CN103756993A - Establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models - Google Patents

Abstract

The invention relates to establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models. The establishment method comprises the following main steps: 1, carrying out drug sensitive tests of 12 antibiotics such as levofloxacin and the like on Shigella, and selecting sensitive strains to carry out an induction experiment; 2, detecting MIC0 of levofloxacin on the primary induction strain; 3, inoculating the single colony into a levofloxacin bacterial liquid culture medium to culture; and 4, taking the bacterial solution in the step 3, transferring to the levofloxacin bacterial liquid culture medium to passage the one generation, taking the obtained bacterial solution, transferring to a levofloxacin liquid culture medium to passage the two generations, sequentially carrying out levofloxacin concentration multiplication culture of bacterial before the drug concentration achieves the sensitive and medium critical value, sequentially carrying out levofloxacin concentration increase culture after the drug concentration achieves the sensitive and medium critical value, carrying out induction culture on the strain for 2 days at the concentration when the induced anti-bacterial drug concentration achieves 2, 4 and 8 mug/ml, carrying out induction culture on the well-growing single colony to obtain the induced Shigella drug-resistance spectral pattern, and carrying out mutation analysis on the induced drug-resistance spectral pattern strain.

Description

The foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model

Technical field

The invention belongs to biological technical field, be specifically related to the method for external structure mediated quinolone resistance shigella model, be specifically related to the method that the many groups of quinolones induction shigella resistance model is set up.

Background technology

The appearance of bacterial drug resistance and spread over the challenge of Shi Dui anti-infective therapy to a great extent, take shigella as example, numerous research has shown that persister frequently appears in shigella at present, and be resistance and produce that speed is fast, resistance scope is wide, resistant rate is high, the equal feature of each bacterial type Antibiotic Resistance, quinolone is as the choice drug of current treatment bacillary dysentery, when last century, came into operation the eighties, Shigella bacterium is to its susceptibility up to more than 95%, and the curative effect of bacillary dysentery was once once taken on a new look.But over nearly 40 years, quinolone antibiotic is widely used, and this medicine together with other antibacterials also for animal infected treatment, bacterium especially Chang Gan section bacterium presents higher resistance to quinolones.

Mutant target gene is the important molecule basis of Shigella bacterium to quinolones, by Chromosome-encoded persister mutant target gene, there is polymorphism, the generation of induction strain multi-drug resistant may be to comprise the active efflux system of AcrAB owing to having activated it, the common existence of active efflux mechanism and mutant target gene causes the resistance to promise ketone of Shigella bacterium medicine

Although there is research, by quinolones, induce shigella, but just induce a strain shigella, form single induction system, the sudden change sequencing of the target gene of resistance to quinolone medicine gyrA, the parC presenting can not be got rid of the impact of self random mutation, and can not explain from consistence and statistics probability and can not describe the problem the accuracy of sudden change sequential.

Summary of the invention

The object of the invention is to overcome above deficiency, a kind of foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model is provided.

A foundation for levofloxacin induction shigella drug-tolerant gene mutation temporal model, main method step is as follows:

(1) with agar diffusion method of the paper, shigella is carried out to Kefzol, cefotaxime, Ampicillin Trihydrate, gentamicin, paraxin, tsiklomitsin, trimethoprim-sulfamethoxazole, Nalidixic Acid, pipemidic acid, Ciprofloxacin, norfloxicin and 12 kinds of antibiotic drug sensitive tests of levofloxacin, take all responsive to 12 kinds of antibacterials is sensitive strain, judgement bacterium sensitivity, medium sensitivity or resistance, sensitive strain is streak culture on bacterium solid culture medium flat board, choose well-grown single bacterium colony and do induction experiment;

(2) with agar dilution, measure levofloxacin (Levofloxacin, LEV) to the primary minimum inhibitory concentration MIC of induction strain ₀, LEV sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2 ~ 8,>=8;

(3) from the bacterium solid culture medium flat board of experiment induction strain, the well-grown single bacterium colony of picking at least five pipe levofloxacin concentrations are 1/4 * MIC ₀bacterial liquid substratum in, 32 ℃-39 ℃ concussion culturing bacterium grow to logarithmic phase OD600=0.6-1;

(4) getting a certain amount of bacterium liquid at least five pipe levofloxacin content in step (3) is 1/4 * MIC ₀bacterial liquid substratum in pass again a generation, and then to get a certain amount of bacterium liquid to levofloxacin concentration be 1/2 * MIC ₀liquid nutrient medium in passed for two generations, before drug level reaches responsive and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled increases progressively cultivation by levofloxacin concentration successively after drug level reaches responsive and resistance threshold value, increases progressively 1 μ g/ml at every turn;

(5) when induction antibacterials concentration reaches 2,4,8 μ g/ml, inducing culture is two days later in this concentration for bacterial strain, wouldn't induce and go down to posterity, first doing at least twice goes down to posterity after cultivation without inductor, be inoculated in containing incubated overnight on the bacterium solid culture medium flat board of same concentrations inductor, select well-grown single bacterium colony and continue inducing culture, finally obtain at least five group shigella inducible resistance spectral patterns; The bacterial strain of this inducible resistance spectral pattern is carried out to gene mutation analysis;

(6) in the process going down to posterity in induction, adopt agar dilution to measure levofloxacin to the experiment induction strain MIC in each generation, LEV sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2 ~ 8, >=8;

(7) build the many groups of levofloxacin induction shigella drug-tolerant gene mutation model, with this, carry out inducible resistance transgenation time series analysis.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, described bacterium solid culture medium is LB solid medium, described bacterial liquid substratum is LB liquid nutrient medium.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, in step (3), concussion culture temperature is 37 ℃.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, in step (3), concussion culturing bacterium grows to OD600=0.9.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, in step (4), getting a certain amount of bacterium liquid to N pipe levofloxacin content in step (3) is 1/4 * MIC ₀lB liquid nutrient medium in, making final concentration is 5 * 10 ⁵cFU/ml.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, final 7 groups of shigellas induction was gone down to posterity for 61 generations.

