CN117866059A - Lysobacter ApeC-like domain protein and application thereof in bacterial binding - Google Patents
Lysobacter ApeC-like domain protein and application thereof in bacterial binding Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a lysobacter ApeC-like domain protein and application thereof in bacterial binding. The present invention clones a bacterial binding protein-LysoACP-like protein, wherein the domain of fragment ApeC-like has similar characteristics to ApeC in invertebrates, retaining four conserved cysteines and three DXED motifs. Research shows that the recombinant protein of LysoACP-like of the lysobacti can well combine a plurality of bacteria and fungi, has broad-spectrum combination activity capability, can agglutinate bacteria, can be used for agglutinating and removing bacteria, and is beneficial to the elimination of pathogenic bacteria by organisms. Therefore, the recombinant LysoACP-like protein of the lysobacti can be used for binding and eliminating bacteria and for preparing medicaments for treating bacterial infectious diseases, and provides a new choice for binding and agglutinating bacterial products and medicament development.
Description
Technical Field
The invention belongs to the technical field of bioengineering. More particularly, it relates to a lysobacter ApeC-like domain protein and its use in bacterial binding.
Background
Lysobacter is a microorganism of the genus Lysobacter (Lysobacter), which is commonly found in plants, crop soil and fresh water, and also in extreme habitats such as up-flow anaerobic sludge blanket. The bacterium of the genus lysobacter has antagonistic activity against microorganisms including other bacteria, fungi, unicellular algae, nematodes and the like, because lysobacter can secrete a variety of extracellular hydrolytic enzyme-cleaving microorganisms, many of which are important applications as biocontrol bacteria in plant disease control. In addition, bacteria of the genus lysobacter are considered as an important resource pool for the production of novel antibiotics, and secondary metabolites produced by them play an important role in antagonizing plant pathogens and biological control, and there are currently few reports of research on the interaction of lysobacter domain proteins with other bacteria.
ApeC (Apextrin C-terminal) is a novel class of protein domains of about 200 amino acids, and a protein containing an ApeC domain is called ACP (ApeC Containing Protein). The prior art originally discovered that this protein was involved in the embryonic development of sea urchins (Heliocidaris erythrogramma), known as the apical extracellular matrix protein (Apextrin). ACP was subsequently found to also be involved in embryo development and larval colonisation by coral (Acropora millepora). Whereas ACPs (apelB and apelP) found in mussels (Mytilus galloprovincialis) are expressed in the highest amounts in blastula and trophoblast stages. In addition, ACP, a pattern recognition protein, is involved in the innate immunity of lower invertebrates, mediating bacterial recognition in cells and activation of NF- κb pathway by specifically recognizing peptidoglycans.
It can be seen that the prior published ACP molecule is applied to the function research of invertebrates, chinese patent CN115974993a discloses that the crassostrea gigas short ApeC domain protein CgACP1 recognizes and bacteria bind in peptidoglycan, and that the published ACP molecule function research of invertebrates is still performed; although there are proteins found in bacteria that contain the ApeC-like domain, there is no study or report on the association of the ApeC domain proteins and genes of bacteria nor on the interaction of ApeC domain proteins with other bacteria.
Disclosure of Invention
The invention aims to overcome the defect of interaction between the existing lysobacter domain protein and other bacteria, and provides a lysobacter ApeC-like domain protein and application thereof in bacterial binding.
It is a first object of the present invention to provide an ApeC-like domain of a lysobacter bacterial binding protein.
A second object of the present invention is to provide a lysoACP-like protein of the ApeC-like domain of lysobacter.
It is a third object of the present invention to provide nucleotides of the LysoACP-like protein.
A fourth object of the present invention is to provide the use of the lysoACP-like protein of the ApeC-like domain of a lysobacter or a recombinant protein thereof.
It is a fifth object of the present invention to provide a bacterial and/or fungal cleaning formulation or binding and agglutinating formulation.
It is a sixth object of the present invention to provide a bacterial removal kit.
It is a seventh object of the present invention to provide a method of binding and/or agglomerating bacteria and/or fungi.
