Morphological responses of Legionella pneumophila biofilm to nanoparticle exposure

Legionella pneumophila is a pathogenic bacterium that forms biofilms in natural and anthropogenic habitats. This feature not only facilitates colonization but also limits the effectiveness of biocides. L. pneumophila was exposed to three sizes of citrate-capped gold nanospheres in both planktonic and biofilm stages. TEM micrographs indicated that gold nanoparticles (AuNPs) adsorbed to the bacterial cell surface, were absorbed into the cells, aggregated within the cells, and integrated into the extrapolymeric matrix of the biofilm. Both 4 and 18 nm, but not 50 nm AuNPs caused an alteration of biofilm morphology. Treatment with 20 nm polystyrene spheres did not induce these changes suggesting that the response was a result of the gold and not just the presence of the nanosphere. The morphological changes observed in the biofilm suggest that aquatic ecosystems may be affected by nanoparticle exposure. This may compromise ecosystem functions such as nutrient cycling facilitated by natural biofilms.     

Discrete nanoparticles induce loss of Legionella pneumophila biofilms from surfaces

Nanoparticles (NPs) have been shown to induce dispersal events in microbial biofilms but the mechanism of the dispersal is unknown. Biofilms contaminate many man-made aquatic systems such as cooling towers, spas and dental lines. Within these biofilms, Legionella pneumophila is a primary pathogen, leading to these environments serving as sources for disease outbreaks. Here we show a reduction in biofilm bio-volume upon treatment with citrate-coated 6-nm platinum NPs, polyethylene glycol (PEG)-coated 11-nm gold NPs, and PEG-coated 8-nm iron oxide NPs. Treatment with citrate-coated 8-nm silver NPs, however, did not reduce biomass. The synthesis of NPs that remain dispersed and resist irreversible aggregation in the exposure media appears to be a key factor in the ability of NPs to induce biofilm dispersal.     

Impact of rifampicin addition to clarithromycin in Legionella pneumophila pneumonia

We evaluated the effectiveness and safety of rifampicin addition to clarithromycin in the treatment of Legionnaires' disease. An observational cohort study was conducted on patients assigned to a Legionnaires' disease outbreak. Of 32 patients with confirmed Legionella pneumonia, 11 received clarithromycin monotherapy and 21 received combination therapy of clarithromycin with rifampicin. Both groups had similar baseline characteristics and all patients were cured. Patients who received rifampicin had a 50% longer length of stay (P=0.035) and a trend towards higher bilirubin levels (P=0.053). Length of stay was directly correlated with the duration of rifampicin treatment (P=0.001). Combination therapy of clarithromycin and rifampicin had no additional benefit compared with clarithromycin monotherapy and could prolong the length of stay owing to possible negative drug interactions that could also affect other antibiotics.     

Bactericidal activity of imipenem compared to erythromycin against intracellular Legionella pneumophila.

Pneumonia is the leading cause of death due to nosocomial infections with mortality ranging from 30 to 70%. Because imipenem has potent in vitro activity against virtually all major causes of nosocomial pneumonia, including P. aeruginosa, S. aureus, members of the family Enterobacteriaceae, and anaerobic organisms, it is used by many physicians as the empirical therapy of choice in severe nosocomial pneumonias. Recognition of Legionella species as nosocomial pathogens has been increasing. The incidence of Legionnaires' disease among patients with nosocomial pneumonia is reported to be as high as 30% (1), but the real prevalence is unknown. Imipenem has bactericidal activity against Legionella in vitro but has not previously been tested for efficacy against intracellular Legionellae.     

