Use of globulol in the preparation of staphylococcus aureus lysine auxotrophs
By co-culturing eucalyptol with Staphylococcus aureus, a lysine-deficient strain was prepared, which solved the problem of establishing a causal relationship between bacterial growth and lysine availability in existing technologies. This provides a highly sensitive growth model tool that significantly affects lysine-related genes and cell wall structure.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-05
AI Technical Summary
Currently, there is no application of eucalyptol in the preparation of lysine-deficient strains of Staphylococcus aureus, which makes it difficult to establish a clear causal relationship between bacterial growth and lysine availability under in vitro conditions, affecting the detection sensitivity and discriminative power of lysine-related effects.
A lysine-deficient Staphylococcus aureus strain was prepared by co-culturing eucalyptol with Staphylococcus aureus. The specific conditions were: eucalyptol concentration of 12.5 μg/mL, culture at 37℃ and 120 rpm/min for 16 h, which consumed endogenous lysine to obtain a growth-inhibiting effect.
Eucalyptol significantly inhibited the growth of Staphylococcus aureus in a lysine-deficient culture system. The growth inhibition was restored after exogenous lysine was added. It significantly affected the mRNA expression of lysine-related genes and cell wall structure, providing a highly sensitive analytical tool for bacterial growth models.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural compound application technology, specifically relating to the application of eucalyptol in the preparation of Staphylococcus aureus lysine auxotypes. Background Technology
[0002] Globulol, commonly known as globulol or (-)-globulol, has the chemical formula C60. 15 H 26 O, with a molecular weight of 222.37, is a colorless crystalline solid with a melting point of 87-88°C at room temperature. This compound is primarily extracted and isolated from the dried fruit of *Eucalyptus globulus*, hence its name. It has also been found in other plants such as hops (*Humulus lupulus*).
[0003] Currently, eucalyptol is mainly used as a standard or reagent in scientific research. Its core research value lies in its antibacterial biological activity. Multiple studies have shown that eucalyptol exhibits significant inhibitory effects on various plant pathogens (such as Xanthomonas vesicatoria, which causes bacterial spot disease) and common test strains (such as Bacillus subtilis).
[0004] Current preparation S. aureus The significance of lysine auxotypes lies in their ability to construct a growth model highly sensitive to lysine supply, enabling the establishment of a clear causal relationship between bacterial growth and lysine availability under in vitro conditions. Because wild-type strains can buffer metabolic stress under conventional nutrient-rich culture conditions through exogenous nutrient supplementation or endogenous reserves, many lysine-related inhibitory effects are masked. Lysine auxotypes can significantly reduce this masking effect, thereby improving the detection sensitivity and discriminative power for lysine-related effects.
[0005] However, there is currently no publicly available information regarding the use of eucalyptol in the preparation of lysine-deficient Staphylococcus aureus strains. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides the application of eucalyptol in the preparation of lysine-deficient Staphylococcus aureus.
[0007] Specifically, this is achieved through the following technical solutions: Application of eucalyptol in the preparation of Staphylococcus aureus lysine auxotype.
[0008] Furthermore, the eucalyptol is (-)-Globulol, with the chemical name (1aR,4R,4aR,7R,7aS,7bS)-1,1,4,7-tetramethyl-2,3,4a,5,6,7,7a,7b-octahydro-1aH-cyclopropa[e]azulen-4-ol.
[0009] Furthermore, the molecular formula of the eucalyptol is C1 15 H 26 O, the specific structural formula is as follows: .
[0010] Furthermore, the preparation of Staphylococcus aureus lysine-deficient type involves the preparation of reagents that induce lysine deficiency in Staphylococcus aureus.
[0011] Furthermore, the preparation of the Staphylococcus aureus lysine auxotype specifically involves co-culturing eucalyptol with Staphylococcus aureus to obtain the Staphylococcus aureus lysine auxotype.
[0012] A formulation for preparing lysine-deficient Staphylococcus aureus contains eucalyptol.