The above object of the present invention is achieved by the following technical scheme:
the invention clones a lysobacter ApeC-like domain protein LysoACP-like in bacteria for the first time, and a sequencing result shows that the protein coded by the gene consists of an N-terminal signal peptide and a C-terminal ApeC domain, and uses the ApeC domain sequence of the Wenchang fish ACP1 as an input sequence, and uses site-specific iterative BLAST (PSI-BLAST), and the full length 1011bp of the lysobacter LysoACP-like gene is obtained through analysis, 336 amino acids are coded, the DNA sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the lysoACP-like protein has four conserved cysteines and three DXED motifs and has similar characteristics with the ApeC in invertebrates.
The invention provides an ApeC-like domain of a bacterial binding protein of lysobacter, the amino acid sequence of which is shown as SEQ ID NO.4, and the nucleotide sequence of the coding ApeC-like domain is shown as SEQ ID NO: 3.
The present invention clones lysoACP-like to colibacillus expression vector pET32a (+), constructs expression plasmid pET32a (+) -lysoACP-like, uses thioredoxin as fusion expression companion, and the system makes the expressed lysoACP-like inclusion body fold correctly into soluble protein through variational renaturation with the help of thioredoxin, and the prepared recombinant protein encodes 311 amino acids, isoelectric point is 4.75, and molecular weight is 35.14 daltons. The LysoACP-like protein can be expressed in E.coli by the expression vector pET32a (+). Research shows that the recombinant LysoACP-like protein can well bind bacteria and fungi, and has broad-spectrum binding capacity; also has the ability to agglutinate bacteria and fungi, and can be used to bind and remove bacteria by binding and agglutinating the bacteria.
Accordingly, the present invention provides the following uses of the LysoACP-like protein of the lysobactin ApeC-like domain or recombinant protein thereof:
use in binding and/or agglutinating bacteria and/or fungi for the purpose of non-disease diagnosis.
Use in the preparation of a bacterial and/or fungal scavenger or binding or agglutinating agent.
The application in preparing medicines for treating bacterial infectious diseases. In particular, the small molecular active substance WAP-8294A produced by the lysobacter has remarkable treatment effect on bacterial infectious diseases caused by methicillin-resistant staphylococcus aureus (MRSA); whereas lysobat can inhibit bacterial peptidoglycan synthesis to inhibit bacterial activity, incorporating the present invention provides a bacterial and/or fungal scavenging agent or binding action, and thus can be used in the manufacture of a medicament for the treatment of bacterial infectious diseases.
The present invention provides a bacterial and/or fungal cleaning formulation or binding, agglutinating formulation comprising the lysobacter ApeC-like domain protein LysoACP-like or recombinant protein thereof.
The invention provides a bacterial removal kit which contains LysoACP-like protein containing an ApeC-like domain of lysobactin or recombinant protein thereof.
The invention also provides a method for binding and/or agglutinating bacteria and/or fungi, which uses a reagent containing lysobacter ApeC-like domain protein lysoACP-like or recombinant protein thereof to treat the bacteria and/or fungi.
Further preferably, the bacteria are one or more of staphylococcus aureus (Staphylococcus aureus), enterococcus faecalis (Enterococcus faecium), escherichia coli (Escherichia coli), vibrio anguillarum (Vibro anguillarum), vibrio parahaemolyticus (Vibro paraheamolyticus) and/or acinetobacter calcoaceticus (Acinetobacter caloacetius); the fungus is Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The invention has the following beneficial effects:
the invention firstly clones and obtains bacterial LysoACP-like protein, wherein the structural domain of a fragment ApeC-like has similar characteristics with ApeC in invertebrate, and four conserved cysteines and three DXED motifs are reserved. Studies show that the lysoACP-like recombinant protein of the lysobacter can well combine a plurality of bacteria and fungi, has broad-spectrum combination activity, can coagulate bacteria, can be used for coagulating and removing bacteria, and is also beneficial to the elimination of pathogenic bacteria by organisms. Therefore, the recombinant LysoACP-like protein of the lysobacti can be used for binding and eliminating bacteria and for preparing medicaments for treating bacterial infectious diseases, and provides a new choice for binding and agglutinating bacterial products and medicament development.