大环内酯类和氟喹诺酮类药物对细胞内嗜肺军团菌的效果分析

目的分析了大环内酯类和氟喹诺酮类药物在抑制巨噬细胞内嗜肺军团菌的疗效。方法选取10株不同嗜肺军团菌血清1型,利用琼脂稀释法对不同嗜肺军团菌血清1型针对大环内酯类和氟喹诺酮类药物的半致死浓度进行分析。THP-1人单核细胞在佛波酯诱导下形成巨噬细胞,然后将不同嗜肺军团菌血清1型与巨噬细胞共同培养,待细胞将巨噬细胞吞噬后,采用半致死浓度不同倍数(1倍、4倍,8倍)的红霉素、阿奇霉素、左氧氟沙星及莫西沙星等药物对胞内的细菌进行抑制,共同培养24 h后,采用平板计数法对军团数量进行计算,用细菌抑制率进行表示。结果红霉素、阿奇霉素、左氧氟沙星及莫西沙星在半致死剂量、4倍半致死剂量、8倍半致死剂量下对细菌的抑制情况如下:红霉素:(51.42±23.19)%、(75.28±19.08)%及(90.52±9.87)%;阿奇霉素:(60.57±23.14)%,(90.25±9.68)%及(98.58±3.89)%;左氧氟沙星:(99.24±0.13)%、(99.56±0.58)%及(99.99±0.01)%;莫西沙星:(99.14±0.25)%、(99.36±0.35)%及(99.89±0.11)%。在不同半致死剂量下氟喹诺酮类药物对巨噬细胞胞内细菌的抑制率明显高于大环内酯类药物,具有统计学意义(P<0.05)。左氧氟沙星和莫西沙星在不同倍数的半致死剂量下对细菌的抑制率没有显著性差异P>0.05),阿奇霉素对巨噬细胞内细菌的抑制率显著性高于于红霉素,具有显著统计学意义(P<0.05)。结论氟唾诺酮类药物抑制胞内抗军团菌效果好于大环内酯类药物,左氧氟沙星和莫西沙星抑制效果相当,阿奇霉素抑制效果明显优于红霉素。     

三种含氯消毒剂杀灭嗜肺军团菌效果的比较

目的　对优氯净、84消毒液、漂白粉杀灭悬液中嗜肺军团菌效果进行比较。方法　应用悬液定量杀菌试验观察优氯净、84消毒液、漂白粉溶液对嗜肺军团菌的杀灭作用。结果　有效氯含量 10 0 mg/L 优氯净、84消毒液、漂白粉溶液分别对嗜肺军团菌作用 1、5、10 min,杀灭率均为 10 0 %。结论　优氯净杀灭悬液中嗜肺军团菌效果最好。     

氨溴索对铜绿假单胞菌生物膜群体感应系统的影响

目的探讨氨溴索对铜绿假单胞菌生物膜群体感应(QS)系统的影响。方法平板法培养成熟的铜绿假单胞菌PAO1野生型菌株和QS系统缺陷株△lasR△rhlR基因缺陷型、△lasI△rhlI基因缺陷型菌株生物膜,分别予生理盐水和氨溴索1.875 g·L~(-1)、3.75 g·L~(-1)作用8 h。通过荧光探针SYTO9/PI标记,激光共聚焦显微镜(CLSM)观察不同浓度氧溴索作用前后不同菌株生物膜的结构变化,利用生物膜图象结构分析软件(ISA)对氨溴索作用前后的生物膜结构参数进行定量分析。结果 PAO1野生型菌株可见大片状生物膜,细菌密集。2个基因缺陷型菌株的生物膜菌落较稀疏,氨溴索作用后不同菌株生物膜厚度、平均扩散距离和结构熵均显著减少,区域孔率增加,有剂量相关性(P<0.05),PAO1野生型菌株生物膜的变化幅度比QS系统缺陷株更大(P<0.05)。结论氨溴索破坏铜绿假单胞菌生物膜的结构,具有拮抗QS系统的特性,高浓度氨溴索(3.75 g·L~(-1))效果更显著。     

Determination of binding parameters of macrolides, lincosamides, and streptogramins to Legionella pneumophila

Parameters of [3H]erythromycin binding to Legionella pneumophila were determined in vitro using both an equilibrium and a kinetic method. Different L. pneumophila serogroups, 1-3, and a virulent strain serogroup, 1, were tested. All strains of bacteria exhibited the same binding pattern, with a dissociation constant of 0.15 microM. Other macrolides, streptogramin B-types, and lincosamides competitively displaced bound erythromycin suggesting that these compounds share common binding sites on the bacteria. Minimum inhibitory concentration (MIC) values for macrolides, streptogramin B-types, and lincosamides were determined with buffered charcoal yeast extract (BCYE) medium. A good correlation (r = 0.994) was found between the corresponding inhibition constants of these antibiotics and their MIC. It was also noted that for lincosamides the microbiological inactivity was associated with a very low bacterium affinity. Thus, it is concluded that binding parameters of these antibiotics reflect their efficacy against L. pneumophila in vitro and may serve as a useful adjunct in developing new compounds.     

