A strain of Enterococcus helixeri with AHL quorum sensing quenching activity and its application as a bacterial biofilm inhibitor

Abstract

The present invention discloses a strain of Enterococcus hirae with AHL quorum sensing quenching effect ( Enterococcus hirae ), belonging to the field of biotechnology. The strain of the present invention was isolated and purified from ginseng rhizosphere soil samples by artificial screening. The strain GS22 was identified as E.hirae The study found that the strain GS22 of the present invention can efficiently degrade the quorum sensing signal molecule AHLs with significant degradation effect, and has great application potential in inhibiting the formation of bacterial biofilms with AHLs-type signal molecules, providing a new way to control the generation of bacterial resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a strain of Enterococcus hiraeum ( Enterococcus hirae ) GS22 and E.hirae Application of the fermentation supernatant of GS22 as a bacterial biofilm inhibitor. Background Art

[0002] Biofilm is a microbial community formed by bacteria in response to adverse environments. The formation of biofilms increases the ability of bacteria to tolerate adverse environments, resist bactericides, and makes it more difficult to remove bacteria from surfaces such as food processing equipment and packaging materials. The bacterial quorum sensing (QS) system is a communication mechanism that exists within and between bacterial species. It can regulate a variety of bacterial biological behaviors and is also related to the formation of bacterial biofilms. There are three main common QS signaling molecules: autoinducing peptides (AIP, 5-10 amino acid ring lactones), N-acyl homoserine lactones (AHLs), and autoinducer 2 (AI-2, furan boric acid diester). Among them, AHLs are the main signal molecules in most Gram-negative bacteria (Khalid SJ, Ain Q, Khan SJ, et al. Targeting Acyl Homoserine Lactones (AHLs) by the quorumquenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa[J]. Saudi Journal of Biological Sciences, 2022, 29(3): 1673-1682.). By inhibiting the AHL-type QS system, it can inhibit and remove the biofilm of Gram-negative bacteria, which is less likely to form bacterial resistance than the traditional method of inhibiting biofilm by antibacterial effect.

[0003] The process of blocking the bacterial QS system by destroying the structure of signal molecules or inhibiting their interaction with receptors is called quorum quenching (QQ). Some bacteria have the ability to secrete quorum quenching enzyme (QQE), which blocks the bacterial QS system through QQE. QQ enzymes mainly include three categories: (1) AHL lactonase, which hydrolyzes the ester bond of the lactone ring to produce acyl homoserine lactone, making short-chain or long-chain AHLs inactive; (2) AHL acylase, which inactivates AHLs by hydrolyzing the amide bond connecting the lactone part and the acyl side chain to produce homoserine lactone and fatty acids; (3) oxidoreductase, which oxidizes or reduces the acyl side chain of AHLs to inactivate AHLs (Fan X, YeT, Li Q, et al. Potential of a quorum quenching bacteria isolate Ochrobactrumintermedium D-2 against soft rot pathogenPectobacterium carotovorum subsp.carotovorum[J]. Frontiers in Microbiology,2020, 11: 898.).

[0004] Hafnia alvei Hafnia alvei ) is a common spoilage bacteria in aquatic products, is a Gram-negative bacterium, has an AHL type QS system, and the present invention relates to H.alvei It has a strong ability to form biofilm and may be a potential threat to the spoilage of aquatic products during storage and transportation. Summary of the invention

[0005] In view of the above problems, the present invention provides an Enterococcus hirae strain ( E.hirae ), using its fermentation supernatant to H.alvei Degradation of AHLs quorum sensing signal molecules achieves inhibition H.alvei Purpose of biofilm.

[0006] In order to achieve the above object, the present invention provides the following technical solutions.

[0007] The present invention provides a E.hirae GS22, characterized in that the strain was deposited in the China General Microbiological Culture Collection Center on April 11, 2023, with the deposit number CGMCC NO: 26984.

[0008] Furthermore, the 16S rDNA sequence thereof is as shown in SEQ ID NO: 1:

[0009]

[0010] Furthermore, the E.hirae Application of GS22 in the preparation of products for degrading AHLs-type quorum sensing signal molecules.

