CN107751089B - A method for screening anti-pandrug-resistant Acinetobacter baumannii drugs using Caenorhabditis elegans - Google Patents
A method for screening anti-pandrug-resistant Acinetobacter baumannii drugs using Caenorhabditis elegans Download PDFInfo
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Abstract
本发明公开了一种利用秀丽隐杆线虫筛选抗泛耐药鲍曼不动杆菌药物的方法。首先构建用于筛选组合物药效的秀丽隐杆线虫‑泛耐药鲍曼不动杆菌感染模型，所述秀丽隐杆线虫具有glp‑ 4;sek‑1双基因突变；秀丽隐杆线虫‑泛耐药鲍曼不动杆菌共培养的培养基组成为20%BHI+5～20μM萘啶酸+5～20μg/ml FeCl3；泛耐药鲍曼不动杆菌浓度为1×106～1×109CFU/mL；细菌感染秀丽隐杆线虫的时长为6～12h；药物治疗感染模型的时长为24～48h。所述模型可用于快速高通量筛选各类化合物或药物或组合物的体内抗菌活性，相比体内动物感染模型，具有制备成本低，周期短，操作简易的巨大优点。相比体外模型，可以筛选排除体内毒性大，代谢差和体内外相关性低的化合物。
The invention discloses a method for screening drugs against pan-drug-resistant Acinetobacter baumannii by using Caenorhabditis elegans. First, a C. elegans-pan-drug-resistant Acinetobacter baumannii infection model for screening the efficacy of the composition was constructed, and the C. elegans had glp -4; sek-1 double gene mutations; C. elegans-pan The medium composition of drug-resistant Acinetobacter baumannii co-culture is 20% BHI+5～20μM nalidixic acid+5～20μg/ml FeCl 3 ; the concentration of pan-resistant Acinetobacter baumannii is 1×10 6 ～1× 10 9 CFU/mL; the duration of bacterial infection of C. elegans is 6 to 12 hours; the duration of drug treatment of infection model is 24 to 48 hours. The model can be used for rapid and high-throughput screening of the in vivo antibacterial activity of various compounds or drugs or compositions. Compared with the in vivo animal infection model, the model has the great advantages of low preparation cost, short cycle and simple operation. Compared with in vitro models, compounds with high toxicity in vivo, poor metabolism and low correlation between in vitro and in vivo can be screened out.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly relates to a method for screening a pan-resistant acinetobacter baumannii medicament by utilizing caenorhabditis elegans.
Background
Acinetobacter baumannii (A), (B), (C), (DAcinetobacter baumannii) Is a non-fermentation gram-negative bacillus, widely exists in the nature and hospital environment, and is one of the most common pathogenic bacteria causing nosocomial infection. The acinetobacter baumannii can cause various infectious diseases including respiratory tract infection, urinary system infection, respiratory tract infection, bacteremia, wound infection, meningitis, ventilator-associated pneumonia and the like, and with the wide use of antibacterial drugs in clinic, the acinetobacter baumannii (MDR-AB) and the wide-range drug resistance (gram-negative bacilli only have polymyxin) with great drug resistance to common antibiotics and even multiple drug resistance (3 types or more of drug resistance which is mainly classified to common antibacterial drugs) and wide-range drug resistance (gram-negative bacilli only have resistance to polymyxin)And tigecycline sensitive) acinetobacter baumannii (XDR-AB), presents a significant challenge to clinical treatment.
The drug resistance of acinetobacter baumannii is mediated by various drug resistance mechanisms, wherein the over-expression of the active efflux pump of bacteria can prevent the antibacterial drug from accumulating in cells to reach the action concentration, and the drug resistance is an important way for generating the drug resistance. In the genus acinetobacter, the resistance-node-differentiation (RND) family plays an important role in the development of bacterial resistance, wherein the AdeABC, adejk, and AdeFGH efflux systems mainly exist in acinetobacter baumannii, and can efflux various antibacterial drugs such as quinolones, aminoglycosides, tetracycline, erythromycin, and the like to cause drug resistance. Quinolone drugs such as ciprofloxacin have been used as first-line antibacterial drugs for treating acinetobacter baumannii infection, but due to extensive drug resistance of baumannii to it, ciprofloxacin for treating drug-resistant acinetobacter baumannii has been basically abandoned in clinical practice. Researches find that the drug resistance mechanism of acinetobacter baumannii to ciprofloxacin is mainly the over-expression of an efflux pump, and the ciprofloxacin is combined with Efflux Pump Inhibitors (EPIs) to play a role in drug-resistant strains again, so that the acinetobacter baumannii drug resistance mechanism becomes a potential effective way for treating acinetobacter baumannii infection.
