CN113564074B

CN113564074B - Myxobacteria and application thereof in preparation of antibacterial drugs

Info

Publication number: CN113564074B
Application number: CN202110820724.0A
Authority: CN
Inventors: 朱红惠; 周杨; 姚青; 蚁烁星
Original assignee: Institute of Microbiology of Guangdong Academy of Sciences
Current assignee: Institute of Microbiology of Guangdong Academy of Sciences
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2023-05-12
Anticipated expiration: 2041-07-20
Also published as: CN113564074A

Abstract

The invention discloses a myxobacteria strain and application thereof in preparation of antibacterial drugs. The invention adopts an escherichia coli induction method to separate and purify a myxobacteria Archangium violaceum-1 from vegetable field pond sediment, and the myxobacteria Archangium violaceum-1 are identified as the purple oocyst bacteria by morphological and molecular biological identification (16S rRNA genes and gyrB genes). The crude extract solution of the fermentation supernatant of the Protocystis violaceum can inhibit the growth of candida albicans, methicillin-resistant staphylococcus aureus and plant pathogenic fungi, and the antibacterial activity shows obvious dosage effect and shows the antibacterial activity of a broad spectrum of the strain 3-1. The myxobacteria 3-1 has good application value in the aspects of clinical infectious microbe inhibition drug development, plant pathogenic fungi biological control bactericides, biopesticides and the like.

Description

Myxobacteria and application thereof in preparation of antibacterial drugs

Technical field:

the invention belongs to the field of microorganisms, and particularly relates to myxobacteria and application thereof in preparation of antibacterial drugs.

The background technology is as follows:

candida albicans (Candida albicans) are conditional pathogenic fungi, and can cause a series of mucosal infection and systemic infection in the process of human immunity hypofunction or interventional operation such as catheter and the like. About 4000 tens of thousands of candida-induced infectious diseases are worldwide, with candida albicans being the most common and fatal, accounting for about 50-70% of candida infection cases and 43% of total mortality, and becoming an important issue in public health systems and healthcare. In the body of infected persons, candida albicans exists in the form of a biological film, so that the drug resistance of the candida albicans is enhanced, and the candida albicans can be even prevented from being attacked by a host immune system, and the control difficulty of the candida albicans is further increased.

The general use of various antibacterial drugs promotes the continuous enhancement of the drug resistance of pathogenic bacteria, in particular to multi-drug resistant strains, which seriously affect the progress of clinical treatment. Methicillin-resistant staphylococcus aureus (Staphylococcus aureus, MRSA) is an important pathogenic bacterium of the prior hospital infection as one of multiple drug-resistant strains, can cause various infectious diseases of human bodies such as blood, lower respiratory tract, skin and the like, and clinical observation shows that the infection rate and the mortality rate of the methicillin-resistant staphylococcus aureus are rising year by year. On the one hand, the difficulty of treating drug-resistant bacteria infection clinically is increased, and on the other hand, the development difficulty of new antibiotics is also increased. The research and development of antibiotics and antibacterial drugs with new chemical structures, new action mechanisms or new action targets is an effective way for treating drug-resistant bacteria infection.

On the other hand, filamentous fungi are coated with hard chitin-containing cell walls, which are significantly different from bacteria and yeasts, and thus have different drug targets. Mould is not only an important pathogenic fungus for clinical infection, but also the most important pathogen causing diseases of food crops, and causes huge economic loss of agriculture. The aggregation of plant pathogenic fungi in soil is an important factor for the occurrence of soil-borne diseases and continuous cropping obstacles, and the difficulty in controlling diseases is high. In addition, almost every crop has several to tens of fungal diseases, often resulting in failure of a single control means. Therefore, broad-spectrum antibacterial agents are of great significance for plant disease control.

The myxobacteria as a novel drug source microorganism can produce secondary metabolites which are rich and various, novel in structure and unique in action mechanism, and are now the second big antibiotic producing bacteria after actinomycetes. Myxobacteria are predatory bacteria capable of killing and lysing predatory bacteria including bacteria and fungi. Secondary metabolites play an important role in the predation process of myxobacteria. The broad predatory spectrum of myxobacteria means that it can be used for inhibition of a variety of pathogenic bacteria. However, the development and utilization of secondary metabolites of myxobacteria is far behind actinomycetes.

