). Take 100 μL of the appropriately diluted bacterial solution and spread it on tryptic soy agar medium. Incubate overnight in a biochemical incubator at 37℃. Observe and count the colonies the next day.

[0056] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 r / min for 10 minutes, and then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution was 64 mg / L vancomycin aqueous solution. The tubes were incubated at room temperature, and samples were taken to determine the culturable count and total viable count.

[0057] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution were detected. The incubation time was determined based on the culturable bacterial count being 0 CFU / mL. 100 μL of bacterial solution was taken and the bacterial count was performed according to the method described in (2) above. The culturable bacterial count in the bacterial solution was detected once every 12 hours until the detected culturable bacterial count was 0 CFU / mL.

[0058] (5) Number of culturable bacteria: The plate technique was used for detection. When the detection result was 0 CFU / mL, the incubation time was 21 days.

[0059] (6) Total viable count determination: 1 mL of bacterial suspension was taken 21 days after stress induction. During the induction process, a large number of bacterial strains died. After induction, the bacterial suspension contained bacteria in three states: dead, culturable, and VBNC. The respiratory cells were stained using the CTC bacterial viability assay kit (Tongren Chemical Research Institute), and the total viable count was determined by flow cytometry. The results are as follows: Figure 1 As shown.

[0060] (7) The content of VBNC bacteria is calculated according to the following formula: VBNC status bacteria = total number of viable bacteria - number of culturable bacteria.

[0061] Comparative Example 3: The resuscitation medium for Comparative Example 3 consisted of sodium pyruvate (1.1 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Sodium pyruvate was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized sodium pyruvate was added.

[0062] Comparative Example 4: The resuscitation medium for Comparative Example 4 consisted of glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The peptone, beef extract, sodium chloride, and water were mixed, and the pH was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Glutamine was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized glutamine was added.

[0063] Comparative Example 5: The resuscitation medium for Comparative Example 5 consisted of recombinant Rpf protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. The recombinant Rpf protein and sodium pyruvate were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized recombinant Rpf protein and sodium pyruvate were added.

[0064] The resuscitation medium obtained in the examples and comparative examples was used to culture Staphylococcus aureus in VBNC state prepared in Example 1. The culture temperature was 37°C and the culture time was 24 h.

[0065] The resuscitation media obtained in the examples and comparative examples were tested: (1) Determination of the composition of the resuscitation medium: Culture medium preparation: Prepare culture media for the examples and comparative examples respectively.

[0066] Bacterial suspension dilution: The total number of viable VBNC-state Staphylococcus aureus working bacterial suspension was adjusted to 10,000 cells / mL using sterile PBS buffer solution.

[0067] Inoculation and Culture: 0.1 mL of VBNC-state Staphylococcus aureus suspension was inoculated into the culture media of the examples and comparative examples, and spread evenly. Three plates were inoculated for each treatment. Incubation was carried out at 37°C for 18-24 hours. 1 mL of the culture was serially diluted 10-fold with sterile PBS buffer (10 mmol, pH 7.2-7.4). 100 μL of the appropriately diluted culture was spread onto tryptic soy agar and incubated overnight at 37°C. Colonies were observed and counted the following day.

[0068] Counting: The number of colonies grown on the plates was counted, and the results are shown in Table 2.

[0069] (2) Verification of VBNC-state Staphylococcus aureus: 1 mL of the VBNC bacterial suspension prepared in the examples and comparative examples was added to tryptic soy medium containing 10 mM sodium pyruvate and tryptic soy medium without sodium pyruvate, respectively, and incubated at 37°C for 24 hours. The results showed that the tryptic soy medium containing sodium pyruvate was significantly turbid, while the tryptic soy medium without sodium pyruvate was clear. It can be seen that the methods of the examples and comparative examples successfully obtained VBNC-state Staphylococcus aureus.

[0070] Further research was conducted using the resuscitation culture medium from Example 1 as an example.

