CN107751089A - It is a kind of that the method for resisting general resistance Acinetobacter bauamnnii medicine is screened using Caenorhabditis elegans - Google Patents
It is a kind of that the method for resisting general resistance Acinetobacter bauamnnii medicine is screened using Caenorhabditis elegans Download PDFInfo
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- CN107751089A CN107751089A CN201710754277.7A CN201710754277A CN107751089A CN 107751089 A CN107751089 A CN 107751089A CN 201710754277 A CN201710754277 A CN 201710754277A CN 107751089 A CN107751089 A CN 107751089A
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- acinetobacter bauamnnii
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- A—HUMAN NECESSITIES
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- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses a kind of method that anti-general resistance Acinetobacter bauamnnii medicine is screened using Caenorhabditis elegans.The general resistance Acinetobacter bauamnnii infection model of Caenorhabditis elegans for screening composition drug effect is built first, and the Caenorhabditis elegans hasglp‑ 4;sek‑1Dual-gene mutation；The culture medium composition that the general resistance Acinetobacter bauamnnii of Caenorhabditis elegans co-cultures is the μ g/ml FeCl of 20%BHI+5~20 μM acidum nalidixicum+5~203；General resistance Acinetobacter bauamnnii concentration is 1 × 106~1 × 109CFU/mL；When a length of 6~12h of bacterium infection Caenorhabditis elegans；When a length of 24~48h of drug therapy infection model.The model can be used for antibacterial activity inside fast high-flux screening classes of compounds or medicine or composition, and compared to internal animal infection modal, with preparing, cost is low, and the cycle is short, operates easy great advantages.Compared to external model, it can screen and exclude that toxicity in vivo is big, the metabolism difference compound low with In vitro-in vivo correlation.
Description
Technical field
The invention belongs to biological technical field.Resist general resistance using Caenorhabditis elegans screening more particularly, to one kind
The method of Acinetobacter bauamnnii medicine.
Background technology
Acinetobacter bauamnnii（Acinetobacter baumannii）It is a kind of non-fermented type gram negative bacilli, extensively
It is present in nature and hospital environment, is to cause one of most common conditioned pathogen of nosocomial infection.Acinetobacter bauamnnii can
Cause related to lung ventilator including respiratory tract infection, urinary system infection contamination, respiratory tract infection, bacteremia, wound infection, meningitis
Multi-infection disease including property pneumonia etc., with antibacterials widely using in clinic, the bacterium is to common antibiotics
Great drug resistance is generated, or even multidrug resistant occurs（3 classes to Common Antibiotics classification or more resistance）
Acinetobacter bauamnnii（MDR-AB）With extensive resistance（Gram-Negative bacillus is only sensitive to polymyxins and tigecycline）'s
Acinetobacter bauamnnii（XDR-AB）, great challenge is brought to clinical treatment.
The drug resistance of Acinetobacter bauamnnii is mediated by a variety of resistance mechanisms, and wherein bacterium Active efflux-pump is excessive
Expression can prevent antibacterials to reach activity from accumulating in the cell, be an important channel caused by drug resistance.Not
In lever Pseudomonas, the differentiation of resistance tubercle（Resistance-nodulation-division, RND）Family is to bacterial drug resistance
Generation plays an important role, and wherein AdeABC, AdeIJK, AdeFGH discharging system is primarily present in Acinetobacter bauamnnii, can arrange outside
A variety of antibacterials such as quinolones, aminoglycoside, tetracycline, erythromycin etc. cause resistance.QNS such as ring third
Sha Xing, once the line antibacterials as treatment Acinetobacter bauamnnii infection, but because Bao Man is to which creating extensive resistance
Property and cause clinical Ciprofloxacin of having abandoned substantially to be used to treat resistance Acinetobacter bauamnnii.Research finds, Acinetobacter bauamnnii
Resistance mechanism to Ciprofloxacin is mainly the overexpression of efflux pump, and Ciprofloxacin is shared into efflux pump inhibitor（efflux
Pump inhibitors, EPIs）It is set to be played a role again to antibody-resistant bacterium, it has also become treatment Acinetobacter bauamnnii infection
Potential effective way.
The research on EPIs is confined to experiment in vitro more at present, and can different EPIs reverse Bao Man not levers in vivo
The drug resistance of bacterium reaches preferable fungistatic effect, if it is the main of current EPIs applications that can produce larger toxic effect and use
Problem, it would be highly desirable to researched and solved by experiment in vivo.Nematode can provide relatively complete experimental animal model, can avoid mouse again
The high price of model, it is time-consuming the shortcomings that, therefore the cost of drug screening can be greatly reduced, be the ideal for large-scale medicine screening
System, favored more and more by scientists in recent years.
