Gram-negative bacteria selective isolation medium and application

Abstract

The application belongs to the technical field of microbial culture, and discloses a selective separation culture medium for gram-negative bacteria and application of the culture medium in rapid separation, purification and identification of gram-negative bacteria in samples such as aquatic products, food, water and soil. The selective separation culture medium takes a perylene quinone photosensitive compound as a key active component.

Description

Technical Field

[0001] This invention belongs to the field of microbial isolation, culture and screening technology, specifically involving a method for preparing selective culture media for bacteria and a technique for isolating and culturing Gram-negative bacteria. Background Technology

[0002] The structure and properties of the cell wall are crucial for distinguishing bacterial biochemical properties. Classifying bacteria based on differences in cell wall chemical composition facilitates further research in molecular biology and physiological metabolism. Currently, Gram staining is a vital technique for differentiating bacterial biochemical properties. However, pure bacterial cultures are required before staining. Furthermore, the staining process is cumbersome, and the results are easily affected by the physiological state of the bacteria, leading to instability. Currently, selective culture, molecular imprinting, and gold nanoparticle reactions are commonly used for bacterial isolation and cell wall property identification (Table 1). Among these, selective plate culture generally uses chemical antibacterial agents to inhibit or promote the growth of Gram-positive or Gram-negative bacteria, resulting in a single species on the plate at the end of the culture. Selective culture methods offer lower cost and lower barriers to entry for screening strains, and have significant potential for efficient isolation or identification of specific bacterial groups from complex samples (e.g., food and soil environmental samples). However, most current selective plates use antibiotics as selective antibacterial agents, which can lead to inactivation and antibiotic resistance during use, affecting their selective performance and stability.

[0003] Table 1. Relevant Inventions for Gram Bacterial Identification

[0004]

[0005]

[0006] Perylene quinone compounds, especially bamboo red fungus extract, are activated by visible light irradiation. They can efficiently transfer electrons to catalyze the generation of singlet oxygen, superoxide anions, and hydroxyl radicals (ROS), selectively attacking the cell membrane and membrane proteins of organisms, thus exhibiting significant photoinduced antibacterial and tumor cell inhibition effects. They have important application value in the fields of photodynamic therapy and medicine. Patent CN1421134A discloses a patent for using bamboo red fungus extract as a photosensitizer for antibacterial activity, confirming the high efficiency of bamboo red fungus extract in antibacterial activity. The antibacterial mechanism of bamboo red fungus extract is physical sterilization, and its mechanism of action is as follows: (1) cell wall rupture; (2) depolarization of cell membrane potential, regulating parameters related to bacterial apoptosis, such as mitochondrial transmembrane potential and DNA rupture; (3) disruption of cell membrane integrity. Compared with chemical bactericides such as antibiotics, bamboo red fungus extract is less likely to induce drug resistance and has a more lasting antibacterial effect. Numerous studies have shown that bamboo red sclerotin is low in toxicity and stable under dark conditions, and is almost non-toxic to humans.

[0007] Bamboo red pigment exhibits significantly different antibacterial activities against Gram-positive bacteria, Gram-negative bacteria, and fungi. The cell walls of Gram-positive bacteria and most fungi are composed of peptidoglycan or glucan and cross-linked chitin, resulting in a porous structure. Under light conditions, reactive oxygen species (ROS) generated by bamboo red pigment can penetrate the cell wall and target the cell membrane, thus exerting a bactericidal effect. However, the cell walls of Gram-negative bacteria have a complex structure consisting of an outer membrane containing lipoproteins, lipopolysaccharides, and phospholipids, a periplasmic membrane containing peptidoglycan, and an inner membrane containing peptidoglycan. The lipopolysaccharides in the cell walls of Gram-negative bacteria can reduce the efficiency of ROS targeting the cell membrane. Compared to Gram-negative bacteria and fungi, bamboo red pigment shows a more significant photoinhibitory effect against Gram-positive bacteria (Yin Hongfu, 2011; Su Yujie, 2011). Furthermore, bamboo red pigment can significantly inhibit the activity of fungi such as Candida albicans and molds. The biofilm formation ability of Candida albicans is reduced by 90%. Therefore, by uniformly dispersing perylene quinone compounds, such as bamboo red mycin, into the culture medium, and utilizing light during the microbial culture process, these compounds can continuously exert their selective inhibitory activity against fungi, actinomycetes, and Gram-negative bacteria, thereby achieving targeted selective culture and isolation of Gram-negative bacteria in the sample.