[0071] (3) Qualitative determination of growth rate of culture medium in Example 1: Adjust the total viable count of VBNC Staphylococcus aureus suspension to 1000 cells / mL using sterile PBS buffer, and adjust the total viable count of Staphylococcus aureus suspension to below 100 cells / mL. Inoculate 1 mL of VBNC Staphylococcus aureus suspension and 1 mL of Staphylococcus aureus suspension into 9 mL of 7.5% sodium chloride broth (commercially available) and the resuscitation medium of this invention, respectively. Inoculate each medium or each component of the medium into three parallel tubes (SaP001, SaP002, SaP003), and incubate at 37°C for 18 hours. Counting: After 18 hours of incubation, take 10 μL of culture from each medium and spread it evenly on a TSA (tryptophan-soybean agar) plate. Count the microbial colonies. If the number of Staphylococcus aureus colonies on the plate is greater than 10 CFU, the resuscitation medium of this invention is considered to have good growth rate.

[0072] (4) Selectivity determination of culture medium in Example 1: 0.1 mL of culture medium containing 1×10 3 -5×10 3CFU of Escherichia coli ATCC25922 was inoculated into 9.9 mL of 7.5% sodium chloride broth and the resuscitation medium of this invention, respectively, in triplicate (SaP001, SaP002, SaP003) and incubated at 37°C for 18 hours. After 18 hours of incubation, 10 μL of culture from each medium was evenly spread onto a TSA plate, and the number of microbial colonies was counted. GB4789.28-2024 states that the optimization of the culture medium should not affect the selectivity of the original national standard culture medium. Therefore, the resuscitation medium of this invention has good selectivity only when the number of Escherichia coli colonies on the TSA plate is <100 CFU.

[0073] The resuscitation medium obtained in Example 1 was prepared into a product, as shown in Table 1.

[0074] Table 1. Components of the finished product: resuscitation culture medium (homogenized bag)

[0075] Table 2 Test results of the examples and comparative examples

[0076] Table 3. Test results of growth rate in Example 1

[0077] Note: a: Turbidity calculation: 0 indicates no turbidity; 1 indicates very slight turbidity; 2 indicates severe turbidity, judged according to the GB4789.28-2024 evaluation standard; b: After enrichment with Staphylococcus aureus ATCC6538 or Escherichia coli ATCC25922, transfer to TSA plates for counting.

[0078] Table 4. Repeatability test results of culture media prepared from three batches of reagent kits.

[0079] Note: a: Turbidity calculation: 0 indicates no turbidity; 1 indicates very slight turbidity; 2 indicates severe turbidity, judged according to the GB4789.28—2024 evaluation standard; b: After enrichment of Staphylococcus aureus ATCC6538 or Escherichia coli ATCC25922, transfer to TSA for counting.

[0080] First, comparing Example 1 with Comparative Examples 1 and 2, it was found that after induction with different solution stresses, Example 1, Comparative Examples 1 and 2 all achieved a culturable Staphylococcus aureus count of 0 CFU / mL. However, the number of Staphylococcus aureus deaths caused by different methods varied, with Example 1 showing the highest average total viable count. Figure 1 It was found that Example 1 not only achieved a culturable Staphylococcus aureus count of 0 CFU / mL, but also increased the number of viable bacteria in the VBNC state. Figure 1 Example 1 showed the highest number of viable bacteria in the VBNC state. Therefore, the method of the present invention, while ensuring the survival rate of Staphylococcus aureus, induces more Staphylococcus aureus ATCC6538 to enter the VBNC state in a shorter time, with a culturable count of 0 cells / mL. Compared with other induction conditions, its VBNC state bacterial concentration is optimal.

[0081] Table 2 shows that in Comparative Example 3, the addition of sodium pyruvate enabled the culture medium to revive Staphylococcus aureus (VBNC), but the revival and enrichment capacity of Comparative Example 3 for VBNC was weaker than that of Example 4, which added Rpf and magnesium sulfate. Comparative Example 5 shows that the simultaneous addition of Rpf, magnesium sulfate, and sodium pyruvate enhanced the revival and enrichment capacity of the culture medium for VBNC. Comparing Examples 1-3 with Comparative Example 5, the experimental results indicate that the simultaneous addition of Rpf, magnesium sulfate, sodium pyruvate, and glutamine significantly improved the revival and enrichment capacity of the culture medium for VBNC, with effects 23.3, 11.1, and 8.9 times that of Comparative Example 5, respectively. Based on this, further research into the synergy between recombinant Rpf protein, magnesium sulfate, sodium pyruvate, and glutamine revealed that glutamine levels increased with increasing sodium pyruvate content, further increasing the number of culturable Staphylococcus aureus. Comparison of Examples 1-2 confirmed that Example 1 had a more significant effect. Therefore, the optimal mass concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