At present, research on EPIs is mostly limited to in vitro experiments, and whether different EPIs can reverse drug resistance of acinetobacter baumannii in vivo to achieve a good bacteriostatic effect or not and whether a large toxic effect is generated or not is a main problem of application of the EPIs at present, and needs to be solved through in vivo experimental research. Nematodes can provide a relatively complete experimental animal model and avoid the disadvantages of high price and time consumption of a mouse model, so that the cost of drug screening can be greatly reduced, and the nematode animal model is an ideal system for large-scale drug screening and is more and more favored by scientists in recent years.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and shortcomings of the anti-pan-drug-resistant acinetobacter baumannii drugs and efflux pump inhibitor research in the prior art, applying ciprofloxacin to the in-vivo infection model by combining a plurality of efflux pump inhibitors through establishing a caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii infection model, screening a combined drug effective to the pan-drug-resistant acinetobacter baumannii, and providing a new method and thought for the clinical treatment of the pan-drug-resistant acinetobacter baumannii.
The invention aims to provide a caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii infection model for drug screening.
The invention also aims to provide a method for screening a pan-resistant acinetobacter baumannii medicament by using the model.
The above object of the present invention is achieved by the following technical solutions:
a caenorhabditis elegans-pan-resistant A. baumannii infection model for drug screening, said caenorhabditis elegans havingglp-4；sek-1Double gene mutation; the composition of the culture medium for the caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii co-culture is 20 percent BHI + 5-20 mu M nalidixic acid + 5-20 mu g/ml FeCl3(ii) a The concentration of pan-drug-resistant acinetobacter baumannii is 1 multiplied by 106～1×109CFU/mL; the duration of the pan-drug-resistant acinetobacter baumannii infected caenorhabditis elegans is 6-12 h; the time for treating the infection model by the medicine is 24-48 h.
When the pan-drug-resistant acinetobacter baumannii antibacterial drugs are screened, the caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii infection model for screening the drug effect of the composition is constructed by selecting the composition of the culture medium for infecting caenorhabditis elegans, the time for infecting caenorhabditis elegans with bacteria and the time for treating the infection model by the composition. The invention discovers that when the BHI culture medium is simply used for co-culturing the caenorhabditis elegans-pan drug-resistant acinetobacter baumannii, no matter what concentration of the BHI liquid culture medium is selected, even the XDR-AB with the highest concentration can not successfully infect all nematodes, about half of the nematodes can wriggle again after being stiff for a period of time, and the method can not be suitable for the purpose of drug screening. Considering that the pathogenicity of acinetobacter baumannii is weak, the pathogenicity of the acinetobacter baumannii needs to be enhanced to improve the infection success rate; because iron ions are virulence factors of acinetobacter baumannii, FeCl with a certain concentration is added into BHI liquid culture medium3The infection effect is obviously improved. However, the above feelingThe staining conditions sometimes cannot be reproduced, and the nematodes sometimes cannot be infected and killed; by analyzing the cause of failure, we speculate that the cleaning is insufficientE.coliOP50 interacts with Bowman to interfere with infection of nematodes by Bowman, so we added a concentration of nalidixic acid to inhibitE.coliThe growth of OP50 has good effect, and the infection success rate is almost 100%.
Preferably, the concentration of the acinetobacter baumannii is 5 x 106～5×108 CFU/mL。
More preferably, the concentration of Acinetobacter baumannii is 5 × 106 CFU/mL。
Preferably, the medium consists of 20% BHI + 5. mu.M nalidixic acid + 10. mu.g/ml FeCl3。
Preferably, the time period of the drug treatment of the infection model is 30-40 h.
