| AIM: Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) are common and important pathogens causing hospital infections. Treatment of staphylococcal infections is becoming very difficult and mortality rates are on-going high because of the increasing emergence of resistance to virtually all of theβ-lactams and a wide variety of antimicrobials. In recent years the number of MRSA and MRSE infections is gradually on the rise. Especially the epidemiology of MRSA may be undergoing a change because MRSA is isolated not only in hospital but also in community. Furthermore community-acquired MRSA (CA-MRSA) prevails and spreads wide with high isolating rate, and a particularly serious aspect of CA-MRSA is that it is capable of causing serious infectious illness in healthy individuals, sometimes even fatalities. The prevalence of CA-MRSA is becoming a serious public health problem, and arousing great concern in the medical profession. Now vancomycin is most effective to MRSA and MRSE, but isolation of vancomycin-resistant S. aureus has been documented. So it is evident that we need to seek a new defending strategy against MRSA and MRSE. Alteration of membrane-bound enzymes known as penicillin-binding proteins (PBPs) has been identified as a primary mechanism of"intrinsic"methicillin resistance of MRSA and MRSE. PBP2a is known to be encoded by a chromosomal gene known as mecA. Our previous study had proved that antisense oligonucleotide targeting mecR1, upstream gene of mecA, can partially reverse the resistance of MRSA to oxacillin. But because we lacked effective drug delivery system, inhibition of PS-ODN was limited. In addition, upstream gene of mecA readily mutates or deletes in clinical isolates of S. aureus, therefore PS-ODN targeting mecR1 is only suitable for MRSA strains harboring complete SCCmec elements. Therefore, the present investigation was designed for two key purposes: 1. According to the characteristics of bacterial cell wall and PS-ODN, we prepare a novel kind of liposome with high efficient transfection of PS-ODN through an improved formulation. 2. To evaluate whether PS-ODN targeting mecA, the downstream gene of SCCmec elements, could absolutely restore the susceptibility of MRSA and MRSE toβ-lactam antibiotics in vitro and in vivo.METHODS:1. Preparation and characterization of a new kind of encapsulating PS-ODN/PEI nanometer particle liposome: Choose mecA mRNA as target gene to design and synthesis PS-ODN. At first we prepared condensed PS-ODN/PEI nanometer particle, and then the liposome was prepared by thin film-dispersion technique with EPC, DMPG and PEG2000-DSPE. The mean diameter of PS-ODN/PEI nanometer particle and liposome were determined by a laser light scattering particle-size analyzer. A gel chromatography was used to separate the non-encapsulated drug from the liposomal dispersion. And then the amount of PS-ODN was monitored by UV absorbance at 260 nm (A260nm) using an UV/Visible Spectrophotometers to calculate encapsulation efficiency of liposome. In vitro release experiments were carried out at different temperature to evaluate the stability of liposome.2. Reversal of resistance of MRSA by anti-mecA mRNA PS-ODN010/PS-ODN853 encapsulated in liposome in vitro and in vivo: Different concentrations (0.7,2,6,18μM) of PS-ODN010 or PS-ODN853 were introduced into MRSA by liposome. The total colony forming unit (CFU) per sample was determined by correcting the colony count for the dilution and the change of MRSA growth rates in the broth medium was monitored by A630 measurements at different time points. Drug-resistant characters of MRSA were evaluated by measuring minimal inhibitory concentration (MIC) of different antibiotics. To determine whether the expression of mecA was inhibited after anti-mecA PS-ODN treatment, real-time PCR was used. Establish BALB/c mouse intraperitoneal model of WHO-2 infection and observe the effect on the survival rate of mouse by PS-ODN853 encapsulated into liposome treatment. In vitro experiments, MRSA stains were treated by PBS, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome (18μM), free PEI (0.2μM), free PS-ODN (18μM) and encapsulated PS-ODN liposome (PS-ODN at a concentration of 0.7, 2, 6 or 18μM), respectively. In vivo study, BALB/c mouse respectively received isotonic sodium chloride solution (control group), 100 mg/kg oxacillin (twice daily, for 7 days), encapsulated PBS liposome (once daily, for 3 days) combined with 100 mg/kg oxacillin(twice daily, for 7 days), 10 mg/kg free PS-ODN853 (once daily, for 3 days) combined with 100 mg/kg oxacillin (twice daily, for 7 days) or 2.5, 5, 10 mg/kg PS-ODN853 encapsulated into liposome (once daily, for 3 days) combined with 100 mg/kg oxacillin(twice daily, for 7 days).3. Reversal of resistance of MRSE by anti-mecA mRNA PS-ODN853 encapsulated in liposome: Different concentrations (0.7,2,6,18μM) of PS-ODN853 were introduced into MRSE by liposome. The total colony forming unit (CFU) per sample was determined by correcting the colony count for the dilution and the change of MRSE growth rates in the broth medium was monitored by A630 measurements at different time points. Drug-resistant characters of MRSE were evaluated by measuring minimal inhibitory concentration (MIC) of different antibiotics. To determine whether the expression of mecA was inhibited after anti-mecA PS-ODN853 treatment, real-time PCR was used. In above experiments, MRSE stains were treated by PBS, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome (18μM), free PEI (0.2μM), free PS-ODN853 (18μM) and encapsulated PS-ODN853 liposome (PS-ODN853 at a concentration of 0.7, 2, 6 or 18μM), respectively.RESULTS:1. Preparation and characterization of a new kind of liposome encapsulating