CN103060224A - Space enterococcus faecium LCT-EF258 strain - Google Patents
Space enterococcus faecium LCT-EF258 strain Download PDFInfo
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Abstract
The invention relates to the biological characteristics, the genome DNA (deoxyribonucleic acid) sequence, the transcriptome and the differential proteomics of an enterococcus faecium LCT-EF258 strain in a space environment, and particularly relates to the effects of the functional sequence and molecules identified by performing comparative analysis with ground enterococcus faecium on micro-organisms in the predetermined space environment, and the applications of the functional sequence and molecules, wherein the comprehensive and accurate mutant genes, differential expression genes and proteins of the enterococcus faecium strain in the space environment can be obtained.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to faecium and biological characteristics thereof, the order-checking of full genomics, transcribe group order-checking and Discrepancy proteome Epidemiological Analysis.
Background technology
Implement gradually with China's manned space program along with human solar-system operation is increasing, spacefarer's health is the key issue that aerospace medicine ensures, wherein pre-bacteriological protection infection is an importance.There is research to find that under simulate microgravity Salmonellas can strengthen the virulence of mouse infection, the immunity degradation of body in the simultaneously solar-system operation, thus cause more easy infection.At present, detected the various bacteria that comprises faecalis in the space station, therefore the research for the space bacterial dissociation is an important topic of space microbe research.At present, the resource of relevant space mutagenesis microorganism lacks very much, has seriously hindered the development in this field.
Faecium is considered to belong in early days the D group streptococcus and belongs to, and along with the development of the every technology of molecular biology, by the homology detection of gene DNA, finds that it belongs to enterococcus spp.Faecalis is a kind of normal microflora in the human body, but when descending, body immunity can cause a disease equally, therefore be a kind of conditionality pathogenic bacterium, often can cause a plurality of systems to infect, showing infection etc. such as human reproductive system, urinary system, abdominal cavity infection, wound, also is one of common flora that causes ward infection simultaneously.In recent years, along with the various enforcements that the inspection of wound property and treatment means arranged and immunosuppressor and antibiotic a large amount of uses, make enterococcal infection more and more.Because the normal natural drug resistance of this kind flora all has resistance to cynnematin, Macrolide, sulfamido, aminoglycoside, the faecalis strain that in recent years tolerates vancomycin increases, clinically so that the doctor is usually at one's wit's end.At present enter the bottleneck phase in the research aspect the intractable faecium infection, well do not studied point of penetration.
Faecium associated biomolecule under the steric requirements is learned the research that proterties (such as virulence and resistance etc.) changes, the molecule mechanism of analyzing its variation be solve that bacterium in the solar-system operation infects and clinical treatment in the effective means of resistance problem.In recent years, genome, transcribe the systems biology research meanses such as group, protein science and information biology and constantly occur, more easily carry out so that carry out the molecular mechanism research of faecium sudden change under the space environment.Can be on the one hand space Biosafety assessment and provide fundamental basis, the research that can be on the other hand clinical intractable infection provides thinking.
Summary of the invention
One object of the present invention provides the faecium LCT-EF258 bacterial strain of a strain space environment
This bacterial strain provided by the invention, its preserving number are CGMCC NO.6525.
One Enterococcus faecalis, Gram-positive is single, chain, agglomerate is arranged; Antibiotic resistance with ground contrast strain is compared, and microbiotic amikacin (amikacin), Ciprofloxacin are tolerated more; Difference without the speed of growth; Can utilize γ-aminobutyric acid and L MALIC ACID to be carbon source, and can not utilize p-hydroxyphenylaceticacid.
Described faecium LCT-EF258 bacterial strain, carry out genome sequencing and the comparative genomics analysis draws mutator gene, see Table 1, space environment causes the variation of SNP on the LCT-EF258 strain gene group, comprise the position of SNP on genome, the gene that mutational site and proteins encoded and note go out; Table 2 comprises the zone of InDel position and generation for space environment causes insertion and disappearance on the LCT-EF258 strain gene group.
Cause isozygotying on the LCT-EF258 strain gene group variation of SNP of table 1 space environment
SNP position mutating alkali yl is with reference to gene I/D annotation database ID mutain Scaffold3_60853 C → A LCT-EF90GL001983 gi|293570275 S → I
Table 2 space environment causes insertion and the disappearance on the LCT-EF258 strain gene group
Described faecium LCT-EF258 bacterial strain, transcribe group order-checking draw difference expression gene (FDR≤0.001 He | log
2Ratio| 〉=2) see attached list 1.
Described faecium LCT-EF258 bacterial strain carries out the Discrepancy proteome Epidemiological Analysis and draws the differentially expressed protein molecule (when albumen abundance difference multiple surpasses more than 2 times; Check by statistics its p-value value less than 0.05; Reach above-mentioned requirements at least twice repetition in repeating for three times) see attached list 2.
