EVALUATING THE EFFICACY OF AMIKACIN IN LOW-CLEARANCE UNILAMELLAR LIPOSOMES IN A S. AUREUS LOCAL INFECTION MODEL

Traditional therapies for Staphylococcal infections such as osteomyelitis or localized abscesses have a difficult time penetrating into tissue sites. To effectively ameliorate these infections, prolonged therapy and/or high doses of antibiotics are frequently required. Aminoglycosides, such as amikacin, are not routinely utilized for treating local infections due to poor efficacy associated with ineffective tissue penetration, toxicity, and poor penetration in an acid millieu. We postulated that a formulation of amikacin in small unilamellar liposomes might readily be engulfed by inflammatory macrophages facilitating drug delivery to the site of infection. This increased drug load to the site of bacterial infection may result in enhanced bactericidal action compared to conventional aminoglycosides. Tissue drug concentrations were determined for liposomal amikacin (L-AN) and conventional amikacin (AN). Plasma amikacin levels were determined for L-AN. The L-AN was very effective at concentrating at the site of infection compared to AN. Following confirmation of adequate tissue drug levels, a rodent subcutaneous abscess infection using S. aureus as the bacterial challenge agent was evaluated. Sprague-Dawley rats were intravenously administered L-AN every other day due to its prolonged half-life, while the comparator agent, AN, was administered daily. Abscess size, weights, severity, histology, and tissue colony counts were examined. In efficacy studies, L-AN was superior to AN in reducing colony counts.     

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.     

Doxorubicin Encapsulated in Long-Circulating Thermosensitive Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (TSL, 180-200 nm in mean diameter), prepared from dipalmitoyl phosphatidyl choline (DPPC)/distearoyl phosphatidyl choline (DSPC) (9:1, m/m) and either 3 mol% of amphipathic polyethylene glycol (PEG) with 1000 in average molecular weight or 6 mol% of ganglioside GMI (GMI), with 95-98% entrapping efficiency by the pH gradient method. 57% or 45% of the entrapped DXR was released from PEG/DPPC/DSPC or G_M1/DPPC/DSPC liposomes, respectively, by incubation with 20% serum at 42°C for 5 min. Inclusion of PEG or G_M1 endowed TSL with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system (RES) uptake over 6 hours after injection. Concomitantly, high DXR level in blood was kept for long time.

Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR-long-circulating TSL was 2 times or 7 times higher than that after treatment with DXR-TSL liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the systemic treatment with DXR-long-circulating TSL and hyperthermia resulted in effective tumor growth retardation and increased survival time. These results indicate that the combination of long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

抗生素的使用情况调查及细菌耐药性分析

目的:分析我院的抗生素的使用频率以及细菌耐药率的变化,为规范临床用药提供参考资料。方法:采用回顾性分析的方法对我院2009年3月-2013年3月收治的8000例住院患者的抗生素使用情况进行调查,并对我院临床上常见革兰阴性菌和阳性菌的耐药率变化进行比较,分析抗生素的使用频率与细菌耐药率变化之间的关系。结果:临床上抗生素的使用频率最大的是β-内酰胺酶抑制剂以及头孢菌素类。金葡菌对环丙沙星的耐药率与青霉素类抗生素的DDDs呈正相关,大肠埃希菌对亚胺培南的耐药率与头孢菌素类抗生素的DDDs呈负相关。结论:抗生素的用药频率与病原菌对抗生素的耐药率有相关性,并且,单一的抗生素并不能引起病原菌的耐药性,而会同时影响其他类型的抗生素的耐药情况。     

The Delivery of Benzyl Penicillin to Staphylococcus aureus Biofilms by Use of Liposomes

The delivery of benzyl penicillin [penicillin G (pen‐G)] encapsulated in cationic liposomes to a pen‐G‐sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter ～ 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC‐chol) at a molar ratio of 1.0 :0.49 :0.43. This composition containing 22 mole% of the cationic lipid DC‐chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561–570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen‐G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen‐G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 × 10^{? 3} mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen‐G. The results were supported by direct measurements of the distribution of pen‐G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen‐G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.     