The invention comprises the following steps:

a first object of the present invention is to provide a myxobacterium Archangium violaceum-1 capable of producing an antibacterial substance, deposited at the cantonese microbiological strain collection center (GDMCC) at day 13, 7, 2021, address: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070, accession number: GDMCC No:61803.

a second object of the present invention is to provide the use of myxobacteria Archangium violaceum-1, or a culture, broth or extract of broth thereof, in the manufacture of a bacteriostatic agent.

The antibacterial drug is a drug for inhibiting Candida albicans, methicillin-resistant staphylococcus aureus Staphylococcus aureus or fusarium oxysporum Fusarium oxysporum f.sp.circumference 4.

The application of the myxobacteria Archangium violaceum 3-1 in preparing the antibacterial medicine is that the myxobacteria Archangium violaceum-1 are cultured to obtain fermentation liquor, then the fermentation liquor is leached by methanol, and the methanol leaching liquor is concentrated and dried to obtain extract which is used for preparing the antibacterial medicine.

Preferably, macroporous resin is added into fermentation broth for culturing myxobacteria Archangium violaceum 3-1, then methanol is used for soaking the macroporous resin, and the methanol extract is concentrated and dried to obtain extract for preparing antibacterial drugs.

A third object of the present invention is to provide a bacteriostatic agent comprising myxobacteria Archangium violaceum-1, or a culture, a bacterial liquid, a fermentation liquid or an extract of a fermentation liquid thereof as an active ingredient.

The extract of the fermentation broth is obtained by culturing myxobacteria Archangium violaceum 3-1, then leaching the fermentation broth with methanol, concentrating and drying the methanol leaching solution to obtain an extract.

Preferably, macroporous resin is added into fermentation broth for culturing myxobacteria Archangium violaceum 3-1, then methanol is used for soaking the macroporous resin, and the methanol extract is concentrated and dried to obtain extract.

The invention adopts an escherichia coli induction method to separate and purify a myxobacteria Archangium violaceum-1 from vegetable field pond sediment, and the myxobacteria Archangium violaceum-1 are identified as the purple oocyst bacteria by morphological and molecular biological identification (16S rRNA genes and gyrB genes). The crude extract solution of the fermentation supernatant of the Protocystis violaceum can inhibit the growth of candida albicans, methicillin-resistant staphylococcus aureus and plant pathogenic fungi, and the antibacterial activity shows obvious dosage effect and shows the antibacterial activity of a broad spectrum of the strain 3-1. The myxobacteria 3-1 has good application value in the aspects of clinical infectious microbe inhibition drug development, plant pathogenic fungi biological control bactericides, biopesticides and the like.

Myxobacteria Archangium violaceum-1 were deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 2021, 7, 13, address: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070, accession number: GDMCC No:61803.

description of the drawings:

FIG. 1 is a morphological feature of myxobacteria Archangium violaceum 3-1; a: colony morphology of violaceum 3-1 grown in VY/2 solid medium for 3 days; b: violaceum 3-1 forms a large number of mature fruit bodies on VY/2 solid medium for 10 days; c: observing the morphology of the A.violaceum 3-1 fruiting body under a stereoscopic microscope; d: violaceum 3-1 rod-shaped vegetative cell crystal violet staining; e: violaceum 3-1 rod-shaped vegetative cells and spherical myxospore crystal violet staining.

FIG. 2 is the inhibition of Candida albicans Candida albicans SC5314 by a crude extract solution of the fermentation supernatant of myxobacterial strain Archangium violaceum 3-1.

FIG. 3 is the inhibition of methicillin-resistant Staphylococcus aureus Staphylococcus aureus GDMCC 1.1263 by a crude extract solution of a fermentation supernatant of a myxobacterial strain Archangium violaceum 3-1.

