| BackgroundPseudomonas aeruginosa and acinitobacter baumannii as the representative of the non-fermentation bacteria is easy to cause hospital acquired infection. In resent years ,as the application of renewal antibiotics,all kinds of immunosuppressant and invasive diagnosis and treatment ,the hospital infection caused by this two kinds of bacterial increased gradually. While the bacterial resistance is more serious recent years, and even appeared resistance to all the anti-Pseudomonas aeruginosa antibiotics besides Polymyxin including Penicillins,Cephalosporins,Single amide classes Flouroquinolones,Aminoglycosides and Carbapenems ,we called it as Pan-grug resistant strains . And in addition to Polymyxin at present all the functionalities of antibiotics is useless ,once patients infected it ,the clinicians became stranded Penicillins. The drug-resistance mechanism of Pseudomonas aeruginosa and acinitobacter baumannii were complicated ,and mainly including following several :(1)the genetic mutation ,insert or missing .(2)change the drug targets.(3)produce inactivated enzymes .These drug-resistant mechanisms common participated in the formation of bacteria resistance issue .At present ,the main constradiction is effective new drug to market rate is low while drug-resistant strains increasing. And now people's research direction mainly has adjusted regimen and research other existing drug's potential antibiotic activity. Different calcium channel blockers coupled with levofloxacin were not identical effects to drug-resistance Pseudomonas aeruginosa in some research. And amlodipine is effective .Also reported respond that adding Macrolides to infections caused by Pseudomonas aeruginosa can improve the clinical cure rate .However , what's the link between these drugs antibacterial effects and the resistance mechanisms were not reported . Therefore it's necessary to carry out a deeper study ,so as to provide theoretical foundation of how treatment of infections caused by drug resistance non-fermentation .ObjectiveTo explore the effects of combined susceptibility and the mechanisms of pan-drug resisitant Pseudomonas aeruginosa and acinitobacter baumannii ,and further more establish in vitro pneumonia model,and carry on the combination,and observe the curative effect,to provide theoretical basis for treatment infections caused by pan-drug resistant strains.Methods1. All strains were identified by VITEK-2 automated system.The combined antimicrobial susceptibility test was evaluated by agar dilution method and chessboard method.25 pan-drug resistance strains (19 strains of Pseudomonas aeruginosa and 6 strains of Acinitobacter baumannii,according to the standard of CLSI 2009 ),were inoculated into different culture medium with different combination of antimicrobial agents ,low concentration group (concentration of antimicrobial agents =1MIC) and high concentration group (concentration of antimicrobial agents=2MIC),For azithromycin ,0.4mg/ml was low concentration group and 3.9mg/ml according to the concentration of peak in blood and the lung tissue after oral intake 500mg azithromycin.2. 25 pan-drug resistance strains were examined by a metallo-beta-lactamas(MBL) E-test.3. Real-time PCR were performed to determinated the xepression of four efflux pumps in 25 pan-drug resistance strains .Four efflux pumps included MexAB-OprM,MexCD-OprJ,MexEF-OprN,and MexXY-OprM。4. One clinical Pseudomonas aeruginosa for strains susceptible to the experiment antimicrobial agents for mouse pneumonia infection .After six-hour infection ,mice were intraperitoneally injected with 0.5ml 0.9% Nacl for control ,mice were intraperitoneally injected with Ceftazidime,Amikacin and Ciprofloxacin ,or Ceftazidime,Amikacin and Ciprofloxacin plus amlodipine respectively for trial. And therapy efficacies were evaluated with bacterial and pathology 1,3,5,7days later.Results1. Synergistic effect of ceftazidim combination with amikacin ,or ciprofloxacine were respectively 84.2% and 36.8%.Through Agar dilution method ,in the low concentration group ,the effects of amikacin combined with ceftazidime ,and azithromycin doxycycline and ciprofloxacin combined with amikacin and ceftazidimes and the strains in the group's combination with high antimicrobial agents concentration were respectively 0,8.0%,0 and 12.0%.After adding amlodipine ,the susceptibility of every combination were respectively 0,0,16.0%and 16.0%(p>0.05)in the low concentration group ;and that were respectively 0,20.0%,0,and 28.0%(p<0.05) in the high concentration group .2. Of the 25 pan-drug resistant strains ,15strains which susceptibility effectively strains belonged to metallo-beta-lactamase negative strains ,and the rest were positive strains .The drug combination of ciprofloxacin and doxycycine group were effective to part of negative enzymes strains ,and were invalid to positive enzymes strains .3. To analyse the efflux pumps gene expression between the combined treatment groups, the strains which effective after adding amlodipine all overexpress for MexAB-OprM,partly overexpress MexCD-OprJ,MexEF-OprN,and MexXY-OprM。And the strains that invalid after adding amlodipine and positive enzymes strains were different degree overexpress every efflux pumps .4. Antimicrobial susceptibility test in vivo showed that whether amlodipine intervention to bacterial ,the was non-significance difference between the two treatment groups to scaveng for bacteria. But had significant difference statistics with control group.Conclusion1. After intervention of amlodipine, the effects of high concentration combination of doxycycine and ciprofloxacin group are significantly effective to part of metallo-beta-lactamase negative strains .2. The efflux pump gene expression was no difference between the strains sensitivity and non-sensitivity in vitro. And amlodipine cannot influence the efflux pump gene expression of pan-drug resistant strains ,maybe just by inhibiting the activity of efflux pump to the effect of antibacterial.3. For Pseudomonas aeruginosa pneumonia,it was still effective to combine antibiotic which ere resistant of drug sensitive test in vitro.After Pseudomonas aeruginosa infection ,there was nothing advantage of amlodipine group compared with non-amlodipine group, suggest that there was insufficient evidence to treat Pseudomonas aeruginosa pneumonia by amlodipine,pneumonia... |
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