Fermentation broth extract of streptomyces m53 and methods of making and using same

Abstract

The present application relates to the technical field of microorganism, and discloses a fermentation broth extract of streptomyces M53 and a preparation method and application thereof.The preparation method of the fermentation broth extract comprises the following steps: inoculating streptomyces M53 into SGTYP liquid culture medium and shaking culture for 72-96 hours to obtain a fermentation broth; extracting the fermentation broth with ethyl acetate, removing the water phase, and obtaining a fermentation broth extract; removing ethyl acetate in the fermentation broth extract to obtain the fermentation broth extract of streptomyces M53; wherein the streptomyces M53 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 65720.The fermentation broth extract prepared by the preparation method has a concentration gradient effect on the swarming movement of green pseudomonas PAO1, can reduce the drug resistance of pseudomonas aeruginosa PAO1 to antibiotics, and can inhibit the production of biofilm and rhamnolipid of the pseudomonas aeruginosa PAO1, and therefore has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a fermentation broth extract of Streptomyces M53, a preparation method thereof, and an application thereof. Background Art

[0002] With the overuse and misuse of antibiotics in clinical practice, agriculture, animal husbandry, aquaculture, and other fields, bacterial resistance is becoming increasingly serious. However, the development of new drug research and development lags far behind. The development of new antimicrobial drugs that are highly effective and less likely to cause cross-resistance is urgent.

[0003] Compared to traditional bactericidal strategies, new drug development strategies targeting pathogenicity impose less selective pressure on bacteria, are less likely to induce drug resistance, and possess greater specificity. They can be used in combination with antibiotics to reduce antibiotic concentrations and enhance efficacy, making them a novel control strategy consistent with sustainable development. The targets of pathogenicity are often related to their swarming behaviors, common of which include swarming and biofilm (BF) formation.

[0004] Surging motility is the flagella-driven, swarming behavior of bacteria across the surface of semisolid culture media. Compared to individual behaviors such as swimming, cells in a surging state exhibit enhanced environmental adaptability through the following characteristics: ① overexpression of flagellar genes to form superflagellar structures; ② bacterial elongation; ③ secretion of surfactants (such as rhamnolipids); and some cells also suppress the expression of outer membrane porins (such as OmpF). Therefore, surging motility, as a specialized bacterial swarm behavior, is closely associated with drug resistance, pathogenicity, and biofilm formation. Importantly, probiotics have not yet been reported to exhibit surging motility. Compared to antibiotics, surging motility inhibitors can selectively inhibit surging motility of pathogens, attenuating their pathogenicity while preserving the probiotic community. Therefore, the development of novel surging motility inhibitors, targeting surging motility rather than directly killing bacteria, is of great research significance.

[0005] Currently, substances known to inhibit surging motility primarily include plant-based natural products and antibiotics operating at subinhibitory concentrations. However, plant-based natural products often require concentrations of several hundred micromolar or even millimolar to exert their effects, their efficiency needs to be further improved, and their molecular mechanisms of surging inhibition remain largely unknown. Furthermore, the working concentrations of antibiotics for surging inhibition are similar to their bactericidal concentrations, and further increasing these concentrations may induce antibiotic resistance in pathogens.

[0006] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a fermentation broth extract of Streptomyces M53 and its preparation method and application. The fermentation broth extract can inhibit the surging movement, drug resistance and biofilm formation of Pseudomonas aeruginosa, and can be used to solve the problem that the existing surging movement inhibitors have low activity efficiency and are prone to cause pathogen resistance.

[0008] The technical solutions of the present invention are as follows:

[0009] In the first aspect, Streptomyces ( Streptomyces A method for preparing a fermentation broth extract of M53 sp., comprising the steps of:

[0010] (1) Inoculate Streptomyces M53 into SGTYP liquid culture medium and shake culture for 72-96 hours to obtain fermentation broth;

[0011] (2) extracting the fermentation broth with ethyl acetate, removing the aqueous phase, and obtaining a fermentation broth extract;

[0012] (3) removing ethyl acetate from the fermentation broth extract to obtain a fermentation broth extract of Streptomyces M53;

[0013] The Streptomyces M53 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65720.

[0014] According to a preferred technical solution, the shaking culture conditions are: temperature 25-28°C, and rotation speed 100-300 rpm.

[0015] In the preferred technical solution, step (1) specifically includes:

[0016] After activating Streptomyces M53 on MA solid medium, take a single colony and add it to 5-15 mL of SGTYP liquid medium and shake it for 20-30 hours to obtain seed liquid;

[0017] The seed liquid is inoculated into a new SGTYP liquid culture medium and shake-cultured for 72 to 96 hours to obtain a fermentation liquid.