More preferably, the drug treatment of the infection model is for a period of 36 hours.
Meanwhile, the application of the model in screening a pan-resistant acinetobacter baumannii compound or drug or composition or the application in screening a low-toxicity efflux pump inhibitor is within the protection scope of the invention.
A method for screening a pan-resistant acinetobacter baumannii drug by using the model comprises the steps of firstly culturingglp-4;sek- 1Performing synchronization treatment on the gene mutant type caenorhabditis elegans to obtain L4-stage nematodes; then the nematodes are added in the mixture containing 1 × 106～1×109Culturing in a liquid culture medium of CFU/mL Acinetobacter baumannii for 6-12 h, cleaning nematodes, adding a to-be-detected drug, culturing for 24-48 h, and observing the survival rate of the nematodes; wherein the 20% BHI liquid culture medium is 20% BHI, 5-20 mu M nalidixic acid and 5-20 mu g/ml FeCl3。
Preferably, the concentration of Acinetobacter baumannii is 5 × 106～5×108 CFU/mL。
More preferably, the concentration of Acinetobacter baumannii is 5 × 106 CFU/mL。
Preferably, the liquid medium is 20% BHI +5 μ M nalidixic acid +10 μ g/ml FeCl3。
Specifically, the components and preparation method of the culture medium are 4.83g of Na2HPO4·12H2O，0.96g KH2PO4，1.60g NaCl，0.04g MgSO4，2.96g BHI，10μM FeCl3Adding distilled water to 400mL, autoclaving at 121 deg.C for 15min, adding nalidixic acid to make the final concentration 5 μ g/mL when the temperature is reduced to below 60 deg.C, and storing at 4 deg.C for use.
Preferably, the culture time after the drug to be detected is added is 30-40 h.
More preferably, the incubation time after addition of the test drug is 36 h.
Preferably, the method is to take XDR-AB single colonies from the bacterial plate and adjust the bacterial concentration to 1.5X 108CFU/mL, washing the synchronized nematodes cultured to L4 stage from the plate, centrifuging for 1min at 500 r/min, repeating for 2-3 times, and suspending the nematodes at a final concentration of 5 × 106After being infected in XDR-AB of CFU/mL for 6h, the nematode is washed by BHI liquid culture medium for at least three times, then the nematode is distributed in 96-well plates, 15-20 nematodes are added to 180 mu L per well, 20 mu L of drug to be detected is added to a treatment group, 20 mu L of polymyxin B of 2 mu g/mL is added to a positive control group, 20 mu L of solvent is added to a negative control group, the nematode is cultured for 30h under the conditions of 25 ℃ and 85% of humidity, and then the survival rate of the nematode is observed under a microscope.
Preferably, the method for synchronously treating the nematode nematodes in the L4 stage comprises the following steps: washing nematodes from an NGM plate by using an M9 buffer solution, collecting the nematodes to a 15mL centrifuge tube, centrifuging for 1min at 500 r/min, taking supernatant to the rest 3.5mL nematode solution, adding 2.5 mL lysate, fully shaking for 3 min, adding M9 buffer solution to 15mL, centrifuging for 1min at 1500 r/min, washing for 5 times, separating to obtain eggs, oscillating at 15 ℃ for 16h to hatch the nematodes in the L1 stage, and placing the eggs on the NGM plateE.coliCulturing OP50 on thallus Porphyrae at 25 deg.C for 48h to obtain synchronized L4 stage nematodes.
A method for screening low-toxicity efflux pump inhibitors by using the model comprises the steps of washing nematodes synchronously cultured to the L4 stage into 15mL centrifuge tubes, centrifuging for 1min at 500 r/min, cleaning for 2-3 times, subpackaging 15-20 nematodes/well into 96-well plates to 180 mu L, respectively adding efflux pump inhibitors with series concentrations, carrying out non-co-culture for 30h under the conditions of 25 ℃ and 85% of humidity, observing the survival rate of the nematodes under a microscope, and judging the toxic effect of each efflux pump inhibitor on normal nematodes.