[0011] Furthermore, the E.hirae Application of GS22 in the preparation of products for inhibiting bacterial biofilms with AHLs-type quorum sensing signal molecules.

[0012] Furthermore, the E.hirae Application of GS22 in the preparation of products for inhibiting the spoilage of aquatic products.

[0013] Furthermore, the application E.hirae GS22 degrades AHLs-type quorum sensing signal molecules.

[0014] The present invention also provides a method for inhibiting bacterial biofilm formation by degrading AHLs quorum sensing signal molecules, characterized in that the method is to use the method described in claim 1 E.hirae The fermentation supernatant or bacterial liquid of GS22 is co-incubated with bacteria to inhibit bacterial biofilm.

[0015] The present invention also provides a kind of E.hirae The application of the fermentation supernatant of GS22 is characterized in that the application comprises:

[0016] I. Application in the preparation of products for degrading AHLs-type quorum sensing signal molecules;

[0017] II. Application in the preparation of products for inhibiting bacterial biofilms with AHLs-type quorum sensing signal molecules;

[0018] III. Application in the preparation of products for inhibiting the spoilage of aquatic products.

[0019] Furthermore, the use of the method in preparing a product for inhibiting the spoilage of aquatic products is E.hirae GS22 degrades AHLs quorum sensing signal molecules.

[0020] The strain of the present invention was isolated and purified from ginseng rhizosphere soil samples by artificial screening. The strain GS22 was identified as E.hirae .

[0021] Colony morphological characteristics of strain GS22: After culturing on nutrient agar plates for 24 hours, the colonies are milky white, raised, smooth and opaque, with neat edges; after culturing in nutrient broth for 24 hours, they are diffusely turbid.

[0022] The morphological characteristics of the bacteria observed under an optical microscope are that the bacteria are spherical.

[0023] The physiological and biochemical characteristics of the strain GS22 are: Gram-positive bacteria,

[0024] The strain GS22 is sensitive to common antibiotics tetracycline, chloramphenicol, kanamycin, gentamicin and streptomycin.

[0025] Experimental studies have shown that the E.hirae GS22 has a significant degradation effect on the quorum sensing signal molecules C4-HSL, C6-HSL, C6-oxo-HSL, and C8-HSL, and can completely degrade the quorum sensing signal molecules C4-HSL, C6-HSL, C6-oxo-HSL, and C8-HSL with an initial concentration of 10μM in 24 hours. It has great application potential in inhibiting the damage of spoilage bacteria mediated by AHLs quorum sensing signal molecules.

[0026] The method for degrading AHLs quorum sensing signal molecules to inhibit bacterial biofilms is to use E.hirae The fermentation supernatant or bacterial liquid of GS22 is used to treat microorganisms to inhibit the formation of bacterial biofilm or remove mature biofilm. The treatment method is as follows: E.hirae GS22 was co-incubated with spoilage bacteria and AHLs quorum sensing signal molecules. E.hirae The optimal pH for GS22 strain to degrade AHLs is 6.5-7.0 and the temperature is 28°C-37°C. The preferred culture medium is LB, and the specific conditions are as follows: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, pH 6.8-7.2, and sterilization at 121°C for 15-25 min. The LB solid culture medium formula is to add 2% (W / V) agar to the liquid culture medium.

[0027] Experiments show that E.hirae GS22 Pair H.alvei The signal molecules produced have obvious degradation effects. At the same time, when using C4-HSL, C6-HSL, C6-oxo-HSL, C8-HSL and other signal molecule standards for experiments, it was found that GS22 could completely degrade the above-mentioned quorum sensing signal molecules with an initial concentration of 10μM at 24h. E.hirae GS22 Pair H.alvei It has a significant inhibitory effect on biofilm formation, motility and siderophore production.

[0028] Compared with the prior art, the present invention has the following beneficial effects.