[0018] In a preferred technical solution, the volume ratio of the seed solution to the new SGTYP liquid culture medium is 1:(50-100).

[0019] In a second aspect, a fermentation broth extract of Streptomyces M53 is provided, wherein the fermentation broth extract is prepared using the preparation method described in the first aspect.

[0020] In a third aspect, there is provided a use of the fermentation broth extract according to the second aspect in preparing a product for inhibiting the surging movement of Pseudomonas aeruginosa.

[0021] In a fourth aspect, there is provided use of the fermentation broth extract according to the second aspect in preparing a product for inhibiting antibiotic resistance of Pseudomonas aeruginosa.

[0022] In a preferred technical solution, the antibiotic is gentamicin.

[0023] In a fifth aspect, there is provided a use of the fermentation broth extract according to the second aspect in preparing a product for inhibiting biofilm formation of Pseudomonas aeruginosa.

[0024] Beneficial Effects: The present invention provides a fermentation broth extract of Streptomyces M53 that inhibits the surging motility of Pseudomonas aeruginosa PAO1 in a concentration-gradient manner, without affecting its swimming, gliding, or growth properties. This specific inhibitor of surging motility is also known to reduce antibiotic resistance in Pseudomonas aeruginosa PAO1 during surging motility and inhibit the formation of biofilms and rhamnolipids. Therefore, the extract has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The morphological observation results and phylogenetic tree of the strain M53 in Example 1 and the solution of the fermentation broth extract of the strain M53 prepared in Example 2 are shown.

[0026] Figure 2 This is a graph showing the results of the M53 fermentation broth extract inhibiting the surging movement of Pseudomonas aeruginosa PAO1 in Example 3.

[0027] Figure 3 This is a result diagram showing that the fermentation broth extract of strain M53 in Example 4 does not affect the growth of Pseudomonas aeruginosa PAO1.

[0028] Figure 4 This is a graph showing the results of the gradient effect of the inhibitory activity of the fermentation broth extract of strain M53 on the surging motility of Pseudomonas aeruginosa PAO1 in Example 5.

[0029] Figure 5 This is a graph showing the result that the fermentation broth extract of strain M53 in Example 6 does not inhibit the swimming and gliding motility of Pseudomonas aeruginosa PAO1.

[0030] Figure 6 This is a graph showing the results of the inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by the fermentation broth extract of strain M53 in Example 7.

[0031] Figure 7 This is a graph showing the results of the M53 fermentation broth extract inhibiting the drug resistance of Pseudomonas aeruginosa PAO1 in Example 8.

[0032] Figure 8This figure shows the result that the addition of rhamnolipid in Example 9 can partially restore the surging motility of Pseudomonas aeruginosa PAO1.

[0033] Figure 9 This is a graph showing the results of the inhibition of rhamnolipid production by the fermentation broth extract of strain M53 in Example 10 by Pseudomonas aeruginosa PAO1. DETAILED DESCRIPTION

[0034] The present invention provides a fermentation broth extract of Streptomyces M53, a preparation method thereof, and an application thereof. To make the objectives, technical solutions, and effects of the present invention clearer and more specific, the present invention is further described in detail below.

[0035] The ocean boasts the richest biological resources on Earth. Marine microorganisms, owing to their unique living environment, characterized by high pressure, high salinity, oligotrophic conditions, low temperatures, limited light, and hypoxia, produce a rich variety of structurally distinct secondary metabolites, many of which possess unique anti-inflammatory, antibacterial, and anti-tumor activities. In recent years, the search for novel active compounds from marine microorganisms has become a hot topic. However, research on novel surging motility inhibitors derived from marine sources remains limited.

[0036] Currently, the new surging motion inhibitors from marine sources generally have the problems of low efficiency and unclear mechanism of action. For example, Document 1 discloses that Payam B et al. Holothuria leucospilota ) extracts, at a concentration of 1 / 2 MIC (15 μg / mL), against Aeromonas hydrophila ( Aeromonas hydrophila ) has an inhibition rate of 77%. Document 2 discloses that Zhang M et al. Fusarium The equisetin isolated from sp. Z10 has an inhibition rate of only 50% on the surging movement of Pseudomonas aeruginosa at a concentration of 300μM. Streptomyces The crude extract extracted with methanol from sp. NIO 10068 can inhibit the surging motility of Pseudomonas aeruginosa by 90% at a concentration of 0.1 mg / mL, but its active ingredients and mechanism of action are still unknown.