The application of the method in-vivo efficacy research and toxicity research under the condition of using the efflux pump inhibitor and the ciprofloxacin together is also within the protection scope of the invention.
The efflux pump inhibitors and ciprofloxacin are applied to an infection model, the survival rate of nematodes is improved to different degrees under different concentrations, wherein the survival rate of the nematodes can be respectively improved by 30-40%, 15-20%, 20-30% and 20% through the low-concentration PA beta N, NMP, omeprazole and reserpine, and the survival rate of the nematodes infected by the high-concentration verapamil can be improved by about 30%. In vitro drug sensitivity and toxicity tests, ciprofloxacin combined with CCCP, omeprazole and verapamil can reduce MIC by 4 times, and PA beta N, NMP and reserpine can reduce MIC by 2 times. The CCCP combined in vitro antibacterial effect is optimal, but the toxicity is large, so that the CCCP combined in vitro antibacterial effect is not suitable for in vivo efficacy research. The result can be used as the starting point of the drug effect of structural optimization of each efflux pump inhibitor, thereby realizing the attenuation and the synergy, effectively inhibiting the ciprofloxacin resistance of acinetobacter baumannii and protecting the final aim of the health of people.
In addition, the invention also provides a drug combination for preventing and treating drug-resistant acinetobacter baumannii, wherein the drug combination is combined by PA beta N and ciprofloxacin, or combined by NMP and ciprofloxacin, or combined by omeprazole and ciprofloxacin, or combined by reserpine and ciprofloxacin.
Compared with the prior art, the invention has the following beneficial effects:
the infection model and the method can be used for rapidly screening in-vivo antibacterial activity of various compounds or medicines or compositions in high flux, and compared with an in-vivo animal infection model, the infection model and the method have the great advantages of low preparation cost, short period and simple and easy operation. Compared with an in vitro model, the method can screen and eliminate compounds with high in vivo toxicity, poor metabolism and low in vitro and in vivo correlation.
Drawings
FIG. 1 is a diagram of the survival status of nematodes; a is the nematode survival rolling state; b is the dead and stiff state of the nematode.
FIG. 2 is a graph showing the results of the drug effect of different concentrations of iron ions on the survival rate of nematodes; wherein PB is polymyxin.
FIG. 3 is a graph showing the survival curves of different concentrations of XDR-AB infected C.elegans in medium (20% BHI, 10. mu. mol/L FeCl3, and 5. mu.g/ml), where PB is polymyxin.
FIG. 4 shows fluorescence of nematodes after infection with DiI-stained A.baumannii.
FIG. 5 is a survival curve of XDR-AB for different lengths of infection versus length of drug intervention, PB being polymyxin.
FIG. 6 is a graph showing the toxicity of each efflux pump inhibitor to a model of nematode infection.
Figure 7 is a graph comparing the in vivo efficacy of each efflux pump inhibitor before and after use in combination with ciprofloxacin.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 establishment of caenorhabditis elegans-pan-drug-resistant Acinetobacter baumannii infection model
1. Test materials
(1) Caenorhabditis elegans (C.) (used in this example)glp-4；sek-1) Escherichia coli (E.coli)E.coli) OP50 was collected from ICU patients in Hospital by general Hospital laboratory of Guangzhou, Legionnaire, Chen 32565, facultative university of Zhongshan Yao, Boehringer-Kunststoff, quality control strain Escherichia coli ATCC25922, and clinically isolated pan-resistant Acinetobacter baumannii (XDR-AB) and identified by VITEK-2 microorganism full-automatic identification drug sensitivity instrument, France Meiriei-ai company.
(2) CCCP (Carbonyl cyanide 3-chlorophenylhydrazone), PA β N (phenyl-arginine- β -naphthylamide), polymyxin B (polymyxin B sulfate salt), nalidixic acid (nalidixic acid), Verapamil (Verapamil hydrochloride), Reserpine (Reserpine), Omeprazole (omaprazole) are all available from Sigma chemical company, usa. NMP (N-methylpyrrolidone [1- (1-naphthylmethyl) -piperazine ]), Ciprofloxacin Hydrochloride (Ciprofloxacin Hydrochloride Monohydrate) were purchased from Alantin reagent company. Fluorescent probe Dil (1, 1-dioctadecyl-3, 3,3 ', 3' -tetramethylindodicarbocyanine) was purchased from Shanghai Michelin Biotech, Inc.
