Antibacterial activity and mechanism of moxifloxacin nanoparticles against drug-resistant Pseudomonas aeruginosa / 药学学报

We prepared moxifloxacin (MXF) loaded nanoparticles by nano-precipitation/self-assembly method, then compared the antibacterial activity of MXF and MXF loaded nanoparticles, and investigated the antibacterial mechanism of MXF loaded nanoparticles against Pseudomonas aeruginosa in vitro. The physicochemical properties such as particle size and zeta potential were investigated by laser particle size analyzer. The in vitro release characteristics were investigated by high performance liquid chromatography (HPLC). The effect of nanoparticles on HBE cells viability was investigated by CCK-8 assay. In addition, the in vitro antibacterial activity was investigated by minimum inhibitory concentration (MIC) assay, biofilm formation assays and transmission electron microscope (TEM) observation, then the antibacterial mechanism was initially explored. The particle size measurement showed that the nanoparticles had a size of 332.5 ± 2.7 nm, a polymer dispersion index (PDI) of 0.125 ± 0.053, a zeta potential of -24.3 ± 1.7 mV, and a uniform particle size distribution, drug loading content was (6.02 ± 1.27) %, encapsulation efficiency was (16.69 ± 1.17) %. The TEM results show that the nanoparticles have a spheroidal structure, and the particle size and distribution are consistent with the particle size measurement results. The nanoparticles can be effectively and rapidly released in phosphate buffer saline (PBS), releasing about 70% in 24 h, and releasing 87% in 72 h, and almost completely releasing the MXF at 120 h. At the same time, compared with moxifloxacin free drug, its MIC value is 8 μg·mL-1, which is 1/2 of MXF solution, and can significantly inhibit the formation of bacterial biofilms. It has well antibacterial activity in vitro and can be targeted to the surface of bacteria to exert its efficacy and improve the antibacterial effect. The moxifloxacin nanoparticles prepared in this study has a uniform particle size distribution, well drug release performance and antibacterial effect, and provides new ideas and strategies for the treatment of bacterial lung infection and the development of new antibacterial nanoformulations.

Metabolic Characteristics of WBC-deplted RBC suspension during Different Storage Stage in MAP Based on UPLC-MS/MS / 中国实验血液学杂志

OBJECTIVE@#To explore the metabolic characteristics and metabolic markers of WBC-depleted RBCs in MAP preservation solution and to analyzed the metabolic profile of RBC in MAP preservation solution by using metabolomics.@*METHODS@#The changes of metabolitcs in 10 U WBC-depleted RBC suspension at 3-different storage period (D 0, D 14 and D 35) were detected by using the UPLC-MS/MS, the charaeteristic ions and metabolic markers of RBC stored in preservation sblution for 0 d, 14 d and 35 days were analyzed by using the principal component analysis(PCA).@*RESULTS@#The number of characteristic ions in RBC and supernatant extracts detected during the initial, middle and final storage could be clearly distinguiseed. The 5 metabolism-related substamces such as lact-c acid, nicotinamide, glucose, 5-htdroxyproline and malic acid showed statistically significant difference in 3 storage period.@*CONCLUSION@#The UPLC-MS/MS method combined with statistical analysis of multivariate data can be used to study the metabolic characteristics of RBC, the different metabolites of RBC in different storage stages can be used as the potential markers for evaluation of guality of RBC in storag period. The results of this study provide a basis for studing the RBC guality changes in storage period.

Study of in vitro endotoxin neutralization effect and antibacterial activity of limulus anti-lipopolysaccharide factor simulated peptide REMP2 / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the role of REMP2 derived from limulus anti-lipopolysaccharide factor in neutralizing endotoxin in vitro and its antibacterial activity.</p><p><b>METHODS</b>(1) REMP2 and PMB in the concentrations of 100.00, 10.00, 1.00, 0.10, 0.01 micromol/L were respectively mixed with LPS (lEU/mL), with PMB as positive control. The LPS concentrations in different specimens were determined by routine method, and the neutralizing percentage was respectively calculated. (2) After adding isotonic saline (NS), the final concentrations of REMP2 and PMB were 10, 20, 40, 80 micromol/L, and the concentration of LPS was 100 microg/L. The murine monocytic macrophages were stimulated with LPS, then cultured with REMP2 and PMB, with NS in culture as negative control. The content of tumor necrosis factor (TNF)-alpha was determined by ELISA kit. (3) The morphologic changes of Escherichia coli. was observed under electron microscope at 10, 20 and 40 minutes after addition of REMP2 to Escherichia coli suspension (with terminal concentration of REMP2 at 40 micromol/L).</p><p><b>RESULTS</b>There were no significant difference in endotoxin-neutralizing percentages between PMB and REMP2 in concentrations of 0.10, 10.00, 100.00 micromol/L (P > 0.05). The contents of TNF-alpha were 1175 +/- 162, 859 +/- 122, 645 +/- 142, 489 +/- 102 ng/L, respectively,after treatment of 10, 20, 40, 80 micromol/L REMP2, which were obviously lower than that of NS (3463 +/- 218 ng/L, P < 0.01). Under transmission electron microscope, the outer and interior membranes of Escherichia coli were obscure and rough, bacterial bodies were swollen with vacuoles in cytoplasm after treatment with REMP2.</p><p><b>CONCLUSION</b>REMP2 has ability of neutralizing endotoxin and also antibacterial activity.</p>