[0008] However, due to the complex reducing components in the culture medium, the efficiency of ROS generation by directly using perylene quinone photosensitive pigments is significantly neutralized and weakened. Furthermore, since the reported photocatalytic antibacterial activities of perylene quinone pigments all occur in an oxygen-rich environment, reactive oxygen species cannot be generated in an anaerobic environment. By using perylene quinone pigments in combination with peroxides, the peroxides can be photocatalytically decomposed to generate hydroxyl radicals, producing more efficient ROS than traditional use of perylene quinone compounds alone. This significantly improves the sensitivity, selectivity, and applicability of selective culture media (especially in anaerobic environments). Simultaneously, the chelating agent eliminates the ineffective decomposition of peroxide components by transition metal ion impurities in the culture medium during preparation and storage.

[0009] This invention innovatively utilizes the synergistic effect of peroxides and perylene quinone compounds in ordinary culture media to selectively isolate and culture Gram-negative bacteria, improving the selectivity and sensitivity of the culture medium and broadening its applicability for microbial screening. The culture process more closely resembles the original culture medium, better preserving the growth state and characteristics of Gram-negative bacteria. Key components include compounds of baicalein, peroxides, and chelating agents, all of which are green and safe, unlikely to induce drug resistance in microorganisms, and will not cause harm to users or the environment. Summary of the Invention

[0010] This invention provides a photodynamic selective separation medium that, through cultivation, efficiently isolates and screens Gram-negative bacteria from complex microbial samples for further purification and identification of the strains.

[0011] To achieve the above objectives, this invention provides a method for preparing and using a functional molecular solid culture medium supplemented with bamboo red pigment A (or) and B, hydrogen peroxide, and sodium EDTA. A solution containing a certain concentration of bamboo red pigment A (or) and B, hydrogen peroxide, and sodium EDTA is dispersed in the culture medium and plates are prepared. Plates inoculated with microbial samples are then cultured under light. At the end of the culture, colonies of Gram-negative bacteria are observed.

[0012] Specifically, the present invention employs the following technical steps.

[0013] A selective isolation medium for Gram-negative bacteria, comprising a combination of perylene quinone compounds and / or their derivatives, peroxides, and metal chelating agents in a basal medium.

[0014] In specific embodiments of the present invention, the basic culture medium includes, but is not limited to: nutrient agar (NA) solid medium, plate counting agar (PCA) solid medium, anaerobic meat liver soup medium with added agar, enhanced clostridium medium (RCM), Pseudomonas selective medium (CFC), lactic acid bacteria medium (MRS), ferrous sulfite agar, and Aeromonas selective medium (RYAN).

[0015] In some specific embodiments of the present invention, the perylene quinone compounds include hypoporelines, hypomycins, celsinochrome, and cercosporin; the derivatives of the perylene quinone compounds include formate, acetate, propionate, isobutyrate, valerate, hexanoate, heptanoate, caprylate, acrylate, benzoate, phenylacetate, phenylbutyrate, tartrate, clavate, citrate, decanoate, fumarate, gluconate, glutamate, glycolate, lactate, laurate, malate, maleate, malonic acid ester, oleate, oxalate, palmitate, pantothenate, and succinate. The peroxide is hydrogen peroxide or calcium peroxide; the associated metal chelating agents include sodium EDTA, sodium NTA, sodium IDS, HEDP, DTPA, etc.

[0016] Preferably, the selective separation culture medium is used under the following conditions: 25-40℃, visible light irradiation; light intensity 10-8000 lux, wavelength range 390-780 nm, aerobic or anaerobic.

[0017] Preferably, the preparation method includes the following steps: adding a composite solution before or after sterilization of the basal culture medium, wherein the composite solution contains perylene quinone compounds, peroxides and metal chelating agents, with final concentrations of 1-1000 μg / mL, 1-1000 μmol / mL and 1-1000 μg / mL, respectively.

[0018] More preferably, the final concentrations of the perylene quinone compound, the peroxide, and the metal chelating agent are 5-50 μg / mL, 10-300 μmol / mL, and 5-600 μg / mL, respectively.

