Investigation of the antibacterial activity and efflux pump inhibitory effect of co-loaded piperine and gentamicin nanoliposomes in methicillin-resistant Staphylococcus aureus

Objective: Antibiotic resistance has stimulated the research for developing novel strategies that can prevent bacterial growth. Methicillin-resistant Staphylococcus aureus (MRSA), regarded as one of the most serious antibiotic-resistant bacteria which has been conventionally recognized as a nosocomial pathogen.

Materials and methods: Nanoliposomal formulations of piperine and gentamicin were prepared by dehydration-rehydration (DRV) method and characterized for size, zeta potential and encapsulation efficiency. Antibactericidal activities of liposomal and free forms were evaluated against MRSA ATCC 43300 by the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and fractional inhibitory concentration index (FICI). The time-kill studies were carried out to evaluate the potency of antibacterial agents. The effect of piperine on bacterial efflux pumps was also investigated.

Results: MIC values of gentamicin and piperine were 32 and 100?µg/mL, respectively. Synergetic effects were observed by the combination of gentamicin and piperine and FICI was determined to be 0.5. Following incorporation of gentamicin into liposomal gentamicin and liposomal combination, the MIC values were reduced 16- and 32-fold, respectively. MBC values of gentamicin reduced 4 and 8 times following incorporation into gentamicin and combination liposomes, respectively. In comparison with vancomycin, liposomal combination was more effective in bacterial inhibition and killing. Liposomal combination was the most effective preparations in time-kill study. Our findings indicated that liposomal piperine was able to inhibit the efflux pump sufficiently.

Conclusion: The results of this study revealed that liposomal combination is a powerful nano-antibacterial agent to eradicate MRSA infection. This dual-loaded formulation was an effective approach for eradication of MRSA.     

抗菌剂对聚乙烯表面生物被膜的抑制

采用共混方法在低密度聚乙烯中分别加入银系抗菌剂和Triclosan制备出两种改性聚乙烯,研究了材料的抗菌性能和抗菌剂对试样表面生物被膜的抑制作用.结果表明,两种改性聚乙烯对大肠杆菌和金黄色葡萄球菌都有良好的抗菌效果;细菌生物被膜的形成主要包括粘附、繁殖和成熟3个阶段,添加Triclosan的试样可以在生物被膜成熟前显著杀灭样品表面的细菌,阻止细菌在其表面粘附和繁殖,从而抑制生物被膜形成.空白试样和银系抗菌剂改性试样表面均有大量细菌粘附并形成生物被膜.     

Atomic force microscopy study of the effect of pulsed electric field on Staphylococcus epidermidis

The effect of a pulsed electric field (PEF) on Staphylococcus epidermidis was investigated by using an atomic force microscopy (AFM) image and force measurement under liquid. The changes in the bacterial envelope were probed in situ before and after applying different dosages of PEF. Our results indicated that PEF induced the changes of bacterial peptidoglycan layer and the exposure of plasma membrane. This conclusion was further confirmed by two control experiments: the effect of the lysozyme or heat on the bacterial envelope. Furthermore, our results demonstrated that AFM analysis including image and force measurement is helpful to explain the relationship between the chemical composition change of the cellular envelope and the external stimulation.     

玄武岩纤维表面改性对生物膜附着性能的影响

采用壳聚糖对玄武岩纤维(BF)进行表面改性处理,研究改性后玄武岩纤维对生物膜附着性能的影响。利用红外光谱、X射线光电子谱仪、扫描电镜等对改性前后玄武岩纤维的表面官能团、成分和形貌进行表征分析,通过接触角测量仪对样品的亲水性进行研究,最后通过挂膜实验,讨论生物膜在改性前后玄武岩纤维上的附着性能。结果表明:采用物理涂覆法可成功制备改性玄武岩纤维(MBF),所制得的MBF表面粗糙度为209.04nm,接触角为66.62°。MBF表面形成的生物膜均匀致密,生物膜附着量明显增大,挂膜率由(129.27±1.23)%增加至(179.92±2.63)%,说明壳聚糖改性玄武岩纤维可以有效提升生物膜的附着性能。     

ZnO films grown by pulsed-laser deposition on soda lime glass substrates for the ultraviolet inactivation of Staphylococcus epidermidis biofilms