The content of the invention
The technical problems to be solved by the invention are to overcome the general resistance Acinetobacter bauamnnii medicine of prior art moderate resistance and outer
The defects of row's pump inhibitor research is present and deficiency, infect mould by establishing Caenorhabditis elegans-general resistance Acinetobacter bauamnnii
Type, Ciprofloxacin is combined into a variety of efflux pump inhibitors applied in this In vivo infection model, screened motionless to general resistance Bao Man
The effective composition of medicine of bacillus, new method and thinking are provided for the clinical treatment of general resistance Acinetobacter bauamnnii.
It is an object of the invention to provide a kind of Caenorhabditis elegans-general resistance Acinetobacter bauamnnii sense for drug screening
Contaminate model.
It is a further object of the present invention to provide the method for resisting general resistance Acinetobacter bauamnnii medicine using the model discrimination.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
A kind of Caenorhabditis elegans-general resistance Acinetobacter bauamnnii infection model for drug screening, the beautiful hidden bar line
Worm hasglp-4；sek-1Dual-gene mutation；The culture medium composition that Caenorhabditis elegans-general resistance Acinetobacter bauamnnii co-cultures
For the μ g/ml FeCl of 20%BHI+5~20 μM acidum nalidixicum+5~203；General resistance Acinetobacter bauamnnii concentration is 1 × 106~1 ×
109CFU/mL；When a length of 6~12h of general resistance Acinetobacter bauamnnii infection Caenorhabditis elegans；Drug therapy infection model
24~48h of Shi Changwei.
The present invention is when screening general resistance Acinetobacter bauamnnii antibacterials, by the training for selecting infection Caenorhabditis elegans
The composition of base, the duration of bacterium infection Caenorhabditis elegans and the duration of composition treatment infection model are supported, is built for screening
The Caenorhabditis elegans of composition drug effect-general resistance Acinetobacter bauamnnii infection model.Present invention discover that use BHI culture mediums merely
When removing to co-culture Caenorhabditis elegans-general resistance Acinetobacter bauamnnii, the BHI fluid nutrient mediums of which kind of concentration no matter are selected, i.e.,
Make to be that the XDR-AB of maximum concentration also can not successfully infect whole nematodes, can be again compacted after about half nematode stiff a period of time
It is dynamic, the purpose of drug screening of the present invention can not be applied to.It is weaker, it is necessary to strengthen it in view of the pathogenicity of Acinetobacter bauamnnii
Pathogenicity is to improve infection success rate；In view of iron ion is the virulence factor of Acinetobacter bauamnnii, thus in BHI fluid nutrient mediums
It is middle to add certain density FeCl3, it has been obviously improved infectious effect.However, above infectious condition can not sometimes reappear, line
Worm still can not infect lethal sometimes；By analyzing failure cause, it is presumed that not cleaning up fullyE.coli OP50
Infection of the Bao Man to nematode is disturbed with Bao Man interactions, therefore we add certain density acidum nalidixicum to suppressE.coliOP50 growth, good effect is obtained, infection success rate is almost 100%.
Preferably, the concentration for stating Acinetobacter bauamnnii is 5 × 106~5 × 108 CFU/mL。
It is highly preferred that the concentration of the Acinetobacter bauamnnii is 5 × 106 CFU/mL。
Preferably, the culture medium composition is the 20%BHI+5 μM of μ g/ml of acidum nalidixicum+10 FeCl3。
Preferably, when a length of 30~40h of the drug therapy infection model.
It is highly preferred that when a length of 36h of the drug therapy infection model.
Meanwhile application of the model in the compound of the anti-general resistance Acinetobacter bauamnnii of screening or medicine or composition
Or the application in hypotoxicity efflux pump inhibitor is screened all falls in the scope of protection of the present invention.