The foundation of above-mentioned levofloxacin induction shigella drug-tolerant gene mutation temporal model, by the analysis of many groups transgenation, find the rule of quinolone medicine mutant target gene sequential, utilize this model to determine quinolone medicine target gene site mutation sequential under LEV inducing action, deduction is shigella quinolone medicine target gene site mutation sequential under extraneous LEV pressure in human body or physical environment, is specially:

(1) method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by LEV:

1) polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification gyrA and parC gene

1. template DNA: boiling method extracts template DNA: get and increase the LB that bacterium arrives logarithmic phase for 6 hours and increase bacterial context soup 1ml in the Eppendorf of 1.5ml pipe, 10000 revs/min centrifugal 1 minute, abandon supernatant, add tri-distilled water 100 μ l, on earthquake device, mix and boil 10 minutes, slightly centrifugal, supernatant liquor is pcr template DNA, and rearmounted-20 ℃ of the DNA label of carrying is saved backup.

2. primer and design of primers

The required primer of amplifying target genes fragment is announced sequence with reference to Fu Shi 2a shigella strain 301 gyrA and parC gene, uses Primer5 software designed, designed:

GyrA gene primer: gyrA full length gene 2628bp, QRDR region is in 166-410 site.GyrA upstream region of gene primer A1 is positioned at gyrA gene 24-43 bit base, downstream primer A2 is positioned at gyrA gene 652-671 position, pcr amplified fragment is positioned at gyrA gene 24-671 position, product 648bp:A1:5 '-TAC ACC GGT CAA CAT TAA GG-3 ', A2:5 '-TTA ATG ATT GCC GCC GTC GG-3 ';

ParC gene primer: parC full length gene 2259bp, QRDR region is in 175-411 site.ParC upstream region of gene primer C1 is positioned at parC gene 88-108 bit base, downstream primer C2 and parC gene 538-556 bit base are complementary, pcr amplified fragment is positioned at parC gene 88-556 site, product 469bp:C1:5 '-GCG TTG CCG TTT ATT GGT GAT-3 ', C2:5 '-GCA GGT TAT GCG GTG GAA T-3 ';

3. pcr amplification scheme

Table 1 gyrA and parC gene PCR amplification system (50 μ l)

Mix slightly centrifugal after, put in temperature cycler, after 94 ℃ of denaturation 5min, circulation; After 94 ℃ of sex change 60s, gyrA, parC be respectively at 56 ℃ and 61 ℃ annealing 45s, then prolong 30s in 72 ℃, carries out altogether 35 circulations, and 72 ℃ are extended 600s.The laggard performing PCR product of loop ends sepharose detects.

4. amplified production detects: (PCR amplified production fragment different concns is different for preparation 1.5% sepharose, fragment is less, concentration is higher), get 5 μ l PCR products and mix with 1 μ l bromjophenol blue load sample damping fluid, application of sample is in sepharose point sample hole, and voltage 5V/cm carries out electrophoresis, electrophoresis liquid is 1 * TBE, stop after electrophoresis, get glue observations photograph in reading glue instrument, standard is 100bp DNA Ladder.

2) the Restriction fragment length polymorphism of gyrA gene (Restriction Fragment Length Polymorphism, RFLP) is analyzed:

1. digestion with restriction enzyme

Nucleic acid fragment is less, and the susceptibility of detection is higher.For the PCR product that is greater than 400bp, should with restriction enzyme, carry out digestion process in advance.Two recognition sites (244thbp and 343thbp) of restricted property restriction endonuclease hinf I in gyrA gene amplification region, this section of PCR product can be digested to three bar segment (221bp, 99bp and 328bp): the enzyme system of cutting is 50 μ l, PCR product: 40 μ l, restriction enzyme damping fluid: 4 μ l, restriction enzyme hinf I 6 μ l, mix and slightly centrifugal, be placed in 2h in 37 ℃ of water-baths; After endonuclease reaction, use sepharose horizontal strip electrophoresis to detect enzyme and cut effect;

2. Cleavage Map: preparation 2% sepharose, to get the above-mentioned PCR product of 7 μ l and mix with 1 μ l load sample damping fluid, in application of sample point sample hole, voltage 5V/cm carries out electrophoresis, and electrophoresis liquid is 1 * TBE.And reading to observe restriction enzyme mapping on glue instrument, take a picture.Numerous research shows, gyrA the 244th bit base sudden change makes Hinf I in this place recognition site forfeiture, thereby only produce two enzymes, cuts product fragment: 320bp and 328bp;

3) single strand conformation polymorphism of parC gene (Single Strand Conformation Polymorphism, SSCP) is analyzed

1. digestion with restriction enzyme: need to process with digestion with restriction enzyme PCR product for the PCR product that is greater than 400bp, produce less DNA fragmentation, then carry out single stranded conformational analysis.The recognition site (279thbp) of parC gene amplification region restricted property restriction endonuclease hinf I, can be digested to this section of PCR product two bar segment (191bp and 278bp), and digestion method is the same;

2. polyacrylamide gel electrophoresis: get 3 μ l enzymes and cut sample and mix with 22 μ l sex change sample solutions, carry out 8% polyacrylamide gel electrophoresis, glue thickness 1.0 mm, long 82mm, voltage 8V/cm, constant voltage electrophoresis 4h.Molecular weight standard 100bp DNA Ladder;

3. silver dyes: after electrophoresis finishes, two sheet glass separately, put into 100 ml silver by gel film and dye stationary liquid and shake 15min, and tri-distilled water is washed 3 times, and then each 2min proceeds to gel film the AgNO of 100ml 0.2 % ₃liquid, washes 3 times with tri-distilled water, and each 30s, adds the about 7-10min of 100ml nitrite ion, when DNA band shows removing, sucks nitrite ion, adds stationary liquid Na ₂cO ₃, standing 2-3min, takes out gel observation.Be placed in observations photograph on x ray view box;

(2) adopt the method for gene sequencing to detect the gene locus suddenling change, find the sequential rule of quinolone medicine target gene gyrA, parC sudden change under LEV induction;

1) mensuration of nucleotide sequence: the primary gyrA of laboratory-induced strain shigella, parC gene PCR amplified production are checked order, separately select part inducible strain, gyrA, parC gene PCR amplified production are checked order.Entrust Beijing six directions Hua Da Gene science company to carry out;

2) compare of analysis of nucleotide sequence: by BLST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared in NCBI.

Adopt technique scheme, the present invention has the following advantages: it is drug-induced until resistance that the present invention uses the same parallel N group of strain shigella sensitive strain (N >=5) to carry out first.The present invention is first by parallel N (N >=5) induction system, the rule of observing target gene gyrA, parC sudden change set up of same strain shigella.N strain sensitive strain has not only formed high density resistance for quinolone to each after single-minded quinolone medicine induction, provide the target gene of resistance to quinolone medicine gyrA, parC sudden change sequential conforming evidence, can describe the problem from consistence and the probability of sudden change sequential, can get rid of self random mutation.In addition N induction system also form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do, in prompting Induction Process, produce crossing drug resistant.It is applied as the rule of finding quinolone medicine mutant target gene sequential, utilize this model to determine quinolone medicine target gene site mutation sequential under LEV inducing action, infer shigella quinolone medicine target gene site mutation sequential under extraneous LEV pressure in human body or physical environment.