[0019] More preferably, the perylene quinone compound is basiloxane A and / or basiloxane B. The peroxide is hydrogen peroxide, and the metal chelating agent is sodium EDTA.

[0020] Most preferably, the final concentrations of the perylene quinone compound, peroxide, and metal chelating agent added are 50 μg / mL, 100 μmol / mL, and 200 μg / mL, respectively.

[0021] As one specific embodiment, the method includes the following steps:

[0022] 1) Dissolve perylene quinone compounds in dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mg / mL, and filter sterilize. Dissolve peroxide and metal chelating agent separately in sterile deionized water to prepare stock solutions with concentrations of 10 mmol / mL and 100 mg / mL, respectively. Store at 4°C protected from light.

[0023] 3) Prepare the basic culture medium;

[0024] 3) Thoroughly mix different volumes of perylene quinone compounds and metal chelating agent stock solutions in the basal culture medium to achieve a final concentration of 1-1000 μg / mL for both perylene quinone compounds and metal chelating agents. Sterilize the culture medium at 115-121℃ for 15-30 min, cool to 50℃, and add different volumes of peroxide stock solution to achieve a final concentration of 1-1000 μmol / mL. Pour plates. Alternatively, sterilize the culture medium at 115-121℃ for 15-30 min, cool to 50℃, add three selective stock solutions, and mix to achieve final concentrations of 1-1000 μg / mL for perylene quinone compounds, 1-1000 μmol / mL for perylene quinone compounds, and 1-1000 μg / mL for metal chelating agents.

[0025] As another inventive point of this invention, this invention also relates to a method of using a selective isolation medium, characterized in that the method is not used for diagnostic purposes, and specifically includes: diluting the sample to be tested and inoculating it into a selective isolation medium, culturing it at 25-37°C under visible light for 24-72 hours, and then observing the growth of the bacterial strain; optionally, performing gene identification on the strain.

[0026] Preferably, the samples are derived from aquatic products, food, soil, and water.

[0027] The technical features and effects of this invention mainly include, but are not limited to, the following aspects:

[0028] This invention provides a selective culture medium that utilizes perylene quinone compounds to photocatalyze peroxides to generate free radicals, selectively killing Gram-positive bacteria, fungi, actinomycetes, and other microorganisms. This enables the targeted and rapid isolation of Gram-negative strains from complex samples for further purification and identification. The selective culture medium of this invention can inhibit Gram-positive bacteria, including but not limited to: *Lactococcus lactis*, *Lactococcus gasseri*, *Enterococcus aureus*, *Enterococcus avium*, *Staphylococcus squirrelii*, *Enterococcus avium*, *Enterococcus hermannii*, *Serratia proteans*, and *Streptomyces*. It also inhibits fungi, including but not limited to *Aspergillus*, *Penicillium*, *Mucor*, *Trichoderma*, *Saccharomyces*, *Candida*, *Candida*, *Branchiolus*, and *Branchiaceae*. Compared to other chemical additives, the three components of this invention offer the following advantages: 1) high selectivity; 2) low dosage; 3) low inactivation rate; 4) no induction of microbial resistance; and 5) natural product origin, making it environmentally friendly. Attached Figure Description

[0029] Figure 1 Growth of *Lactococcus lactis* on a culture medium containing 50 μmol / mL hydrogen peroxide and 100 μg / mL sodium EDTA. The figures indicate the concentrations of 0, 0.1, 1, 10, 50, 100, and 200 μg / mL of rutin.

[0030] Figure 2 The bacterial colony growth of aquatic products on plates and MacConkey plates with different concentrations of bamboo red fungicide were shown. The culture medium contained 100 μmol / mL hydrogen peroxide and 200 μg / mL sodium EDTA. The images indicate bamboo red fungicide A concentrations of 0, 5, 7.5, 10, 12.5, 15, and 200 μg / mL.

[0031] Figure 3Colonies of Acinetobacter baumannii grown on rubigin-hydrogen peroxide-EDTA-PCA. Detailed Implementation

[0032] The experimental design and implementation of the above examples are all based on achieving the purpose of this invention and are explained accordingly. Specific implementation methods and operating conditions are not limited by this invention. Other experts and scholars in the art may make improvements and optimizations without departing from the spirit and principle of this invention, but all such improvements and optimizations should be included within the scope of protection of this invention.

