| BACKGROUD AND AIMPseudomonas aeruginosa and Escherichia coli was the most frequentpathogenic bacteria in clinical infectious diseases. Because of long-term usingand abusing antibiotic, which induced more and more bacterial drug resistanceproblems.Multiple-drug resistant Pseudomonas aeruginosa (MDR-PA) has highinfection rate and case-fatality rate, it was an independent risk factor to induceempyrosis patient died. The isolating rate of fluoroquinolones-resistantEscherichia coli (FREC) was continual rising with using FQNS. So it wasimminent for us to look for a new strategy to control infection of MDR-PA andFREC. Efflux pump was a key factor in MDR-PA and FREC. MexAB-OprMwas the most efflux pump in MDR-PA, among the total, oprM gene codogenicouter membrane protein OprM was played an important role in MDR-PA. But in FREC, AcrAB-TolC was the major efflux pump. AcrAB was a significanceproteinum to determine Escherichia coli resistant to FQNS. In this study, oprMand acrB mRNA was a target gene, reversal MDR-PA and FREC drug-resistantby inhibiting efflux pump though thio-oligodeoxynucleotide.METHODS1. Preparation of polyethyleneimine (PEI) and phosphorothioateoligodeoxynucleotide (PS-ODNs) nanometer particle liposome: Choose oprM astarget gene to design and synthesis PS-ODNs (PS-ODNs617) and acrB mRNAas target gene to design and synthesis PS-ODNs (PS-ODNs831). At first, PEIand PS-ODNs was condensed into PEI-ODN nanometer through particleelectrostatic interactions, then the liposome was prepared by thin film-dispersiontechnique with EPC, DMPG and PEG2000-DSPE in 14:0.9:1 molar ratio. Themean diameter of PEI-ODN nanometer particle and PEI-ODN nanometerparticle liposome were determined by a laser light scattering particle-sizeanalyzer. The gel chromatography was used to separate the non-encapsulatedPS-ODNs from the liposomal dispersion. And then the amount of PS-ODNs wasmonitored by UV absorbance at 260nm (A260nm) using an UV/VisibleSpectrophotometers to calculate encapsulation efficiency of liposome. Thestability of liposome was evaluated at different temperature in vitro.2. Reversal of resistance of MDR-PA by anti-oprM PS-ODNs617:PS-ODNs617 of different concentrations (3μg/ml,10μg/ml,30μg/ml and100μg/ml) were introduced into MDR-PA by PEI-ODN nanometer particleliposome. After induced, the total colony forming unit (CFU) ofMDR-PA070801 was determined by correcting the colony count for the dilution.The growth inhibiting effect of piperacillin to MDR-PA070801 was determinedby using absorb in A630nm. Drug-resistant character of different MDR-PA strains was evaluated by measuring minimal inhibitory concentration (MIC) ofdefferent antibiotics. To quantify the expression of oprM in MDR-PA070801,real-time RT-PCR was used. The initial template amount was monitored byvarying fluorencence intensity with SYBR Green I in close tubes. The relativeexpression levels of oprM in comparison with 16SrRNA were calculated andused to determine whether the expression of oprM was inhibited after anti-oprMPS-ODNs617 treatment. MDR-PA070801 strains were divided into controlgroup, blank liposome group, envelop random chain liposome group (100μg/mlPS-ODNs0701), free PEI (5.5μg/ml), free PS-ODNs617 group (100μg/mlPS-ODNs617) and envelop PS-ODNs617 liposome treated groups (3,10,30,100μg/ml PS-ODNs617).3. Reversal of resistance of FREC070701 by anti-acrB PS-ODNs831:PS-ODNs831 of different concentrations (3μg/ml,10μg/ml,30μg/ml and100μg/ml) were introduced into competent FREC070701 by PEI-ODNnanometer particle liposome. After induced, the total colony forming unit (CFU)per sample was determined by correcting the colony count for the dilution. Thegrowth inhibiting effect of ciprofloxacin and levofloxacin to FREC070701 wasdetermined by using absorb in A630nm. Drug-resistant character of FREC070701strains was evaluated by measuring minimal inhibitory concentration (MIC) ofciprofloxacin and levofloxacin. To quantify the expression of acrB inFREC070701, real-time RT-PCR was used. The initial template amount wasmonitored by varying fluorencence intensity with SYBR Green I in close tubes.The relative expression levels of acrB in comparison with 16SrRNA werecalculated and used to determine whether the expression of acrB was inhibitedafter anti-acrB PS-ODNs831 treatment. FREC070701 strains