Figure of description

Fig. 1 is embodiment 1gyrAPCR amplified fragments (648bp) electrophorogram;

Lanes-strain：M-100bp DNA Ladder 1- S51573 2- Y2-15 3-Y2-42 4-Y5-15 5-Y5-42 6-Y7-15 6-Y7-42；

Fig. 2 is embodiment 1 parC pcr amplification product electrophoresis result (469bp) figure;

Lanes-strain：M-100bp DNA Ladder 1- S51573 2- Y2-15

3-Y2-42 4-Y5-15 5-Y5-42 6-Y7-15 7-Y7-42；

Fig. 3 is the gyrAPCR amplified fragments hinf I restriction enzyme mapping of embodiment 1.

Lanes-strain：M-100bp DNA Ladder 1- Y2-28 2- Y2-42

3-Y2-48 4-Y5-32 5-Y5-40 6-Y7-41 7-Y7-53 8-S51573；

8 is not mutated strain, produces three enzymes and cuts product (99bp, 221bp, 328bp) (221bp and 328bp are overlapping); All the other are mutant strain, produce two enzymes and cut product (320bp, 328bp);

Fig. 4 is the parCPCR amplified fragments hinf I restriction enzyme mapping of embodiment 1;

After cutting, there is 191bp and 278bp fragment Lanes-strain:M-100bp DNA Ladder 1-Y2-28 2-Y2-42 3-Y2-48 4-Y5-32 5-Y5-40 6-Y7-41 7-Y7-53 8-Y1-43 in parC PCR product hinf I enzyme;

Fig. 5 is that the parC PCR-RFLP-SSCP of embodiment 1 analyzes: parC PCR-RFLP-SSCP analyzes and shows many strand bands; 1 is not mutated strain; 2,3,4,5,6,7,8 is mutant strain, its strand band position different from remaining person (gap is greater than 3 millimeters).Lanes-strain：M-MarkerⅠ1-Y2-3 2-Y2-39 3-Y2-43 4-Y3-59 5-Y5-60 6-Y7-51 7-Y7-60 8-Y3-54；

Fig. 6 is Y2-42gyrA and standard nucleic acid sequence alignment figure in BLAST in embodiment 1;

Fig. 7 is Y2-61parC and standard nucleic acid sequence alignment figure in BLAST in embodiment 1;

Fig. 8 is Y2-43 gyrA83 in embodiment 1,87 change figure, note: Serine (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC);

Fig. 9 is Y5-40 gyrA87 position change figure in embodiment 1, note: aspartic acid (GAC)-87 → tyrosine (TAC);

Figure 10 is that in embodiment 1, Y5-59 gyrA177 position changes, note: glycine (GGT)-177 → aspartic acid (GAT);

Figure 11 is 80,84 changes of Y2-43 parC in embodiment 1, note: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC);

Figure 12 is 183 changes of Y2-61 parC in embodiment 1, note: Histidine (CAT)-183 → aspartic acid (AAT).

Embodiment

(1) bacterium source

(1) shigella of the present invention adopts shigella sonnei, shigella sonnei name is called Shigella sonnei, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its deposit number is: CGMCCNo:7632, preservation date is: on May 20th, 2013, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica's postcode 100101.

(2) drug sensitive test Quality Control bacterial strain intestinal bacteria ATCC 25922 is purchased from Clinical Laboratory center, Henan Province, and S51573 is from purchased from Chinese drug inspection office.

(2) substratum using in the present invention's experiment, microbiotic preparation and main agents

(1) LB substratum preparation: LB liquid nutrient medium: take 1.0 g Tryptoness, 0.5 g yeast soaks powder, 1.0 g sodium-chlor, are dissolved in 100 ml distilled water, and with 10 mol/L NaOH adjust pHs to 7.2,121 ℃ of autoclaving 20 min, put 4 ℃ and save backup.

LB solid medium: take 1.0 g Tryptoness, 0.5 g yeast soaks powder, 1.0 g sodium-chlor, 1.5 g agar powders, are dissolved in 100 ml distilled water, with 10 mol/L NaOH adjust pHs to 7.2,121 ℃ of autoclaving 20 min, while being cooled to approximately 50 ℃, pour plate is standby.

(2) M-H solid medium preparation: take 4.2g M-H agar heating for dissolving in 100ml distilled water, 120 ℃ of autoclaving 20min, to be cooled to 50 ℃, standby in the aseptic plate of impouring.

(3) levofloxacin microbiotic stoste (2560 μ g/ml, 100ml), microbiotic pulvis quality is calculated as follows: quality (mg)=solvent (ml) * concentration (μ g/ml)/analysis usefulness (μ g/mg)=100ml * 2560 μ g/ml/9730/mg=26.31mg.Adopt ten thousand/analytical balance accurately to take 26.31mg medicine powder, be dissolved in 1/2 volume and go out in tri-distilled water and 0.1mol/L NaOH solution, with 0.22 μ m membrane filtration degerming, rearmounted-80 ℃ of preservations of packing, used up in 6 months.

(4) main agents: 12 kinds of microbiotic such as Kefzol (CFZ), cefotaxime (CTX), Ampicillin Trihydrate (AMP), gentamicin (GM), paraxin (C), tsiklomitsin (TE), trimethoprim-sulfamethoxazole (SMZ/TMP), Nalidixic Acid (NAL), pipemidic acid (PI), Ciprofloxacin (CIP), norfloxicin (NOR) and levofloxacin (LEV) all originate from Beijing the Temple of Heaven Pharmaceutical Biotechnology development company.Induced drug levofloxacin is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.PCR related reagent: comprise that PCR Mix and DNA Marker 100bp Ladder are all purchased from Lai Feng bio tech ltd, Shanghai.Substratum related reagent: MH agar is purchased from Qingdao high-tech park hypo Bioisystech Co., Ltd, and Tryptones and yeast soak powder purchased from Britain Oxoid company.Other common agents are domestic analytical pure level reagent.The experiment material and the method that are not specifically noted belong to known technology, at biological technical field common tool book, for example, molecular cloning experiment guide (J. Pehanorm Brooker and D.W Russell work, the third edition, Science Press, 2002), in can find.