Drawings
FIG. 1 is a graph showing the result of the analysis of the structure prediction of LysoACP-like protein of lysobacter.
FIG. 2 is a graph showing the results of comparison of lyso-ACP-like proteins from lyso-bacillus with ACP proteins from other invertebrate species.
FIG. 3 is a diagram showing construction of recombinant expression plasmid pET32a (+) -LysoACP-like of LysoACP-like protein (A. PET32a (+) plasmid map; B. LysoACP-like fragment insertion position sketch).
FIG. 4 shows the recombinant protein LysoACP-like induced expression and purification electrophoresis (lane 1: protein Marker; lane 2: non-induced TRX-LysoACP-like ultrasonic precipitation; lane 3: induced LysoACP-like ultrasonic mixture; lane 4: induced LysoACP-like ultrasonic supernatant; lane 5: induced LysoACP-like ultrasonic precipitation; lane 6: purified TRX-LysoACP-like).
FIG. 5 is a graph showing the results of a bacterial binding assay for recombinant protein LysoACP-like.
FIG. 6 is a graph showing the results of bacterial agglutination experiments with recombinant protein lysoACP-like.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The following examples describe methods for replication of pET32a-LysoACP-like expression plasmids by Sambrook et al (Sambrook et al 1989, molecular cloning Spring Harbor Labroratory Press. USA): caCl is adopted 2 The method of (2) transforming E.coli.DH5 or BL21 (DE 3) strain with the plasmid, culturing the transformed strain in LB medium containing ampicillin (100 g/mL), and extracting the plasmid with Omega kit.
Example 1 determination of LysoACP-like protein Gene sequence and structural analysis of LysoACP-like protein Gene
Extracting RNA of the lysobacter to perform amplification sequencing analysis, and then using the ApeC domain sequence of the Wenchang fish ACP1 (KM 017614.1) as an input sequence, performing homology search on a bacterial genome database of NCBI by using PSI-BLAST to obtain a lysoACP-like sequence (WP_ 139215448.1). This sequence was then sent to a biological organism (Shanghai, china) for gene synthesis. Analysis shows that the total length of the lyso-ACP-like gene of the lyso-bacillus is 1011bp, the coding is 336 amino acids, the DNA sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The ApeC-like fragment sequence coded by the LysoACP-like gene is shown as SEQ ID NO.3, and the amino acid sequence of the fragment protein is shown as SEQ ID NO. 4.
The synthesized target fragment is connected with pUC57 Simple Vector cloning Vector, then is transformed into escherichia coli DH5 alpha, and recombinant clones are selected for sequencing. The sequencing results were predictive analyzed using SMART software and the results are shown in FIG. 1, which shows that the protein encoded by this gene is composed of an N-terminal signal peptide and a C-terminal ApeC domain and is designated as Lysobacillus Lysoap-like. Subsequent comparison of known ACP protein homologous sequences of different invertebrates using MEGA analysis software, results are shown in fig. 2, which shows that the C-terminal 200 amino acids of ACP proteins are more conserved among different species, while bacteria retain the four cysteines and three DXED motifs therein.
EXAMPLE 2 expression and purification of LysoACP-like fusion proteins from lysobactin
1. Construction of recombinant pET32a-lysoACP-like expression plasmid
The recombinant expression vector pET32a (+) -LysoACP-like was obtained by fusing the gene fragment obtained in example 1 with Thioredoxin (TRX) tag and carrying 6 XHis tag according to the UC57 Simple plasmid containing the LysoACP-like gene as a template, preparing a recombinant protein of LysoACP-like from Abmart corporation (removing signal peptide), using pET32aa (+) vector and E.coli expression system, and finally purifying and dialyzing by Ni colum. The molecular weight of the recombinant protein obtained was 47.12 daltons.