[0037] Document 1: Payam B, Soltani M, Mehrgan MS, et al. Saponins from seacucumber disrupt aeromonashydrophila quorum sensing to mitigate pathogenicity[J]. AMB Express, 2025, 15(1): 43.

[0038] Literature 2: Zhang M, Wang M, Zhu X, et al. Equisetin as potential quorumsensing inhibitor of pseudomonas aeruginosa [J]. Biotechnology Letters, 2018,40(5): 865-870.

[0039] Document 3: Naik DN, Wahidullah S, Meena R M. Attenuation of pseudomonas aeruginosa virulence by marine invertebrate–derived streptomyces sp[J].Letters in Applied Microbiology, 2013, 56(3): 197-207.

[0040] Based on this, the present invention isolated a Streptomyces strain ( Streptomyces sp.) M53, and studied the surging motion of its fermentation broth extract.

[0041] The embodiment of the present invention provides Streptomyces ( Streptomyces A method for preparing a fermentation broth extract of M53 sp., comprising the steps of:

[0042] (1) Inoculate Streptomyces M53 into SGTYP liquid culture medium and shake culture for 72-96 hours to obtain fermentation broth;

[0043] (2) extracting the fermentation broth with ethyl acetate, removing the aqueous phase, and obtaining a fermentation broth extract;

[0044] (3) removing ethyl acetate from the fermentation broth extract to obtain a fermentation broth extract of Streptomyces M53;

[0045] The Streptomyces M53 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65720.

[0046] Specifically, it was found that the fermentation broth extract prepared by the embodiment of the present invention has an antibacterial effect on Pseudomonas aeruginosa ( Pseudomonas aeruginosa) PAO1 had no bactericidal effect, but at a lower concentration (10 μg / mL), it significantly inhibited the surging motility of P. aeruginosa PAO1. It had no inhibitory effect on other types of motility of P. aeruginosa PAO1, such as swimming and twitching. This indicates that the fermentation broth extract is a highly effective and specific inhibitor of surging motility. Furthermore, the fermentation broth extract significantly inhibited other pathogenic factors of P. aeruginosa PAO1, such as rhamnolipid production and biofilm formation, and also reduced P. aeruginosa PAO1's resistance to antibiotics. These results suggest that the fermentation broth extract of Streptomyces M53 has promising application prospects and is expected to be developed into a novel strategy for the control of pathogenic bacteria in the future.

[0047] In one embodiment, the shaking culture conditions are: temperature 25-28° C., and rotation speed 100-300 rpm.

[0048] In one embodiment, step (1) specifically includes:

[0049] After activating Streptomyces M53 on MA solid medium, take a single colony and add it to 5-15 mL of SGTYP liquid medium and shake it for 20-30 hours to obtain seed liquid;

[0050] The seed liquid is inoculated into a new SGTYP liquid culture medium and shake-cultured for 72 to 96 hours to obtain a fermentation liquid.

[0051] In one embodiment, the volume ratio of the seed solution to the new SGTYP liquid culture medium is 1:(50-100).

[0052] The embodiment of the present invention provides a fermentation broth extract of Streptomyces M53, which is prepared using the preparation method described above.

[0053] An embodiment of the present invention provides use of the fermentation broth extract described above in preparing a product for inhibiting the surging movement of Pseudomonas aeruginosa.

[0054] An embodiment of the present invention provides the use of the fermentation broth extract described above in preparing a product for inhibiting antibiotic resistance of Pseudomonas aeruginosa.

[0055] In one embodiment, the antibiotic is gentamicin.

[0056] An embodiment of the present invention provides use of the fermentation broth extract described above in preparing a product for inhibiting biofilm formation of Pseudomonas aeruginosa.

[0057] In one embodiment, the Pseudomonas aeruginosa is Pseudomonas aeruginosa PAO1.

[0058] The present invention will be further described below with reference to specific examples.