Embodiment 1: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: 1. pair evaluation, the shigella sonnei of preservation carries out Kefzol, cefotaxime, Ampicillin Trihydrate, gentamicin, paraxin, tsiklomitsin, trimethoprim-sulfamethoxazole, Nalidixic Acid, pipemidic acid, Ciprofloxacin, 12 kinds of antibiotic drug sensitive tests of norfloxicin and levofloxacin, with Quality Control bacterial strain in contrast, with ruler measurement, comprise drug sensitive test paper inhibition zone diameter, using be invisible to the naked eye bacterium obviously growth be limited as the edge of antibacterial ring, adopt (the National Committeefor Clinical Laboratory Standards of standard committee of the state-run clinical labororatory of the U.S., NCCLS) guide of formulating, judgement bacterium is responsive, medium sensitivity or resistance.By the shigella sonnei of evaluation, preservation being carried out to above-mentioned drug sensitive test, verify that it is all responsive to 12 kinds of antibacterials, using this bacterial strain as experiment sensitive strain, sensitive strain is streak culture on LB flat board, and single bacterium colony that picking growth conditions is good is done induction experiment; 2. with agar dilution, measure levofloxacin to the primary MIC of experiment induction strain ₀its Plays is controlled bacterium and is adopted escherichia coli ATCC 25922, < < drug sensitive test experimental standard > > standard interpretation with reference to National Committee of Clinical Laboratory Standards (NCCLS) in 1999 publication: levofloxacin (Levofloxacin, LEV) sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2～8,>=8, record MIC ₀=0.125 μ g/ml; 3.LEV is dissolved in 0.lmol/L NaOH solution, with sterile purified water, is diluted to and needs after 10 times of concentration, and-20 ℃ of of short duration preservations, were finished in one month, before using, with LB meat soup, is diluted to the induced concentration needing; 4. from the LB flat board of experiment induction strain, well-grown single bacterium colony to the 7 pipe levofloxacin concentration of picking is in the LB liquid nutrient medium of 0.03125 μ g/ml, and 37 ℃ of concussion culturing bacterium grow to mid-log phase OD600=0.9; 5. get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 7 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 μ g/ml and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn; 6. when induction antibacterials concentration reaches 2,4,8 μ g/ml, inducing culture is two days later in this concentration for bacterial strain, wouldn't induce and go down to posterity, first doing 4 times goes down to posterity after cultivation without inductor, be inoculated in the LB solid medium incubated overnight containing same concentrations inductor, select well-grown single bacterium colony and continue inducing culture, final 7 groups of shigellas induction was gone down to posterity for 61 generations;

7. with agar dilution, measure levofloxacin to the experiment induction strain MIC in each generation, its Plays is controlled bacterium and is adopted escherichia coli ATCC 25922, the < < drug sensitive test experimental standard > > standard interpretation of reference National Committee of Clinical Laboratory Standards (NCCLS) in 1999 publication: LEV sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2～8, >=8;

8. the method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC;

1) polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification gyrA and parC gene: 1. template DNA: boiling method extracts template DNA: get and increase the LB that bacterium arrives logarithmic phase for 6 hours and increase bacterial context soup 1ml in the Eppendorf of 1.5ml pipe, 10000 revs/min centrifugal 1 minute, abandon supernatant, add tri-distilled water 100 μ l, on earthquake device, mix and boil 10 minutes, slightly centrifugal, supernatant liquor is pcr template DNA, and rearmounted-20 ℃ of the DNA label of carrying is saved backup.2. primer and design of primers: the required primer of amplifying target genes fragment is announced sequence with reference to Fu Shi 2a shigella strain 301 gyrA and parC gene, use Primer5 software designed, designed: gyrA gene primer: gyrA full length gene 2628bp, QRDR region is in 166-410 site.GyrA upstream region of gene primer A1 is positioned at gyrA gene 24-43 bit base, downstream primer A2 is positioned at gyrA gene 652-671 position, pcr amplified fragment is positioned at gyrA gene 24-671 position, product 648bp:A1:5 '-TAC ACC GGT CAA CAT TAA GG-3 ', A2:5 '-TTA ATG ATT GCC GCC GTC GG-3 ', parC gene primer: parC full length gene 2259bp, QRDR region is in 175-411 site.ParC upstream region of gene primer C1 is positioned at parC gene 88-108 bit base, downstream primer C2 and parC gene 538-556 bit base are complementary, pcr amplified fragment is positioned at parC gene 88-556 site, product 469bp:C1:5 '-GCG TTG CCG TTT ATT GGT GAT-3 ', C2:5 '-GCA GGT TAT GCG GTG GAA T-3 '.3. pcr amplification scheme: table 1 gyrA and parC gene PCR amplification system (50 μ l)

Mix slightly centrifugal after, put in temperature cycler, after 94 ℃ of denaturation 5min, circulation; After 94 ℃ of sex change 60s, gyrA, parC be respectively at 56 ℃ and 61 ℃ annealing 45s, then prolong 30s in 72 ℃, carries out altogether 35 circulations, and 72 ℃ are extended 600s.The laggard performing PCR product of loop ends sepharose detects.4. amplified production detects: (PCR amplified production fragment different concns is different for preparation 1.5% sepharose, fragment is less, concentration is higher), get 5 μ l PCR products and mix with 1 μ l bromjophenol blue load sample damping fluid, application of sample is in sepharose point sample hole, and voltage 5V/cm carries out electrophoresis, electrophoresis liquid is 1 * TBE, stop after electrophoresis, get glue observations photograph in reading glue instrument, standard is 100bp DNA Ladder.

2) the Restriction fragment length polymorphism of gyrA gene (Restriction Fragment Length Polymorphism, RFLP) is analyzed: 1. digestion with restriction enzyme: nucleic acid fragment is less, and the susceptibility of detection is higher.For the PCR product that is greater than 400bp, should with restriction enzyme, carry out digestion process in advance.Two recognition sites (244thbp and 343thbp) of restricted property restriction endonuclease hinf I in gyrA gene amplification region, this section of PCR product can be digested to three bar segment (221bp, 99bp and 328bp): the enzyme system of cutting is 50 μ l, PCR product 40 μ l, restriction enzyme damping fluid 4 μ l, restriction enzyme hinf I 6 μ l, mix and slightly centrifugal, be placed in 2h in 37 ℃ of water-baths.After endonuclease reaction, use sepharose horizontal strip electrophoresis to detect enzyme and cut effect.2. Cleavage Map: preparation 2% sepharose, to get the above-mentioned PCR product of 7 μ l and mix with 1 μ l load sample damping fluid, in application of sample point sample hole, voltage 5V/cm carries out electrophoresis, and electrophoresis liquid is 1 * TBE.And reading to observe restriction enzyme mapping on glue instrument, take a picture.Numerous research shows, gyrA the 244th bit base sudden change makes Hinf I in this place recognition site forfeiture, thereby only produce two enzymes, cuts product fragment: 320bp and 328bp.