(3) LB (Luria-Bertani) solid Medium: 4.2g of LB powder and 3g of agar powder, adding 200mL of distilled water, sterilizing at 121 ℃ for 15min under high pressure, and storing at 4 ℃ for later use. Escherichia coli OP 50: escherichia coli OP50 was inoculated on LB solid medium and incubated at 37 ℃ in a carbon dioxide incubator for 24 hours.
(4) Ngm (nematode growth medium) medium: 1.5 g of tryptone, 10.8 g of agar powder and 1.80 g of NaCl, adding 585mL of distilled water, then carrying out autoclaving at 121 ℃ for 15min, and adding 660 mu L of cholesterol with the concentration of 5mg/mL and 1mol/L of CaCl for filtration and sterilization when the temperature is reduced to below 60 DEG C2660 μ L, 1mol/L MgSO4660 μ L, and phosphate buffer (K) was added2HPO4And KH2PO4) 13mL of the strain is distributed in culture dishes with the diameter of 9cm, after the plate is cooled and solidified, a single colony of Escherichia coli OP50 is taken and coated on a culture medium, and after incubation for 24 hours at 37 ℃, the strain is stored at 4 ℃ for later use.
(5) 20% BHI liquid medium (serum-heart infusion medium): 4.83g Na2HPO4·12H2O，0.96g KH2PO4，1.60g NaCl，0.04g MgSO42.96g BHI, distilled water to 400mL, autoclaving at 121 ℃ for 15min, and cooling for use.
（6）20%BHI+FeCl3+ nalidixic acid liquid medium: 4.83g Na2HPO4·12H2O，0.96g KH2PO4，1.60g NaCl，0.04g MgSO4，2.96g BHI，10μM FeCl3Adding distilled water to 400mL, autoclaving at 121 deg.C for 15min, adding nalidixic acid to make the final concentration 5 μ g/mL when the temperature is reduced to below 60 deg.C, and storing at 4 deg.C for use.
(7) CAMHB medium: 6.6g CAMHB, 300mL distilled water, 121 ℃ high pressure sterilization for 15min, 4 ℃ storage for standby.
(8) M9 buffer: 3.02g Na2HPO4·12H2O，0.6g KH2PO4，1g NaCl，0.024g MgSO4Adding 200mL of distilled water, and autoclaving at 121 ℃ for 15min for later use.
(9) Nematode lysate: 0.4g NaOH, 2mL HClO solution, 4mL distilled water, now prepared.
2. Culture and synchronized treatment of nematodes
Choose to useglp-4;sek-1The genetically mutant C.elegans, compared to N2 wild-type nematodes, was unable to lay eggs at 25 ℃ and was immunodeficient. The nematodes were cultured according to the international standard procedures. Four days prior to the nematode infection experiment, synchronized nematodes were prepared: washing nematodes from an NGM plate by using an M9 buffer solution, collecting the nematodes to a 15mL centrifuge tube, centrifuging for 1min at 500 r/min, taking supernatant to the rest 3.5mL nematode solution, adding 2.5 mL lysate, fully shaking for 3 min, adding M9 buffer solution to 15mL, centrifuging for 1min at 1500 r/min, washing for 5 times, separating to obtain eggs, oscillating at 15 ℃ for 16h to hatch the nematodes in the L1 stage, and placing the eggs on the NGM plateE.coliCulturing OP50 on thallus Porphyrae at 25 deg.C for 48h to obtain synchronized L4 stage nematodes.
3. Preparation of the bacterial suspension
Recovering XDR-AB strain frozen at-80 deg.C, streak-culturing in LB solid medium, and culturing at 37 deg.C for 24 hr.