A kind of method for being resisted general resistance Acinetobacter bauamnnii medicine using above-mentioned model discrimination, is cultivated firstglp-4;sek- 1Genic mutation type Caenorhabditis elegans, resynchronization handle to obtain L4 phase nematodes；Then by nematode containing 1 × 106~1 × 109
Nematode is cleaned after cultivating 6~12h in the fluid nutrient medium of CFU/mL Acinetobacter bauamnniis, adds medicine to be measured, cultivates 24~48h
Nematode survival rate is observed afterwards；Wherein, the 20%BHI fluid nutrient mediums are the μ g/ml of 20%BHI+5~20 μM acidum nalidixicum+5~20
FeCl3。
Preferably, the concentration of the Acinetobacter bauamnnii is 5 × 106~5 × 108 CFU/mL。
It is highly preferred that the concentration of the Acinetobacter bauamnnii is 5 × 106 CFU/mL。
Preferably, the fluid nutrient medium is the 20%BHI+5 μM of μ g/ml of acidum nalidixicum+10 FeCl3。
Specifically, the composition and collocation method of the culture medium are 4.83g Na2HPO4·12H2O, 0.96g KH2PO4,
1.60g NaCl, 0.04g MgSO4, 2.96g BHI, 10 μM of FeCl3, add distilled water to 400mL, 121 DEG C of autoclavings
15min, when temperature is reduced to below 60 DEG C, adding acidum nalidixicum makes its final concentration of 5 μ g/mL, is saved backup in 4 DEG C.
Preferably, the incubation time added after medicine to be measured is 30~40h.
It is highly preferred that the incubation time added after medicine to be measured is 36h.
Preferably, methods described is that XDR-AB single bacterium colonies are taken from Bacterial Plate, adjustment bacteria concentration most 1.5 ×
108CFU/mL, culture after synchronization to the nematode of L4 phases is washed down from flat board, 500 r/min, centrifuges 1min, repeats 2~3
It is secondary, nematode is suspended in final concentration of 5 × 106In CFU/mL XDR-AB infect 6h after, with BHI fluid nutrient mediums clean to
Few three times then to put nematode distribution in 96 orifice plates, 15~20/hole adds to 180 μ L, and treatment group adds 20 μ L medicine to be measured,
Positive controls add the 2 μ g/mL μ L of polymyxin B 20, and negative control group adds the μ L of solvent 20, in 25 DEG C, humidity 85%
Under conditions of cultivate 30h, then under the microscope observe nematode survival rate.
Preferably, the method for the L4 phases nematode nematode synchronization process is：Nematode is washed from NGM plates with M9 buffer solutions
It is lower to collect to 15mL centrifuge tubes, 500 r/min centrifugation 1min, take supernatant to add splitting for 2.5 mL to remaining 3.5mL nematode liquid
The min of liquid shake well 3 is solved, adds M9 buffer solutions to centrifuge 1min to 15 mL, 1500 r/min, washs 5 times, isolated worm's ovum,
15 DEG C of vibration 16h egg hatches are placed on NGM flat boards to the nematode of L1 phasesE.coliOn OP50 lawns, 25 DEG C of cultures
48h, obtain synchronized L4 phases nematode.
A kind of method using above-mentioned model discrimination hypotoxicity efflux pump inhibitor, the nematode by synchronizing culture to the L4 phases
It is washed till in 15mL centrifuge tubes, 500 r/min, centrifuges 1min, cleans 2~3 times, 15~20/hole is dispensed into 96 orifice plates to 180
μ L, the efflux pump inhibitor of series concentration is added respectively, it is non-under conditions of being 85% in 25 DEG C, humidity to co-culture culture 30h,
Nematode survival rate is observed under microscope, judges toxic action of each efflux pump inhibitor to normal nematode.
The above method under the conditions of efflux pump inhibitor and Ciprofloxacin are used in conjunction with inside drug efficacy study and toxicity grind
The application studied carefully is also in the scope of the present invention.
Each efflux pump inhibitor joint Ciprofloxacin is applied in infection model, to the survival rate of nematode under various concentrations
There is different degrees of raising, nematode survival rate can be respectively increased for wherein PA β N of low concentration, NMP, Omeprazole, reserpine
30%~40%, 15%~20%, 20~30%, 20%, the survival rate for infecting nematode can be improved 30% left side by the Verapamil of high concentration
It is right.In vitro in drug sensitive test and toxicity test, Ciprofloxacin is combined with CCCP, Omeprazole and Verapamil can reduce by 4 times
MIC, PA β N, NMP and reserpine can reduce by 2 times of MIC.The external Combination best results of wherein CCCP, but toxicity is larger
It is unsuitable for internal drug efficacy study.Result above can carry out the drug effect starting point of structure optimization as each efflux pump inhibitor, so as to real
Existing attenuation synergistic, effectively contain the ciprofloxacin resistance of Acinetobacter bauamnnii, protect the final goal of people's health.