[0029] The present invention has found that E.hiraeIt can efficiently degrade AHLs-type quorum sensing signal molecules with significant degradation effect. It has great application potential in inhibiting the formation of bacterial biofilms with AHLs-type signal molecules, and provides a new way to control the development of bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 For the strain GS22 of the present invention H.alvei Diagram for the determination of AHLs signaling molecule degradation activity.

[0031] Figure 2 The figure is a colony morphology diagram of the strain GS22 of the present invention on LB medium.

[0032] Figure 3 The figure is a colony morphology diagram of the strain GS22 of the present invention on blood agar medium.

[0033] Figure 4 It is an optical microscope picture of strain GS22 of the present invention.

[0034] Figure 5 It is a phylogenetic tree analysis diagram of the strain GS22 of the present invention.

[0035] Figure 6 This is a diagram showing the drug sensitivity test results of the strain GS22 of the present invention.

[0036] Figure 7 This is a plate diagram showing the degradation activity of the strain GS22 of the present invention on different AHLs.

[0037] Figure 8 This is to determine the enzyme production location of the strain GS22 of the present invention.

[0038] Fig. 9 This is to determine the production type and thermal stability of the strain GS22 of the present invention.

[0039] Fig.10 For the strain GS22 of the present invention H.alvei Biofilm inhibition and removal capabilities.

[0040] Fig.11 For the strain GS22 of the present invention H.alvei Biofilm inhibition and removal ability observed under optical microscope.

[0041] Fig.12 For the strain GS22 of the present invention H.alvei Figure 2. The inhibitory effect on motor performance.

[0042] Fig.13 The figure is an agarose gel electrophoresis diagram of PCR amplification of the strain GS22 of the present invention using different QQE primers. DETAILED DESCRIPTION

[0043] The following examples will contribute to the understanding of the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents. The present invention is further described below in conjunction with specific examples and the accompanying drawings, 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.

[0044] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0045] Example 1 E.hirae Acquisition and identification of strain GS22.

[0046] 1. Isolation of strain GS22 and strain degradation H.alvei Determination of the signaling molecule capacity of AHLs.

[0047] (1) Soil sample collection: Soil samples collected from the rhizosphere of ginseng were used as microbial sources.

[0048] (2) Strain isolation and purification: isolation was performed using the dilution and plate spreading method.

[0049] Screen strains from ginseng root soil samples. 10g sample, 90g sterile water, mix the sample with sterile water, beat to mix, let stand for 5-10min, then take 100µL of the mixture and transfer it to LB solid medium for coating. After coating, place it in an incubator at a suitable temperature for 24-48h, then purify the strains of different forms on the medium, pick a single colony and inoculate it in LB broth for 24-48h, then transfer the bacterial solution to 30% glycerol and store it at -80℃.

[0050] (3) Strain degradation H.alvei Determination of the signaling ability of AHLs: Using the reporter strain Bacillus violaceus CV026 ( Chromobacterium violaceum CV026) to evaluate the effects of strains isolated from soil samples on H.alvei Degradation of AHLs produced.

[0051] H.alvei Extraction of AHLs signal molecules produced by strains. H.alveiIncubate for 24 hours and centrifuge at 10,000 rpm for 10 minutes. Then, mix the supernatant with an equal volume of acidified ethyl acetate [0.1% (v / v) glacial acetic acid] and place it in a separatory funnel. After 3 hours of extraction, pour out the upper layer of ethyl acetate, add new ethyl acetate [0.1% (v / v) glacial acetic acid], and repeat the above operation. Repeat this process 2 to 3 times, and mix the entire ethyl acetate fraction. The mixed ethyl acetate fraction is evaporated to dryness using a vacuum rotary evaporator (35°C, 150rpm) and redissolved in an appropriate amount of methanol to obtain H.alvei The AHLs signal molecule extract produced by the strain was stored in a -20℃ refrigerator in a sterile centrifuge tube.