4. Establishment of nematode-pan-drug-resistant acinetobacter baumannii infection model
(1) Effect of Medium composition and Acinetobacter baumannii concentration on nematode infection model
XDR-AB (5X 10) was used at different concentrations8，5×107，5×106，5×105 CFU/mL，E.coliOP50 control group) in different liquid media: (different concentrations of BHI, FeCl3And nalidixic acid) to infect caenorhabditis elegans, record the survival status of the nematode, and determine appropriate infection conditions. In the liquid culture medium, the survival nematodes are in a sinusoidal state,pharyngeal muscles move and XDR-AB infected dead nematodes are in a linear catalepsy state (see FIG. 1).
As a result:
when BHI with different concentrations is selected as a liquid culture medium, even the XDR-AB with the highest concentration cannot successfully infect all nematodes, about half of the nematodes can wriggle again after being stiff for a period of time, and when a BHI liquid culture medium of 20 percent is selected, the infection effect is relatively better than that of other concentrations. Considering that the pathogenicity of acinetobacter baumannii is weak, the pathogenicity of acinetobacter baumannii needs to be enhanced to improve the success rate of infection. Since iron ions are virulence factors of Acinetobacter baumannii, we added FeCl at different concentrations to 20% BHI broth3When the concentration of XDR-AB is 5X 106 The infection effect is better when CFU/ml is used. FeCl3At concentrations of 10, 20 and 40. mu. mol/L, there was no significant difference in nematode survival curves (as shown in FIG. 2). In view of saving raw materials, we chose 10. mu. mol/L as the preferred formulation. Then the infection conditions are sometimes not reproducible, and the nematodes are sometimes not infected and killed. By analyzing the cause of failure, we speculate that the cleaning is insufficientE.coliOP50 interacts with Bowman to interfere with infection of nematodes by Bowman, so we added nalidixic acid at a minimum inhibitory concentration of 5 μ g/ml to inhibit nematode infestationE.coliThe growth of OP50 has good effect, and the infection success rate is almost 100%.
Preferably 20% BHI, 10. mu. mol/L FeCl3And 5 mug/ml nalidixic acid as a liquid medium composition, and the influence of different bacterial concentrations on the survival rate of the nematode is examined. As the concentration of acinetobacter baumannii decreased, nematode survival time was prolonged (as shown in fig. 3). The half-lethal duration (time for half to die, LT 50) of nematodes infected with different bacterial concentrations is significantly different at 5X 108LT50 was 12h in CFU/mL Acinetobacter baumannii infection at 5X 107LT50 was 18h in CFU/mL Acinetobacter baumannii infection at 5X 106LT50 was 30h in CFU/mL Acinetobacter baumannii infection at 5X 105LT50 is 42h in CFU/mL acinetobacter baumannii infection, and according to pathogenicity and test schedule of acinetobacter baumannii with different concentrations, 5 × 10 is selected in the following experiments6CFU/mL Acinetobacter baumannii was used as the infection concentration.
To determine that the mortality of C.elegans in the broth was due to accumulation of A.baumannii in the nematode, we cultured the nematodes in M9 broth containing 20% BHI of A.baumannii for 6h, and observed under a microscope that the entire intestinal tract of the nematode was significantly swollen. To further confirm that the swelling of the nematode gut is due to colonization by Acinetobacter baumannii, the experiment used Dil (1, 1-dioctadecyl-3, 3,3 ', 3' -tetramethylindodicarbocyanine) cell membrane red fluorescent probe to mark Acinetobacter baumannii to track the possible infection route of Acinetobacter baumannii, cultured the nematodes in M9 liquid medium containing 20% BHI of fluorescent-marked bacteria for 6h, observed under a fluorescent microscope, that the whole gut of the nematodes fed with Acinetobacter baumannii is obviously swollen and filled with strong red fluorescent bacteria (FIG. 4)
(2) Effect of different infection durations and treatment observation durations on nematode infection models
The length of time for bacterial infection of nematodes and the length of treatment with antibacterial agents are key factors affecting the survival rate of nematodes, and in order to determine the optimal length of infection and the optimal length of treatment, we designed the length of infection of nematodes to be 3h, 6h and 12h, and after washing, PB was added to the positive control wells and the optimal length of treatment was observed (FIG. 5). Log-rank Test shows that when the acinetobacter baumannii is infected for 3h, the nematode can almost normally live, a better infection effect is not achieved (x 2=3.154, P > 0.05), the influence of infection for 6h and infection for 12h on the survival rate of the nematode is not obviously different (x 2=0.669, P > 0.05), and in combination with the influence of the acinetobacter baumannii on the lethality of the nematode and the experimental progress in different infection time periods, 6h is finally selected as the infection time period, and 36h is selected as the treatment observation time period of the antibacterial drug (x 2=46.56, P < 0.0001).
