| 1.BackgroundPseudomonas aeruginosa,a kind of non-fermented gram-negative bacilli,is an important opportunistic pathogen in the hospital.Carbapenem antimicrobial agents were once used as first-line drug for Gram-negative bacteria because of their extended-spectrum antibacterial activity.However,with its widely clinical used,carbapenem resistant strains were appeared.In particular,infections caused by CRPA have been reported to increase the morbidity and mortality of patients and pose a serious threat to human health.It is difficult to guarantee clinical anti-infection effect by using an antibiotic alone.In vivo and in vitro experiments have proved that the combination of two drugs and even three drugs can achieve a better therapeutic effect.In order to help the clinical diagnosis and treatment of XDR-GNB infection,chose a reasonable way of administration,22 clinicians in China,clinical microbiologists and multi-disciplinary experts such as clinical pharmacologists have reached a consensus related to XDR-GNB infection[9].To deal with XDRPA infection,this consensus recommends the combination of two drugs and three drugs.At present,many non-antimicrobial agents,such as some ion channel modulators acting on human cells,cyclooxygenase inhibitors,histone deacetylase inhibitors,traditional Chinese medicine commonly used as antipyretic detoxification drugs.The antimicrobial activity of traditional Chinese medicine and its monomers have been proved to increase the sensitivity of antifungal drugs.It is reported in the literature that some traditional Chinese medicine extracts can reverse the drug resistance of bacteria.2.ObjectiveThe purpose of this study was to verify the correlation between drug resistance of CRPA strains and carbapenem and biofilm,and to find a new approach to fight CRPA by recommending combined regimen that Chinese experts have agreed and non-consensus regimen for the treatment of XDRPA.3.Methods3.1 Strains collection,MICs determination and drug resistance backgroundDetermination of susceptibility of experimental strains to common clinical drugs Six multidrug resistant P.aeruginosa strains isolated from Qianfoshan hospital in Shandong Province and identified by ATB instrument.According to the method recommended by CLSI M100-S25 for the determination of bacterial sensitivity,micro broth dilution method was used.Thirteen kinds of anti-PA drugs commonly used were prepared into 2560μg/m L with suitable solvent.The antimicrobial agents were diluted by double dilution method,and then added to the 96 well plates from the second column to eleventh,the concentration from low to high,showing a series of gradient concentrations.Column 1 was used as growth control group and column 12 as blank control group.The 96-well plate containing bacteria and drugs,and incubated overnight in a constant temperature incubator at 35℃.The concentration of the drug that could completely inhibit the growth of bacteria was taken as the minimum inhibitory concentration.Carba NP test was used to test whether the bacteria produced carbapenem.We selected the single colony of strains which was activated and inoculated on CAMHB solid medium.The supernatant was isolated and centrifuged,adding phenol red solution(0.05%,PH 7.8±0.1)to the reaction system,imipenem(0.6 mg/m L)and Zn SO4(0.1mmol/L),a total of 100μL solution was obtained.The blank control group was completely consistent with the experimental group except imipenem.After incubated at 35℃ for 2 hours,the color of the system was observed every 0.5 h and compared with the negative control group in white background to determine the production of carbapenem.The biofilm forming ability of the experimental strain was determined by the method of crystal violet staining.The experimental strains dissolved in 4 m L LB medium,concussion cultured for 24 h at 35℃,adjusting the concentration of bacteria to 5×105 CFU/m L,add 200μL per well to 96 well plate,the blank control group was only given sterile LB medium,each strain set 3 holes,the whole system is cultured in a constant temperature incubator at 35℃.After 24 hours,the culture medium was abandoned and the crystal violet solution was added.Then dyeing 15 min at room temperature,washed with PBS phosphate buffer for 3 times.Drying at room temperature,95% ethanol was added and the absorbance value at 590 nm was measured,and the average value of 3 complex holes was obtained.Results: the average value of the blank control hole was 3 times the standard deviation of the blank control as the cut off value.The A590 nm and Ac values of the experimental group were compared to judge the ability of bacterial membrane formation.3.2 Evaluation of the effect of the drug-combinations against XDRPA recommended by Chinese experts on CRPADilution all drugs of each drug combination by using broth microdilution method,and then add the diluted drugs to the 96-well plate according to the concentration of the liquid from high to low.Next add the adjusted concentration of bacteria,resulting in a final concentration of 5×105 CFU/m L,sterile CAMHB liquid medium as negative control group.Using FICI model to determine whether each drug combination has synergistic effect.First of all,PA11 