Abstract

We found that a ZnO film of 2 μm thickness which was laser-deposited at room temperature onto a plain soda lime glass substrate, exhibits notable antibacterial activity against a biofilm of Staphylococcus epidermidis when back-illuminated by a UVA light source with a peak emission wavelength of about 365 nm. X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible absorption spectroscopy, Raman spectroscopy and x-ray photoemission spectroscopy (XPS) were used to characterize the ZnO films before and after the interactions with the biofilm and the ultraviolet light, respectively. The as-deposited film was highly textured with the wurtzite (0002) in-plane orientation (c-axis perpendicular to ZnO surface) and had a surface rms roughness of 49.7 nm. In the as-deposited film, the Zn to O ratio was 1 to 0.95. After the UV and biofilm treatments, the ZnO film surface had become rougher (rms roughness 68.1 nm) and presented uniform micron-sized pitting randomly distributed, while the zinc to oxygen ratio had become 1 to 2.2. In this case, both the UV-visible and Raman spectra pointed to degradation of the structural quality of the material. On the strength of these data, we propose a model for the mediation of the bactericidal activity in which the photogeneration of highly oxidizing species and the presence of active surface defect sites both play an important role. This study is of particular interest for the acute problem of disinfection of pathogenic biofilms which form on medical device/implant surfaces.     

ZnO films grown by pulsed-laser deposition on soda lime glass substrates for the ultraviolet inactivation of Staphylococcus epidermidis biofilms

We found that a ZnO film of 2 μm thickness which was laser-deposited at room temperature onto a plain soda lime glass substrate, exhibits notable antibacterial activity against a biofilm of Staphylococcus epidermidis when back-illuminated by a UVA light source with a peak emission wavelength of about 365 nm. X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible absorption spectroscopy, Raman spectroscopy and x-ray photoemission spectroscopy (XPS) were used to characterize the ZnO films before and after the interactions with the biofilm and the ultraviolet light, respectively. The as-deposited film was highly textured with the wurtzite (0002) in-plane orientation (c-axis perpendicular to ZnO surface) and had a surface rms roughness of 49.7 nm. In the as-deposited film, the Zn to O ratio was 1 to 0.95. After the UV and biofilm treatments, the ZnO film surface had become rougher (rms roughness 68.1 nm) and presented uniform micron-sized pitting randomly distributed, while the zinc to oxygen ratio had become 1 to 2.2. In this case, both the UV-visible and Raman spectra pointed to degradation of the structural quality of the material. On the strength of these data, we propose a model for the mediation of the bactericidal activity in which the photogeneration of highly oxidizing species and the presence of active surface defect sites both play an important role. This study is of particular interest for the acute problem of disinfection of pathogenic biofilms which form on medical device/implant surfaces.     

基于流式分析技术的奶粉中金黄色葡萄球菌活菌快速定量检测方法研究

建立了一种奶粉中金黄色葡萄球菌活菌快速定量检测新方法.以异硫氰酸荧光素偶联的金黄色葡萄球菌多克隆抗体特异性标记金黄色葡萄球菌,再通过碘化丙啶来区分死活菌,建立流式分析法,并应用于奶粉样品中金黄色葡萄球菌活菌定量检测.结果表明,该流式分析法特异性好;当人工污染奶粉样品中金黄色葡萄球菌浓度在2.72 × 103～2.80 ...     

Carriage of Staphylococcus aureus in the nose of patients on regular dialysis treatment using hemodialysis catheters

In the hemodialysis population, the incidence of Staphylococcus aureus colonization has been documented to be as high as 80%; effective prophylaxis of vascular access infection and bacteremia is a worthwhile goal in the management of hemodialysis population. Surveillance of 50 hemodialysis patients for S. aureus‐positive nasal cultures was performed by monthly nasal swabs over a 12‐month period. All patients were performing dialysis using hemodialysis catheters thrice weekly. All positive cultures were treated with a prophylactic antibiotic regimen. Thirty‐one patients (62%) had one or more positive cultures. The surveillance period was longer in the S. aureus nasal carriers (p < 0.01). The frequency of positive cultures correlated with the duration of surveillance (p < 0.05). The incidence of S. aureus bacteremia was greater in patients with three or more positive cultures (p < 0.05). This study suggests that continuous surveillance for S. aureus nasal colonization is essential to properly identify all hemodialysis patients using catheters at risk of developing S. aureus bacteremias.     