Description of drawings
Fig. 1 faecium LCT-EF258 bacterial strain gramstaining result
Fig. 2 faecium LCT-EF258 bacterial strain Physiology and biochemistry qualification result
Fig. 3 faecium LCT-EF258 strain growth curve
Fig. 4 faecium LCT-EF90 strain growth curve
The GC content of Fig. 5 LCT-EF258 bacterial strain and Depth association analysis figure
The two-dimentional collinearity figure of Fig. 6 LCT-EF258 bacterial strain and ground control group LCT-EF90
Sequenced genes coverage figure is organized in transcribing of Fig. 7 LCT-EF258 bacterial strain
Fig. 8 transcribes the difference expression gene quantity that the group order-checking is identified
The differential expression protein quantity that Fig. 9 Differential Proteomic is identified
The annex explanation
The differentially expressed protein information that annex 2 faecium LCT-EF258 bacterial strain quantitative proteomics are analyzed
Embodiment
Used experimental technique in the following embodiment if no special instructions, is ordinary method.Used material, reagent in the following embodiment if no special instructions, all can obtain from commercial channels.
Embodiment one: the gramstaining of faecium LCT-EF258 bacterial strain
Get the 50ul sample centrifugal, abandon supernatant, add sterilized water 50ul, draw 20ul and on slide glass, be fixed; Just dye, draw gramstaining I (Viola crystallina) 2 and on slide specimen, carry out just dying 1min, then wash dye liquor with tap water; Mordant dyeing, 2 coverings of absorption gramstaining II (iodine liquid) are coated with face and dye approximately 1min, then wash dye liquor with tap water, draw the moisture that is coated with on the face with thieving paper; Decolouring is drawn gramstaining III (alcohol) 2 and is dropped on the face of being coated with approximately 30s, and water flushing slide glass is drawn the moisture that is coated with on the face with thieving paper; Redye, draw gramstaining IV (sarranine) 2 and drop on the face of being coated with approximately 1min, water flushing slide glass is drawn the moisture that is coated with on the face with thieving paper; Observe, find the target visual field with the microscope low power lens first, and then observe with oily mirror (100 *), judge and record the result, take pictures, see Fig. 1, Gram-positive is single, chain, agglomerate arrangement.
Embodiment two: the antibiotics sensitivity of faecium LCT-EF258 bacterial strain detects
Preparation LB solid medium 25L falls 1500 flat boards behind the bacterium of having gone out, dry, and is for subsequent use; The inclined-plane of these single bacterium is taken out in the preparation of bacteria suspension, and prepares to fill the 1.5ml centrifuge tube of 1ml stroke-physiological saline solution, dips proper amount of strains with the bamboo let of sterilization in the 1.5ml centrifuge tube from the inclined-plane, mixing, accent bacteria concentration approximately 10
7～10
8Dilution spread is drawn the bacteria suspension 100 μ l that get ready and is coated with, and as much as possible coating is even, and the single bacterium of every strain is coated with 6 flat boards, whenever is painted with a strain bacterium, just carries out next step (pasting susceptibility sheet); Paste the susceptibility sheet, select microbiotic to carry out drug sensitive test, to filter out the bacterial strain of drug-resistant mutation; After having pasted the susceptibility sheet, flat board is placed on 37 ℃ of lower 18～24h of cultivation; Observations is cultivated 18～24h with flat board, takes out the antibacterial situation of observing dull and stereotyped upper susceptibility sheet, and the size (diameter cm) of record inhibition zone, and wherein susceptibility sheet diameter is about 0.6cm.To amikacin (amikacin) and Ciprofloxacin tolerance, without inhibition zone, and the inhibition zone of ground control group is respectively 1.2cm and 1.4cm.
Embodiment three: the Physiology and biochemistry identification experiment of faecium LCT-EF258 bacterial strain
Collecting cells, the inclined-plane of taking-up bacterial strain dips bacterial classification inoculation (4ml system) in the LB liquid nutrient medium with the sterilization bamboo let, cultivates 24h.Then get the centrifugal 5min of 2ml bacterium liquid 6000r/s, abandon the supernatant substratum, stay centrifugal thalline, then the physiological saline with sterilization washs thalline, the centrifugal 5min of 6000r/s, again supernatant discarded, with the Biolog inoculation liquid thalline that suspends, go to again in the inoculation liquid of 15ml at last again.Transfer to concentration approximately 10
7～10
8Inoculation, to inoculating in the ware, concussion is evenly with above bacteria suspension, carry out application of sample with 12 roads (or the 8 roads) volley of rifle fire, each Biolog plate hole application of sample 100ul, totally 96 holes, after adding sample, cover Biolog plate lid, be placed on 37 ℃ of lower cultivations, 24h and 48h observe, and record result, see Fig. 2 and table 3, can find out that from figure neutralization table main difference is that LCT-EF258 can utilize γ-aminobutyric acid and L MALIC ACID to be carbon source, and can not utilize p-hydroxyphenylaceticacid.