The construction process of the pET32a (+) -lysoACP-like is shown in fig. 3A, and the sequence in the constructed expression vector pET32a (+) -lysoACP-like is identified to be correct through sequencing, which shows that the pET32a (+) -lysoACP-like vector is constructed successfully (fig. 3B).
2. Expression and purification of lysobacter LysoACP-like fusion proteins
The constructed pET32a (+) -LysoACP-like plasmid is transformed into escherichia coli BL21 (DE 3), and the optimal culture conditions of the genetically engineered bacteria are as follows: inoculating 1 μl of the strain into 40mL of LB culture solution (tryptone 10g, yeast 5g, sodium chloride 10g, ultrapure water to 1L) containing ampicillin, shaking at 37deg.C and 220rpm overnight; 10mL of the bacterial liquid is inoculated in 400mL of LB culture liquid containing ampicillin in the next day, and shaking is carried out at 220rpm at 37 ℃ until OD 600 =0.6; 1M IPTG was added to the culture solution at a ratio of 1:1000, and the final concentration was 1mM, and induction was performed at 30℃for 4 hours; after completion of the expression, the cells were collected by centrifugation at 6000rpm for 5min and resuspended in 20mL of 20mM Tris buffer (pH 8.0).
Then carrying out ultrasonic pyrolysis precipitation on the genetically engineered bacteria, and crushing the bacteria by using an ultrasonic crusher at the power of 300W for 5s, stopping for 3s and carrying out ice bath ultrasonic treatment for 30 min; subsequently, the supernatant was collected for subsequent purification by centrifugation at 12000rpm for 30min at 4℃and the remaining inclusion bodies were purified according to 1g of bacteria: the washing was performed with a washing solution at a ratio of 5 mL. The method comprises the following steps: washing solution A (50mM Tris,1mMEDTA,PH7.0-8.5), washing solution B (2M urea, 50mM Tris,1mMEDTA,0.1%Triton-100, PH7.0-8.5), washing solution C (2M urea, 50mM Tris,1mMEDTA,PH7.0-8.5). The heavy suspension is blown by a pipetting gun for each cleaning, and centrifuged at 12000rpm for 5min; finally, 1g of sediment is carried out: binding Buffer for inclusion body histidine tag protein purification was added in a ratio of 10mL, and the pellet was resuspended and blown off using a pipette.
Then carrying out denaturation and renaturation of inclusion bodies: soaking a dialysis bag (Thermo SnakeskinTM) with 8M urea at 4 ℃ overnight, and filling denatured protein supernatant into the dialysis bag and clamping; the dialysis bag was transferred to buffer A (50 mM Tris-Cl, pH6.0, 150mM NaCl,2mM reduced Glutathione (GSH), 0.2mM oxidized glutathione (GSSG), 10% glycerol, 6M urea) and dialyzed at 4℃for 12h; the dialysis bag was then transferred to buffer B (50 mM Tris-Cl, pH6.0, 150mM NaCl,2mM GSH,0.2mM GSSG,10% glycerol, 4M urea), dialyzed at 4℃for 12h, then transferred to buffer C (50 mM Tris-Cl, pH6.0, 150mM NaCl,2mM GSH,0.2mM GSSG,10% glycerol, 2M urea), dialyzed at 4℃for 12h, finally transferred to buffer D (50 mM Tris-Cl, pH6.0, 150mM NaCl,10% glycerol), dialyzed at 4℃for 12h, and fresh dialysis buffer D was replaced, and the previous step was repeated. Recovering dialyzed protein, packaging, and storing at-80deg.C.
Purification and concentration of proteins: purifying the protein by using a Ni column, respectively loading the ultrasonic supernatant after centrifugation and the inclusion body protein after renaturation into a PBS (1 mL in volume of beads) after pre-equilibrium, uniformly mixing and incubating for 40min; after the incubation is finished, collecting the flow-through, washing the chromatographic column with PBS, and washing 100 column volumes; washing the chromatographic column with PBS containing 20mM imidazole, and washing the column for 20 column volumes; finally, 5-10mL PBS containing 250mM imidazole is used for eluting the target protein, and the eluent is collected. 1mL of eluent was added each time, elution was repeated, 10. Mu.L of eluent was added to 100. Mu.L of Braford G250 each time the eluent was collected, and color change was observed, and the elution of protein was blue. 1mL of the eluent was repeatedly added until no more protein eluted.