The nosocomial transmission rate of animal-associated ST398 meticillin-resistant Staphylococcus aureus

The global epidemiology of meticillin-resistant Staphylococcus aureus (MRSA) is characterized by different clonal lineages with different epidemiological behaviour. There are pandemic hospital clones (hospital-associated (HA-)MRSA), clones mainly causing community-acquired infections (community-associated (CA-)MRSA, mainly USA300) and an animal-associated clone (ST398) emerging in European and American livestock with subsequent spread to humans. Nosocomial transmission capacities (R_A) of these different MRSA types have never been quantified. Using two large datasets from MRSA outbreaks in Dutch hospitals (dataset 1, the UMC Utrecht for 144 months; dataset 2, 51 hospitals for six months) and a recently developed mathematical model, we determined the genotype-specific R_A for ST398 and non-ST398 isolates (categorized as HA-MRSA), using observational data, the detection rate of MRSA carriage and the discharge rate from hospital as the input. After detection of 42 MRSA index cases in dataset 1 (all non-ST398 MRSA) 5076 people were screened, yielding 30 secondary cases. In dataset 2, 75 index cases (51 non-ST398 MRSA and 24 ST398) resulted in 7892 screened individuals and 56 and three secondary cases for non-ST398 MRSA and ST398, respectively. The ratio between discharge and the detection rate was 2.7. R_A values (95% confidence interval (CI)) were 0.68 (0.47–0.95) for non-ST398 MRSA in dataset 1, 0.93 (0.71–1.21) for non-ST398 MRSA in dataset 2 and 0.16 (0.04–0.40) for ST398. The R_A ratio between non-ST398 MRSA and ST398 was 5.90 (95% CI 2.24–23.81). ST398 is 5.9 times less transmissible than non-ST398 MRSA in Dutch hospitals, which may allow less stringent transmission-control measures for ST398 MRSA.     

环境因素对细菌附着的影响

生物污损是船舶及海洋工程面临的主要问题,其中由细菌附着形成的生物膜难以避免,且具有介导无脊椎污损生物幼虫附着的特性,尤为棘手。因此,控制海洋细菌生物膜的形成成为防污研究的重要课题。本文利用海洋细菌Bacillus subtilis和Escherichia coli设计试验,考察环境因素和基底材料对细菌生物膜形成的影响,揭示细菌生物膜的形成规律。研究结果表明:温度、盐度等环境因素对Bacillus subtilis和Escherichia coli附着形成生物膜的影响极显著(方差检验结果即P<0.01),通过控制环境因素可以干扰生物膜的形成。     

Chitosan formulations for steroid delivery: effect of formulation variables on in vitro characteristics

Chitosan film formulations for steroid delivery after craniomaxillofacial surgery were formulated by using three different types of chitosan with respect to their molecular weight as low, medium and high. Film formulations were prepared by casting/solvent evaporation technique. In vitro characterization, film thickness, equilibrium swelling degree, in vitro release profiles and surface morphologies were investigated. For two different types of crosslinkings, the release of dexamethasone sodium phosphate (DSP) can be extended as the molecular weight increases. As a result, chitosan film formulations should be beneficial for steroid delivery for a certain time after craniomaxillofacial surgery.     

Modelling the combined effect of surface roughness and topography on bacterial attachment

Bacterial attachment is a complex process affected by flow conditions,imparted stresses,and the sur-face properties and structure of both the supporting material and the cell.Experiments on the initial attachment of cells of the bacterium Streptococcus gordonii (S.gordonii),an important early coloniser of dental plaque,to samples of stainless steel (SS) have been reported in this work.The primary aim motivating this study was to establish what affect,if any,the surface roughness and topology of sam-ples of SS would have on the initial attachment of cells of the bacterium S.gordonii.This material and bacterium were chosen by virtue of their relevance to dental implants and dental implant infections.Prior to bacterial attachment,surfaces become conditioned by the interfacing environment (salivary pellicle from the oral cavity for instance).For this reason,cell attachment to samples of SS pre-coated with saliva was also studied.By implementing the Extended Derjaguin Landau Verwey and Overbeek(XDLVO) theory coupled with convection-diffusion-reaction equations and the surface roughness infor-mation,a computational model was developed to help better understand the physics of cell adhesion.Surface roughness was modelled by reconstructing the surface topography using statistical parame-ters derived from atomic force microscopy (AFM) measurements.Using this computational model,the effects of roughness and surface patterns on bacterial attachment were examined quantitatively in both static and flowing fluid environments.The results have shown that rougher surfaces (within the sub-microscale) generally increase bacterial attachment in static fluid conditions which quantitatively agrees with experimental measurements.Under flow conditions,computational fluid dynamics (CFD) simula-tions predicted reduced convection-diffusion inside the channel which would act to decrease bacterial attachment.When combined with surface roughness effects,the computational model also predicted that the surface topographies discussed within this work produced a slight decrease in overall bacterial attachment.This would suggest that the attachment-preventing effects of surface patterns dominate over the adhesion-favourable sub-microscale surface roughness;hence,producing a net reduction in adhered cells.This qualitatively agreed with experimental observations reported here and quantitatively matched experimental observations for low flow rates within measurement error.     