[0013] A method for preparing Staphylococcus aureus lysine auxotype involves co-culturing Staphylococcus aureus with eucalyptol to obtain the Staphylococcus aureus lysine auxotype.
[0014] Furthermore, the method involves co-culturing eucalyptol with Staphylococcus aureus, with the eucalyptol concentration being 12.5 μg / mL.
[0015] Furthermore, the co-culture conditions of the eucalyptol and Staphylococcus aureus are: 12.5 μg / mL eucalyptol, 37℃, 120 rpm / min, culture for 16 h.
[0016] Furthermore, the concentration of Staphylococcus aureus was 1×10⁻⁶. 5 CFU / mL.
[0017] In summary, the beneficial effects of this invention are as follows: This invention provides for the first time the application of eucalyptol in the preparation of lysine-deficient Staphylococcus aureus. Eucalyptol of this invention is a natural monomeric component from Eucalyptus plants and is a natural sesquiterpene compound. After pretreatment according to this invention to consume endogenous lysine... S.aureus Eucalyptol significantly inhibited the growth of lysine-deficient culture systems. In the same control system, this growth inhibition was significantly restored after the addition of exogenous lysine. Attached Figure Description
[0018] Figure 1 Eucalyptol effects S. aureus The growth curve.
[0019] Figure 2 Eucalyptol effects S. aureus Box plot of lysine in post-metabolomics.
[0020] Figure 3 Eucalyptol effects S. aureus Results of lysine content detection.
[0021] Figure 4 Exogenous lysine deficiency-replenishment pair S. aureus The influence of the growth curve.
[0022] Figure 5 Exogenous lysine deficiency-replenishment pair S. aureus The influence of mRNA expression of related genes during PG and lysine biosynthesis.
[0023] Figure 6 Eucalyptol effects S. aureus TEM image.
[0024] Figure 7 Under the action of eucalyptol S. aureus The impact of PG leakage during CCW autolysis.
[0025] Figure 8 Under the action of eucalyptol S. aureus The effect of PG content.
[0026] Figure 9 PG replenishment S. aureus The impact.
[0027] ("#", "*", ") " indicates P < 0.05; "##" and "**" indicate P <0.01. Detailed Implementation
[0028] The specific embodiments of the present invention will be described in further detail below, but the present invention is not limited to these embodiments. Any improvements or substitutions based on the basic spirit of these embodiments shall still fall within the scope of protection claimed by the claims of the present invention.
[0029] 1. Preparation of eucalyptol: Accurately weigh eucalyptol and dissolve it in DMSO to prepare a concentration of 2 mg / mL. Filter the solution through a 0.22 µm sterile microporous membrane and store at 4 °C for later use.
[0030] 2. Eucalyptol and S. aureus Co-culture method S. aureus Growth curve detection and results: Set up 1 / 2×, 1×, 2×, 4×, and 8× MIC concentrations of eucalyptol (Note: 1× MIC concentration is 12.5 μg / mL), and a blank control group was also set up. S. aureus Final concentration 1×10 5 CFU / mL. Samples were taken at 37 ℃ and 120 rpm / min at 0, 2, 4, 6, 8, 10, 12, and 24 h, and the corresponding OD values were read using a microplate reader. 600 Plot bacterial growth curves.
[0031] The bacterial growth curve is shown in the attachment. Figure 1 As shown, the blank control group and the 1 / 2×MIC eucalyptol group S. aureus OD after 4 h of incubation 600 The value begins to increase logarithmically, ≥1×MIC of eucalyptol OD 600 The value change occurred after 8 hours and showed a concentration-dependent effect; the OD value of ≥2×MIC eucalyptol was... 600 The value change occurred around 12 hours later.