FIG. 4 shows inhibition of the filamentous plant pathogenic fungus Fusarium oxysporum Fusarium oxysporum f.sp.cube race 4 by a crude extract solution of a fermentation supernatant of a myxobacterial strain Archangium violaceum 3-1.

Detailed Description

The following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1: isolation and purification of strains

1.1 sample collection and pretreatment

Collecting sediment from a pond of a rape field in a pond of a West village in an east town pond of a Cheng Hai region of Shanzhi, filtering out water, and naturally airing at room temperature; a further portion of the fresh sediment sample was added to sterile deionized water at 1:10 (v/v) and subjected to shaking for 1h, centrifuged to obtain a supernatant, and filtered through a 0.22 μm filter to prepare a leaching solution. The characteristics of the myxobacteria that can produce stress-resistant fruiting bodies are utilized to inhibit the interference of other bacteria, fungi and protozoa by air-drying treatment.

1.2 isolation, purification and preservation of myxobacterial Strain 3-1

10g of air-dried sediment sample is taken and 15mL of 100 mug ml is added ^-1 Filtering sterilized cycloheximide, soaking overnight, centrifuging, and discarding supernatant. Water agar medium (i.e., WCX medium containing CaCl) using Escherichia coli as substrate ₂ ·2H ₂ O1 g/L and agar 15g/L, the balance being water; pH 7.2), coliform bacteria are separated, a 'field' shape is drawn on the surface of the WCX culture medium, and sediment samples with the size of soybean grains are respectively inoculated in four blank spaces of the 'field' shape. Culturing at 30deg.C for 7 days, observing with a stereomicroscope, separating and picking fruiting body, and purifying (VY/2 culture medium containing 5g/L yeast and CaCl) ₂ ·2H ₂ O 1g/L，VB ₁₂ 0.5mg/L and 15g/L of agar, the balance being water; pH 7.2). The fruiting body or colony edge was repeatedly transferred with VY/2 until pure strain was observed with naked eyes. The obtained pure strain is inoculated in nutrient broth culture medium (containing 10g/L peptone, 3g/L beef extract powder and 5g/L NaCl, the balance being water; pH 7.2), and the culture is subjected to overnight culture and purification at 30 ℃, if the culture solution is clear, the myxobacteria is the pure strain. Transferring the purified myxobacterial strain onto a VY/2 solid culture medium until fruiting body is formedScraping after maturation for preparing glycerol seed and freeze-dried seed, and preserving glycerol seed at-80deg.C and freeze-dried seed at 4deg.C. Thus, strain 3-1 was obtained.

Example 2: identification of Strain Archangium violaceum 3-1

2.1 morphological and cultural characterization observations of strains

Strain 3-1 shows a sliding expansion transparent colony on a VY/2 solid culture medium, and the whole colony is radial; aggregate formation was observed beginning at about 5 days of growth on VY/2 solid medium, with the mature fruiting bodies being spherical, dark purple brown. Gram staining was negative (fig. 1). The morphological characteristics above indicate that the strain meets the basic characteristics of myxobacteria.

2.2 Strain identification based on 16S rRNA Gene and gyrB Gene sequences

Scraping fresh thalli 3-1 growing on a VY/2 solid culture medium for 3 days, adding a small amount of sterile water, treating at 90 ℃ for 10min, and centrifuging to obtain a supernatant serving as a template for PCR reaction. The PCR reaction system (25. Mu.L) was 12.5. Mu.L buffer, 1. Mu.L upstream and downstream primers, 1. Mu.L template, and 9.5. Mu.L sterile deionized water. The reaction procedure: 95℃for 5min,95℃for 30s,56℃for 30s,72℃for 90s,30 cycles, 72℃for 10min. The PCR products were subjected to agarose electrophoresis to determine that no errors occurred and then submitted to sequencing by Jin Weizhi Biotechnology Co.