The Physiology and biochemistry qualification result of table 3:LCT-EF258 bacterial strain
| Numbering | Substrate | The LCT-EF258 bacterial strain | Contrast LCT-EF90 bacterial strain |
| B8 | N-acetyl-D-galactosamine | +/- | - |
| C8 | The L-rhamnosyl | +/- | - |
| D4 | Inositol | +/- | - |
| E9 | Serine | - | +/- |
| F3 | Galactonolactone | +/- | - |
| F4 | Maltonic acid | - | +/- |
| F6 | Glucuronamide | - | +/- |
| G1 | P-hydroxyl-toluylic acid | - | + |
| G3 | The D-ALPHA-Hydroxypropionic acid methyl esters | +/- | - |
| G5 | Citric acid | - | +/- |
| G8 | L MALIC ACID | + | - |
| H2 | γ-aminobutyric acid | + | - |
Embodiment four: the growth curve of faecium LCT-EF258 bacterial strain
The activation bacterial strain is cultivated 1d in 15ml test tube (4ml substratum), then be seeded in the 100 hole perforated plates, carries out the mensuration of growth curve in Bioscreen, measures 24h, reading of data; With the calculating of the resulting OD value of the every strain bacterium value of averaging, and then curve plotting, see Fig. 3 and Fig. 4, as can be seen from Figure under the space environment LCT-EF258 bacterial strain with ground LCT-EF90 strain growth curve indifference.
Embodiment five: the gene order-checking of faecium LCT-EF258 bacterial strain and icp gene group analysis
Cultivate faecium and extract genomic dna, at first adopting ultrasonic method will detect qualified DNA sample interrupts at random, obtain a series of dna fragmentations, after T4DNA polysaccharase, Klenow archaeal dna polymerase and T4 polynueleotide kinase etc. are processed, reclaim the purpose fragment, obtain sequencing library, utilize the Genome Analyzer II order-checking of Illumina; The reads that order-checking obtains after finishing assembles and analyzes after processing after filtration; Adopt Glimmer3.0 software annotation gene and analyze SNP, Indel, gene rearrangement and structure variation etc.The SNP sudden change of isozygotying of finding sees Table 1, Indel analysis and does not find considerable change.The Quality Control of order-checking is that GC content and Depth association analysis statistical graph are seen Fig. 5, and wherein X-coordinate is GC content, and ordinate zou is mean depth.Without calculating its GC content and mean depth of repeating, this scatter diagram is similar to Poisson's distribution take 500bp as window, i.e. GC is without obvious skewed popularity in the order-checking process.The two-dimentional collinearity figure that sees Fig. 6 is analyzed in collinearity and rearrangement, transverse axis is the reference sequences genome among the figure, the longitudinal axis is institute's cls gene group, the level that color is more shallow or vertical straight line represent cutting apart between each Scaffold, the nucleotide sequence that red lines represent institute's cls gene group is in normal chain, and the blue nucleotide sequence of institute's cls gene group that represents is in minus strand.
Embodiment six: faecium LCT-EF258 bacterial strain transcribe the group sequencing analysis
After extracting the total RNA of faecium LCT-EF258 bacterial strain, behind test kit removal rRNA.Add fragmentation buffer mRNA is broken into the short-movie section, take mRNA as template, with the synthetic article one cDNA chain of hexabasic basic random primer (random hexamers), then add the synthetic second cDNA chain of damping fluid, dNTPs, RNase H and DNA polymerase I, doing terminal reparation through QiaQuick PCR test kit purifying and after adding the EB buffer solution elution, adding polyA and connect sequence measuring joints, then carrying out clip size with agarose gel electrophoresis selects, carry out at last pcr amplification, the sequencing library of building up Illumina HiSeq
TM2000 check order.The original series data are removed statistics rRNA statistics after the impurity data, estimate distribution gene coverage, the statistics gene expression amount difference of Reads on genome, analyzed difference expression gene etc., see Table 3, table 4, Fig. 7 and Fig. 8, difference expression gene for details see attached table 1,497 of down-regulated genes, 20 of up-regulated genes.Fig. 7 is that the gene sequencing coverage refers to the per-cent that each gene is covered by reads, and its value equals the base number of unique mapping reads covering in the gene with the ratio of all base numbers of gene coding region.