The collected protein solution was left for SDS-PAGE analysis. Concentration of proteins was performed using Millipore ultrafiltration centrifuge tubes. Washing a newly purchased Millipore ultrafiltration centrifuge tube with precooled distilled water for several times, adding the purified protein solution into the ultrafiltration centrifuge tube, centrifuging at 4000g for 15min at 4 ℃, and collecting the supernatant (about 1 mL); the above steps are repeated until all liquid is concentrated. The mixture was stored in a 4℃refrigerator for a short period of time with distilled water, and in a-20℃refrigerator for a long period of time with 20% ethanol. Finally, the protein concentration was determined using Pierce BCA Protein Assay Kit.
The measurement results are shown in FIG. 4, which shows that the strain has obvious specific expression product bands in ultrasonic cleavage supernatant and precipitation after induction, the molecular weight accords with a predicted theoretical value of 47.12kD, and the purified lysoACP-like recombinant protein of the lysobacter is obtained, and the protein exists in supernatant and inclusion bodies, and exists in a majority of supernatant forms.
EXAMPLE 3 bacterial binding and agglutination Activity assay of LysoACP-like recombinant proteins of lysobacti
The bacteria used in this example include: gram positive bacteria staphylococcus aureus (Staphylococcus aureus), acinetobacter calcoaceticus (Acinetobacter caloacetius), enterococcus faecalis (Enterococcus faecium) and Escherichia coli (Escherichia coli) are all purchased from the Guangdong microorganism strain collection center, and vibrio anguillarum (Vibro anguillarum) and vibrio parahaemolyticus (Vibro paraheamolyticus) are also purchased from the China center for marine microorganism strain collection; the fungus is Saccharomyces cerevisiae (Saccharomyces cerevisiae), purchased from the Guangdong microbiological strain collection center.
1. Bacterial binding Activity assay of lysobacter LysoACP-like recombinant proteins
Adding 2X 10 to 1mL PBS 7 CFU/g bacteria and 5. Mu.g LysoACP-like protein were incubated at 4℃for 1h. The samples were then centrifuged at 15000 Xg for 10min, the pellet washed four times with PBST (0.1% Tween-20, v/v), suspended in 80. Mu.L PBS and 20. Mu.L 5 Xloading buffer, and then boiled at 100℃for 10min. Western blot analysis was performed on binding proteins using a mouse anti-6 xhis mAb (sigma) with TRX as a blank.
As shown in FIG. 5, the recombinant LysoACP-like protein can bind to different bacteria and has a binding capacity, but has weak binding to Escherichia coli and Vibrio anguillarum, and has a certain broad spectrum as a whole.
2. Analysis of bacterial agglutination Activity of LysoACP-like recombinant proteins of lysobacti
Inoculating 2 μL of seed retaining bacteria of staphylococcus aureus, fecal field coccus, acinetobacter calcoaceticus, escherichia coli, vibrio parahaemolyticus, vibrio anguillarum and saccharomyces cerevisiae respectively into 5mL of a proper liquid culture medium, and culturing overnight under proper conditions, wherein the vibrio anguillarum is cultured overnight with seawater culture medium 2216E (Soy palo, china) and YPD culture medium (10 g yeast, 20g peptone, 20g glucose and 1L) for saccharomyces cerevisiae at 28 ℃, and the staphylococcus aureus, the fecal field coccus and the escherichia coli are cultured overnight with LB culture medium at 37 ℃; acinetobacter calcoaceticus was cultured overnight at 28℃with LB medium; vibrio parahaemolyticus was cultured overnight at 37℃with LB30 medium (10 g peptone, 10g yeast, 30g sodium chloride, water to 1L). 1.5mL of cells (OD) were collected the next day 600 >1.5 6000g, centrifuging for 5min, and washing with sterile PBS for 3 times; subsequently, 50. Mu.L of FITC 10mg/mL was added to each bacterium, the volume was made up to 1mL with PBS, and incubated at room temperature in the dark and shaken for about 3 hours; sterile PBS cleaning thalli3-6 times until the solution is colorless, and suspending bacteria in 1mL PBS solution for later use; adjusting the concentration of bacteria to OD 600 =2.0, 50 μl of FITC-labeled bacteria (final concentration about 1×10 7 CFU/mL bacteria or 1X 10 6 CFU/mL yeast) and 10 μg target protein were incubated in 96-well plates, supplemented with PBS to 100 μl, thoroughly mixed, left standing at room temperature for incubation for 2h at dark, and then observed for agglutination activity under a fluorescence microscope and photographed, with TRX protein as a negative control.