Liposomes in the Treatment of Parasitic,Viral, Fungal and Bacterial Infections

Abstract

The applicability of liposomes as carriers of immunomodulatory agents or antibiotics for improvement of treatment of severe infections is under investigation. The use of “classical'’ liposomes for targeting of macrophage modulators to enhance non-specific host resistance to infections caused by a variety of micro-organisms shows good results. The therapeutic prospects of “classical'’ liposomes as carriers of antibiotics are good, however are limited to the treatment of intracellular infections in mononuclear phagocyte system (MPS) tissues. The recent development of liposome formulations with reduced affinity to the MPS and long circulation half-lives creates new possibilities for obtaining improved delivery of antibiotics to infected tissues in general including infections in non-MPS tissues.     

Enhanced Delivery and Antitumor Effect of Doxorubicin Encapsulated in Long-Circulating Liposomes

Abstract

Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) or ganglioside GM1 (GM1) have been tested for their utility as enhanced delivery system of doxorubicin (DXR) in vivo. DXR was entrapped into liposomes by pH gradient method.

The long-circulating LUV (200 nm in size) composed of DSPC/CH (1:1, m/m) and either 6 mol% of DSPE-PEG1000 or GM1 entrapped DXR with >95% in trapping efficiency. DXR-long-circulating LUVs were administered to leukemic (LI210) mice via the tail vein at a dose of 5mg DXR/kg. The high blood concentration was kept for long time, and significantly increased survival time was observed as compared with free DXR and DXR-LUV. The data indicated that DXR was slowly released from long-circulating LUV during that stayed in bloodstream for long time. Administration of DXR-long-circulating SUV (100 nm) to the colon 26 bearing mice produced the increased DXR level in tumor compared with bear SUV or free drug did, respectively, and resulted in effective tumor growth retardation and increased survival time. DXR was delivered to tumor by accumulation of SUVs themselves.

Long-circulating thermosensitive liposomes (TSL) were prepared from DPPC /DSPC (9:1, m/m) and 3-6 mol% of PEG1000 or GM1. DXR was entrapped with >95% in trapping efficiency. Accumulation of DXR into tumor tissue by local hyperthermia after injection of DXR-long-circulating TSL to colon 26 bearing mice was significantly higher man that of DXR-bare TSL or free DXR, and resulted in effective tumor growth retardation and increased survival time. It was suggested that the entrapped DXR was efficiently released from long-circulating TSL by hyperthermia at the tumor site and entered the tumor tissue by simple diffusion.     

Anaerobic Bacterial Flora of Intra-abdominal Infections and their Antimicrobial Susceptibility Pattern in Kuwait

Clinical samples obtained from 200 patients with intra-abdominal infections were investigated for the presence of anaerobic bacteria. The majority of samples were from patients with appendicitis (108, 54%) followed by peritoneal abscess/peritonitis (37, 18.5%). A total of 153 anaerobes were isolated from 83 culture positive specimens with an isolation rate of 1.8 per sample. Ninety (59%) yielded Bacteroides fragilis group and B. fragilis stricto sensu accounted for half of them. Other isolates were 36 (23.5%) Prevotella species and 15 (9.8%)Peptostreptococcus micros . The susceptibility of the 153 isolates against eight antibiotics was determined by the E-test. All the isolates were susceptible to metronidazole, MIC₉₀s varying between 1–2 μg/mL. ThePrevotella spp., Peptostreptococcus spp., Fusobacterium spp. and Porphyromonas spp. were all susceptible to clindamycin (MIC₉₀s=0.25–2 μg/mL respectively), imipenem (MIC₉₀s=0.12–0.5μg/mL respectively) and meropenem (MIC₉₀=0.25 μg/mL each). About 25% of the B. fragilis group were resistant to clindamycin with MIC more than 256 μg/mL. Piperacillin-tazobactam also exhibited excellent in vitro activity against all the isolates (MIC₉₀=0.25 μg/mL).     