In summary, the preferred operation steps are: XDR-AB single colonies were removed from the bacterial plate and the bacterial concentration was adjusted to 0.5 McLeod units (approximately 1.5X 10) in 20% BHI using a turbidimeter8CFU/mL), the nematodes cultured to L4 after synchronization were washed off the plate and centrifuged (500 r/min, 1 min)) Repeating the above steps for 2-3 times, and suspending the nematodes in the solution at a final concentration of 5 × 106After being infected in XDR-AB of CFU/mL for 6h, the nematode is washed by BHI liquid culture medium for at least three times, the nematode is distributed in a 96-well plate, 15-20 nematodes are added to 180 mu L per well, 20 mu L of drug to be detected (dissolved in 1% DMSO) is added to a treatment group, 20 mu L of polymyxin B (dissolved in 1% DMSO) is added to a positive control group, 20 mu L of 1% DMSO is added to a negative control group, the nematode is cultured for 30h under the conditions of 25 ℃ and 85% humidity, and the survival rate of the nematode is observed under a microscope.
Example 2 toxicity testing of efflux Pump inhibitors
1. Washing the nematodes synchronously cultured to the L4 stage into a 15mL centrifuge tube by using a 20% BHI liquid culture medium, washing for three times at 500 r/min for 1min, subpackaging 15-20 nematodes/well into a 96-well plate to 180 mu L, respectively adding serial concentrations of an efflux pump inhibitor CCCP, PA beta N (5 mu g/mL, 10 mu g/mL, 15 mu g/mL, 20 mu g/mL, 25 mu g/mL, 30 mu g/mL, 35 mu g/mL, 40 mu g/mL), NMP, omeprazole, verapamil, reserpine (10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL, 50 mu g/mL, 60 mu g/mL, 70 mu g/mL, 80 mu g/mL), 20 mu L, non-co-culturing for 30h under the condition of 25 ℃ and 85% humidity, observing the survival rate of the nematodes under a microscope, and judging the toxic effect of each efflux pump inhibitor on normal nematodes.
2. Results
The 6 efflux pump inhibitors CCCP, NMP, PA beta N, omeprazole, verapamil and reserpine have different toxic effects on nematodes, wherein significant toxic effects start to appear on nematodes when the concentration of CCCP is greater than 5 mug/mL, the concentration of PA beta N is greater than 25 mug/mL and the concentration of reserpine is greater than 30 mug/mL (figure 6), and the survival rate of nematodes starts to decline. Omeprazole, verapamil and NMP have small toxic effects, have no influence on the survival rate of nematodes within the range of 80 mug/mL, but the active survival state of the nematodes is obviously reduced when the concentration is more than 60 mug/mL after observation.