[0033] Example 1

[0034] Selective inhibition of Lactococcus lactis by bamboo red pigment-hydrogen peroxide-EDTA-PCA medium.

[0035] A strain of Gram-positive *Lactococcus lactis* was isolated from commercially available chilled tilapia and preserved in glycerol tubes for later use. PCA medium was prepared by adding *Lactococcus faecium* extract at final concentrations of 0.1, 1, 10, 50, 100, and 200 μg / mL, sterilizing at 121°C for 20 min, and then cooling. Under aseptic conditions, hydrogen peroxide at final concentrations of 0, 50, 100, and 200 μmol / mL and sodium EDTA at a final concentration of 100 μg / mL were added, mixed thoroughly, and poured into plates. The activated *Lactococcus lactis* was cultured and diluted to a concentration of 1.0 × 10⁻⁶. 8 The inoculum was spread onto selective plates and cultured anaerobically at 37°C for 48 hours under white light intensity of 1100-1300 lux. The results are shown in Table 2 and [Table data missing]. Figure 1 .

[0036] Table 2. Growth of Lactococcus lactis on bamboo red pigment-hydrogen peroxide-EDTA-PCA.

[0037]

[0038]

[0039] "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0040] Example 2

[0041] Selective inhibition of Lactococcus lactis by bamboo red pigment-hydrogen peroxide-EDTA-PCA

[0042] A strain of Gram-positive *Lactococcus lactis* was isolated from commercially available chilled tilapia and preserved in glycerol tubes for later use. PCA medium was prepared by adding *Lactococcus faecium* extract to a final concentration of 50 μg / mL. After sterilization at 121°C for 20 min and cooling, hydrogen peroxide at final concentrations of 0, 50, 100, and 200 μmol / mL and sodium EDTA at a final concentration of 100 μg / mL were added under sterile conditions and mixed thoroughly before being poured into plates. The activated *Lactococcus lactis* was then serially diluted with sterile physiological saline to different concentrations (10 μmol / mL, ... 2 10 3 10 4 10 5 10 6 10 7 10 8 CFU / mL was used to inoculate selective plates and incubate anaerobically at 37°C for 48 h under white light intensity of 1100-1300 lux. The results are as follows:

[0043] Table 3. Growth of Lactococcus lactis on polysaccharide-hydrogen peroxide-EDTA-PCA at different concentrations.

[0044]

[0045] *: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0046] Example 3

[0047] The selective effect of bamboo red fungicide-hydrogen peroxide-EDTA culture medium components on bacteria in fish tissue

[0048] Ten Gram-positive and Gram-negative bacteria derived from chilled tilapia fillets (Table 4) were preserved in glycerol tubes for later use.

[0049] The strains used in the examples in Table 4

[0050]

[0051] Prepare PCA medium and nutrient agar medium, sterilize at 121℃ for 20 min, (1) add bamboo red fungus A to a final concentration of 50 μg / mL to prepare bamboo red fungus-PCA medium (A) and bamboo red fungus-nutrient agar medium (B). (2) Add bamboo red fungus A to a final concentration of 50 μg / mL and 100 μmol / mL hydrogen peroxide to prepare bamboo red fungus-hydrogen peroxide-PCA medium (C) and bamboo red fungus-hydrogen peroxide-nutrient agar medium (D). (3) Add bamboo red fungus A to a final concentration of 50 μg / mL, 100 μmol / mL hydrogen peroxide and 200 μg / mL sodium EDTA to prepare bamboo red fungus-hydrogen peroxide-EDTA-PCA medium (E) and bamboo red fungus-hydrogen peroxide-EDTA-nutrient agar medium (F). Pour plates to obtain bamboo red fungus plates. The activated indicator bacteria were diluted with sterile physiological saline at a concentration of 1.0 × 10⁻⁶. 6 CFU / mL concentration was plated and inoculated onto (A)-(F) plates, and cultured aerobically at 37℃ for 48 h under white light intensity of 1100-1300 lux. The results are as follows:

[0052] Table 5. Bacterial growth on different culture media

[0053]

[0054]

[0055] "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies; Gram-positive bacteria (G+), Gram-negative bacteria (G-).