3) single strand conformation polymorphism of parC gene (Single Strand Conformation Polymorphism, SSCP) analyze: 1. digestion with restriction enzyme: for the PCR product that is greater than 400bp, need to process with digestion with restriction enzyme PCR product, produce less DNA fragmentation, then carry out single stranded conformational analysis.The recognition site (279thbp) of parC gene amplification region restricted property restriction endonuclease hinf I, can be digested to this section of PCR product two bar segment (191bp and 278bp), and digestion method is the same.2. polyacrylamide gel electrophoresis: get 3 μ l enzymes and cut sample and mix with 22 μ l sex change sample solutions, carry out 8% polyacrylamide gel electrophoresis, glue thickness 1.0 mm, long 82mm, voltage 8V/cm, constant voltage electrophoresis 4h.Molecular weight standard 100bp DNA Ladder.3. silver dyes: after electrophoresis finishes, two sheet glass separately, put into 100 ml silver by gel film and dye stationary liquid and shake 15min, and tri-distilled water is washed 3 times, and then each 2min proceeds to gel film the AgNO of 100ml 0.2 % ₃liquid, washes 3 times with tri-distilled water, and each 30s, adds the about 7-10min of 100ml nitrite ion, when DNA band shows removing, sucks nitrite ion, adds stationary liquid Na ₂cO ₃, standing 2-3min, takes out gel observation.Be placed in observations photograph on x ray view box.

(8) adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

1) mensuration of nucleotide sequence: the primary gyrA of laboratory-induced strain, parC gene PCR amplified production are checked order, separately select part inducible strain, gyrA, parC gene PCR amplified production are checked order.Entrust Beijing six directions Hua Da Gene science company to carry out.2) compare of analysis of nucleotide sequence: by blast program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared in NCBI.

Experimental result: experiment induction strain parallel induction 7 times, is labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7.Each induction was gone down to posterity for 61 generations, levofloxacin final concentration 18 μ g/ml, levofloxacin to each group induction strain wherein number generation and final MIC value in Table 2, increase along with inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Table 2 levofloxacin is MIC value for bacterial strain to each

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.7 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do, the results are shown in Table 3.

Table 3 inducible resistance bacterial strain is to 10 kinds of antibiotic MIC

Note: CFZ Kefzol, CTX cefotaxime, AMP amoxycilline Trihydrate bp, GM gentamicin, C paraxin, TE tsiklomitsin, SMZ trimethoprim-sulfamethoxazole, NOR norfloxicin, CIP Ciprofloxacin, LEV levofloxacin; Various kinds of drug upper right side is labeled as the resistance MIC dividing value to bacterium separately, 1 >=32 μ g/ml wherein, 2 >=64 μ g/ml, 3 >=32 μ g/ml, 4 >=8 μ g/ml, 5 >=32 μ g/ml, 6 >=16 μ g/ml, 7 >=32 μ g/ml, 8 >=8 μ g/ml, 9 >=4 μ g/ml, 10 >=8 μ g/m

Y1-Y7 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production, the results are shown in Figure 1,2.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and the endonuclease bamhi that produces two 300bp left and right is shown in Fig. 3.8 is not mutated strain, produces three enzymes and cuts product (99bp, 221bp, 328bp) (221bp and 328bp are overlapping); All the other are mutant strain, produce two enzymes and cut product (320bp, 328bp).ParC PCR-RFLP analyzes, and sees Fig. 4, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes, and sees Fig. 5, occurs different single stranded conformationals in persister.

The gyrA of experiment induction strain experiment induction parallel 7 strains of strain (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared, comparison result is as Fig. 6, and 7.Fig. 6 is Y2-42gyrA and standard nucleic acid sequence alignment, and replacing appears in the 83rd, 87 bit bases, Serine (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC); Fig. 7 is Y2-61parC and standard nucleic acid sequence alignment, 80th, replacing appears in 84,183 bit bases, Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Comparison situation in the drawings vacancy part can be shown.

Each induction system is all induced 61 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 7 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y2 induction system of take describes change process in detail as example: the 28th generation when levofloxacin final concentration be 5 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 28th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 33rd generation, when levofloxacin final concentration is 8 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=16 μ g/ml of levofloxacin to induction strain.In the 43rd generation, when levofloxacin final concentration is 11 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=32 μ g/ml of levofloxacin to induction strain.In the 53rd generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 61st generation, working as levofloxacin final concentration is 18 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Fig. 8-12 all, in gyrA and parC QRDR region, are seen in above mutational site.In induction target gene gyrA, parC, amino acid sites change detection case gathers in Table 4.

Table 4 each MIC value and target position detected result for inducible strain LEV

Note: * is inductor concentration (μ g/ml); Blank for not detecting; "-" is detect but do not change

Each MIC value for inducible strain LEV of table 4 and target position detected result (continuous upper table)

Note: * is inductor concentration (μ g/ml); Blank for not detecting; "-" is detect but do not change.