奈诺沙星对嗜肺军团菌体外抗菌活性的研究

目的探索奈诺沙星对不同来源的嗜肺军团菌菌株体外药物敏感性,为临床用药提供参考。方法分析奈诺沙星、左氧氟沙星、阿奇霉素分别对环境及临床分离的嗜肺军团菌菌株体外药物敏感性,采用微量肉汤稀释法测定三种药物的最低抑菌浓度(MIC)和最低杀菌浓度(MBC),进行对比评价。结果体外药物敏感性检测结果显示奈诺沙星、左氧氟沙星、阿奇霉素对嗜肺军团菌的MIC(mg/L)分别为0.031~0.5、0.004~0.5、0.062~4 mg/L,MIC50分别为0.062、0.031、1 mg/L;MIC90分别为0.125、0.5、4 mg/L,MBC分别为0.062~1、0.008~2、0.125~8 mg/L,MBC50分别为0.125、0.125、2 mg/L,MBC90分别为0.5、1、8 mg/L。结论奈诺沙星对嗜肺军团菌有较强的抑菌和杀菌作用,明显强于阿奇霉素,略优于左氧氟沙星。     

Long-term stability of PBCA nanoparticle suspensions

In this study, the stability of poly(butyl cyanoacrylate) (PBCA) nanoparticle suspensions was examined for up to 1 year by measuring the nanoparticle sizes. The nanoparticles were prepared with different stabilizers (dextran 70.000, poloxamer 188, or polysorbate 85), and the particle size was determined before and after purification by centrifugation and after dilution with different solutions (0.1 N HCl, 0.01 N HCl, H ₂O, and PBS). The most constant sizes were with the untreated acidic nanoparticle suspensions. In all other cases, agglomeration of the particles occurred: the extent of this agglomeration and the time at which the agglomeration occurred depended on the experimental conditions. Nanoparticle polymer degradation, as indicated by size decrease, was not observed. Thus, PBCA nanoparticles can be stored as suspensions, making the lyophilization and the sometimes problematic resuspension by ultrasonication, unnecessary, which is advantageous for clinical applications.     

The surfactant dipalmitoylphophatidylcholine modifies acute responses in alveolar carcinoma cells in response to low‐dose silver nanoparticle exposure

Nanotechnology is a rapidly growing field with silver nanoparticles (AgNP) in particular utilized in a wide variety of consumer products. This has presented a number of concerns relating to exposure and the associated toxicity to humans and the environment. As inhalation is the most common exposure route, this study investigates the potential toxicity of AgNP to A549 alveolar epithelial carcinoma cells and the influence of a major component of lung surfactant dipalmitoylphosphatidylcholine (DPPC) on toxicity. It was illustrated that exposure to AgNP generated low levels of oxidative stress and a reduction in cell viability. While DPPC produced no significant effect on viability studies its presence resulted in increased reactive oxygen species formation. DPPC also significantly modified the inflammatory response generated by AgNP exposure. These findings suggest a possible interaction between AgNP and DPPC causing particles to become more reactive, thus increasing oxidative insult and inflammatory response within A549 cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Current understanding of the toxicological risk posed to the fetus following maternal exposure to nanoparticles

Introduction: With the broad use of nanotechnology, the number and variety of nanoparticles that humans can be exposed to has further increased. Consequently, there is growing concern about the potential effect of maternal exposure to various nanoparticles during pregnancy on a fetus. However, the nature of this risk is not fully known.

Areas covered: In this review, materno-fetal transfer of nanoparticles through the placenta is described. Both prenatal and postnatal adverse effects, such as fetal resorption, malformation and injury to various organs in mice exposed to nanoparticles are reviewed. The potential mechanisms of toxicity are also discussed.

Expert opinion: The toxicology and safe application of recently developed nanoparticles has attracted much attention in the past few years. Although many studies have demonstrated the toxicology of nanoparticles in various species, only a small number of studies have examined the effect on a fetus after maternal exposure to nanoparticles. This is particularly important, because the developing fetus is especially vulnerable to the toxic effects of nanoparticles during fetal development due to the unique physical stage of the fetus. Nanoparticles may directly or indirectly impair fetal development and growth after maternal exposure to nanoparticles.     

A new biocompatible nanoparticle delivery system for the release of fibrinolytic drugs

The preparation of novel biocompatible polymeric nanoconstructs suitable to load sensitive bioactive protein agents is reported. Nanoparticles were prepared as based on hybrid polymeric matrices consisting of synthetic bioerodible alternating copolymers of maleic anhydride and n-butylvinylether hemiesterified with 2-methoxyethanol and grafted with poly(ethylene glycol) segments and monoclonal antibody single chain fragment specific for fibrin clot.