[0032] 3. Effects of Eucalyptol S. aureus Methods and results for detecting post-lysine Activated S.aureus Inoculate the culture medium into 7.5% sodium chloride tryptone soybean broth and culture until the logarithmic growth phase. Collect the bacterial cells by centrifugation and wash with PBS for later use. Add eucalyptus oil solution to sterilized fresh 7.5% sodium chloride tryptone soybean broth to a final concentration of 1×MIC. DMSO serves as a control group. Add [missing information - likely a specific ingredient or ingredient] to each culture medium. S.aureus The bacterial suspension concentration was then adjusted to 1×10⁻⁶. 5 After incubation at 37 ℃ and 120 rpm for 20 h at CFU / mL, the bacterial cells were collected by centrifugation and washed three times with PBS buffer. After flash freezing in liquid nitrogen, the samples were sent to Suzhou Panomick Biomedical Technology Co., Ltd. for analysis using non-targeted metabolomics technology. Metabolomics results showed that lysine was downregulated after treatment with eucalyptol (see attachment). Figure 2 .
[0033] 4. Optimal use of eucalyptol and S. aureus Co-culture conditions, methods and results The basic system, amino acid composition, carbon source, and vitamin composition of the custom culture medium formulation are shown in Table 1. A lysine-deficient group was set up. - ), groups lacking lysine + 1 / 2×, 1×MIC eucalyptol, and respectively selected S. aureus Single colonies were cultured in 7.5% sodium chloride broth to the logarithmic growth phase, then cultured for two generations in lysine-free CDM to pre-starve the endogenous lysine reserves before being transferred to the experimental system. S. aureus The final concentration is 1×108 CFU / mL, 0, 1 / 2×, and 1×MIC eucalyptol were incubated at 37 ℃ and 120 rpm / min for 12, 14, 16, 20, and 24 h. Lysine content in each group was detected using a lysine content assay kit to screen eucalyptol for... S. aureus Optimal co-culture conditions. Results are attached. Figure 3 The results showed that eucalyptol and S. aureus The optimal co-culture conditions were 1×MIC of eucalyptol at 37 °C and 120 rpm / min for 16 h.
[0034] 5 S. aureus Evidence of lysine deficiency: 5.1 Experimental methods and results of exogenous lysine deficiency-replenishment The basic system, amino acid composition, carbon source, and vitamin composition of the custom culture medium formulation are shown in Table 1. A complete custom culture medium group (complete CDM) and a lysine-deficient group (Lys) were set up. - ), Lysine deficiency + MIC eucalyptol group (Lys - +MIC), Lysine deficiency +MIC eucalyptol + lysine supplementation group (Lys - +MIC+ Lys), pick them out respectively S. aureus Single colonies were cultured in 7.5% sodium chloride broth to the logarithmic growth phase, then cultured for two generations in lysine-free CDM to pre-starve the endogenous lysine reserves before being transferred to the experimental system. S. aureus The final concentration is 1×10 5 CFU / mL, 37 ℃, 120 rpm / min. Samples were taken at 0, 1, 3, 6, 9, 12, and 24 hours for the former (pre-starvation treatment); and at 0, 1, 3, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 24 hours for the latter (pre-starvation treatment). The corresponding OD values were read using a microplate reader. 600 A growth curve was plotted. The results are shown in the attachment. Figure 4 The results showed that the complete group OD without prior starvation or drug treatment was intact. 600 The value began to increase after 9 hours, and the OD values of the remaining three groups... 600 The OD value of the eucalyptol group of bacteria began to increase after 12 hours. 600 The values were not significantly different from those of other groups. P >0.05); while the OD of the untreated group and the dependent group after starvation treatment were >0.05). 600 The value began to increase at 9 h. In a culture system lacking exogenous lysine, the OD of eucalyptol at the MIC concentration decreased after 18 h. 600 The value has only just begun to increase; while the OD of exogenous lysine replenishment... 600The value began to increase significantly after 14 hours. P <0.01).