Example 3 in vitro drug susceptibility testing of ciprofloxacin and various efflux pump inhibitors
1. Method for measuring Minimum Inhibitory Concentration (MIC) of ciprofloxacin antibacterial drug by trace broth dilution method
Escherichia coli ATCC25922, BowmanThe Actinomyces ATCC19606 was tested as a quality control strain in the same lot. Preparing 5120 mu g/mL antibacterial drug stock solution, taking 13 sterile test tubes, preparing ciprofloxacin hydrochloride with series concentrations by using a CAMHB culture medium, wherein the concentration of ciprofloxacin hydrochloride is 512 mu g/mL, 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL, 1 mu g/mL, 0.5 mu g/mL, 0.25 mu g/mL and 0.125 mu g/mL, and 100 mu L of each antibiotic drug stock solution is put in a 96-well plate; freshly cultured colonies were collected using a photometric turbidimeter and the concentration of the bacterial suspension was adjusted to 0.5 McLee units (approximately 1.5X 10)8CFU/mL), 100. mu.L of each well was added to give a final concentration of 5X 106CFU/mL, ciprofloxacin concentration of each well was 256. mu.g/mL, 128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.0625. mu.g/mL, and the wells were incubated in an incubator at 35 ℃ for 16 to 20 hours, and the results were evaluated in μ g/mL according to CLSI (clinical and Laboratory Standards institute) standard. And (3) determining the MIC value of each efflux pump inhibitor and the MIC value of ciprofloxacin added with the efflux pump inhibitor, respectively adding each efflux pump inhibitor according to the toxicity test result, and comparing the change of the MIC value of the strain to the antibacterial agent before and after adding the efflux pump inhibitor.
2. Results
As shown in the following table 1, when CIP is used alone, the MIC value of XDR-AB reaches 256 mu g/mL, when CCCP and PA beta N are used alone, the MIC value is 15 mu g/mL and 30 mu g/mL, when NMP, omeprazole, verapamil and reserpine are used alone, the obvious bacteriostatic action on XDRAB is not generated, and after the EPIs in the toxicity range are added, the MIC value of CIP is reduced by 2-4 times.
TABLE 1
Example 4 in vivo efficacy of Ciprofloxacin (CIP) in combination with different EPIs against infected nematodes
1. And (3) obtaining the infected nematodes by non-coculture of the synchronized nematodes according to the established infection model, treating the infected nematodes by combined medication, determining the drug concentration within a toxic range, setting the drug concentrations of the high group, the low group and the medium group, observing the survival rate of the nematodes after 30h, and judging the in-vivo treatment effect of different EPIs on the infected nematodes by combining CIP under different drug concentrations. Infection experiments were performed 3 times independently, with positive and negative control experiments performed simultaneously.
2. Results
After the treatment drug is added for 30 hours, the CCCP is combined with CIP under the conditions of 5 mug/mL, 2.5 mug/mL and 1 mug/mL, the effect is not ideal, and the survival rate of the nematodes is almost zero. As shown in FIG. 7, the combination of PA β N and CIP can significantly improve the therapeutic effect of CIP in infected nematodes, wherein different concentrations of PA β N have significant differences (P < 0.0001) in the combination effect. The PA beta N has better combined action (P is less than 0.0001) with the CIP when the concentration is lower, and the survival rate of the infected nematodes can be improved by 30-40% compared with that when the CIP is singly used. The omeprazole combined CIP with different concentrations has obvious difference on the treatment effect of the nematode infection (P is less than 0.0001), wherein the omeprazole combined treatment effect with low concentration (P is less than 0.0001) is better, and the survival rate of the nematode infection can be improved by 20 to 30 percent compared with the survival rate of the nematode infection when the CIP is singly used. When NMP, reserpine and CIP with different concentrations are used for treating the infected nematodes, no significant difference (P is more than 0.05) is found, in order to reduce the potential drug toxicity caused by the combined use of the drugs and the EPIs, the survival rate of the infected nematodes after being treated is improved by 15-20 percent (P is less than 0.0001) compared with that of the infected nematodes after being used by combining the low-concentration 20 mug/mL NMP and CIP, and the survival rate of the infected nematodes after being treated is improved by 20 percent (P is less than 0.0001) compared with that of the infected nematodes after being used by combining the low-concentration 10 mug/mL reserpine and CIP. When the verapamil with different concentrations is used in combination with the CIP, the treatment effect of the verapamil at high concentration is obviously better (P is less than 0.0001), and the survival rate of the infected nematodes after the verapamil with the high concentration of 60 mug/mL is used in combination with the CIP can be improved by about 30%. The result can be used as the starting point of the drug effect of structural optimization of each efflux pump inhibitor, thereby realizing the attenuation and the synergy, effectively inhibiting the ciprofloxacin resistance of acinetobacter baumannii and protecting the final aim of the health of people.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
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