In vitro antimicrobial effect of encapsulated vancomycin on internalized Staphylococcus aureus within endothelial cells

Vancomycin (VCN) is a glycopeptide antibiotic that is effective in the treatment of gram-positive bacterial infections, but mainly reserved for methicilin resistant Staphylococcus aureus. It is, however, ineffective against intracellular bacteria and hence a particulate form of VCN would be required. Bovine serum albumin (BSA) microspheres of VCN with a mean particle size of 5 ± 1.6 μm were used. Human microvascular endothelial cells internalized both S. aureus and VCN microspheres in a time and concentration-dependent manner, however, the uptake was inhibited by cytochalasin D. Action of VCN on S. aureus in the intracellular microenvironment decreased the bacterial load considerably.     

Pulmonary dispersion formulations: The impact of dispersed powder properties on pressurized metered dose inhaler stability

Suspension-type metered dose inhaler formulations characteristically have a high degree of dispersion instability. This may occur as the result of any of a large number of formulation issues including phase separation, particle growth, agglomeration/flocculation, moisture ingress, and particle interactions with both the canister material and other particles. Many of these undesirable instabilities may arise as a result of the physical and chemical properties of the dispersed powder in the liquid propellant system. As such, this review provides a detailed understanding of the characteristics of the dispersed phase in the liquid propellant system necessary to ensure stability of the final formulation.     

The impact of manufacturing variables on in vitro release of clobetasol 17-propionate from pilot scale cream formulations

Objectives: The purpose of the study was to evaluate the effect of different homogenization speeds and times, anchor speeds and cooling times on the viscosity and cumulative % clobetasol 17-propionate released per unit area at 72?h from pilot scale cream formulations. A 2⁴ full factorial central composite design for four independent variables were investigated.

Materials and methods: Thirty pilot scale batches of cream formulations were manufactured using a Wintech® cream/ointment plant. The viscosity and in vitro release of CP were monitored and compared to an innovator product that is commercially available on the South African market, namely, Dermovate® cream.

Results and discussion: Contour and three-dimensional response surface plots were produced and the viscosity and cumulative % CP released per unit area at 72?h were found to be primarily dependent on the homogenization and anchor speeds. An increase in the homogenization and anchor speeds appeared to exhibit a synergistic effect on the resultant viscosity of the cream whereas an antagonistic effect was observed for the in vitro release of CP from the experimental cream formulations. The in vitro release profiles were best fitted to a Higuchi model and diffusion proved to be the dominant mechanism of drug release that was confirmed by use of the Korsmeyer–Peppas model.

Conclusion: The research was further validated and confirmed by the high prognostic ability of response surface methodology (RSM) with a resultant mean percentage error of (±SD) 0.17?±?0.093 suggesting that RSM may be an efficient tool for the development and optimization of topical formulations.     