In addition, the present invention also provides a kind of drug regimen for being used to prevent and treat resistance Acinetobacter bauamnnii, the drug regimen
It is combined for PA β N and Ciprofloxacin, or NMP is combined with Ciprofloxacin, or Omeprazole and Ciprofloxacin, or reserpine and ring third
Sha Xing is combined.
Compared with prior art, the invention has the advantages that：
The infection model and method of the present invention can be used for inside fast high-flux screening classes of compounds or medicine or composition
Antibacterial activity, compared to internal animal infection modal, with preparing, cost is low, and the cycle is short, operates easy great advantages.Compared to body
External model, big, the metabolism difference compound low with In vitro-in vivo correlation that exclude toxicity in vivo can be screened.
Brief description of the drawings
Fig. 1 is the survival condition figure of nematode；A is that nematode survival rolls up state；B is nematode death spasticity.
Fig. 2 is pharmacodynamic results figure of the different iron concentrations to nematode viability rate；Wherein PB is polymyxins.
Fig. 3 is culture medium（20%BHI, 10 μm of ol/L FeCl3 and 5 μ g/ml）Middle various concentrations XDR-AB infection is beautiful hidden
The survivorship curve of rhabditida, wherein PB are polymyxins.
Fig. 4 is the metainfective fluorescing matter of Acinetobacter bauamnnii that nematode is dyed by DiI.
Fig. 5 is the different infection durations of XDR-AB and pharmaceutical intervention duration survivorship curve, and PB is polymyxins.
Fig. 6 is toxicity profile of each efflux pump inhibitor to nematode infections model.
Fig. 7 is that each efflux pump inhibitor is combined inside front and rear figure compared with drug effect with Ciprofloxacin.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The foundation of the Caenorhabditis elegans of embodiment 1-general resistance Acinetobacter bauamnnii infection model
1st, test material
（1）The Caenorhabditis elegans used in the present embodiment（glp-4；sek-1）, EHEC（E.coli）OP50 is by middle mountain
University Medical institute is old to inherit light professor and give, Quality-control strains Escherichia coli ATCC25922, the general resistance Bao Man being clinically separated not lever
Bacterium（XDR-AB）Gathered by clinical laboratory of Guangzhou General Hospital Guangzhou Military Command from hospital ICU patient, using French Mei Liai companies
The full-automatic identification and susceptibility instrument identification of VITEK-2 microorganisms.
（2）CCCP（Carbonyl cyanide 3-chlorophenyl hydrazone）, PA β N（phenyl-
arginine-β-naphthylamide）, polymyxin B（Polymyxin B sulfate salt）, acidum nalidixicum
（nalidixic acid）, Verapamil（Verapamil hydrochloride）, reserpine（Reserpine）, Omeprazole
（Omeprazole）It is purchased from Sigma chemical reagents corporations of the U.S..NMP（1-METHYLPYRROLIDONE [1-（1-
naphthylmethyl）-piperazine]）, ciprofloxacin hydrochloride（Ciprofloxacin Hydrochloride
Monohydrate）Purchased from Aladdin Reagent Company.Fluorescence probe Dil（The double octadecyls -3,3,3 ' of 1,1-, 3 '-tetramethyl Yin
The carbon cyanines of diindyl two）Purchased from Shanghai Mike's woods biochemical technology Co., Ltd.
（3）LB（Luria-Bertani）Solid medium：LB powder 4.2g, agar powder 3g, add distilled water 200mL, 121 DEG C
4 DEG C save backup after autoclaving 15min.Escherichia coli OP50：Escherichia coli OP50 is seeded on LB solid mediums, in
24h is incubated in 37 DEG C of carbon dioxide incubators.
（4）NGM（nematode growth medium）Culture medium：The g of tryptone 1.5, agar powder 10.8 g, NaCl
1.80 g, add 121 DEG C of autoclaving 15min after distilled water 585mL, when temperature is reduced to below 60 DEG C, add filtration sterilization
5mg/mL cholesterol 660 μ L, 1mol/L CaCl2660 μ L, 1mol/L MgSO4660 μ L, and add phosphate buffer
（K2HPO4And KH2PO4）13mL, in separating device diameter 9cm culture dish, after flat board cooled and solidified, take Escherichia coli OP50 mono-
Individual bacterium colony is coated on culture medium, and after 37 DEG C are incubated 24h, 4 DEG C save backup.