[0059] In the embodiment of the present invention, the culture medium involved is prepared with deionized water, and the formula is as follows:

[0060] Marine Agar 2216 (MA) solid medium: Protein peptone 5g / L, Yeast extract 1g / L, Iron citrate citrate) 0.1 g / L, sodium chloride (NaCl) 19.45 g / L, magnesium chloride (MgCl2) 5.98 g / L, sodium sulfate (Na2SO4) 3.24 g / L, calcium chloride (CaCl2) 1.8 g / L, potassium chloride (KCl) 0.55 g / L, sodium carbonate (Na2CO3) 0.16 g / L, potassium bromide (KBr) 0.08 g / L, strontium chloride (SrCl2) 0.034 g / L, boric acid (H3BO3) 0.022 g / L, sodium silicate (Na2SiO3) 0.004 g / L, sodium fluoride (NaF) 0.0024 g / L, ammonium nitrate (NH4NO3) 0.0016 g / L, disodium hydrogen phosphate (Na2HPO4) 0.008 g / L, agar (Agar) 15 g / L.

[0061] LA solid medium: Tryptone 10 g / L, yeast extract 1 g / L, sodium chloride (NaCl) 5 g / L, agar 15 g / L.

[0062] LB liquid medium: Tryptone 10 g / L, yeast extract 1 g / L, sodium chloride (NaCl) 5 g / L.

[0063] SGTYP liquid medium: starch 5 g / L, glucose 5 g / L, tryptone 1 g / L, bacterial peptone 1 g / L, yeast extract 1 g / L, sea salt 17 g / L.

[0064] Example 1 Isolation and identification of strain M53

[0065] Strain M53 was isolated from the root soil of Acanthus truncatus in Mai Po Mangrove Nature Reserve, Hong Kong, China in October 2016. Strain M53 was scraped onto MA solid culture medium and streaked, then cultured at 28°C for 48 hours. During this period, the morphology of the colonies was observed and recorded, and the surface hyphae were picked onto a glass slide and stained with a Gram staining kit. The hyphae morphology was observed under an optical microscope. It can be observed that the colonies are dry with neat edges, milky white in color, and smooth and opaque on the surface; then they develop into a fuzzy shape, the color changes from milky white to brownish red, and are not easy to pick up. Figure 1 As shown in a. Observation under a 100× oil microscope showed that the hyphae were fine and dense, with abundant branches and intertwined. The 16S rRNA sequence of strain M53 (SEQ ID NO.1) was uploaded to the sequence comparison website (https: / / www.ezbiocloud.net / ), and the strain with the highest similarity was found to be Streptomyces cavourensis BBRC 13026, phylogenetic tree as Figure 1 As shown in c. Based on the 16S rRNA sequence alignment, colony and hyphae morphology, and Gram color results, strain M53 was identified as Streptomyces ( Streptomyces sp.).

[0066] Strain M53 has been deposited in Guangdong Provincial Microbial Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 65720 and the deposit date as January 6, 2025. Its classification name is Streptomyces sp.

[0067] Example 2 Preparation of the fermentation broth extract of strain M53

[0068] Take a single colony from the MA solid culture medium containing strain M53 and transfer it to 10 mL of SGTYP liquid culture medium, and shake at 28°C (200 rpm) for 24 hours to obtain seed liquid. Take 10 mL of seed liquid and add it to 1 L of SGTYP liquid culture medium at a ratio of 1:100, and shake at 28°C (200 rpm) for 96 hours to obtain fermentation liquid. The fermentation liquid is extracted three times with 1 L of ethyl acetate and dried in a centrifugal concentrator at a temperature of 30-40°C. Finally, dimethyl sulfoxide (DMSO) is used to adjust the volume to 200 mg / mL to obtain a solution of the fermentation liquid extract. The solution of the fermentation liquid extract is as follows. Figure 1 As shown in b.

[0069] Example 3: The extract from the fermentation broth of strain M53 inhibits the surging motility of Pseudomonas aeruginosa PAO1

[0070] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, 1 μL was dripped into the surging motility medium containing fermentation broth extract (100 μg / mL), the surging motility medium containing DMSO (0.2% (v / v)) (negative control group), and the surging motility medium without addition (blank control group), and cultured upright at 37°C for 14 h. The photos were taken and recorded. The results are shown in the figure. Figure 2 shown. Figure 2 In the figure, a is the colony morphology, b is the statistical result of surging movement distance; each experiment was repeated three times, **** represents p <0.0001. Figure 2 As can be seen, P. aeruginosa PAO1 in the negative and blank control groups had already moved to the edge of the plate by 14 hours, exhibiting a typical "tentacle"-like colony morphology. However, at 100 μg / mL of the fermentation broth extract, the surging motility of P. aeruginosa PAO1 was completely inhibited. Therefore, the fermentation broth extract of strain M53 has inhibitory activity against the surging motility of P. aeruginosa PAO1.