PS-ODN/PEI nanometer particle: The encapsulation efficiencies of liposomes were found to be 79.7%±2.69%. The representative particle size of PS-ODN/PEI nanometer particle and liposomes were 82.0±21.0 nm and 217.1±78.6nm, respectively. The stability study of liposomes showed that, the liposomes retained 76.32% and 70.1% of the encapsulated drug until 14 days at 4℃and room temperature, respectively. Though the results obtained at 4℃and at room temperature were statistically comparable, larger fluctuations were observed with liposome-encapsulated PS-ODN maintained at room temperature than at 4℃. Analysis of the samples from liposomes preparations collected at different time points for 2 days maintained at 37℃showed sustained significant release of PS-ODN, and at second day, about 40% of the drug was released from the liposomes, which indicated PS-ODN could slowly release from liposomes at 37℃.2. Reversal of resistance of MRSA by anti-mecA mRNA PS-ODN010/PS-ODN853 encapsulated in liposome in vitro and in vivo:⑴Compared to the untreated group (control), the number of MRSA colonies (WHO-2 or MRSA071001) was decreased significantly in free anti-mecA PS-ODN010/PS-ODN853 treated group and all encapsulated anti-mecA PS-ODN010/PS-ODN853 liposome treated groups (P < 0.01). However, compared to control, the CFU counts of MRSA were not influenced in encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome and free PEI treated group (P > 0.05). Compared with free PS-ODN, PS-ODN encapsulated into liposome (at different concentration) could decreased the CFU significantly (P < 0.01), although free PS-ODN also decreased the CFU.⑵Results showed marked growth inhibition of MRSA cells by oxacillin treated with PS-ODN010/PS-ODN853 encapsulated into liposomes as compared to cells grown in control, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome, free PS-ODN and free PEI treated group. At the same time, the growth of MRSA was inhibited in a concentration-dependent manner when cells were treated with different concentrations PS-ODN encapsulated in liposomes.⑶Compared to untreated group, The MICs of six antibiotics for all MRSA strains were reduced obviously in encapsulated PS-ODN853 liposome treated groups.⑷PS-ODN853 encapsulated into liposome significantly inhibited the expression of mecA mRNA concentration-dependently. The PS-ODN203 showed no effects on the expression of mecA.⑸In vivo study, 26.7%, 46.7% and 53.3% mouse survived in encapsulated anti-mecA PS-ODN853 (2.5, 5, 10 mg/kg) liposome treated group, while 100% mouse died in other control groups. Compared to control group, the CFU of WHO-2 strain in blood samples reduced significantly in encapsulated anti-mecA PS-ODN853 (2.5, 5, 10 mg/kg) liposome treated group.3. Reversal of resistance of MRSE by anti-mecA mRNA PS-ODN853 encapsulated in liposomes in vitro:⑴Compared to the untreated group (control), the number of MRSE070901 colonies was decreased significantly in free anti-mecA PS-ODN853 treated group and all encapsulated anti-mecA PS-ODN853 liposome treated groups (P < 0.01). However, compared to control, the CFU counts of MRSE070901 were not influenced in encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome and free PEI treated group (P > 0.05). Compared with free PS-ODN853, PS-ODN853 encapsulated into liposome (at different concentration) could decreased the CFU significantly (P < 0.01), although free PS-ODN853 also decreased the CFU.⑵Results showed marked growth inhibition of MRSE070901 cells by oxacillin treated with PS-ODN853 encapsulated into liposomes as compared to cells grown in control, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome, free PS-ODN853 and free PEI treated group. At the same time, the growth of MRSE070901 was inhibited in a concentration-dependent manner when cells were treated with different concentrations PS-ODN853 encapsulated in liposomes.⑶Compared to untreated group, The MICs of six antibiotics for MRSE070901 were reduced obviously in encapsulated anti-mecA PS-ODN853 liposome treated groups.⑷PS-ODN853 encapsulated into liposomes significantly inhibited the expression of mecA mRNA concentration-dependently. The PS-ODN203 showed no effects on the expression of mecA. CONCLUSION:1. Anti-mecA mRNA PS-ODN010 and PS-ODN853 encapsulated into liposome selectively inhibited the expression of mecA mRNA, and increased the killing effect ofβ-lactam antibitics against MRSA standard strain WHO-2 and clinical strains, as well as conversing phenotype of antibiotic resistance of MRSA to drug sensitivity.2. We first observed that anti-mecA mRNA PS-ODN853 encapsulated into liposome could restore WHO-2 strain sensitivity to oxacillin in mouse intraperitoneal model of WHO-2. Therefore oxacillin rescued 53.3% of infected animals.3. Anti-mecA mRNA PS-ODN853 encapsulated into liposomes selectively inhibited the expression of mecA mRNA, and increased the killing effect ofβ-lactam antibitics against a MRSE clinical strain MRSE070901, as well as conversing phenotype of antibiotic resistance of MRSE to drug sensitivity.4. We first prepared a novel liposome which encapsulated PS-ODN/PEI nanometer particle. This kind of liposome was stable and easy to obtain with high encapsulation efficiency. We certified liposome could availably delivery PS-ODN/PEI nanometer particle into MRSA and MRSE cells, and inhibited the expression of mecA mRNA. This kind of liposome encapsulating PS-ODN/PEI nanometer particle could to be an efficient vector in delivering PS-ODN into bacteria.5. The blockade of resistant gene of bacteria by antisense drug might be a new viable strategy to preserve the efficacies of existingβ-lactam antibiotics to resistant bacteria.
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