The 16S rRNA gene and gyrB gene sequences were aligned for BLAST homology on EzBioCloud (https:// www.ezbiocloud.net /), respectively. The results showed that strain 3-1 had the highest similarity to model strain Archangium violaceum DSM 14727, with sequence similarity of 99.28% and 98.16% for the 16S rRNA gene and gyrB gene, respectively. The sequences of the 16S rRNA gene and the gyrB gene are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

The strain was identified as Archangium violaceum by the morphological and molecular biological identification described above, and was deposited at the Guangdong province microbiological strain collection center (GDMCC) at 7.13 in 2020, accession number 3-1: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070, accession number: GDMCC No:61803.

example 3: preparation of strain Archangium violaceum-1 by liquid fermentation and fermentation supernatant fluid crude extract

The fermentation medium is MD1 liquid (casein peptone 0.6%, soluble starch 0.2%, mgSO by mass fraction) ₄ ·7H ₂ O0.2％，CaCl ₂ ·2H ₂ O0.04%, pH 7.2), 2% of macroporous resin XAD-16 was added before inoculation, and Archangium violaceum-1 seed liquid was inoculated in an inoculum size of 2% by volume. 140rpm, shake culturing at 30deg.C for 7d, filtering with gauze to collect resin, adding 2 times of methanol, shake leaching at 30deg.C for 1 hr, and filtering to collect methanol; the leaching process was repeated 3-4 times until the methanol was colorless. Combining the collected methanol on a rotary evaporator at 35 ℃ for 40r min ^-1 And (5) evaporating to dryness under reduced pressure. 4.5L of the strain is fermented to obtain 2.5873g of crude extract. Dissolving the above crude extract with small amount of Dimethyl Sulfoxide (DSMO), and diluting with sterile deionized water to concentration of 100, 50, 25, 12.5, 6.25 and 3.125mg ml ^-1 Is prepared from the crude extract solution of (a) and (b).

Example 4: inhibition of candida albicans Candida albicans SC5314 by bacterial strain Archangium violaceum 3-1 fermentation supernatant crude extract solution

C.albicans SC5314 cells cultured overnight on YPD solid medium (glucose 20g/L, yeast extract 10g/L, peptone 20g/L, agar 2%) were scraped off, sterile deionized water was added to prepare a bacterial suspension, and the bacterial suspension was dipped with a cotton swab and spread on fresh YPD solid medium. After the plates were dried, 3 sterile filter paper sheets with a diameter of 0.5cm were spread, and 20. Mu.l of each concentration of the crude extract solution was added to each filter paper sheet. As positive and negative controls, geneticin G418 (100. Mu.g/ml) and aqueous DSMO were simultaneously set, respectively. After the filter paper is dried, the filter paper is placed at 30 ℃ for culture, and after 12 hours, the size of the inhibition zone is observed and measured. The results show that the fermentation broth extract of the strain 3-1 obviously inhibits the growth of C.albicans SC5314, and the inhibition has a dose effect. When the concentration of the extract of the fermentation liquid is 100, 50, 25, 12.5, 6.25 and 3.125mg ml ^-1 The zone of inhibition diameters were 29.3mm, 24.6mm, 22.5mm, 21mm, 15.8mm and 12.6mm, respectively (FIG. 2).

Example 5: inhibition of methicillin-resistant staphylococcus aureus Staphylococcus aureus GDMCC 1.1263 by bacterial strain Archangium violaceum 3-1 fermentation supernatant crude extract solution

S.aureus GDMCC 1.1263 cells cultured overnight on TSA solid medium (tryptone 15g/L, soytone 5g/L, naCl 5g/L, agar 1.5%, pH 7.3) were scraped off, sterile deionized water was added to make a bacterial suspension, and the bacterial suspension was dipped with a cotton swab and spread on fresh TSA solid medium. After the plates were dried, 3 sterile filter paper sheets with a diameter of 0.5cm were spread, and 20. Mu.l of each concentration of the crude extract solution was added to each filter paper sheet. Vancomycin (100. Mu.g/ml) and aqueous DSMO were simultaneously set as positive and negative controls, respectively. After the filter paper is dried, the filter paper is placed at 30 ℃ for culture, and after 12 hours, the size of the inhibition zone is observed and measured. The results show that the higher concentration of the strain 3-1 fermentation broth extract obviously inhibits the growth of the test MRSA bacteria, and the inhibition effect has a dosage effect. When the concentration of the extract of the fermentation liquid is 100, 50, 25 and 12.5mg ml ^-1 The diameter of the inhibition zone was 8.2mm, 10.5mm, 12.6mm and 13.5mm, respectively (FIG. 3).