The prepared nanoparticles were loaded with proteolytic enzymes (trypsin and urokinase), encapsulating up to 2500 UI of urokinase/mg of dried nanoparticles. The release of the enzyme from nanoparticles resulted time controlled and it was assessed that in case of administration of urokinase-loaded nanoparticles, the enzyme would preserve its thrombolytic properties more efficiently in respect to free drug administration. Moreover, the nanoparticles showed a good in vitro biocompatibility, suitable for biomedical applications. The stability (shelf life) of the prepared nanostructured dosage forms was evaluated. The drug-loaded nanoparticles resulted stable under stressed conditions (35 °C for 13 weeks) in a lyophilized form and preserved their morphological and functional characteristics when stored in suspension for 18 months at 4 °C.     

Pulmonary exposure to metallic nanomaterials during pregnancy irreversibly impairs lung development of the offspring

Due to the growing commercial applications of manufactured nanoparticles (NPs), toxicological studies on NPs, especially during the critical window of development, are of major importance. The aim of the study was to assess the impact of respiratory exposure to metallic and metal oxide NPs during pregnancy on lung development of the offspring and to determine the key parameters involved in lung alterations. Pregnant mice were exposed to weekly doses of 100?μg (total dose 300?μg) of titanium dioxide (TiO₂), cerium oxide (CeO₂), silver (Ag) NPs or saline solution by nonsurgical intratracheal instillation. The offspring lungs were analyzed at different stages of lung development: fetal stage (gestational day 17.5), pulmonary alveolarization (post-delivery day 14.5) and lung maturity (post-delivery day 49.5). Regardless of the type of NP, maternal exposure during gestation induced long-lasting impairment of lung development of the offspring. This effect was accompanied by: i) decreased placental efficiency together with the presence of NPs in placenta, ii) no increase of inflammatory mediators present in amniotic fluid, placenta or offspring lungs and iii) decreased pulmonary expression of vascular endothelial growth factor-α (VEGF-α) and matrix metalloproteinase 9 (MMP-9) at the fetal stage, and fibroblast growth factor-18 (FGF-18) at the alveolarization stage. Respiratory exposure to metallic NPs during pregnancy induces stereotyped impairment of lung development with a lasting effect in adult mice, independently of the chemical nature of the NP.     

Gold nanoparticle trafficking of typically excluded compounds across the cell membrane in JB6 Cl 41-5a cells causes assay interference

Abstract

Nanoparticles (NP) often interfere with the mechanism and interpretation of high throughput in vitro toxicity assays. This interference may occur at any time during the assay and spans most NP systems. This study reports on a specific type of gold NP assay interference, where unmodified gold NPs were able to traffic certain assay molecules that contained primary amines across the cell membrane resulting in false positive results for toxicity assays. The enhanced assay molecule permeability was eliminated when the gold NP surface was both sterically and chemically blocked by polyethylene glycol (PEG). The results support the growing consensus that appropriate controls and assay validation should occur prior to interpretation of results of assays using NP.     

静脉注射用胰岛素三嵌段共聚物纳米粒的制备

目的研究静脉注射用胰岛素三嵌段共聚物纳米粒(Ins-PELGE-NPs)的制备工艺,并考察其影响因素。方法通过复乳/溶剂扩散法制备Ins-PELGE-NPs,并考察其形态、粒径分布、包封率、zeta电位等。结果载药量为4.48%~4.67%的Ins-PELGE-NPs在透射电镜下均呈球形或近球形,分布均匀,平均粒径为140 nm,平均包封率为94%~98%,平均zeta电位为-21.7±4.3 mV。结论所用制备工艺简单、包封率高,可用于系列注射用三嵌段共聚物纳米粒的制备。     

纳米颗粒介导的传输系统用于结直肠癌光动力治疗的研究进展

结直肠癌主要是起源于结直肠表面黏膜上皮细胞的腺癌,已成为第四大最常见和第三大最致命的恶性肿瘤。手术切除是结直肠癌的主要治疗方法,放疗和化疗是晚期结直肠癌转移病人的治疗策略,此外,细胞治疗、基因治疗、免疫治疗和靶向治疗在结直肠癌治疗中也表现出了突出的潜力。然而,上述策略由于存在转移复发、系统毒性、多机制耐药、非特异性强和获益人群有限等问题,应用局限。因此有必要探索一种新的非侵入性、对肿瘤细胞具有高度选择性和对正常组织最小毒性的治疗策略来应对这些挑战。光动力疗法联合纳米材料作为一种治疗结直肠癌的新手段,有望克服这些挑战。该研究综述了光动力疗法的原理及纳米颗粒在结直肠癌光动力疗法中的应用,以期为科研与临床提供一种思路。     

Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung

Particulate nanocarriers have been praised for their advantageous drug delivery properties in the lung, such as avoidance of macrophage clearance mechanisms and long residence times. However, instilled non-biodegradable polystyrene nanospheres with small diameters and thus large surface areas have been shown to induce pulmonary inflammation. This study examines the potential of biodegradable polymeric nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and the novel PLGA derivative, diethylaminopropylamine polyvinyl alcohol-grafted-poly(lactic-co-glycolic acid) (DEAPA-PVAL-g-PLGA), to provoke inflammatory responses in the murine lung after intratracheal instillation. Lactate dehydrogenase (LDH) release, protein concentration, MIP-2 mRNA induction, and polymorphonucleocyte (PMN) recruitment in the bronchial alveolar lavage fluid (BALF) were used to evaluate an inflammatory response in Balb-C mice. Two sizes of polystyrene (PS) nanospheres (diameters: 75 nm and 220 nm) were included in the study for comparison. All nanoparticle suspensions were instilled at concentrations of 1 microg/microl and 2.5 microg/microl, representative of an estimated "therapeutic dose" and a concentrated "dose" of particles. In all experiments, the 75 nm PS particles exhibited elevated responses for the inflammatory markers investigated. In contrast, biodegradable particles of comparable hydrodynamic diameter showed a significantly lower inflammatory response. The most marked differences were observed in the extent of PMN recruitment. While the 75 nm and 220 nm PS nanospheres exhibited 41 and 74% PMN within the total BALF cell population after 24 h, respectively, PMN recruiting in lungs instilled with both types of biodegradable particles did not exceed values of the negative isotonic glucose control. In conclusion, evidence suggests that biodegradable polymeric nanoparticles designed for pulmonary drug delivery may not induce the same inflammatory response as non-biodegradable polystyrene particles of comparable size.     

Internal benchmarking of a human blood-brain barrier cell model for screening of nanoparticle uptake and transcytosis

Transport of drugs across the blood-brain barrier, which protects the brain from harmful agents, is considered the holy grail of targeted delivery, due to the extreme effectiveness of this barrier at preventing passage of non-essential molecules through to the brain. This has caused severe limitations for therapeutics for many brain-associated diseases, such as HIV and neurodegenerative diseases. Nanomaterials, as a result of their small size (in the order of many protein-lipid clusters routinely transported by cells) and their large surface area (which acts as a scaffold for proteins thereby rendering nanoparticles as biological entities) offer great promise for neuro-therapeutics. However, in parallel with developing neuro-therapeutic applications based on nanotechnology, it is essential to ensure their safety and long-term consequences upon reaching the brain. One approach to determining safe application of nanomaterials in biology is to obtain a deep mechanistic understanding of the interactions between nanomaterials and living systems (bionanointeractions). To this end, we report here on the establishment and internal round robin validation of a human cell model of the blood-brain barrier for use as a tool for screening nanoparticles interactions, and assessing the critical nanoscale parameters that determine transcytosis.     

Cardiopulmonary effects induced by occupational exposure to titanium dioxide nanoparticles

Although some toxicological studies have reported that exposure to titanium dioxide nanoparticles (nano-TiO₂) may elicit adverse cardiopulmonary effects, related data collected from human are currently limited. The purpose of this study is to explore cardiopulmonary effects among workers who were exposed to nano-TiO₂ and to identify biomarkers associated with exposure. A cross-sectional study was conducted in a nano-TiO₂ manufacturing plant in eastern China. Exposure assessment and characterization of TiO₂ particles were performed in a packaging workshop. Physical examination and possible biomarkers for cardiopulmonary effects were examined among 83 exposed workers and 85 controls. In packaging workshop, the total mass concentration of particles was 3.17?mg/m³. The mass concentration of nanoparticles was 1.22?mg/m³ accounting for 39% of the total mass. Lung damage markers (SP-D and pulmonary function), cardiovascular disease markers (VCAM-1, ICAM-1, LDL, and TC), oxidative stress markers (SOD and MDA), and inflammation markers (IL-8, IL-6, IL-1β, TNF-α, and IL-10) were associated with occupational exposure to nano-TiO₂. Among those markers, SP-D showed a time (dose)–response pattern within exposed workers. The data strongly suggest that nano-TiO₂ could contribute, at least in part, to the cardiopulmonary effects observed in workers. The studied markers and pulmonary function tests may be useful in health surveillance for workers exposed to nanomaterials.