were divided intocontrol group, blank liposome group, envelop random chain liposome group (100μg/ml PS-ODNs0701), free PEI (5.5μg/ml), free PS-ODNs831 group(100μg/ml PS-ODNs831) and envelop PS-ODNs831 liposome treatedgroups(3,10,30,100μg/ml PS-ODNs831).RESULTS1. Preparation of PEI-ODN nanometer particle liposome: The envelopmentefficiencies of liposome were found to be 79.7±2.69%. The size of thenanometer particle liposome was 217.1±78.6nm. The stability study of liposomewas indicated that the liposome still retained 76.32% and 70.1% of the envelopedrug until day 14 at 4℃and room temperature respectively. The samples fromliposome preparations were collected at different time points for 2 daysmaintained at 37℃showed that significant release of PS-ODNs, and at secondday, about 40% of the drug was released from the liposome, which indicatedthat PS-ODNs could slowly release from liposome at 37℃.2. Reversal of resistance of MDR-PA by envelop anti-oprM PS-ODNs617liposome: The numbers of MDR-PA070801 colonies on the Mueller-Hinton agarcontaining piperacillin (150μg/ml) were significantly decreased in all envelopanti-oprM PS-ODNs617 liposome treated groups, in concentration dependentmanner with 3,10,30,100μg/ml of PS-ODNs617 respectively (P<0.01), whileCFU of MDR-PA070801 was not influenced in blank liposome group, enveloprandom chain PS-ODNs0701 liposome group, free PEI group. CFU ofMDR-PA070801 was only slight low in free PS-ODNs617 group whilecompared with control group. Piperacillin could inhibit growth ofMDR-PA070801 in all envelop anti-oprM PS-ODNs617 liposome treatedgroups, in concentration dependent manner with 3,10,30,100μg/ml ofPS-ODNs617 respectively, but in other treated groups, piperacillin was no orlittle growth inhibiting effect to MDR-PA070801. MICs of all envelop anti-oprM PS-ODNs617 liposome treated groups were obviously decreased,which were lower than control group and other groups. Envelop anti-oprMPS-ODNs617 liposome treated groups inhibited mRNA expressions of oprM inconcentration-dependently manner. The other treated groups showed no or littleeffects on the expression oprM.3.Reversal of resistance of FREC070701 by envelop anti-acrBPS-ODNs831 liposome: The numbers of FREC070701 colonies on theMueller-Hinton agar containing ciprofloxacin (6μg/ml) and levofloxacin(12μg/ml) were significantly decreased in all envelop anti-acrB PS-ODNs831liposome treated groups, in concentration dependent manner with 3, 10, 30,100μg/ml of PS-ODNs831 respectively (P<0.01), while CFU of FREC070701was not influenced in blank liposome group, envelop random chainPS-ODNs0701 liposome group, free PEI group . CFU of FREC070701 was onlyslight decreased in free PS-ODNs831 group while compared with control group.ciprofloxacin and levofloxacin could inhibit growth of FREC070701 in allenvelop anti-acrB PS-ODNs831 liposome treated groups, in concentrationdependent manner with 3,10,30,100μg/ml of PS-ODNs831 respectively, but inother treated groups, ciprofloxacin and levofloxacin was no or little growthinhibiting effect to FREC070701. MICs of all envelop anti-acrB PS-ODNs831liposome treated groups were obviously decreased, which were lower thancontrol group and other groups. Envelop anti-acrB PS-ODNs831 liposometreated groups inhibited mRNA expressions of acrB inconcentration-dependently manner. The other treated groups showed no or littleeffects on the expression of acrB.CONCLUSION1. The liposome encapsulating PEI-ODN nanometer particle was stable and easy to obtain with high encapsulation efficiency. Pharmacodynamicsexperiment certified liposome could availably delivery PEI-ODN nanometerparticle into MDR-PA and FREC cells.2. Envelop anti-oprM PS-ODNs617 liposome selectively inhibitedexpression of oprM mRNA, and inhibited activity of efflux pump ofMexAB-OprM, then increased the killing effect of different antibiotics againstMDR-PA, as well as conversing phenotype of antibiotic resistance of MDR-PAto drug sensitivity.3. Envelop anti-acrB PS-ODNs831 liposome selectively inhibitedexpression of acrB mRNA, and inhibited activity of efflux pump ofAcrAB-TolC, then increased the killing effect of ciprofloxacin and levofloxacinagainst FREC070701, as well as recovery sensitivity of FREC070701 tociprofloxacin and levofloxacin.
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