Reproduction of the murine endotoxin shock model in D-galactosamine-sensitized KunMing mice / 中华烧伤杂志

<p><b>OBJECTIVE</b>To reproduce a Kunming murine endotoxin shock model suitable for the anti-endotoxin pharmaceutical research.</p><p><b>METHODS</b>Kunming mice were challenged with an intraperitoneal (i. p.) injection of different doses of D-galactosamine (D-Gal) and endotoxin (LPS) and divided into 10 groups: i.e, group 1 [with injection of isotonic saline solution (NS) and LPS]; group 2 (with injection of NS and 90mg/kg LPS), group 3 (with injection of NS and 500mg/kg D-Gal), group 4 (with injection of 500mg/kgD-Gal and 25 microg /kg LPS), group 5 (with injection of 500mg/kg D-Gal and 50 microg/kg LPS), group 6(with injection of 500mg/kg D-Gal and 250 microg/kg LPS), group 7( with injection of NS and 600mg/ kg D-Gal), group 8 (with injection of 600mg/kg D-Gal and 10 microg/kg LPS), group 9( with injection of 600mg/kg D-Gal and 25 microg/kg LPS), group 10 (with injection of 600mg/kg D-Gal and 50 microg/kg LPS). The death of the mice were observed and the mortality rate was recorded at 48 post-injection hour (PIH). The dose of D-Gal and LPS which caused 100% lethality was chosen for the subsequent experiment to serve as control group (with injection of NS and 600mg/kg D-Gal), LPS group (with injection of 600mg/kg D-Gal and 580mg/kg LPS for later experiment). The venous blood of the mice were collected for the detection of serum content of TNF-alpha with ELISA method at 30, 75 and 120 post-injection minutes (PIM). The tissues of lung, liver, intestine were also harvested at 5 PIH for the pathological examination.</p><p><b>RESULTS</b>The lethality of mice was 100% in the groups 2, 6 and 10 (P < 0.01). The serum content of TNF-alpha was maintained in a low level in control group, but it increased remarkably in LPS group, and it reached peak at 75 PIM (6365 +/- 2087ng/L, P < 0.01). Obvious inflammatory reaction was observed in the lung, liver and intestine in LPS group, while only mild inflammatory reaction was observed in liver in control group.</p><p><b>CONCLUSION</b>The Kunming mice showed signs of endotoxin shock after D-galactosamine presensitizing and endotoxin challenge, and it is suitable for anti-endotoxin pharmaceutical research.</p>

Determination of prulifloxacin active metabolite in human plasma and urine by RP-HPLC / 中国感染与化疗杂志

Objective To establish a RP-HPLC method for determining the concentration of prulifloxacin active metabolite in human plasma and urine.Methods The supernatant obtained by centrifugation after the sample was precipitated with methanol- acetonitrile (1:1) was chromatographically separated on a Diamonsil C_(18)(250 mm?4.6 mm,5?m) using a mobile phase con- sisting of acetonitrile and 0.05 mol/L potassium dihydrogen phosphate (pH2.2) containing 1% tetrabutylammonium bromide. The solutions of 20:80 (V/V) and 12:88 (V/V) at a flow rate of 1.0 mL/min and 1.6 mL/min were used for plasma and u- rine, respectively.Then the samples were assayed at wavelength of Ex 280 nm and Em 425 nm.Results The linear range for prulifloxacin active metabolite in plasma and urine were 0.005-5 mg/L (r=0.9999) and 0.05-5 mg/L(r=0.9999)with a low- er limit of quantitation of 0.002 mg/L and 0.01 rag/L, respectively.In plasma, the relative recovery ranged from 100.64% to 101.00% at the concentration of 5.00, 0.50 and 0.05 mg/L and within-day and between-day precisions were less than 2.5% and 4.6% respectively.Meanwhile, the relative recovery ranged from 97.20% to 100.20% at the concentration of 2.50, 0.50 and 0.10 mg/L in urine.The within-day and between-day precisions were lower than 1.3% and 4.3%, respectively.The method had been successfully used for the pharmacokinetic studies of a prulifloxacin formulation after oral administration to healthy volunteers.Conclusions The present method is simple, rapid, accurate, reproducible and suitable for the pharmacoki- netic study of prulifloxacin in humans.