[0056] Example 4

[0057] The selective effect of bamboo red pigment concentration on bacteria in fish tissue in the formula bamboo red pigment-hydrogen peroxide-EDTA-PCA

[0058] PCA medium containing 10, 20, 30, 40, and 50 μg / mL of bamboo red fungus A was prepared. After sterilization at 121℃ for 20 min, hydrogen peroxide (to a final concentration of 100 μmol / mL) and EDTA sodium salt (to a final concentration of 200 μg / mL) were added under aseptic conditions upon cooling, and the medium was poured into plates. The activated indicator bacteria were diluted with sterile physiological saline and inoculated onto bamboo red fungus A plates. The plates were then incubated aerobically at 37℃ for 48 h under white light intensity of 1100-1300 lux. The results are as follows:

[0059] Table 6 shows the growth of indicator bacteria on selective plates containing different concentrations of bamboo red mycotoxin.

[0060]

[0061] *: "+" indicates the presence of bacterial colonies, and "-" indicates the absence of bacterial colonies.

[0062] Example 5

[0063] Selective culture of microbial communities in aquatic products using bamboo red mycin-hydrogen peroxide-EDTA-PCA plates.

[0064] PCA media containing 0, 5, 7.5, 10, 12.5, and 15 μg / mL of bamboo red fungus A were prepared. After sterilization at 121℃ for 20 min, hydrogen peroxide (to a final concentration of 100 μmol / mL) and sodium EDTA (to a final concentration of 200 μg / mL) were added under aseptic conditions upon cooling, and the mixtures were poured into plates. MacConkey agar (MAC) plates were also prepared. Commercially available chilled tilapia fillets were chopped, homogenized with sterile physiological saline, and then enriched. The enriched bacterial solutions were diluted and inoculated onto bamboo red fungus A-hydrogen peroxide-EDTA-PCA and MacConkey agar plates, respectively. The plates were cultured at 37℃ under aerobic light at 1100-1300 lux for 48 h. The results are shown in Table 6. Figure 2 .

[0065] Table 6. Colony growth on bamboo red pigment-hydrogen peroxide-EDTA-PCA and MacConkey agar plates.

[0066]

[0067] When using a bamboo red fungicide-hydrogen peroxide-EDTA-PCA plate culture enrichment solution containing 50 μg / mL bamboo red fungicide A, only one distinct colony was obtained. Figure 3

[0068] According to BLAST comparison in the NCBI database, this strain belongs to Acinetobacter baumannii. This bacterium is a Gram-negative pathogen in farmed aquatic animals. It is not easily destained during Gram staining and is easily stained purple, thus being identified as a Gram-positive bacterium.

[0069] While the specific embodiments of the present invention have been described above in conjunction with examples, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A selective isolation medium for Gram-negative bacteria, characterised in that: The basal culture medium contains a combination of bamboo red fungus A, hydrogen peroxide, and sodium EDTA. The basal culture medium is PCA solid medium. The concentration of bamboo red fungus A is 50 μg / mL, the concentration of hydrogen peroxide is 100 μmol / mL, and the concentration of sodium EDTA is 200 μg / mL. The Gram-negative bacteria are Proteus vulgaris, Serratia marcescens, or Acinetobacter baumannii.

2. The selective separation culture medium according to claim 1, characterized in that, The conditions for using the selective separation medium are: aerobic culture at 25-37℃, culture under visible light; light intensity 1100-1300 lux, wavelength range 390-780 nm.

3. The method for preparing a selective separation medium according to claim 1 or 2, characterized in that, The process includes the following steps: after sterilizing the basal culture medium, a compound solution is added, wherein the components of the compound solution include bamboo red fungus A, hydrogen peroxide and sodium EDTA; the concentration of bamboo red fungus A is 50 μg / mL, the concentration of hydrogen peroxide is 100 μmol / mL, and the concentration of sodium EDTA is 200 μg / mL.

4. Use of the selective separation medium according to claim 1 or 2 or of the selective separation medium obtained by the method according to claim 3, characterized in that, The method is not for diagnostic purposes and specifically includes: diluting the sample to be tested and inoculating it into the selective separation medium, culturing it aerobically at 25-37℃ under visible light with a light intensity of 1100-1300 lux, observing the growth of the strain after 24-72 hours, and identifying the gene of the strain; the sample is derived from aquatic products, food, soil, and water.

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