Example 6: inhibition of the filamentous plant pathogenic fungi Fusarium oxysporum Fusarium oxysporum f.sp.circumference race 4 by the crude extract solution of the fermentation supernatant of the strain Archangium violaceum-1

F.oxysporum bacterial blocks are selected and inoculated in the center of PDA solid culture medium (peeled potato 200G/L, glucose 20G/L, agar 2%), when the diameter of fungus bacterial plaque reaches 2cm, 4 holes with the diameter of 0.5mm are punched at the position 1cm away from the edge of a bacterial colony in the cross direction, 20 mu L of crude extract solution with different concentrations is added into each hole, and meanwhile, geneticin G418 (100 mu G/ml) and DSMO aqueous solution are respectively arranged as positive and negative controls. Culturing at 30deg.C, observing and measuring the size of inhibition zone after 12 hr, and calculating inhibition rate. The result shows that the 3-1 fermentation broth extract can inhibit the growth of the filamentous fungus fusarium oxysporum, and the inhibition effect has a dosage effect. When the concentration of the extract of the fermentation liquid is 100, 50, 25, 12.5, 6.25 and 3.125mg ml ^-1 The inhibition rates were 14.1%, 23.3%, 29.4%, 35.6%, 41.7% and 44.8%, respectively (fig. 4).

Sequence listing

<110> the institute of microbiology of the academy of sciences of Guangdong province (microbiological analysis and detection center of Guangdong province)

<120> a myxobacterium and application thereof in preparation of antibacterial drugs

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<212> DNA

<213> myxobacteria 3-1 (Archangium violaceum)

<400> 1

agtcgagcgc gaatggagca atcctagtag agcggcgcac gggtgcgtaa cacgtggata 60

atctgcctgg gtgtctggga taaccagtcg aaagattggc taataccgga taagcccccg 120

ggagcttcgg ctcctgaggg aaaaggtggc ctctgtatac aagctatcac atccagatga 180

gtccgcggcc catcagctag ttggcggggt aatggcccac caaggcgacg acgggtagct 240

ggtctgagag gacgatcagc cacactggaa ctgagacacg gtccagactc ctacgggagg 300

cagcagtggg gaattttgcg caatgggcga aagcctgacg cagcaacgcc gcgtgtgtga 360

tgaaggtctt cggattgtaa agcactttcg accgggacga aaacccctag cctaacacgc 420

tggggcttga cggtaccggg agaagaagca ccggctaact ctgtgccagc agccgcggta 480

atacagaggg tgcaagcgtt gttcggaatt attgggcgta aagcgcgtgt aggcggcttt 540

gcaagtcggg tgtgaaagcc ctcagctcaa ctgaggaagt gcgcccgaaa ctgcagagct 600

tgagtgccgg agagggtggc ggaattcccc aagtagaggt gaaattcgta gatatgggga 660

ggaacaccgg tggcgaaggc ggccacctgg acggtaactg acgctgagac gcgaaagcgt 720

ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga gaactaggtg 780

tcgtgggtgt tgacccccgc ggtgccgtag ctaacgcatt aagttctccg cctgggaagt 840

acggtcgcaa gactaaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900

tggtttaatt cgacgcaacg cgcagaacct tacctggtct tgacatcctc ggaatccctc 960

agagatgagg gagtgcccgc aagggaaccg agagacaggt gctgcatggc tgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aaccctcgcc tttagttgcc 1080