To sum up, 7 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 2: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 32 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=1.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 5 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 2 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 5 groups of shigellas induction was gone down to posterity for 60 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 5 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5.Each induction was gone down to posterity for 60 generations, levofloxacin final concentration 17 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.5 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.Y1-Y5 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 5 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 60 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 5 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y3 induction system of take describes change process in detail as example: the 30th generation when levofloxacin final concentration be 6 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 30th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 34th generation, when levofloxacin final concentration is 8 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=16 μ g/ml of levofloxacin to induction strain.In the 44th generation, when levofloxacin final concentration is 11 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=32 μ g/ml of levofloxacin to induction strain.In the 54th generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 60th generation, working as levofloxacin final concentration is 17 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 5 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

The foundation of embodiment 3 levofloxacin induction shigella drug-tolerant gene mutation temporal models: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 39 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.6.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 10 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 3 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 10 groups of shigellas induction was gone down to posterity for 59 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 10 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10.Each induction was gone down to posterity for 59 generations, levofloxacin final concentration 17 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.10 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y10 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 10 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 59 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 10 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y5 induction system of take describes change process in detail as example: the 29th generation when levofloxacin final concentration be 6 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 29th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 32nd generation, when levofloxacin final concentration is 7 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=8 μ g/ml of levofloxacin to induction strain.In the 41st generation, when levofloxacin final concentration is 10 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=16 μ g/ml of levofloxacin to induction strain.In the 52nd generation, working as levofloxacin final concentration is 15 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 59th generation, working as levofloxacin final concentration is 17 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 10 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 4: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 33 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.7.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 15 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 5 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 15 groups of shigellas induction was gone down to posterity for 62 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 15 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15.Each induction was gone down to posterity for 62 generations, levofloxacin final concentration 18 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.15 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y15 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction parallel 15 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 62 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 15 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y10 induction system of take describes change process in detail as example: the 29th generation when levofloxacin final concentration be 6 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 29th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 34th generation, when levofloxacin final concentration is 8 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=8 μ g/ml of levofloxacin to induction strain.In the 39th generation, when levofloxacin final concentration is 9 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=16 μ g/ml of levofloxacin to induction strain.In the 53rd generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 62nd generation, working as levofloxacin final concentration is 18 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 15 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 5: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 34 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.7.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 20 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 6 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 20 groups of shigellas induction was gone down to posterity for 63 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 20 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20.Each induction was gone down to posterity for 63 generations, levofloxacin final concentration 19 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.20 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y20 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 20 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 63 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 20 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y15 induction system of take describes change process in detail as example: the 33rd generation when levofloxacin final concentration be 8 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 33rd generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 43rd generation, when levofloxacin final concentration is 11 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=32 μ g/ml of levofloxacin to induction strain.In the 54th generation, when levofloxacin final concentration is 16 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=128 μ g/ml of levofloxacin to induction strain.In the 59th generation, working as levofloxacin final concentration is 17 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 61st generation, working as levofloxacin final concentration is 18 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 20 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 6: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 35 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.8.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 25 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 7 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 25 groups of shigellas induction was gone down to posterity for 64 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 25 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25.Each induction was gone down to posterity for 64 generations, levofloxacin final concentration 19 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.25 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y25 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 25 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 64 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 25 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y20 induction system of take describes change process in detail as example: the 28th generation when levofloxacin final concentration be 5 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=4 μ g/ml of levofloxacin to induction strain.In the 28th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 40th generation, when levofloxacin final concentration is 9 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=16 μ g/ml of levofloxacin to induction strain.In the 48th generation, when levofloxacin final concentration is 13 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=64 μ g/ml of levofloxacin to induction strain.In the 54th generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 64th generation, working as levofloxacin final concentration is 19 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 25 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 7: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 36 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.8.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 30 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 8 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 30 groups of shigellas induction was gone down to posterity for 64 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 30 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25, Y26, Y27, Y28, Y29, Y30.Each induction was gone down to posterity for 65 generations, levofloxacin final concentration 20 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.30 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y30 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 30 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25, Y26, Y27, Y28, Y29, Y30), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 65 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 30 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y25 induction system of take describes change process in detail as example: the 30th generation when levofloxacin final concentration be 6 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=4 μ g/ml of levofloxacin to induction strain.In the 30th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 41st generation, when levofloxacin final concentration is 10 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=16 μ g/ml of levofloxacin to induction strain.In the 52nd generation, when levofloxacin final concentration is 15 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=64 μ g/ml of levofloxacin to induction strain.In the 54th generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 62nd generation, working as levofloxacin final concentration is 18 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 30 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Embodiment 8: the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model: method steps is with embodiment 1, wherein bacterium solid culture medium adopts LB dull and stereotyped, bacterial liquid substratum adopts LB liquid culture, and in step (4), 38 ℃ of concussion culturing bacterium grow to logarithmic phase OD600=0.9.Get in the LB liquid nutrient medium that in step (4), a certain amount of bacterium liquid to 35 pipe levofloxacin content is 0.03125 μ g/ml and pass again a generation, and then get in the LB liquid nutrient medium that a certain amount of bacterium liquid to levofloxacin concentration is 0.0625 and passed for two generations, before drug level reaches levofloxacin sensitivity and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled, after drug level reaches levofloxacin sensitivity and intermediary's threshold value, successively levofloxacin concentration is increased progressively to cultivation, increase progressively 1 μ g/ml at every turn.When induction antibacterials concentration reaches 2,4,8 μ g/ml, bacterial strain inducing culture in this concentration two days later, wouldn't be induced and go down to posterity, and first does 9 times and goes down to posterity after cultivation without inductor, is inoculated in the LB solid plate incubated overnight containing same concentrations inductor.Select well-grown single bacterium colony and continue inducing culture, final 35 groups of shigellas induction was gone down to posterity for 66 generations.The method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by MIC.Adopt the method for gene sequencing to detect the gene locus suddenling change, find the rule of quinolone medicine target gene gyrA, parC sudden change.

Experimental result: 35 groups of experiment induction strain parallel inductions, are labeled as respectively Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25, Y26, Y27, Y28, Y29, Y30, Y31, Y32, Y33, Y34, Y35.Each induction was gone down to posterity for 66 generations, levofloxacin final concentration 20 μ g/ml, along with the increase of inductor levofloxacin concentration, levofloxacin can, by the highest 128 μ g/ml～256 μ g/ml that rise to of 0.125 μ g/ml, be original 1024～2048 times to the MIC of inducible strain.

Use levofloxacin time Mlc as initial concentration, successfully to induce and build mediated quinolone resistance shigella model.35 strain sensitive strains have not only formed high density resistance for quinolone to each after the induction of single-minded quinolone medicine, and form to quinolones structure and mechanism of action on the multidrug resistant of other a few class antimicrobial drugs of all haveing nothing to do.

Y1-Y35 through levofloxacin induction, obtain each for bacterial strain, all amplify respectively the fragment of 648bp and 469bp length, be respectively gyrA gene, parC gene PCR amplified production.GyrA PCR-RFLP analyzes demonstration, and quinolones is not occurred to the strain of going down to posterity of resistance all occurs after enzyme is cut that 221bp, 99bp and tri-enzymes of 328bp cut product fragment; The restriction enzyme mapping that quinolones is occurred to the strain of going down to posterity of resistance changes, and produces the endonuclease bamhi of two 300bp left and right.ParC PCR-RFLP analyzes, and consistent single stranded conformational appears in quinolone sensitive strain.PCR-SSCP analyzes in persister and occurs different single stranded conformationals.