Isolation and analysis of the drug resistance of the flavobacterium and its production of beta-lactamases / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the drug resistance of flavobacterium and its ability to produce BLA (beta-lactamases) and ESBLs (Extended-spectrum beta-lactamases).</p><p><b>METHODS</b>The production of BLA and ESBLs from 6 clinical isolated flavobacterium strains was determined by nitrocefin disc test and double-disc synergy method, respectively. The antibiotic susceptibilities of the strains were determined by Kirby-Bauer disc diffusion test and the agar dilution method and the MIC was assessed.</p><p><b>RESULTS</b>All the six flavobacteria were BLA-producing strains and more than 80% of them were ESBLs-producing, and they were highly resistant to beta-lactamase antibiotics (MIC 32 - 256 mg/L), but susceptible to fluoroquinolones and cephalosporin with beta-lactamase inhibitors (MIC 0.125 - 8 mg/L).</p><p><b>CONCLUSION</b>Most of the flavobacteria in nosocomial infections were beta-lactamase-producing and were highly resistant to beta-lactamase antibiotics. Fluoroquinolones and beta-lactamase antibiotics with lactamase inhibitors should be the first choice for the management of infection caused by flavobacterium.</p>

Influence of intra-abdominal hypertension on the intestinal permeability and endotoxin/bacteria translocation in rabbits / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe different degrees of intra-abdominal pressure and different duration on the intestinal permeability and endotoxin/bacteria translocation in rabbit model, so as to explore the mechanism of the development of abdominal compartment syndrome (ACS) and MODS.</p><p><b>METHODS</b>Rabbit model of intra-abdominal hypertension was established by injection of gaseous nitrogen into the peritoneal cavity. Thirty-nine New Zealand white rabbits were employed in the study. The change in intestinal permeability was determined by fluorescein isothiocyanate dextran (FITC-D) and two kinds of molecular probes of type II horseradish peroxidase (HRP-II). The effects of intra-abdominal hypertension on the endotoxin/bacteria translocation were also detected.</p><p><b>RESULTS</b>The contents of FITC-D and HRP-II in portal veins increased evidently (P < 0.01) when intra-abdominal pressure (IAP) was higher than 20 mmHg. The endotoxin (ET) content in portal vein in rabbits with IAP of 10 mmHg for 1, 2 and 4 hours exhibited no difference compared with that in normal control, while the ET content increased obviously after 1 hour with IAP of 20 mmHg and increased thereafter along with the prolongation of IAP, and increase in pressure. The bacterial translocation rates were 33.3%, 66.7% and 100% when IAP was maintained at 20 mmHg for 1, 2 and 4 hours, respectively, and there was evidence of bacterial translocation to the liver. The rate of bacterial translocation to intestinal mesenteric lymph nodes was 100% when IAP was 30 mmHg for 1 and 2 hours. There was no bacterial translocation to the spleen in all experimental rabbits.</p><p><b>CONCLUSION</b>Intestinal mucosal permeability increased significantly with increased endotoxin content in portal vein when IAP was higher than 20 mmHg. At the sane time, the bacteria could be translocate to intestinal mesenteric lymph nodes and liver, which might be constitute one of the important factors leading to the development of ACS and MODS.</p>

Preliminary study of the lethal effects of E. coli DNA on mice / 第三军医大学学报

Objective　To investigate the role of bacterial DNA in systemic inflammatory response syndrome (SIRS). Methods　A total of 100 mice of Kunming species were divided into ten groups: E.coli DNA (30, 20, 10, 5 and 1 mg/kg ), 30 mg/kg of CT DNA, 60Co DNA, DNased DNA, organic residue of DNA extraction and sterile water control. The last two were pre-treated with D-galactoamine (600 mg/kg intra peritoneally). Animals were administratively injected via tail vein. General physical condition and the death rate of mice were observed within 48 h. Results　①Obvious lethal effect of double strand E.coli DNA on mice were observed with a dose-effect correlation, LD50=11.51 mg/kg. ②NO difference in death rate was found in the group of 30 mg/kg E.coli DNA with or without 60Co irradiation (10/10 and 8/10,P>0.05). ③No rats died in the group of DNased DNA, organic residue of DNA extraction and calf thymic DNA (0/10). Conclusion　Bacterial DNA may play an important role in the development of SIRS.

Preliminary study of the lethal effects of E. coli DNA on mice / 第三军医大学学报

Objective　To investigate the role of bacterial DNA in systemic inflammatory response syndrome (SIRS). Methods　A total of 100 mice of Kunming species were divided into ten groups: E.coli DNA (30, 20, 10, 5 and 1 mg/kg ), 30 mg/kg of CT DNA, 60Co DNA, DNased DNA, organic residue of DNA extraction and sterile water control. The last two were pre-treated with D-galactoamine (600 mg/kg intra peritoneally). Animals were administratively injected via tail vein. General physical condition and the death rate of mice were observed within 48 h. Results　①Obvious lethal effect of double strand E.coli DNA on mice were observed with a dose-effect correlation, LD50=11.51 mg/kg. ②NO difference in death rate was found in the group of 30 mg/kg E.coli DNA with or without 60Co irradiation (10/10 and 8/10,P>0.05). ③No rats died in the group of DNased DNA, organic residue of DNA extraction and calf thymic DNA (0/10). Conclusion　Bacterial DNA may play an important role in the development of SIRS.