（5）20%BHI fluid nutrient mediums（brain-heart infusion medium）：4.83g Na2HPO4·12H2O,
0.96g KH2PO4, 1.60g NaCl, 0.04g MgSO4, 2.96g BHI, add distilled water to 400mL, 121 DEG C of autoclavings
15min, cooling are standby.
（6）20%BHI+FeCl3+ acidum nalidixicum fluid nutrient medium：4.83g Na2HPO4·12H2O, 0.96g KH2PO4, 1.60g
NaCl, 0.04g MgSO4, 2.96g BHI, 10 μM of FeCl3, add distilled water 121 DEG C of autoclaving 15min, to treat temperature to 400mL
When degree is reduced to less than 60 DEG C, adding acidum nalidixicum makes its final concentration of 5 μ g/mL, is saved backup in 4 DEG C.
（7）CAMHB culture mediums：6.6g CAMHB, 300mL 121 DEG C of autoclaving 15min of distilled water are added, in 4 DEG C of preservations
It is standby.
（8）M9 buffer solutions：3.02g Na2HPO4·12H2O, 0.6g KH2PO4, 1g NaCl, 0.024g MgSO4, add and steam
It is standby after distilled water 200mL, 121 DEG C of autoclaving 15min.
（9）Nematode lysate：0.4g NaOH, 2mL HClO solution, 4mL distilled water, matching while using.
2nd, the culture of nematode and synchronization process
Fromglp-4;sek-1Genic mutation type Caenorhabditis elegans, N2 wild type nematodes are compared, having can not produce at 25 DEG C
Ovum, and there is immunodefiiciency.Nematode is cultivated according to international standard program.Prepare to synchronize within four days before nematode infections experiment
Nematode：Nematode is washed into lower collect to 15mL centrifuge tubes, 500 r/min from NGM plates with M9 buffer solutions to centrifuge 1min, take supernatant
Liquid adds the 2.5 mL min of lysate shake well 3, adds M9 buffer solutions to 15 mL, 1500 r/ to remaining 3.5mL nematode liquid
Min centrifuges 1min, washs 5 times, isolated worm's ovum, and 15 DEG C of vibration 16h egg hatches are placed on NGM to the nematode of L1 phases
Flat boardE.coliOn OP50 lawns, 25 DEG C of culture 48h, synchronized L4 phases nematode is obtained.
3rd, the preparation of bacteria suspension
The XDR-AB bacterial strains recovery at -80 DEG C will be frozen, line is incubated at LB solid mediums, and 37 DEG C of culture 24h are standby.
4th, the foundation of nematode-general resistance Acinetobacter bauamnnii infection model
（1）Culture medium forms and influence of the Acinetobacter bauamnnii concentration to nematode infections model
Using the XDR-AB of various concentrations（5×108, 5 × 107, 5 × 106, 5 × 105 CFU/mL,E.coliOP50 control groups）
In different fluid nutrient mediums：（The BHI of various concentrations, FeCl3And acidum nalidixicum）Caenorhabditis elegans is infected, records the life of nematode
State is deposited, it is determined that suitable infectious condition.In liquid medium within, sinuous state is presented in living nematodes, and pharyngeal muscle moves, and
The dead linear type spasticity of nematode of XDR-AB infection（See Fig. 1）.
As a result：
When select various concentrations BHI for fluid nutrient medium when, also can not successfully infect whole even if the XDR-AB of maximum concentration
Nematode, about half nematode can wriggle again after stiff a period of time, when selecting 20%BHI fluid nutrient mediums, its infectious effect compared with
Other concentration are relatively preferable.It is weaker in view of the pathogenicity of Acinetobacter bauamnnii to be infected, it is necessary to strengthen its pathogenicity with improving
Success rate.In view of iron ion is the virulence factor of Acinetobacter bauamnnii, we add different dense in 20%BHI fluid nutrient mediums
The FeCl of degree3, when XDR-AB concentration is 5 × 106 During CFU/ml, infectious effect is preferable.FeCl3Concentration is 10,20 and 40 μ
During mol/L, there was no significant difference for the survivorship curve of nematode（As shown in Figure 2）.In view of saving raw material, we select 10 μm of ol/
L is as optimum ratio.Then above infectious condition can not sometimes reappear, and nematode still can not infect lethal sometimes.Pass through
Failure cause is analyzed, it is presumed that not cleaning up fullyE.coliOP50 and Bao Man interactions disturb Bao Man to line
The infection of worm, therefore we add acidum nalidixicum that minimal inhibitory concentration is 5 μ g/ml to suppressE.coliOP50 growth, is obtained
Good effect is arrived, infection success rate is almost 100%.