The anti-tumor effects of cordycepin-loaded liposomes on the growth of hepatoma 22 tumors in mice and human hepatoma BEL-7402 cells in culture

Liposomes have successfully been used for decades to encapsulate and protect drugs that are prone to deactivation in the body. The present study aimed to demonstrate the use of liposomes to encapsulate cordycepin, an adenosine analog that quickly loses its activity in vivo. The cordycepin-loaded liposomes were prepared by the ammonium sulfate gradient approach, and its in vitro and in vivo antitumour activities were evaluated using BEL-7402 cells and hepatocellular carcinoma H22 transplanted tumors, respectively. An MTT assay was used to observe the cytotoxicity of cells treated with cordycepin and cordycepin-loaded liposomes in vitro. High-content screening (HSC) was carried out using Hoechst 33342 to detect apoptotic cells and the ratio of cells in different cell cycle stages. The data demonstrated that both the cordycepin and the cordycepin-loaded liposomes resulted in clear cytotoxicity with IC50 values of 18.97 and 29.39?μg/mL, respectively. The latter showed significantly strong inhibitory effects on H22 tumor growth in mice, while the former did not show any inhibitory effects on tumor growth. In addition, the HSC assay showed that the cordycepin-loaded liposomes resulted in a higher rate of apoptosis than the cordycepin alone in BEL-7402 cells. Further data analysis revealed that the cells treated with cordycepin-loaded liposomes were predominately arrested at the G2/M phase (p?p?in vivo anti-tumor activity of cordycepin.     

The effect of pressure on GRIN lenses

The changes of the optical power of Gradient Index (GRIN) lenses occurring in hydrostatic pressures of the range of 0-2 GPa are investigated. The measurements of the position of the waist of the semiconductor laser beam revealed the increase of the optical power of the lenses with increasing pressure. The use of the special plano-concave lenses insensitive to the changes of the refractive index of the pressure medium allowed to attribute the changes to the increasing index gradient in GRIN material. The effect has been explained within the frame of Mueller’s theory of photoelasticity. The findings have been then confirmed in experiments with the plano-planar and plano-convex lenses of the same materials.     

The effect of ionizing radiation on metoprolol

The influence of ionising radiation on physico-chemical properties of metoprolol tartrate (MT) in solid phase was studied. The compound was irradiated by radiation produced by a beam of high-energy electrons in an accelerator, in doses from 25 to 400 kGy, and the possible changes in the samples were detected by organoleptic analysis (colour, forms, clarity), chromatographic and spectrometric methods. Already at the standard sterilisation dose of 25 kGy, the presence of free radicals (0.3764 × 10¹⁶ spin/g) and a decrease in the melting point by 1°C were noted. At higher doses of irradiation products of radiolysis appeared (100 kGy) and the colour was changed from white to pale cream (200 kGy). Our observation was that with increasing mass loss of MT after irradiation with 100, 200 and 400 kGy, the concentration of free radicals increased from 1.0330 to 1.6869 × 10¹⁶ spin/g. The radiolytic yield of total radiolysis was 4.54 × 10⁷ mol/J for 100 kGy, 7.42 × 10⁷ mol/J for 200 kGy and 4.74 × 10⁷ mol/J for 400 kGy. No significant changes were observed in the character of FT–IR spectra, but in UV an increase in intensity of the band at the analytical wavelength was noted. As follows from the results MT shows high radiochemical stability for the typical sterilisation doses 25–50 kGy, and will probably be able to be sterilised by radiation in the dose of 25 kGy.     

The effect of crystalline structure on molecular effect in ion-induced electron emission

The results of experimental and theoretical study of the molecular effect in ion-induced electron emission from single crystal and polycrystalline materials are presented. It has been found that the electron emission yield γ₂ under N₂⁺ bombardment of a single crystal shows a dependence on ion incidence angle θ, which is analogous to the anisotropy of the yield γ₁ under N⁺ impact. The ratio R₂(θ)=γ₂/2γ₁<1 exhibits, for single crystals, a drastic angular dependence, which was successfully described on the basis of electron sweeping-out mechanism by conjunction of both the sharp channeling at θ<ψ_L and the shadowing-caused bell-like pattern at θ>ψ_L, the Lindhard angle.     

The effect of vacuum on fatigue crack growth

The paper reviews the modern concepts of the vacuum effect on fatigue crack growth under cyclic loading. It reports and discusses experimental data (obtained by the author and others) on the crack growth rate at separate stages of the process, the size and structural characteristics of the plastic zone and the fracture micromechanisms for different metals and alloys in air and in vacuum. The relationship between the macroscopic and microscopic characteristics of crack growth in both environments is considered. The idea is developed that the vacuum effect on the kinetics of crack propagation is associated with changes in the process of dislocation motion in the plastic zone (in contrast to atmospheric conditions) and the mechanism of the effect is sensitive to the fatigue mechanism of metals and alloys.