[0082] The growth rate and selectivity of the resuscitation medium of the present invention were tested, and the results are shown in Table 3. Staphylococcus aureus, VBNC Staphylococcus aureus, and Escherichia coli were inoculated into 7.5% sodium chloride broth and the resuscitation medium of the present invention, respectively. After 18 hours of incubation, the turbidity was observed. Table 3 shows that Staphylococcus aureus showed slight turbidity in the 7.5% sodium chloride broth and severe turbidity in the resuscitation medium of the present invention; while VBNC Staphylococcus aureus and Escherichia coli did not show turbidity in the 7.5% sodium chloride broth, indicating that VBNC Staphylococcus aureus and Escherichia coli did not grow in the 7.5% sodium chloride broth, further verifying that the induction method of the present invention successfully prepared VBNC-state Staphylococcus aureus; VBNC Staphylococcus aureus also showed severe turbidity in the resuscitation medium of the present invention. Referring to the semi-quantitative detection method in GB4789.28-2024, the cultured bacterial suspension after 18 hours of incubation was inoculated onto TSA plates for counting. This method requires that after amplification from the liquid medium, 10 μL of the culture suspension be spread onto a TSA plate for counting; the colony count should be no less than 10 CFU, demonstrating that the medium has a good growth rate. The results show that VBNC Staphylococcus aureus was freed from the VBNC state, and Staphylococcus aureus successfully grew. In summary, the resuscitation medium of this invention not only has a good growth rate but also good selectivity.

[0083] Table 4 shows that three boxes of pilot-scale product were randomly selected from each of the three batches for intra-batch and inter-batch repeatability testing. The results showed that after enrichment of Staphylococcus aureus on the resuscitation medium, TSA counting was performed, and colonies with >10 CFU appeared on all plates, indicating good growth rate of Staphylococcus aureus on the three batches of resuscitation medium. Therefore, the resuscitation medium of this invention has good repeatability.

[0084] In summary, the asepticity, nutritional capacity, and selectivity of the resuscitation medium of the present invention all meet the requirements for food microbiology testing.

Claims

1. A resuscitation medium for VBNC state Staphylococcus aureus, characterized in that, The resuscitation medium comprises Rpf recombinant protein, MgSO4, proteose peptone, beef extract and sodium chloride, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, proteose peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L):(0.5 g / L-0.7 g / L):(9 g / L-11 g / L):(4 g / L-6 g / L):(70 g / L-80 g / L). The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.

1.

2. The resuscitation medium for VBNC state Staphylococcus aureus according to claim 1, characterized in that, The resuscitation medium further comprises sodium pyruvate and glutamine, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, proteose peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L):(0.5 g / L-0.7 g / L):(0.27 g / L-2.2 g / L):(1 g / L-3 g / L):(9 g / L-11 g / L):(4 g / L-6 g / L):(70 g / L-80 g / L).

3. The resuscitation medium for VBNC state Staphylococcus aureus according to claim 2, characterized in that, The mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, proteose peptone, beef extract and sodium chloride is 100 ng / L:0.6 g / L:1.1 g / L:2 g / L:10 g / L:5 g / L:75 g / L.

4. The resuscitation medium for VBNC state Staphylococcus aureus according to claim 1, characterized in that, The pH value of the resuscitation medium is 7.2-7.

6.

5. Use of the resuscitation medium according to any one of claims 1 to 4 for culturing Staphylococcus aureus in a VBNC state, characterized in that, The VBNC state Staphylococcus aureus is cultured in the resuscitation medium, the culture temperature is 36-38℃, and the culture time is 18-48 h.

6. A method of inducing VBNC state Staphylococcus aureus in the use of claim 5, characterized by, The induction method comprises: The pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3 using glacial acetic acid, the solution is uniformly mixed, and the Staphylococcus aureus not in the VBNC state is transferred into the uniformly mixed solution, and incubated at a temperature of 2-8℃ for 22-24 h.

7. The method of inducing VBNC state Staphylococcus aureus according to claim 6, characterized in that, The pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3 using glacial acetic acid.

8. The method of inducing VBNC state Staphylococcus aureus of claim 6, wherein, The mass concentration of the vancomycin in the solution containing vancomycin and sodium chloride is 60-68 mg / L.

9. The method of inducing VBNC state Staphylococcus aureus of claim 6, wherein, The mass concentration of the sodium chloride in the solution containing vancomycin and sodium chloride is 140-160 g / L.

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