[0052] Will be from H.alvei The extracted AHLs were added to the bacterial solution obtained by separation and screening, and AHLs were co-incubated with sterile water as a negative control at 28°C for 24 hours. The co-incubated material was centrifuged (10000r / min, 10min, 4°C), the supernatant was taken, and then filtered with a 0.22µm filter membrane. The presence of AHLs in the co-incubated material was detected using CV026 using the agar diffusion method. Take the CV026 bacterial solution cultured overnight, add (1%~2% inoculation volume) to LB semi-solid culture medium (the amount of agar added is 1%, w / v), mix well, and pour into a plate with an Oxford cup. After it solidifies, remove the Oxford cup, and then add 100µL of filtered supernatant to the hole, place it in a 28°C incubator and culture it for 24-48h. The degradation of AHLs is judged by observing the formation of color change circles around the holes. The more AHLs content, the larger the diameter of the color change circle. The positive bacteria can be screened out based on the experimental results. H.alvei The strains producing AHLs had the best degradation effect, such as Figure 1 As shown, named GS22.

[0053] 2. Identification and phylogenetic tree analysis of strain GS22.

[0054] (1) Colony morphology: The above strain GS22 was streaked onto LB solid medium and cultured in a 37°C biochemical incubator for 24 h. The colonies were convex, smooth and opaque, with neat edges. Figure 2 As shown in the figure, strain GS22 showed diffuse turbidity in LB liquid medium, grew well at 37°C, and was facultative anaerobic. Figure 3 As shown, blood agar plates showed that the strain was not hemolytic.

[0055] (2) Morphological characteristics of bacteria: Figure 4 As shown, the bacteria are spherical.

[0056] (3) 16S rDNA sequence and phylogenetic tree analysis: The length of the strain 16S rDNA gene sequence was 1471 bp. After comparison with the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), it was found that the strain GS22 was E.hirae The homology is high (>99%), and its phylogenetic tree is as follows Figure 5 shown.

[0057] In summary, strain GS22 was identified as E.hirae , and deposited in the China General Microbiological Culture Collection Center with the accession number CGMCC NO: 26984. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences.

[0058] Example 2 Antibiotic sensitivity analysis of strain GS22.

[0059] In order to better study the aquatic preservation potential of strain GS22 obtained in Example 1, the biological characteristics of the strain were studied in depth. The sensitivity of strain GS22 to different antibiotics was studied experimentally.

[0060] The experimental results showed that strain GS22 was sensitive to five antibiotics, namely chloramphenicol (30 μg), kanamycin (30 μg), tetracycline (30 μg), streptomycin (10 μg) and gentamicin (10 μg), and was resistant to penicillin (10 μg), ampicillin (10 μg) and clarithromycin (15 μg). Figure 6 shown.

[0061] Example 3 Analysis of the degradation ability of strain GS22 on different AHLs.

[0062] C4-HSL, C6-oxo-HSL and C8-HSL with a final concentration of 10 μM were incubated with 800 μL of overnight culture of the strain at 28°C for 24 h, and AHLs were incubated with 800 μL of sterile water at 28°C for 24 h as a negative control. The incubation was centrifuged (10000 r / min, 10 min, 4°C), the supernatant was taken, and the supernatant was filtered through a 0.22 μm membrane, and the degradation of AHLs in the incubation was detected by agar diffusion method.

[0063] The experimental results showed that strain GS22 had a significant degradation effect on the quorum sensing signal molecules C4-HSL, C6-HSL, C6-oxo-HSL, and C8-HSL. It could completely degrade the quorum sensing signal molecules C4-HSL, C6-HSL, C6-oxo-HSL, and C8-HSL with an initial concentration of 10 μM within 24 hours. Figure 7 shown.

[0064] Example 4: Determination of the location, type and thermal stability of the quorum sensing quenching enzyme produced by strain GS22.

[0065] 1. The location of the quorum sensing quenching enzyme produced by strain GS22 was determined.

[0066] Crude cell extract (CCE) and cell-free supernatant (CFS) of strain GS22 were taken, and the location of the enzyme produced by strain GS22 was determined by agar diffusion method. Strain GS22 was cultured overnight, and then the bacterial solution was centrifuged and the supernatant was filtered through a 0.22µm filter to obtain CFS; the bacterial cells were resuspended in sterile water, then ultrasonicated, centrifuged (10000 rpm, 10 min, 4℃) and the supernatant was filtered through a 0.22µm filter to obtain CCE. 800µL of CFS and CCE were mixed with AHLs respectively and incubated at 28℃ for 24h. AHLs were incubated with sterile water as a negative control. The remaining AHLs in the incubation were detected by agar diffusion method to determine whether the quorum sensing quenching enzyme was secreted outside the cell.