Table 3: sample LCT-EF258 and with reference to the statistics of genome alignment
Table 4: sample LCT-EF258 and the statistics of comparing with reference to gene
Each row implication of above table is as follows:
Embodiment seven: the Discrepancy proteome Epidemiological Analysis of faecium LCT-EF258 bacterial strain
From faecium LCT-EF258 sample, extract albumen; Protein sample after extracting is carried out reductive alkylation process, opened disulfide bond is so that follow-up abundant enzymolysis protein; 2D quant kit method with GE company is carried out the concentration determination of protein; Equal-volume carries out SDS (sodium laurylsulfonate) electrophoresis; Enzymolysis protein; With iTRAQ reagent mark peptide section; Peptide section behind the mark is carried out balanced mix; Use strong cation exchange chromatography (Strong Cation Exchange Choematography, SCX) to carry out pre-separation to mixed peptide section; Carry out liquid phase tandem mass spectrum (liquid chromatography coupled with tandem mass spectrometry, LC-MS/MS) analysis.To the source document of machine under the mass spectrum, carry out peak identification, obtain the peak tabulation; Set up reference database, carry out the evaluation of peptide section and protein; The last relation of the relative content of each albumen between each sample (when albumen abundance difference multiple surpasses more than 2 times relatively; Check by statistics its p-value value less than 0.05; Reach above-mentioned requirements at least twice repetition in repeating for three times), thus some interested important albumen obtained, and differential protein for details see attached table 2 raises the down-regulation protein statistics and sees Fig. 9,25 of upregulated proteins, 57 of down-regulation proteins.
Described faecium LCT-EF258 bacterial strain can be applied to screening and identification clinical antibiotics resistance target spot according to change and the molecule mechanism of antibiotic resistance, for the diagnosis and treatment product of developing clinical intractable faecium infection provides reference.
Explanation about the LCT-EF258 bacterial strain of preservation
A. depositary institution's title and the address of bacterial classification
Title: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
B. hand over the date of mechanism's preservation
On September 5th, 2012
C. the preserving number that gives of preservation mechanism
CGMCC No.6525
D. Classification And Nomenclature faecium Enterococcus Faecium
Claims (6)
1. space faecium LCT-EF258, its preserving number is: CGMCC NO.6525.
2. an Enterococcus faecalis according to claim 1 is characterized in that: Gram-positive is single, chain, agglomerate is arranged; Antibiotic resistance with ground contrast strain is compared, and microbiotic amikacin (amikacin), Ciprofloxacin are tolerated more; Difference without the speed of growth; Can utilize γ-aminobutyric acid and L MALIC ACID to be carbon source, and can not utilize p-hydroxyphenylaceticacid.
3. faecium LCT-EF258 bacterial strain according to claim 1 carries out genome sequencing and the comparative genomics analysis draws mutator gene.
4. faecium LCT-EF258 bacterial strain according to claim 1 is transcribed the group order-checking and is drawn difference expression gene.
5. faecium LCT-EF258 bacterial strain according to claim 1 carries out the Discrepancy proteome Epidemiological Analysis and draws the differentially expressed protein molecule.
6. faecium LCT-EF258 bacterial strain according to claim 1 can be applicable to screening and identification clinical antibiotics resistance target spot.
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| CN109637585A (en) * | 2018-12-27 | 2019-04-16 | 北京优迅医学检验实验室有限公司 | The antidote and device of depth is sequenced |
Citations (4)
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|---|---|---|---|---|
| EP0508701A2 (en) * | 1991-04-08 | 1992-10-14 | Quest International B.V. | Probiotic |
| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
| CN102031253A (en) * | 2010-12-16 | 2011-04-27 | 复旦大学 | Molecular marker method for identifying indica type rice and japonica rice by using rice grain |
| US20110104134A1 (en) * | 2008-07-04 | 2011-05-05 | Nippon Meat Packers, Inc. | Novel lactic acid bacterium having high immunoglobulin-a-inducing ability |
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2012
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0508701A2 (en) * | 1991-04-08 | 1992-10-14 | Quest International B.V. | Probiotic |
| US20110104134A1 (en) * | 2008-07-04 | 2011-05-05 | Nippon Meat Packers, Inc. | Novel lactic acid bacterium having high immunoglobulin-a-inducing ability |
| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
| CN102031253A (en) * | 2010-12-16 | 2011-04-27 | 复旦大学 | Molecular marker method for identifying indica type rice and japonica rice by using rice grain |
Non-Patent Citations (1)
| Title |
|---|
| 袁杰利 等: "屎肠球菌活菌制剂的研究", 《中国微生态学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109637585A (en) * | 2018-12-27 | 2019-04-16 | 北京优迅医学检验实验室有限公司 | The antidote and device of depth is sequenced |
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