血清降钙素原鉴别病毒性感染、一般性细菌感染和重症细菌性感染的临床应用价值

目的:研究血清中降钙素原在病毒性感染、一般性细菌感染和重症细菌性感染患者中的应用价值。方法:选择2014年6月~2016年12月在我院重症医学科进行诊治的感染患者60例,按照患者感染的严重程度分为病毒性感染患者(A组)15例,一般细菌感染组(B组)22例以及重症细菌性感染组(C组)23例。三组患者均采取纠正水电解质平衡、抗生素抗感染和营养支持等对症治疗,并在入院后第1、3、5、7 d检测患者的血清降钙素原水平,比较三组治疗前后血清降钙素原水平的差异,观察三组血清降钙素原≥0.5μg/L的例数。结果:B组血清降钙素原≥0.5μg/L的例数为20例,占90.91%,C组血清降钙素原≥0.5μg/L的例数为21例,占91.30%,B组和C组之间相比无明显差异(P0.05),但A组血清降钙素原≥0.5μg/L的例数为3例,占20.00%,明显低于B组和C组血清降钙素原≥0.5μg/L的例数,差异均具有统计学意义(P0.05);C组入院后第1、3、5、7 d的血清降钙素原水平均明显高于B组的血清降钙素原水平,差异均具有统计学意义(P0.05);重症细菌性感染患者中,血清降钙素原≥0.5μg/L的患者存活率明显低于血清降钙素原0.5μg/L的患者(P0.05)。结论:血清中降钙素原水平对于重症感染患者的临床诊断和治疗具有较好的应用价值,对于医生判断重症感染患者的预后情况具有很好的效果,值得临床广泛应用推广。     

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.     

Novel Neo Glycolipid: Formulation into Pegylated Cationic Liposomes and Targeting of DNA Lipoplexes to the Hepatocyte-Derived Cell Line HepG2

Liver parenchymal cells are an important target for the treatment of several metabolic and viral disorders. Corrective gene delivery for this purpose is an avenue that is receiving increasing attention. In the present study, we report a novel neo glycolipid that may be formulated into cationic liposomes with or without poly(ethylene glycol) decoration. Lipoplexes formed with plasmid DNA are nuclease resistant and are targeted to the human hepatoblastoma cell line HepG2 by selective asialoglycoprotein receptor mediation. Transfection levels achieved by lipoplexes containing the targeting ligand cholesteryl-3β-N-(4-aminophenyl-β-D-galactopyranosyl) carbamate were sixfold greater than those obtained with similar but untargeted lipoplexes.     

降钙素原和超敏C-反应蛋白在病毒和细菌感染早期鉴别中的临床应用

为探讨降钙素原(PCT)和超敏C-反应蛋白(hs-CRP)在病毒感染和细菌感染患者鉴别诊断中的价值,对我院276例患者,包括病毒感染组(64例)、细菌感染组(164例)和非感染组(48例)进行降钙素原和超敏C-反应蛋白水平的检测,并对三组间的差异进行分析。结果显示,细菌感染组和病毒感染组PCT、hs-CRP水平显著性高于非感染组(p<0.05);细菌感染组PCT、hs-CRP水平显著性高于病毒感染组(p<0.05)。工作特征曲线及Logistic回归分析结果显示,PCT在鉴别诊断细菌感染和病毒感染的效果高于hs-CRP。研究结果表明,降钙素原、超敏C-反应蛋白可对病毒感染和细菌感染进行早期的鉴别诊断,而降钙素原的效果较好。     