[0067] The experimental results show that Figure 8 As shown, CFS can completely degrade AHLs ( Figure 8 a), while CCE hardly degrades AHLs ( Figure 8 b), therefore, it is judged that QQE is secreted outside the cell and mainly exists in CFS.

[0068] 2. Determination of the type and thermal stability of quorum sensing quenching enzyme produced by strain GS22.

[0069] In order to determine whether the QQ activity of the selected AHLs-degrading strains is caused by lactonase, an acidification experiment was performed. Lactonase will open the lactone ring of AHLs and destroy the structure of AHLs, while under acidic conditions, the lactone ring will close and re-form AHLs. First, AHLs were added to 500µL of bacterial solution with QQ activity at a final concentration of 10µM, and then incubated in an incubator at 28°C for 24h. The co-incubation was then centrifuged (10000rpm, 10min, 4°C) to take the supernatant, and the supernatant was filtered through a 0.22µm filter. A portion of the supernatant was adjusted to pH 2.0 and incubated in an incubator at 30°C for 24h. The changes of AHLs after acidification were detected by agar diffusion method. At the same time, in order to verify the stability of QQE, it was placed in a 95°C water bath for 10min, and then incubated with AHLs for 24h, and the agar diffusion method was used to detect whether AHLs were degraded after co-incubation.

[0070] The experimental results show that Fig. 9As shown, after acidification, the color change zone was regenerated but was significantly smaller than that of the control group ( Fig. 9 b), it can be judged that the QQE of GS22 is mainly composed of AHLs acylase, and other types of QQE may also be included; after 10 minutes in a 95℃ water bath, QQE loses its activity ( Fig. 9 c).

[0071] Example 5 strain GS22 H.alvei The impact of biofilm.

[0072] 1. Strain GS22 H.alvei Inhibition of biofilm formation and ability to remove established biofilm.

[0073] Biofilm inhibition experiment: 100 μL of strain GS22 CFS was transferred to a 96-well plate, and then 100 μL of LB broth was added, and activated H.alvei In the well plate, culture overnight at 28℃, and then stain after certain treatment; for the biofilm removal experiment, add 100μL LB broth to the well plate and inoculate H.alvei 28℃ overnight culture, then add strain GS22 CFS, culture at 28℃ overnight, and stain after certain treatment; both experiments were performed with H.alvei The bacterial solution was used as a positive control, and LB was used as a negative control. Before staining, the free cells and excess culture medium adhering to the biofilm were washed away, washed twice with PBS, air-dried for 30 minutes, and 200 μL of 0.1% crystal violet stain was added for 20 minutes. The excess stain was washed away and air-dried for 30 minutes. 200 μL of anhydrous ethanol was added for decolorization for 20 minutes, and the OD was measured with an enzyme-labeled instrument. 595 nm The biofilm inhibition rate and clearance rate are calculated using the following formulas.

[0074] The biofilm morphology was observed under an optical microscope. The specific operation was as follows: Place the sterile cell slide in a 12-well plate, add LB broth and add the activated H.alvei , add strain GS22 CFS at the same time, culture at 28℃ for 48h, remove bacterial suspension, take out cell slides from 12-well plate, wash thoroughly with sterile PBS to wash away floating bacteria. Then fix with methanol for 15-20min, stain with 0.1% crystal violet stain for 20min, rinse with sterile PBS to remove excess stain, and air dry for 30min. Place the glass slide under an optical microscope for observation.