gcgcaagcgg atctctagag ggactgccgg tgttaaaccg gaggaaggtg gggatgacgt 1140

caagtcctca tggcctttat gaccagggct acacacgtgc tacaatggcc ggtacaacgc 1200

gtcgccaacc cgcgaggggg agctaatcgc ataaaaccgg tctcagttca gattggagtc 1260

tgcaactcga ctccatgaag gcggaatcgc tagtaatcgc ggatcagcac gccgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtcgat tgctccagaa 1380

gtcatcccac caagg 1395

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ggcgtcacct gcgtcaactt cctgtccgag tggttcaagg tccgcgtgca gcgcaacggc 60

aaggtgtacg agcagtcgta cgcccgcggc gtgtccaatg gctcgccggc cctcgtgggc 120

gagacggaca agcgcggcac gctcatctgg ttcaagccag acaccaccgt catggagacg 180

gtggacttca acttcgacac gctcagccag cgcatgcggg agctcgcgtt cctcaacgcc 240

ggcctgcgca tcgtcatccg cgacatgcgc atcggcaagg agcacgactt caagttcgac 300

ggcggcatcg tctccttcgt cgagtacatc aacaaggcga agcaggcgct caacgacaag 360

cccatccact tccgcacgga gaaggagggc gtgtcgctgg agatcgccct gcagtggaac 420

gacggctacg acgagcgcat cttcaccttc gccaacaaca tcaacacgca cgagggtggc 480

agccacctgt ccggcttcaa ggcggcgctc acgcgcacgc tcaacagcta cgccgagaag 540

ggcgggcagt ggaaggacct gaaggagacc cccacgggcg aggacgcgcg cgagggtctg 600

gccgccgtca tctccgtgaa gctctccaac ccccagttcg agggccagac gaagacgaag 660

ctgggcaaca gcgaggtgaa gggcctggtc gagcagatgg tgaatgatca gctcgccacc 720

ttcctcgagg agaacccggt cgtcagcaag aagatcgtcg ccaagattgg cgatgccacg 780

cgggcccgca tcgccgcgcg caaggcgcgt gagacggtgc ggcgcaaggg cgtgctggac 840

ggcggctcgc tgccgggcaa gctcgccgac tgccagagcc gcgaccccag cgagagcgag 900

ctctacatcg tcgagggtga ctccgcaggt ggctcggcca agcagggccg cgaccggcgc 960

aaccaggcca cctcccgct 979

Claims

1. Rhodocystis praecox (S.purpureus)Archangium violaceum) 3-1, accession number: GDMCC No:61803.

2. use of the rhodocystis, or a culture, a bacterial solution, a fermentation solution or a methanol extract of a fermentation solution thereof according to claim 1 for the preparation of a bacteriostatic agent which inhibits candida albicansCandida albicansMethicillin-resistant staphylococcus aureusStaphylococcus aureusOr Fusarium oxysporumFusarium oxysporumf. sp, cube race 4.

3. The use according to claim 2, wherein the use of the rhodocystis 3-1 in the preparation of a bacteriostatic agent is to culture the rhodocystis 3-1 to obtain a fermentation broth, then leaching the fermentation broth with methanol, concentrating and drying the methanol leaching broth to obtain an extract, and then preparing the bacteriostatic agent.

4. The use according to claim 3, wherein the macroporous resin is added to the fermentation broth of the culture of the rhodocystis 3-1, then the macroporous resin is soaked in methanol, and the methanol extract is concentrated and dried to obtain an extract for preparing the antibacterial medicament.

5. An antibacterial agent comprising the rhodocystis 3-1 or a culture, a bacterial solution, a fermentation solution or a methanol extract of the fermentation solution of claim 1 as an active ingredient.

6. The antibacterial agent according to claim 5, wherein the extract of the fermentation broth is obtained by culturing the rhodocystis purpureus 3-1, and then leaching the fermentation broth with methanol, and concentrating and drying the methanol leaching solution to obtain an extract.

7. The antibacterial agent according to claim 6, wherein macroporous resin is added to the fermentation broth for culturing the rhodocystis 3-1, then the macroporous resin is soaked in methanol, and the methanol extract is concentrated and dried to obtain an extract.