The gyrA of experiment induction strain parallel 35 groups (numbering is followed successively by Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25, Y26, Y27, Y28, Y29, Y30, Y30, Y31, Y32, Y33, Y34, Y35), the order-checking of parC PCR amplified production, base sequence is compared all identical with S51573.The result of analyzing for bacterial strain PCR-RFLP-SSCP according to each, selecting bacterial strain checks order, gyrA, parC pcr amplification product are analyzed, in NCBI, by BLAST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared.Each induction system is all induced 66 generations of going down to posterity, and each generation induction strain all amplifies gyrA and parC gene.The Induction Process of 35 induction systems is along with the increase of inductor concentration, suddenly change on target position mutational site quantity and relate on target position enzyme number and all show consistence of system separately, therefore the Y30 induction system of take describes change process in detail as example: the 31st generation when levofloxacin final concentration be 7 μ g/ml, first gyrA the 83rd amino acids changes, the MIC=8 μ g/ml of levofloxacin to induction strain.In the 30th generation, induced strain sequencing result and former generation sequence to compare, and gyrA gene generation Serine (TCG)-83 → leucine (TTG) changes; ParC carries out PCR-RFLP-SSCP analysis, and conformation does not change, and changing does not appear in the order-checking of parC sequence.In the 40th generation, when levofloxacin final concentration is 9 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC gene PCR-RFLP-SSCP analyzes, and conformation changes, and the point mutation of parC gene base is Serine (AGC)-80 → arginine (AGA), the MIC=16 μ g/ml of levofloxacin to induction strain.In the 49th generation, when levofloxacin final concentration is 13 μ g/ml, occur gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC) changes; ParC PCR-RFLP-SSCP conformation does not change, but parC gene sequencing is found following base point mutation: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).The MIC=64 μ g/ml of levofloxacin to induction strain.In the 54th generation, working as levofloxacin final concentration is 16 μ g/ml, the MIC=128 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT); The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC).In the 63rd generation, working as levofloxacin final concentration is 19 μ g/ml, the MIC=256 μ g/ml of levofloxacin to induction strain.There is gyrA genonema propylhomoserin (TCG)-83 → leucine (TTG), aspartic acid (GAC)-87 → glycine (GGC), glycine (GGT)-177 → aspartic acid (GAT) changes; The following base point mutation of parC gene: Serine (AGC)-80 → arginine (AGA), L-glutamic acid (GAA)-84 → aspartic acid (GAC), l-asparagine (CAT)-183 → aspartic acid (AAT).Above mutational site is all in gyrA and parC QRDR region.

To sum up, 35 laboratory-induced systems all show that levofloxacin Induction Process first appears as gyrA point mutation, along with induced liquid concentration increases, occur parC point mutation afterwards, and the sequential in mutational site is: gyrA-83, gyrA-87, parC-80, parC-84, gyrA-177, parC-183 position, along with point mutation or many target position enzyme mutant, drug-resistant intensity improves constantly.

Claims (7)

1. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model, is characterized in that, main method step is as follows:

With agar diffusion method of the paper, shigella is carried out to Kefzol, cefotaxime, Ampicillin Trihydrate, gentamicin, paraxin, tsiklomitsin, trimethoprim-sulfamethoxazole, Nalidixic Acid, pipemidic acid, Ciprofloxacin, norfloxicin and 12 kinds of antibiotic drug sensitive tests of levofloxacin, take all responsive to 12 kinds of antibacterials is sensitive strain, judgement bacterium sensitivity, medium sensitivity or resistance, sensitive strain is streak culture on bacterium solid culture medium flat board, choose well-grown single bacterium colony and do induction experiment;

With agar dilution, measure levofloxacin to the primary minimum inhibitory concentration MIC of induction strain ₀, levofloxacin sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2 ~ 8,>=8;

From the bacterium solid culture medium flat board of experiment induction strain, the well-grown single bacterium colony of picking at least five pipe levofloxacin concentrations are 1/4 * MIC ₀bacterial liquid substratum in, 32 ℃ ~ 39 ℃ concussion culturing bacterium grow to logarithmic phase OD600=0.6 ~ 1;

Getting a certain amount of bacterium liquid at least five pipe levofloxacin content in step (3) is 1/4 * MIC ₀bacterial liquid substratum in pass again a generation, and then to get a certain amount of bacterium liquid to levofloxacin concentration be 1/2 * MIC ₀liquid nutrient medium in passed for two generations, before drug level reaches responsive and intermediary's threshold value, the culturing bacterium of successively levofloxacin concentration being doubled increases progressively cultivation by levofloxacin concentration successively after drug level reaches responsive and resistance threshold value, increases progressively 1 μ g/ml at every turn;

When induction antibacterials concentration reaches 2,4,8 μ g/ml, inducing culture is two days later in this concentration for bacterial strain, wouldn't induce and go down to posterity, first doing at least twice goes down to posterity after cultivation without inductor, be inoculated in containing incubated overnight on the bacterium solid culture medium flat board of same concentrations inductor, select well-grown single bacterium colony and continue inducing culture, finally obtain at least five group shigella inducible resistance spectral patterns, the bacterial strain of this inducible resistance spectral pattern is carried out to gene mutation analysis;

In the process going down to posterity in induction, adopt agar dilution to measure levofloxacin to the experiment induction strain MIC in each generation, levofloxacin sensitivity, intermediary, resistance standard are followed successively by MIC(μ g/ml)≤2,2 ~ 8, >=8;

Build the many groups of levofloxacin induction shigella drug-tolerant gene mutation model, with this, carry out inducible resistance transgenation time series analysis.

2. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, is characterised in that: described bacterium solid culture medium is LB solid medium, and described bacterial liquid substratum is LB liquid nutrient medium.

3. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, is characterised in that: in step (3), concussion culture temperature is 37 ℃.

4. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, is characterised in that: in step (3), concussion culturing bacterium grows to OD600=0.9.

5. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, is characterised in that: in step (4), getting a certain amount of bacterium liquid to N pipe levofloxacin content in step (3) is 1/4 * MIC ₀lB liquid nutrient medium in, making final concentration is 5 * 10 ⁵cFU/ml.

6. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, is characterised in that: final 7 groups of shigellas induction was gone down to posterity for 61 generations.