[0071] Example 4 The extract from the fermentation broth of strain M53 does not affect the growth of Pseudomonas aeruginosa PAO1

[0072] In order to exclude the possibility that the fermentation broth extract of strain M53 has a bactericidal effect and thus causes the inhibition of surging motility, this example uses growth curve determination and dilution spread plate colony counting method to detect the effect of the fermentation broth extract on the growth of Pseudomonas aeruginosa PAO1.

[0073] The seed solution of Pseudomonas aeruginosa PAO1 that was cultured overnight at 37°C (200 rpm) was diluted with LB liquid medium. The volume ratio of the seed solution of Pseudomonas aeruginosa PAO1 to LB liquid medium was 1:100. The fermentation broth extract was then supplemented with final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. DMSO (0.1% (v / v) and 0.2% (v / v)) was added to the negative control group, and no DMSO was added to the blank control group. The mixed system was incubated at 37°C and 200 rpm, and the OD was measured at 4, 7, 10, 12, 14, and 24 hours, respectively. 600 , the experimental results are as follows Figure 3 As shown. Figure 3It can be seen that the growth of P. aeruginosa PAO1 was not inhibited by the fermentation broth extract at concentrations of 25 to 400 μg / mL. Therefore, the fermentation broth extract of strain M53 has no bactericidal activity against P. aeruginosa PAO1 and does not inhibit surging motility by inhibiting PAO1 growth. It is speculated that the inhibitory activity is a non-antibiotic surging substance.

[0074] Example 5 The inhibitory activity of the fermentation broth extract of strain M53 on the surging motility of Pseudomonas aeruginosa PAO1 showed a gradient effect

[0075] To further explore whether the fermentation broth extract of strain M53 has a concentration gradient effect in inhibiting the surging motility of Pseudomonas aeruginosa PAO1, a series of concentration gradients were designed in this example.

[0076] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, 1 μL was dripped into surging culture medium containing fermentation broth extract (5 μg / mL, 7.5 μg / mL, 8.125 μg / mL, 8.75 μg / mL and 10 μg / mL), surging culture medium containing DMSO (0.2% (v / v)) (negative control group) and surging culture medium without addition (blank control group), and cultured upright at 37°C for 14 h. Photos were taken and recorded. The results are shown in the figure. Figure 4 shown. Figure 4 In the figure, a is the colony morphology, and b is the statistical result of the inhibition rate. Figure 4 After 16 hours of incubation, P. aeruginosa PAO1 colonies in the blank and negative controls moved to the edge of the plate and exhibited a typical "tentacle" morphology. In stark contrast, increasing the concentration of the fermentation broth extract between 5µg / mL and 10µg / mL significantly inhibited the surging motility of P. aeruginosa PAO1. At concentrations of 7.5µg / mL and 8.125µg / mL, the surging motility of P. aeruginosa PAO1 was only slightly inhibited. However, at 10µg / mL, the surging motility of P. aeruginosa PAO1 was almost completely inhibited, with an inhibition rate approaching 100%. The inhibitory effect of the fermentation broth extract of strain M53 on the surging motility of P. aeruginosa PAO1 was concentration-dependent.

[0077] Example 6 The extract from the fermentation broth of strain M53 does not inhibit the swimming and gliding motility of Pseudomonas aeruginosa PAO1

[0078] Swimming motility is driven by the rotation of monopolar flagella, mediating the rapid diffusion of bacteria in liquid environments. Slippery motility, on the other hand, utilizes the periodic extension and contraction of type IV pili to assist bacteria in short-distance movement across solid interfaces. To investigate whether the fermentation broth extract of strain M53 affects swimming motility by inhibiting flagellar function or inhibiting slippery motility by interfering with pili assembly, this example measured the swimming and slippery motility of Pseudomonas aeruginosa PAO1 in the presence of the fermentation broth extract.

[0079] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, take 1 μL dropwise and inoculate in swimming motility medium containing fermentation broth extract (100 μg / mL), gliding motility medium containing fermentation broth extract (100 μg / mL), swimming motility medium containing DMSO (0.1% (v / v)) (negative control group), gliding motility medium containing DMSO (0.1% (v / v)) (negative control group), swimming motility medium without additives (blank control group) and gliding motility medium without additives (blank control group). The swimming motility experiment was cultured upright at 37°C, and the gliding motility experiment was cultured inverted at 37°C. No more than 2 cells were stacked together. Photos were taken and recorded after 14 hours. The results are shown in the figure. Figure 5 shown. Figure 5 In the figure, a is the colony morphology in the swimming motility experiment, c is the statistical result of the inhibition rate in the swimming motility experiment; b is the colony morphology in the gliding motility experiment, d is the statistical result of the inhibition rate in the gliding motility experiment. Figure 5 The results indicate that the 100 µg / mL fermentation broth extract did not inhibit the swimming and surging motility of Pseudomonas aeruginosa PAO1. Therefore, it is speculated that the fermentation broth extract of strain M53 is a specific inhibitor of surging motility.