[0035] Table 2. Complete Custom Culture Medium (CDM) Components
[0036] a Content per 1000 mL of culture medium 5.2 Detection methods and results of exogenous lysine deficiency-replenishment related protein mRNA (Due to the lack of commercially available protein antibodies, qRT-PCR was used to indirectly detect changes in the mRNA levels of relevant proteins.) Searches were conducted on NCBI. S. aureus The gene sequences of MurB, DapA, DapB, Alr, FemB, DapE, and LysA related to PG and lysine biosynthesis were obtained, and primers were designed. Primers were synthesized by Qingke Biotechnology, and the primer sequences are shown in Table 2. RNA was extracted according to the EZNATM Bacterial RNA Kit procedure and ScanDrop was used. 2 The purity and concentration of the product were determined using an ultra-micro analyzer. Genomic DNA residues were removed according to the instructions of the StarScript II RT Mix with gDNA Remover kit, and the DNA was reverse transcribed into cDNA. The changes in mRNA levels of these related proteins in the lysine complementation experiment were detected using qRT-PCR. Results are shown in the appendix. Figure 5 As shown, under the combined effects of exogenous lysine deficiency and eucalyptol, the mRNA levels of DapB, LysA, DapE, MurB, and FemB were significantly or extremely significantly increased. P <0.05, P <0.01), while the mRNA levels of DapA and Alt decreased significantly or extremely significantly ( P <0.05, P <0.01); After recombinant exogenous lysine, the mRNA levels of DapB, LysA, DapE, and MurB were significantly or extremely significantly restored. P <0.05, P <0.01, reaching the level of the normal group ( P >0.05); the mRNA levels of DapE and MurB were also restored with extremely significant or significant differences ( P <0.05, P <0.01, but did not reach the level of the normal group ( P <0.05, P<0.01). However, the mRNA levels of DapA, Alr, and FemB did not recover. P >0.05).
[0037] Table 2 qRT-PCR primer sequences and annealing temperatures
[0038] 5.3 Electron microscopy and its effects on PG and backfill: experimental methods and results exist S. aureus Bacterial suspension OD 600 When the concentration was 0.3, 1×MIC eucalyptol was added. A blank control was also set up. The OD of the cultured bacterial culture was measured. 600 When the bacterial cell concentration was approximately 0.7, the cells were collected, washed, and centrifuged, then the supernatant was discarded. The cells were fixed overnight at 4 °C with glutaraldehyde. After discarding the fixative, the embedded samples were sectioned according to the instructions of the GENMED transmission electron microscopy (TEM) slide preparation kit and using a UC Enuity microtome. Images were taken and observed using an HT 7800 transmission electron microscope (80 kV). The results are shown in the appendix. Figure 6 As shown, under the action of 1×MIC eucalyptol S. aureus Cell wall TEM results as follows Figure 6 As shown. S. aureus The cells are smooth and flat, individually distributed, and regularly round or oval in shape. Oval cells have regularly spaced, low-light-density septa in the center, with uniform light density appearing as bands. These septa either traverse the entire cell or are connected to the cell wall at their ends, sometimes breaking in the middle. The cell wall edges are clear and have uniform light density. This is in contrast to cells treated with eucalyptol. S. aureus Cells are distributed individually or adhere to each other. The cells are irregular in shape, ranging from nearly round, oval, to dumbbell-shaped. More than 80% of the cells have septa. Their optical density is uneven, the cell wall edges are rough and feathery, the local outlines are blurred, and the optical density is reduced.
[0039] Eucalyptol effects S. aureus Determination of PG content in the supernatant: A blank group and a 1×MIC eucalyptol group were set up to prepare... S. aureus Coarse cell wall components (CCW) were resuspended in 0.05M potassium phosphate buffer (pH=7.20) and the OD of the solution was adjusted. 600 ≈0.60, incubated at 37 ℃ with constant temperature and shaking. Samples were taken every 1 h, and the PG content in the supernatant was determined within 6 h according to the ELISA kit procedure. (Under the action of eucalyptol) S. aureus The results of PG leakage during CCW autolysis are shown in the attachment. Figure 7 As shown, the concentration curve of PG in the supernatant of the blank group changed little over time, and there was no significant difference within 6 hours. P>0.05). Compared with the blank group, the change in PG concentration in the supernatant from 1 h to 6 h under 1×MIC eucalyptol treatment was significant or highly significant. P <0.05, P <0.01).