It is preferred that 20%BHI, 10 μm of ol/L FeCl3Formed with 5 μ g/ml acidum nalidixicum for fluid nutrient medium, investigate bacterium not
With influence of the concentration to nematode viability rate.With the reduction of Acinetobacter bauamnnii concentration, the nematode survival time has extended（Such as figure
Shown in 3）.Median lethal duration of the nematode under the infection of different bacteria concentrations（Time for half to die, LT50）Have obvious
Difference, 5 × 108LT50 is 12h in the infection of CFU/mL Acinetobacter bauamnniis, 5 × 107CFU/mL Acinetobacter bauamnniis
Infection in LT50 be 18h, 5 × 106LT50 is 30h in the infection of CFU/mL Acinetobacter bauamnniis, 5 × 105 CFU/mL
LT50 is 42h in the infection of Acinetobacter bauamnnii, according to the pathogenicity of various concentrations Acinetobacter bauamnnii and tests scheduling,
Test below and choose 5 × 106CFU/mL Acinetobacter bauamnnii is as infection concentration.
To determine that the lethal of Caenorhabditis elegans is due in the online polypide of Acinetobacter bauamnnii in liquid medium within
Accumulation, we are the 6h in the M9 fluid nutrient mediums of the 20%BHI containing Acinetobacter bauamnnii by culture of nematodes, observe under the microscope,
The whole enteron aisle of nematode substantially expands.Further to confirm that the expansion of nematode enteron aisle is due to the field planting of Acinetobacter bauamnnii, this
Experiment Dil（The double octadecyls -3,3,3 ' of 1,1-, the carbon cyanines of 3 '-tetramethyl indoles two）Cell membrane red fluorescence probe marks Bao
Graceful acinetobacter calcoaceticus, to follow the trail of the possible infection path of Acinetobacter bauamnnii, by culture of nematodes 20% of the bacterium containing fluorescence labeling
6h is cultivated in BHI M9 fluid nutrient mediums, in fluorescence microscopy Microscopic observation, the nematode of feeding Acinetobacter bauamnnii can be observed
Whole enteron aisle substantially expands, and is filled with strong red fluorescence bacterium（Fig. 4）
（2）The influence of difference infection duration and Ureteral Calculus duration to nematode infections model
Bacterium infection nematode duration and the treatment duration for adding antibacterials are the key factors for influenceing nematode viability rate, to determine
Optimal infection duration and treatment duration, the infection duration that we design nematode is respectively 3h, 6h and 12h, positive right after cleaning
PB is added according to hole, observes optimal treatment duration（Fig. 5）.Log-rank Test are shown when infecting Acinetobacter bauamnnii 3h, line
Worm almost can be not reaching to preferable infectious effect with normal existence（χ 2=3.154, P ＞ 0.05）, and infect 6h and infection 12h
The no notable difference of influence to the survival rate of nematode（χ 2=0.669, P ＞ 0.05）, with reference to Acinetobacter bauamnnii in different senses
Influence when contaminating duration to the lethal ability of nematode and experiment progress, final choice 6h select 36h as anti-as infection duration
The Ureteral Calculus duration of bacterium medicine（χ 2=46.56, P ＜ 0.0001）.
To sum up, preferable operating procedure is：XDR-AB single bacterium colonies are taken from Bacterial Plate, luminosity is used in 20%BHI
Bacteria concentration is adjusted to 0.5 maxwell unit than turbid instrument（About 1.5 × 108CFU/mL）, by culture after synchronization to the nematode of L4 phases
Wash down, centrifuge from flat board（500 r/min, 1min）, repeat 2~3 times, nematode be suspended in final concentration of 5 × 106 CFU/
After infecting 6h in mL XDR-AB, cleaned at least three times, nematode distribution is put in 96 orifice plates, 15~20 with BHI fluid nutrient mediums
Bar/hole adds to 180 μ L, and treatment group adds 20 μ L medicine to be measured（It is dissolved in 1%DMSO）, 2 μ g/mL's of positive controls addition is more viscous
The μ L of rhzomorph B 20（It is dissolved in 1%DMSO）, the negative control group addition μ L of 1%DMSO 20, cultivated under conditions of being 85% in 25 DEG C, humidity
30h, nematode survival rate is observed under the microscope.