[0080] Example 7: The extract from the fermentation broth of strain M53 inhibits the formation of biofilms of Pseudomonas aeruginosa PAO1

[0081] Biofilms are macromolecular complexes formed by Pseudomonas aeruginosa PAO1 to protect against adverse external conditions. Biofilms are closely linked to drug resistance. To investigate whether the fermentation broth extract of strain M53 reduces drug resistance by inhibiting biofilm formation in Pseudomonas aeruginosa PAO1, this example examined biofilm formation in Pseudomonas aeruginosa PAO1 after treatment with the fermentation broth extract.

[0082] Pseudomonas aeruginosa PAO1 was cultured in LB liquid medium at 37°C overnight with shaking (200 rpm). The bacterial solution was added to LB liquid medium supplemented with 5g / L glucose at a volume ratio of 1:20, and different concentrations of fermentation broth extract (50, 100, 200, and 400µg / mL) were added. DMSO (0.1% (v / v) and 0.2% (v / v)) were added to the negative control group, and no DMSO was added to the blank control group. The above mixed bacterial solution was statically cultured at 37°C for 16 hours in a 24-well plate (1mL / well). The upper liquid was aspirated, heat-fixed at 70°C for 30 minutes, and then stained with 0.1% (w / v) crystal violet for 7 minutes. After staining, the plate was washed with water three times, air-dried, and then the bottom biofilm was washed with 30% (v / v) acetic acid to remove it. After drying, the biofilm on the wall was dissolved with 30% (v / v) acetic acid and placed in a 96-well plate to measure the OD. 550 , the results are as follows Figure 6 shown. Figure 6 In the figure, a is the morphology of Pseudomonas aeruginosa PAO1 biofilm, b is the quantitative determination result of Pseudomonas aeruginosa PAO1 biofilm, and c is the inhibition rate of fermentation broth extract on Pseudomonas aeruginosa PAO1 biofilm. Figure 6 The results show that the fermentation broth extract of strain M53 significantly inhibited biofilm formation by P. aeruginosa PAO1. A 50 µg / mL fermentation broth extract had no significant effect on biofilms formed by P. aeruginosa PAO1 on the sidewalls of 24-well plates, but biofilm formation gradually decreased with increasing concentration. The fermentation broth extract of strain M53 exhibited approximately 38% and 52% inhibition rates against P. aeruginosa PAO1 biofilm formation at 100 µg / mL and 200 µg / mL, respectively. Increasing the concentration of the fermentation broth extract to 400 µg / mL also increased the inhibition rate to 60%. Therefore, the fermentation broth extract of strain M53 exhibits concentration-dependent inhibitory activity against P. aeruginosa PAO1 biofilm formation.

[0083] Example 8: Inhibition of drug resistance of Pseudomonas aeruginosa PAO1 by extract from fermentation broth of strain M53

[0084] Increased drug resistance is a typical characteristic of bacteria entering a surging state of motility. When bacteria enter this state, their drug resistance typically increases significantly. To verify whether the fermentation broth extract of strain M53 could reduce drug resistance in Pseudomonas aeruginosa PAO1 by inhibiting surging motility, this example combined the fermentation broth extract of strain M53 with gentamicin to analyze whether the minimum inhibitory concentration (MIC) of the antibiotic changed before and after treatment.

[0085] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, 1 μL was dripped into surging motility medium containing gentamicin (0.5, 1, 2, 4, 8, 16 μg / mL), swimming motility medium containing gentamicin (0.5, 1, 2, 4, 8, 16 μg / mL), surging motility medium without addition (blank control group), and swimming motility medium (blank control group), respectively. After upright culture at 37°C for 14 h, photos were taken and recorded. The results are shown in the figure. Figure 7 As shown in a and b. Figure 7 In the figure, a is the colony morphology, and b is the statistical result of the inhibition rate. Figure 7 As shown in Figures a and b, P. aeruginosa PAO1 in the blank control group reached the edge of the plate after 14 hours. When 0.5 µg / mL gentamicin was added, the surging motility of P. aeruginosa PAO1 was only slightly inhibited (inhibition rate approximately 30%), while the same concentration of gentamicin significantly inhibited its swimming motility (inhibition rate approximately 90%). When 1 µg / mL gentamicin was added, the inhibition rate of surging motility of P. aeruginosa PAO1 was approximately 40%, while at the same concentration of gentamicin, its swimming motility was completely inhibited, and P. aeruginosa PAO1 was unable to grow. These results are consistent with expectations, indicating that P. aeruginosa PAO1's resistance to gentamicin is significantly enhanced during the surging motility state.