[0040] Eucalyptol effects S. aureus Post-PG content determination: Extraction was performed using the trichloroacetic acid method. S. aureus PG. S. aureus The initial bacterial concentration was 1×10⁻⁶. 8 CFU / mL, bacteria (control group) were treated with 1 / 2×MIC, 1×MIC eucalyptol and DMSO for 12 h, and then collected by centrifugation. The extraction steps mainly included deteichoic acid removal, protein removal, lipid removal, dehydration with anhydrous ethanol, and drying at 70℃ to finally obtain PG. The results are shown in the appendix. Figure 8 As shown, eucalyptol treatment S. aureus After 12 hours, compared with the control group, the PG content at 1 / 2 × MIC showed no significant change. P >0.05), the PG content in the 1×MIC group was significantly reduced ( P <0.05).
[0041] Exogenous PG replenishment S. aureus Effect on growth: Self-extracted PG solution was added to the bacterial suspensions of each group to achieve final PG concentrations of 0 and 800 mg / mL. The bacterial suspensions containing different PG concentrations were then incubated with 0, 1 / 2×, and 1×MIC eucalyptol at 37 °C and 250 rpm / min for 2 h. Bacterial abundance was determined by plate counting. Results are shown in the appendix. Figure 9 As shown, supplementing with 800 mg / mL PG significantly increased the colony count of cultured bacteria. P <0.01), at all 1 / 2 ×MIC and 1 ×MIC concentrations, eucalyptol significantly reduced colony counts in a dose-dependent manner. P <0.01), and the bacterial colony count increased significantly after the bacteria were treated with eucalyptol and then replenished with exogenous PG. P <0.01).
[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the equivalent meaning and scope of the claims be included within the protection scope of the present invention.
Claims
1. Application of eucalyptol in the preparation of lysine-deficient Staphylococcus aureus.
2. The application of eucalyptol as described in claim 1 in the preparation of Staphylococcus aureus lysine-deficient strains, characterized in that, The eucalyptol is (-)-Globulol, with the chemical name (1aR,4R,4aR,7R,7aS,7bS)-1,1,4,7-tetramethyl-2,3,4a,5,6,7,7a,7b-octahydro-1aH-cyclopropa[e]azulen-4-ol.
3. The application of eucalyptol as described in claim 1 in the preparation of Staphylococcus aureus lysine-deficient strains, characterized in that, The molecular formula of the eucalyptol is C 15 H 26 O, the specific structural formula is as follows: 。 4. The application of eucalyptol as described in claim 1 in the preparation of Staphylococcus aureus lysine-deficient strains, characterized in that, The preparation of lysine-deficient Staphylococcus aureus involves the preparation of reagents that induce lysine deficiency in Staphylococcus aureus.
5. The application of eucalyptol as described in claim 1 in the preparation of Staphylococcus aureus lysine-deficient strains, characterized in that, The preparation of the Staphylococcus aureus lysine auxotype specifically involves co-culturing eucalyptol with Staphylococcus aureus to obtain the Staphylococcus aureus lysine auxotype.
6. A formulation for preparing lysine-deficient Staphylococcus aureus, characterized in that, It contains eucalyptol.
7. A method for preparing Staphylococcus aureus lysine-deficient strains, characterized in that, The lysine-deficient Staphylococcus aureus was obtained by co-culturing eucalyptol with Staphylococcus aureus.
8. The method for preparing Staphylococcus aureus lysine-deficient lysine using eucalyptol as described in claim 7, characterized in that, The method involves co-culturing eucalyptol with Staphylococcus aureus, with a eucalyptol concentration of 12.5 μg / mL.
9. The method for preparing Staphylococcus aureus lysine-deficient lysine using eucalyptol as described in claim 7, characterized in that, The co-culture conditions for eucalyptol and Staphylococcus aureus were: 12.5 μg / mL eucalyptol, 37℃, 120 rpm / min, for 16 h.
10. The method for preparing Staphylococcus aureus lysine-deficient lysine using eucalyptol as described in claim 7, characterized in that, Staphylococcus aureus concentration 1×10 5 CFU / mL.