The toxicity test of the efflux pump inhibitor of embodiment 2
1st, synchronizing culture to the nematode of L4 phases is washed till in 15mL centrifuge tubes with 20%BHI fluid nutrient mediums, 500 r/min,
1min is cleaned three times, and 15~20/hole dispenses into 96 orifice plates the efflux pump inhibitor for 180 μ L, adding series concentration respectively
CCCP, PA β N（5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL）,
NMP, Omeprazole, Verapamil, reserpine（10μg/mL、20μg/mL、30μg/mL、40μg/mL、50μg/mL、60μg/mL、
70μg/mL、80μg/mL）20 μ L, non-co-cultivation 30h under conditions of being 85% in 25 DEG C, humidity, nematode are observed under the microscope and is deposited
Motility rate, judge toxic action of each efflux pump inhibitor to normal nematode.
2nd, result
6 kinds of CCCP, NMP, PA β N, Omeprazole, Verapamil and reserpine efflux pump inhibitors to the toxic action of nematode not
Together, wherein when CCCP concentration is more than 5 μ g/mL, PA β N concentration is more than 30 μ g/mL more than the concentration of 25 μ g/mL and reserpine
When start obvious toxic action occur to nematode（Fig. 6）, nematode viability rate begins to decline.Omeprazole, Verapamil and NMP
Toxic action it is smaller, nematode viability rate is not influenceed in the range of 80 μ g/mL, but find to be more than 60 μ g/ when concentration after observing
Nematode viability active state is decreased obviously during mL.
The drug sensitivity testing in vitro of the Ciprofloxacin of embodiment 3 and each efflux pump inhibitor
1st, micro-broth dilution method antibacterials Ciprofloxacin minimal inhibitory concentration（MIC）
With EHEC ATCC25922, Acinetobacter bauamnnii ATCC19606 is tested for same batch as Quality-control strains.Prepare
5120 μ g/mL antibacterials storing solution, 13 sterile test tubes are taken, the hydrochloric acid ring third of series concentration is prepared with CAMHB culture mediums
Husky μ g/mL of star 512,256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL,
2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, 100 μ L are respectively taken in 96 orifice plates；Use light
Degree takes the bacterium colony of fresh cultured, adjustment bacteria suspension concentration is 0.5 maxwell unit than turbid instrument（About 1.5 × 108CFU/mL）, take
100 μ L, which add each hole, makes bacterium final concentration of 5 × 106CFU/mL, each hole Ciprofloxacin Concentration are 256 μ g/mL, 128 μ g/
mL、64 μg/mL、32 μg/mL、16 μg/mL、8 μg/mL、4 μg/mL、2 μg/mL、1 μg/mL、0.5 μg/mL、0.25
μ g/mL, 0.125 μ g/mL, 0.0625 μ g/mL, 16~20 h of incubation in 35 DEG C of incubators are placed in, result evaluation is according to CLSI
（Clinical and Laboratory Standards Institute）Standard, unit are μ g/mL.Determine each efflux pump suppression
The MIC value that the MIC value and Ciprofloxacin of preparation are added after efflux pump inhibitor, it is separately added into according to toxicity test result each outer
Pump inhibitor is arranged, and compares change of the bacterial strain to antibacterials MIC value before and after addition efflux pump inhibitor.
2nd, result
It is as shown in table 1 below, when CIP is used alone, to XDR-AB MIC value up to 256 μ g/mL, when CCCP, PA β N is used alone
MIC value is that 15 μ g/mL, 30 μ g/mL, NMP, Omeprazole, Verapamil and reserpine be not obvious to XDRAB when being used alone
Bacteriostasis, after adding the EPIs in toxicity range, CIP MIC value declines 2~4 times.
Table 1
The Ciprofloxacin of embodiment 4（CIP）Combine different EPIs to drug effect inside infection nematode
1st, synchronized nematode is obtained according to established infection model by non-co-cultivation infecting nematode, drug combination treatment sense
Nematode is contaminated, drug concentration is determined in toxicity range, sets high, normal, basic three groups of drug concentrations, and the survival rate of nematode is observed after 30h,
Judge that different EPIs combines CIP to therapeutic action inside infection nematode under different pharmaceutical concentration.Infection experiment independently enters
Row 3 times, while carry out positive control experiment and negative control experiment.