[0086] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, 1 μL was dripped into the surging motility medium and cultured at 37°C for 14 h for phenotypic induction. Using a sterile toothpick, motile cells 2-3 mm from the edge of the motility area were picked and inoculated into the center of the surging motility medium containing fermentation broth extract (7.5 μg / mL), surging motility medium containing gentamicin (1, 2 μg / mL), surging motility medium containing gentamicin (1, 2 μg / mL) and fermentation broth extract (7.5 μg / mL), surging motility medium containing gentamicin (1, 2 μg / mL) and DMSO (0.1% (v / v)), surging motility medium containing DMSO (0.1% (v / v)) (negative control group) and surging motility medium without addition (blank control group). After incubation at 37°C for 14 h, photos were taken and the movement radius was measured to calculate the inhibition rate. The results are shown in the figure. Figure 7 As shown in c and d. Figure 7 In the figure, c is the colony morphology, and b is the statistical result of inhibition rate. Figure 7 As shown in Figures c and d, the combination of fermentation broth extract and gentamicin can significantly reduce the drug resistance of Pseudomonas aeruginosa PAO1: when 1µg / mL gentamicin is combined with 7.5µg / mL fermentation broth extract, the inhibition rate of surging motility of Pseudomonas aeruginosa PAO1 is 60%, which is significantly higher than the inhibition rate of gentamicin alone at the same concentration (about 30%). p <0.01); when 2µg / mL gentamicin was combined with 7.5µg / mL fermentation broth extract, the surging motility of P. aeruginosa PAO1 was completely inhibited, and colony growth was almost nonexistent. This inhibition rate was significantly higher than that of gentamicin alone at the same concentration (inhibition rate of approximately 40%). Therefore, the fermentation broth extract of strain M53 can reduce the drug resistance of P. aeruginosa PAO1 by inhibiting surging motility, and its combination with low-concentration gentamicin significantly enhances its efficacy.

[0087] Example 9 Addition of rhamnolipids can partially restore the surging motility of Pseudomonas aeruginosa PAO1

[0088] Pseudomonas aeruginosa PAO1 requires the secretion of the surfactant rhamnolipid to reduce surface tension to initiate surging motility. To investigate whether the inhibition of surging motility in P. aeruginosa PAO1 is related to rhamnolipid, this example adds different concentrations of rhamnolipid to the surging motility experiment to study the changes in surging motility of P. aeruginosa PAO1.

[0089] Pseudomonas aeruginosa PAO1 was activated in LA solid medium and cultured at 37°C for 24 h. The activated Pseudomonas aeruginosa PAO1 was collected with a sterile inoculation loop and diluted to 1×10 9 CFU / mL, 1 μL was dripped into surging motility medium containing fermentation broth extract (10, 20 μg / mL) and rhamnolipid (0.1, 1 mg / mL), surging motility medium containing fermentation broth extract (10, 20 μg / mL), surging motility medium containing DMSO (0.1% (v / v)) and rhamnolipid (0.1, 1 mg / mL), surging motility medium containing DMSO (0.1% (v / v)), surging motility medium containing rhamnolipid (0.1, 1 mg / mL) and surging motility medium without additives, and cultured upright at 37°C for 14 h. Photos were taken and recorded. The results are shown in Figure 4. Figure 8 As shown. Figure 8As shown, 10µg / mL and 20µg / mL of fermentation broth extract completely inhibited the surging motility of P. aeruginosa PAO1. Supplementation with 0.1mg / mL rhamnolipids partially restored the surging motility of P. aeruginosa PAO1 previously inhibited by 10µg / mL of fermentation broth extract, while the surging motility of P. aeruginosa PAO1 remained unrestored by 20µg / mL of fermentation broth extract. Supplementation with 1mg / mL of rhamnolipids further restored the surging motility of P. aeruginosa PAO1 previously inhibited by fermentation broth extract, while the surging motility of P. aeruginosa PAO1 in the control group without fermentation broth extract was partially inhibited under these conditions. Therefore, the addition of rhamnolipids can partially restore the surging motility of P. aeruginosa PAO1 previously inhibited by fermentation broth extract, but high concentrations of rhamnolipids also inhibit normal surging motility of P. aeruginosa PAO1.