关节镜清理联合抗菌药物治疗膝关节革兰阳性菌感染的疗效观察

目的:探讨关节镜清理联合抗菌药物治疗膝关节革兰阳性菌感染患者的疗效,为临床合理治疗提供参考依据。方法:选取2017年4月～2019年7月在我院治疗的经细菌学检查确诊为革兰阳性菌感染的膝关节炎症患者128例,随机分为观察组和对照组,各64例,观察组接受关节镜下膝关节清理术联合万古霉素或替考拉宁治疗,对照组接受万古霉素或替考拉宁治疗。分析患者治疗前病原菌分布情况,比较两组临床疗效、不良反应发生情况及复发率,比较两组患者的膝关节症状评分、体征评分。结果:128例革兰阳性菌感染患者中,共检出病原菌128株,金黄色葡萄球菌、表皮葡萄球菌、凝固酶阴性葡萄球菌及肠球菌分别占23.71%、25.77%、24.74%、20.62%。观察组的优良率为95.31%(61/64),高于对照组的81.25%(52/64),两组优良率比较,差异有统计学意义(P0.05)。治疗后观察组的膝关节症状评分、体征评分均低于对照组(P0.05)。观察组的复发率低于对照组(P0.05),而两组不良反应发生率比较差异无统计学意义(P0.05)。结论:采用关节镜清理联合抗菌药物治疗膝关节革兰阳性菌感染患者疗效确切,可改善患者症状积分和体征积分,降低复发率,安全可靠。     

Bacterial infection of human hematopoietic stem cells induces monocytic differentiation

Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34(+) hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.     

应用RAPD分析技术研究生姜青枯病的土壤防治

利用随机扩增多态性技术(RAPD)研究生姜连作地、施加有机肥后的生姜连作地及新开地中土壤微生物多样性与生姜青枯病的关系, 结果发现, 连作地的青枯病发病率为100%, 新开地没有发病, 而施加有机肥的连作地生姜青枯病发病率降至37%。发现施加有机肥连作地的真菌、放线菌数量增加, 平板上不同菌落形态的真菌、细菌、放线菌增加明显。多样性指数分析发现Shannon指数、Simpson指数等均显著增加。随机扩增条带出现连作地的条带丰富度较少、新开地的条带最多、施加有机肥连作地的RAPD条带明显多于连作地, 接近新开地, 并出现了一些连作地没有的特异条带。结果表明, 施加有机肥可以改善土壤微生物群落结构。     

Efficient Encapsulation of Plasmid DNA in Anionic Liposomes by a Freeze/Thaw-Extrusion Procedure

Abstract

In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity during extrusion. A solution containing supercoiled plasmid pCMVβ (7164 base pairs) was added to dry lipid films, and after freezing and thawing, the suspension was extruded through a filter with 0.2 μm pores. About 20% of the DNA became encapsulated, as evidenced by protection from degradation by endonuclease added externally. Plasmid isolated from the liposomes was structurally intact, and had essentially the same transfection activity as untreated DNA. These results show that plasmid DNA can be reliably and efficiently encapsulated in anionic liposomes by freeze/thaw and extrusion.     

大鼠肝移植术后腹腔细菌感染模型的建立与评价

目的建立大鼠肝移植术后腹腔感染的模型。方法构建DA大鼠到LEW大鼠的肝移植模型,采用腹腔内细菌注射的方法建立感染模型,通过对大鼠肝功、血气、血细胞计数等各项指标的检测对模型进行综合评价。结果肝移植术后5 d注射细菌,大鼠死亡率高,不利后续研究;术后3 d注射细菌,并选定5×105cfu/mL为最终注射浓度,感染后大鼠的7d存活率累计可达到37.5%左右,随之感染的加重,大鼠状态逐渐变差,直肠温度不断升高,WBC计数也随之增加,pH下降,大鼠出现代谢性酸中毒,肝功能损害进行性加重,肝实质的损害重于胆道的损伤,大约在感染5 d左右相继死亡,多器官病理分析表明,大鼠死亡原因为肝损害,不并发肺脏、肾脏损害。结论采用的腹腔内大肠埃希菌注射建立肝移植术后腹腔细菌感染的模型是比较成功的,可用于相关领域的研究。     

Analysis of Bacterial Community in the Ginseng Soil Using Denaturing Gradient Gel Electrophoresis (DGGE)

The objective of this work was to determine the shifts in the PCR-DGGE profiles of bacterial communities associated with the rhizosphere soil of ginseng at varying age levels. Differences in the dominance of intense DNA bands in the DGGE profile was observed over the age of the plants indicating the fluctuation in the microbial community structure. The bacterial orders of actinomycetales of Actinobacteria and Spingomonadales and Rhizobiales of α-Proteobacteria were predominant in the ginseng soil.