As shown in FIG. 6, the recombinant LysoACP-like protein was observed under a fluorescence microscope to effectively agglutinate E.coli, vibrio anguillarum and Saccharomyces cerevisiae.
Taken together, the results show that the first cloned LysoACP-like protein of the invention into the bacterium, wherein the domain of the fragment ApeC-like has similar characteristics to ApeC in invertebrates, retains four conserved cysteines and three DXED motifs. Researches show that the lysobacter LysoACP-like recombinant protein can well combine a plurality of bacteria and fungi, has broad-spectrum combination activity capability, can agglutinate bacteria, can be used for agglutinating and removing bacteria, and is beneficial to the elimination of pathogenic bacteria by organisms. Therefore, the recombinant LysoACP-like protein of the lysobacti can be used for binding and eliminating bacteria and for preparing medicaments for treating bacterial infectious diseases, and provides a new choice for binding and agglutinating bacterial products and medicament development.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An ApeC-like domain of a bacterial binding protein of lysobacter, characterized in that it has an amino acid sequence as shown in SEQ ID No. 4.
2. The lysobacter ApeC-like domain protein lysoACP-like is characterized in that the amino acid sequence of the lysoACP-like protein is shown as SEQ ID NO. 2.
3. A nucleotide encoding the LysoACP-like protein of claim 2, wherein said nucleotide sequence is set forth in SEQ ID No. 1.
4. Use of the lysobacter ApeC-like domain protein LysoACP-like or a recombinant protein thereof according to claim 2 for binding and/or agglutinating bacteria and/or fungi for the purpose of non-disease diagnosis.
5. Use of the lysobacter ApeC-like domain protein LysoACP-like or recombinant protein thereof of claim 2 for the preparation of a bacterial and/or fungal scavenger or binding or agglutinating agent.
6. Use of the lysobacter ApeC-like domain protein LysoACP-like or recombinant protein of claim 2 in the manufacture of a medicament for the treatment of a bacterial infectious disease.
7. A bacterial and/or fungal cleaning agent or binding or agglutinating agent, characterized in that it comprises the lysobacter ApeC-like domain protein LysoACP-like or recombinant protein thereof.
8. A bacterial removal kit, comprising a LysoACP-like protein or recombinant protein thereof, which comprises a lysobactin ApeC-like domain.
9. A method for binding and/or agglutinating bacteria and/or fungi, characterized in that the bacteria and/or fungi are treated with a reagent comprising the lysobacter ApeC-like domain protein LysoACP-like or a recombinant protein thereof.
10. The use according to claims 4 to 6, or the formulation according to claim 7, or the kit according to claim 8, or the method according to claim 9, wherein the bacterium is one or more of staphylococcus aureus (Staphylococcus aureus), acinetobacter calcoaceticus (Acinetobacter caloacetius), enterococcus faecalis (Enterococcus faecium), escherichia coli (Escherichia coli), vibrio anguillarum (Vibro anguillarum) or vibrio parahaemolyticus (Vibro paraheamolyticus); the fungus is Saccharomyces cerevisiae (Saccharomyces cerevisiae).
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