[0090] Example 10: The extract from the fermentation broth of strain M53 inhibits the production of rhamnolipids by Pseudomonas aeruginosa PAO1

[0091] The results of Example 9 indicate that the inhibition of surging motility of Pseudomonas aeruginosa PAO1 may be related to the production of rhamnolipids. Therefore, this example measured the rhamnolipid production of Pseudomonas aeruginosa PAO1 under the action of the fermentation broth extract of strain M53.

[0092] Pseudomonas aeruginosa PAO1 was activated into LA solid medium and cultured at 37°C for 24 hours. A single colony of Pseudomonas aeruginosa PAO1 was scraped and inoculated into LB liquid medium. After shaking culture at 37°C (200rpm) for 16 hours, it was transferred to M9 medium supplemented with 2% (w / v) glycerol, 0.05% (w / v) glutamic acid, and 0.05% (w / v) Triton X-100 at a volume ratio of 1:100. Fermentation broth extract (50, 100, 200 μg / mL) or 0.1% (v / v) DMSO (negative control group) was added, and no addition was used as a blank control group. After shaking culture at 37°C (200rpm) for 24 hours, the OD was measured. 600 The supernatant was collected by centrifugation and extracted three times with 3 volumes of ethyl acetate. The extract was dried by centrifugal concentrator and dissolved in 1 mL of water. After dissolution, 100 μL was transferred to a new EP tube, and then 800 μL of 60% (w / v) dilute sulfuric acid and 100 μL of orcinol reagent were added. The reaction was carried out at 80°C for 30 minutes. After the reaction was completed, the tube was allowed to stand at room temperature for 15 minutes and the OD was measured. 421 , through OD 421 / OD 600 The ratio was used to evaluate rhamnolipid production, and the results were as follows: Figure 9 shown. Figure 9 Each experiment was repeated three times, **** represents p <0.0001. Figure 9It can be seen that after treatment with 50µg / mL of fermentation broth extract, rhamnolipid production in P. aeruginosa PAO1 decreased by approximately 50%; after treatment with 100µg / mL of fermentation broth extract, rhamnolipid production decreased by approximately 75%. As the fermentation broth extract concentration increased to 200µg / mL, rhamnolipid production in P. aeruginosa PAO1 decreased by 85%. Therefore, the fermentation broth extract of strain M53 can significantly reduce rhamnolipid production in P. aeruginosa PAO1, thereby inhibiting surging motility.

[0093] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Streptomyces ( Streptomyces sp . ) A method for preparing a fermentation broth extract of M53, characterized in that: Including steps: (1) Inoculate Streptomyces M53 into SGTYP liquid culture medium and shake culture for 72-96 hours to obtain fermentation broth; (2) extracting the fermentation broth with ethyl acetate, removing the aqueous phase, and obtaining a fermentation broth extract; (3) removing ethyl acetate from the fermentation broth extract to obtain a fermentation broth extract of Streptomyces M53; The Streptomyces M53 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65720.

2. The preparation method according to claim 1, characterized in that The shaking culture conditions are: temperature 25-28° C., rotation speed 100-300 rpm.

3. The preparation method according to claim 1, characterized in that Step (1) specifically includes: After activating Streptomyces M53 on MA solid medium, take a single colony and add it to 5-15 mL of SGTYP liquid medium and shake it for 20-30 hours to obtain seed liquid; The seed liquid is inoculated into a new SGTYP liquid culture medium and shake-cultured for 72 to 96 hours to obtain a fermentation liquid.

4. The preparation method according to claim 3, characterized in that The volume ratio of the seed solution to the new SGTYP liquid culture medium is 1:(50-100).

5. A fermentation broth extract of Streptomyces M53, characterized in that The fermentation broth extract is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the fermentation broth extract according to claim 5 in preparing a product for inhibiting the surging movement of Pseudomonas aeruginosa.

7. Use of the fermentation broth extract according to claim 5 in the preparation of a product for inhibiting antibiotic resistance of Pseudomonas aeruginosa.

8. The use according to claim 7, characterized in that The antibiotic is gentamicin.

9. Use of the fermentation broth extract according to claim 5 in preparing a product for inhibiting biofilm formation of Pseudomonas aeruginosa.

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