2nd, result
After adding medicine 30h, CCCP combines CIP under conditions of 5 μ g/mL, 2.5 μ g/mL, 1 μ g/mL, and effect is paid no attention to
Think, nematode survival rate is almost nil.As shown in fig. 7, PA β N can significantly improve CIP in infection nematode body after combining with CIP
There were significant differences to combined effect by the PA β N of therapeutic action, wherein various concentrations（P ＜ 0.0001）.During PA β N low concentrations with
CIP has preferable synergy（P ＜ 0.0001）, than be used alone CIP when infect nematode survival rate can improve 30%~40%.
The omeprazole CIP of various concentrations has significant difference to the therapeutic action for infecting nematode（P ＜ 0.0001）, wherein low dense
The omeprazole therapeutic effect of degree is more preferable（P ＜ 0.0001）, than be used alone CIP when infect nematode survival rate can improve
20%~30%.And when the NMP of various concentrations, reserpine and CIP therapeutic alliances infection nematode, do not find significant difference（P ＞
0.05）, be used in combination to reduce medicine and EPIs and bring potential drug toxicity, we with the μ g/mL of low concentration 20 NMP with
After CIP is used in combination, infection nematode improves 15%~20% through treating survival rate than single drug（P ＜ 0.0001）, the μ of low concentration 10
After g/mL reserpine is used in combination with CIP, infection nematode survival rate after treatment improves 20% than single drug（P ＜
0.0001）.When being used in combination using Verapamil and the CIP of various concentrations, the therapeutic action of Verapamil is obvious during high concentration
Preferably（P ＜ 0.0001）, the survival rate that postoperative infection nematode is used in combination with CIP for the μ g/mL of high concentration 60 Verapamil can improve
30% or so.Result above can carry out the drug effect starting point of structure optimization as each efflux pump inhibitor, so as to realize attenuation synergistic,
The effectively ciprofloxacin resistance of containment Acinetobacter bauamnnii, protect the final goal of people's health.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although by upper
Embodiment is stated the present invention is described in detail, it is to be understood by those skilled in the art that can in form and
Various changes are made in details to it, the spirit and scope limited without departing from appended claims of the present invention.
Claims (10)
- A kind of 1. Caenorhabditis elegans-general resistance Acinetobacter bauamnnii infection model for drug screening, it is characterised in that institute Stating Caenorhabditis elegans hasglp-4;sek-1Dual-gene mutation；Caenorhabditis elegans-general resistance Acinetobacter bauamnnii co-cultures Culture medium composition be the μ g/ml FeCl of 20%BHI+5~20 μM acidum nalidixicum+5~203；General resistance Acinetobacter bauamnnii concentration is 1 ×106~1 × 109CFU/mL；When a length of 6~12h of general resistance Acinetobacter bauamnnii infection Caenorhabditis elegans；Drug therapy When a length of 24~48h of infection model.
- 2. the answering in the compound or medicine or composition of the anti-general resistance Acinetobacter bauamnnii of screening of model described in claim 1 With.
- 3. application of the model described in claim 1 in hypotoxicity efflux pump inhibitor is screened.
- A kind of 4. method for resisting general resistance Acinetobacter bauamnnii medicine using model discrimination described in claim 1, it is characterised in that Cultivate firstglp-4;sek-1Genic mutation type Caenorhabditis elegans, resynchronization handle to obtain L4 phase nematodes；Then by nematode Containing 1 × 106~1 × 109Nematode is cleaned after cultivating 6~12h in the fluid nutrient medium of CFU/mL Acinetobacter bauamnniis, addition is treated Medicine is surveyed, nematode survival rate is observed after cultivating 24~48h；Wherein, the fluid nutrient medium be 20%BHI+5~20 μM acidum nalidixicum+ 5~20 μ g/ml FeCl3。
- 5. according to the method for claim 4, it is characterised in that the concentration of the Acinetobacter bauamnnii is 5 × 106~5 × 108 CFU/mL。
- 6. according to the method for claim 5, it is characterised in that the concentration of the Acinetobacter bauamnnii is 5 × 106 CFU/ mL。
- 7. according to the method for claim 4, it is characterised in that the fluid nutrient medium is the 20%BHI+5 μM of μ of acidum nalidixicum+10 g/ml FeCl3。
- 8. according to the method for claim 4, it is characterised in that the incubation time added after medicine to be measured is 30~40h.
- 9. according to the method for claim 8, it is characterised in that the incubation time added after medicine to be measured is 36h.
- 10. any one of claim 4~9 methods described under the conditions of efflux pump inhibitor and Ciprofloxacin are used in conjunction with inside Application in drug efficacy study and toxicity research.
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