7. the foundation of levofloxacin induction shigella drug-tolerant gene mutation temporal model as claimed in claim 1, it is characterized in that: by the analysis of many groups transgenation, find the rule of quinolone medicine mutant target gene sequential, utilize this model to determine quinolone medicine target gene site mutation sequential under LEV inducing action, deduction is shigella quinolone medicine target gene site mutation sequential under extraneous LEV pressure in human body or physical environment, is specially:

(1) method that adopts gyrA gene PCR-rflp analysis and parC gene PCR-RFLP-SSCP to analyze detects the induction strain filtering out by LEV;

1) polymerase chain reaction (PCR) amplification gyrA and parC gene:

1. template DNA: boiling method extracts template DNA: get and increase the LB that bacterium arrives logarithmic phase for 6 hours and increase bacterial context soup 1ml in the Eppendorf of 1.5ml pipe, 10000 revs/min centrifugal 1 minute, abandon supernatant, add tri-distilled water 100 μ l, on earthquake device, mix and boil 10 minutes, slightly centrifugal, supernatant liquor is pcr template DNA, and rearmounted-20 ℃ of the DNA label of carrying is saved backup;

2. primer and design of primers: the required primer of amplifying target genes fragment is announced sequence with reference to Fu Shi 2a shigella strain 301gyrA and parC gene, use Primer5 software designed, designed:

GyrA gene primer: gyrA full length gene 2628bp, Quinolone resistance determining region (QRDR) is in 166-410 site; GyrA upstream region of gene primer A1 is positioned at gyrA gene 24-43 position, and downstream primer A2 is positioned at gyrA gene 652-671 position, and pcr amplified fragment is positioned at gyrA gene 24-671 position, product 648bp:

A1：5’-TAC ACC GGT CAA CAT TAA GG-3’，

A2：5’-TTA ATG ATT GCC GCC GTC GG-3’，

ParC gene primer: parC full length gene 2259bp, QRDR region is in 175-411 site; ParC upstream region of gene primer C1 is positioned at parC gene 88-108 bit base, and downstream primer C2 and parC gene 538-556 bit base are complementary, and pcr amplified fragment is positioned at parC gene 88-556 site, product 469bp:

C1：5’-GCG TTG CCG TTT ATT GGT GAT-3’，

C2：5’-GCA GGT TAT GCG GTG GAA T-3’，

3. pcr amplification scheme

Table 1 gyrA and parC gene PCR amplification system (50 μ l)

Mix slightly centrifugal after, put in temperature cycler, after 94 ℃ of denaturation 5min, circulation; After 94 ℃ of sex change 60s, gyrA, parC be respectively at 56 ℃ and 61 ℃ annealing 45s, then prolong 30s in 72 ℃, carries out altogether 35 circulations, and 72 ℃ are extended 600s; The laggard performing PCR product of loop ends sepharose detects;

4. amplified production detects: preparation 1.5% sepharose, getting 5 μ l PCR products mixes with 1 μ l bromjophenol blue load sample damping fluid, application of sample is in sepharose point sample hole, voltage 5V/cm carries out electrophoresis, electrophoresis liquid is 1 * TBE, stop after electrophoresis, get glue observations photograph in reading glue instrument, standard is 100bp DNA Ladder;

2) the Restriction fragment length polymorphism analysis of gyrA gene

1. digestion with restriction enzyme: nucleic acid fragment is less, and the susceptibility of detection is higher; For the PCR product that is greater than 400bp, should with restriction enzyme, carry out digestion process in advance; Two recognition sites (244thbp and 343thbp) of restricted property restriction endonuclease hinf I in gyrA gene amplification region, can be digested to this section of PCR product three bar segment (221bp, 99bp and 328bp):

The enzyme system of cutting is 50 μ l

PCR product 40 μ l

Restriction enzyme damping fluid 4 μ l

Restriction enzyme hinf I 6 μ l

Mix and slightly centrifugal, be placed in 2h in 37 ℃ of water-baths;

After endonuclease reaction, use sepharose horizontal strip electrophoresis to detect enzyme and cut effect;

2. Cleavage Map: preparation 2% sepharose, getting the above-mentioned PCR product of 7 μ l mixes with 1 μ l load sample damping fluid, in application of sample point sample hole, voltage 5V/cm carries out electrophoresis, and electrophoresis liquid is 1 * TBE, and is reading to observe restriction enzyme mapping on glue instrument, take a picture, numerous research shows, gyrA the 244th bit base sudden change makes Hinf I in this place recognition site forfeiture, thereby only produce two enzymes, cuts product fragment: 320bp and 328bp;

3) single-strand conformation polymorphism analysis of parC gene

1. digestion with restriction enzyme: need to process with digestion with restriction enzyme PCR product for the PCR product that is greater than 400bp, produce less DNA fragmentation, then carry out single stranded conformational analysis; The recognition site 279thbp of parC gene amplification region restricted property restriction endonuclease hinf I, can be digested to two bar segment 191bp and 278bp by this section of PCR product, and digestion method is the same;

2. polyacrylamide gel electrophoresis: get 3 μ l enzymes and cut sample and mix with 22 μ l sex change sample solutions, carry out 8% polyacrylamide gel electrophoresis, glue thickness 1.0 mm, long 82mm, voltage 8V/cm, constant voltage electrophoresis 4h; Molecular weight standard 100bp DNA Ladder;

3. silver dyes: after electrophoresis finishes, two sheet glass separately, put into 100 ml silver by gel film and dye stationary liquid and shake 15min, and tri-distilled water is washed 3 times, and then each 2min proceeds to gel film the AgNO of 100ml 0.2 % ₃liquid, washes 3 times with tri-distilled water, and each 30s, adds the about 7-10min of 100ml nitrite ion, when DNA band shows removing, sucks nitrite ion, adds stationary liquid Na ₂cO ₃, standing 2-3min, takes out gel observation; Be placed in observations photograph on x ray view box;

(2) adopt the method for gene sequencing to detect the gene locus suddenling change, find the sequential rule of quinolone medicine target gene gyrA, parC sudden change under LEV induction;

1) mensuration of nucleotide sequence: the primary gyrA of laboratory-induced strain shigella, parC gene PCR amplified production are checked order, separately select part inducible strain, gyrA, parC gene PCR amplified production are checked order; Entrust Beijing six directions Hua Da Gene science company to carry out;

2) compare of analysis of nucleotide sequence: by BLST program, surveyed sequence results and the gyrA retrieving from GenBank, parC gene standard sequence are compared in NCBI.

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