[0075] The experimental results showed that strain GS22 could significantly inhibit H.alveiBiofilm formation, at the same time, can be removed after the biofilm is formed, but the inhibition ability is better than the removal ability, the inhibition rate is 55.7%, and the removal rate is 50%. Fig.10 As shown; strain GS22 was observed under an optical microscope H.alvei The results showed that the control group had large and dense biofilms, while after being treated with strain GS22, the colonies were dispersed and no dense biofilms were formed. H.alvei The ability to remove biofilms. After the biofilms were formed, they were obviously detached after being treated with strain GS22 CFS. The results showed that QQ bacteria had a certain ability to remove biofilms. Fig.11 shown.

[0076] 2. Strain GS22 H.alvei The impact on athletic ability.

[0077] The effects of GS22 on H.alvei Effect of clustering and swimming motility. Clustering medium: 1g peptone, 0.5g sodium chloride, 0.4g agar, 0.5g fructose and 100mL distilled water. Swimming medium: 1g tryptone, 0.5g sodium chloride, 0.3g agar, 100mL distilled water. Add strain GS22 CFS to the culture dish and pour the prepared medium into the culture dish. After cooling, H.alvei Inoculate in the center of the clustering and swimming plates, and use the plate without active substance as a control. Place the clustering and swimming plates in a 28°C incubator and observe the migration diameter of the strain after 24 hours of incubation.

[0078] The experimental results show that H.alvei On the motility and clustering plates, the bacteria diffused from the inoculation point to the surrounding area and moved over a large distance, indicating that they had strong motility. When treated with strain GS22, the movement distance of the bacteria showed a significant decrease. Obviously, GS22 significantly inhibited H.alvei The swarming and swimming movements of Fig.12 shown.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0080] Example 6 PCR amplification of QQE homologous genes in strain GS22.

[0081] Three pairs of primers were designed (Table 1), and PCR amplification was used to examine whether strain GS22 contained homologous genes for AHL lactonase (aiiA) and acylase (pvdQ and quiP). The PCR amplification conditions were 94°C initial denaturation for 5 min, 94°C (45s), 44°C (45s), 72°C (1min) for 5 cycles; 94°C (45s), 53°C (45s), 72°C (1min), 30 cycles, and then primer extension at 72°C for 8 min. 1% agarose gel electrophoresis was then used to verify the presence of the target band and determine the size of the PCR amplification product.

[0082] The experimental results showed that strain GS22 may have the aiiA gene with an amplicon size of 257 bp and the pvdQ gene homolog with an amplicon size of 1411 bp. Fig.13 shown.

[0083] Table 1 Primers for quorum sensing quenching enzyme genes (lactonase and acylase)

[0084] .

Claims

1. One plant E.hirae GS22, characterized by: The strain was deposited in the China General Microbiological Culture Collection Center on April 11, 2023, with the deposit number CGMCC NO: 26984.

2. As claimed in claim 1 E.hirae Application of GS22 in the preparation of products for degrading quorum sensing signal molecules AHLs.

3. As claimed in claim 1 E.hirae Application of GS22 in the preparation of products for inhibiting bacterial biofilms with AHLs-type signaling systems.

4. As claimed in claim 1 E.hirae Application of GS22 in the preparation of products for inhibiting the spoilage of aquatic products.

5. The use according to claim 4, characterized in that The application is E.hirae GS22 degrades AHLs-type quorum sensing signal molecules.

6. A method for degrading AHLs quorum sensing signal molecules and inhibiting the formation of biofilm of bacteria having AHLs quorum sensing signal molecules, characterized in that: The method is as described in claim 1 E.hirae The fermentation supernatant or bacterial liquid of GS22 is co-incubated with bacteria to inhibit or eliminate bacterial biofilm.

7. A method according to claim 1 E.hirae The use of the fermentation supernatant of GS22 is characterized in that: The application is: I. Application in the preparation of products for degrading AHLs-type quorum sensing signal molecules; II. Application in the preparation of a product for inhibiting bacterial biofilms having AHLs-type quorum sensing signal molecules; III. Application in the preparation of products for inhibiting the spoilage of aquatic products.

8. The use according to claim 7, characterized in that In the preparation of the product for inhibiting the spoilage of aquatic products E.hirae GS22 degrades AHLs-type quorum sensing signal molecules.

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