was used as the representative strain,all combinations were recommended for preliminary activity screening,9 drug combinations with synergistic effect were tested in the results of primary screening,and the combined activity of 6 strains of CRPA was further tested,and the synergistic antibacterial effect of drug combination was determined by FICI model.3.3 In vitro combined effect of non-consensus recommended drug combination on CRPAIn this part of the experiment,the microchessboard dilution method was used to evaluate the activity of some antimicrobial drugs and some non-PA drugs which were not recommended by consensus.Antimicrobial combinations include: imipenem+ levofloxacin;imipenem+azithromycin;ceftazidime+levofloxacin;ceftazidime+azithromycin;fosfomycin+rifampicin.Non-PA drugs include: iron ion interference drugs gallium nitrate,gallium chloride;bacterial protein synthesis inhibitor linezolid;cyclooxygenase inhibitor licofelone;phenylbutyric acid;mucolysis drug ambroxol;ion channel regulator carbamazepine;Ion channel regulator drugs verapamil,amlodipine,amiodarone,flunarizine 5-HT uptake inhibitor fluoxetine,sertraline;traditional Chinese medicine extract luteolin,rhein,butyphthalide.The synergistic effect of combinations were evaluated by the FICI model.4.Result4.1 Collection of strains,MIC determination and drug resistance background analysisThe results of drug sensitivity test showed that 6 P.aeruginosa strains were resistant to imipenem,meropenem and biapenem,then 6 strains could be identified as CRPAs.In the case of carbapenem resistance,only polymyxin B and colistin still had good antibacterial activity,and no colistin resistant bacteria appeared.In the Carba NP test,the color of the control group was red,and the resistant strain showed yellow,which proved that 6 strains of CRPA could produce carbapenems.In crystal violet staining,all 6 strains of CRPA had A590nm>Ac,which could prove that the selected strains were positive in biofilm formation.In conclusion,imipenem,meropenem and biapenem showed high drug resistance in this experiment.In this experiment,the antimicrobial activity to CRPA was only colistin and polymyxin B.The production of carbapenem and the formation of biofilm were the important reasons for carbapenem resistance of P.aeruginosa.The results of Carba NP test and crystal violet staining showed that both carbapenase and biofilm production of CRPA strain were positive.It can be concluded that carbapenase and biofilm formation are partly responsible for CRPA production.4.2 In vitro effect of Chinese consensus recommended combination of drugs against XDRPA on CRPAThe effect of the drug combination scheme recommended by Chinese experts on six CRPA strains selected in this experiment shows both synergy and addition,and some combinations show independent effects.Among them,the best combination was the combination of two β-lactam drugs,which showed different degrees of synergistic effect against six CRPA strains in this experiment.The synergistic rate of ceftazidime+cefoperazone sulbactam was 100% and ceftazidime+piperacillin tazobactam was 50%.The second was the combination of β-lactam and other drugs,the synergistic rate of aztreonam and fosfomycin was 66.7%.4.3 In vitro combined effect of non-consensus recommended drug combination on CRPAThe results of non-consensus recommended drug combination on CRPA showed that the combination of fosfomycin and rifampicin has a synergistic effect on 6 CRPA strains.The synergistic rates of ceftazidime with levofloxacin and azithromycin were 50%,and imipenem with levofloxacin and azithromycin were 33.3% respectively.Some non-antimicrobial agents can reduce the minimum inhibitory concentration of antimicrobial agents in varying degrees.The results showed that the drugs of non-gram-negative bacilli,licofelone,linazolamine 4-phenylbutyric acid,carbamazepine,Fluoxetine can reduce the MIC value of imipenem by at least one gradient concentration.The ion channel blockers flunarizine and amiodarone combined with imipenem had antagonistic effects.Most other non-antimicrobial agents combined with imipenem did not increase their antimicrobial activity and showed no effect.The MIC values of gallium nitrate and gallium chloride monopharmaceuticals ranged from 4μg/m L to 8μg/m L,and the antimicrobial activity was significant,but neither of them could enhance the effect of antimicrobial agents.5.ConclusionThe formation of carbapenem and biofilm is partly responsible for the production of CRPA.The combined antimicrobial effect of the recommended regimen was partly synergistic,in which two β-lactams had better synergistic effect,among which ceftazidime and cefoperazone-sulbactam had better antibacterial activity.The combination of phosphomycin and rifampicin,non-resistant to Pseudomonas aeruginosa,showed a synergistic effect of 66.7% on 6 strains of drug-resistant bacteria.Most non-antimicrobial agents combined with imipenem did not increase their antimicrobial activity and showed no effect.The antimicrobial effect of gallium nitrate and gallium chloride is significantly.But neither drug could enhance the effect of antimicrobial agents. |
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