Diffusion of ofloxacin in the endocarditis vegetation assessed with synchrotron radiation UV fluorescence microspectroscopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique i.v. injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510-560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.     

Synchrotron radiation infrared microspectroscopy to assess the activity of vancomycin against endocarditis vegetation bacteria

Infrared microspectroscopy was used to show that vancomycin alters infrared spectra of endocarditis vegetation bacteria, and that vancomycin effects on bacterial biochemical contents are unevenly distributed between peripheral and central areas of bacterial masses. Infrared microspectroscopy is useful to study the activity of antibacterial agents against bacteria in tissues.     

"Difficult to treat infections" pharmacokinetic and pharmacodynamic factors--a review

"Difficult to cure infections" are characterized by poor penetration of antibiotics into infected vegetations, altered metabolic state of bacteria within the vegetation, absence of adequate host defense/cellular response. These infections typically include endocarditis, urinary tract infections (infected urinary tract stones), abscesses, infected fibrin clots (septic thromboemboli, haematomas, catheter-related infections) and foreign body infections. Four main aspects are discussed for the influence on human therapy: 1. the kinetics of antibiotic diffusion into vegetations 2. the specificity of some pharmacodynamic aspects and pharmacokinetic regimes 3. fibrin as one of the main constituents associated with infectious processes and 4. synergistical activities of antibiotic combinations on bacterial vegetations.     

Long-wavelength fluorescence of tyrosine and tryptophan solutions

We report in this paper the presence of fluorescence bands of tryptophan and tyrosine solutions centered above 550 nm. This long-wavelength fluorescence is of much lower intensity, (0.4-2.7)%, than the UV fluorescence of these aromatic aminoacids. The basic characteristic of these fluorescence bands are: (a) tyrosine: lambda em = 600 nm with two excitation peaks centered at 453 nm and 550 nm (b) tryptophan: lambda em = 675 nm with two excitation peaks centered at 455 and 560 nm. It has been found that irradiation of tyrosine solutions with a potent UV lamp promotes an important increase of absorption at 310 nm and above 400 nm.     

Effect of antibacterials on surface properties and adhesion to uroepithelial cells in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The effect of sub-inhibitory and inhibitory concentrations of antimicrobials including aminoglycosides, third generation cephalosporins and quinolones on the surface properties and adhesion of Klebsiella pneumoniae to uroepithelial cells (UECs) was examined. Antibiotics, ceftazidime and ofloxacin at 1/4, 1/8 × MIC and minimum inhibitory concentration (MIC) induced filament formation in bacteria, however cells treated with amikacin were similar in length to control organisms but showed a rough topology under the scanning electron microscope. An increase in bacterial hydrophobicity and decrease in uronic acid content were noted in the presence of ceftazidime and ofloxacin at MIC and sub-MIC level. However, amikacin at MIC level caused decreased hydrophobicity of the cells and the uronic content remained the same. This study clearly indicates that, although ceftazidime and ofloxacin brought about profound changes in cell surface characteristics, these changes did not result in any advantage to the bacterial cell in terms of adhesion. In contrast, with amikacin, which did not show any appreciable change in cell morphology or surface topology, exposure markedly increased the adherence of bacteria to UECs, indicating that the prophylactic use of this antibiotic not only induces resistance in bacteria but can also promote the colonization of UECs.     

小麦叶面积指数与冠层反射光谱的定量关系

在分析不同氮素水平下小麦叶面积指数（LAI）和冠层光谱反射率随生育期变化模式的基础上,确立了LAI与冠层光谱反射率及光谱参数的相关关系,提出了小麦LAI的敏感光谱参数及预测方程.结果表明,小麦LAI和近红外短波段（760～1 220 nm）反射率都随施氮量的增加呈上升趋势,可见光波段反射率则相反;从拔节期到成熟期,LAI和近红外短波段反射率均表现为先上升后下降的趋势,而可见光波段（460～710 nm）反射率随生育期的推进先降低后升高,以孕穗期反射率最低,近红外长波段区域（1 480～1 650 nm）反射率的变化与可见光部分相同.LAI与可见光波段反射率呈负相关,与近红外短波段反射率呈极显著正相关,其中以810 nm相关性最好.可以选择RVI(810,510)和DVI(810,560)作为反演小麦LAI的光谱参数.另外,在证明垂直植被指数PVI和转换型土壤调整指数TSAVI对LAI预测能力的同时,发现利用RVI(810,510)、DVI(810,560)和PVI 3个植被指数共同推算小麦LAI的准确度更高.     

Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.     

OBSERVATTVNS ON THE UNDERWATER LIGHT FIELD OF TWO HWERTROPHIC,SOUTH AFRICAN IMPOUNDMENTS

SUMMARY

The spectral composition of the underwater light field was examined in two hypertrophic South African Impoundments (Hartbeespoort and Roodeplaat Dams) under a range of inorganic turbidities and chlorophyll α concentrations. The data indicated that inorganic turbidity and gilvin were dominant to chlorophyll in regulating underwater light attenuation during the present study. Under all conditions the wavelengths between 405 and 510 nm were attenuated more rapidly than near UV and the wavelengths above 510 nm and the 623 nm component penetrated deepest. Under low turbidities the 546 nm wavelength was the next most penetrating component, but its attenuation increased with increasing turbidity. This characteristic of the underwater light field may be important to the cyanobacteria which dominate in these hypertrophic lakes.     

水稻叶片全氮浓度与冠层反射光谱的定量关系

利用数学统计方法分析了不同施氮水平和不同水稻品种群体叶片全氮浓度（LNC）与冠层反射光谱的定量关系,建立了水稻群体叶片全氮浓度的光谱监测模型.结果表明：基于原始反射率构造的光谱参数与叶片全氮浓度的相关程度均高于原始反射率,近红外波段（760～1 220 nm）与可见光波段510、560、680及710 nm组成的比值植被指数、差值植被指数和归一化植被指数与群体叶片全氮浓度呈极显著正相关,其中与归一化植被指数（NDVI）的相关性最好;对拟合较好的6个两波段组合参数及4个特征光谱参数的预测标准误（SE）和决定系数（R²）进行比较后,选取参数NDVI (1220, 710)为反演群体叶片全氮浓度的最佳光谱参数,方程为LNC=3.2708 × NDVI (1220,710) + 0.8654.利用不同粳稻品种、水分和氮肥处理的试验数据对监测模型进行了检验,估计的根均方差（RMSE）均小于20%,预测值和实测值的拟合R²为0.674～0.862,拟合斜率为0.908～1.010,RMSE为11.315%～19.491%,表明模型预测值与实测值之间符合度较高,对不同栽培条件下的水稻群体叶片全氮浓度具有较好的预测性.     

Anti-fibrin antibody binding in valvular vegetations and kidney lesions during experimental endocarditis.

In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)-labeled anti-fibrin antibody one or three days later. When rabbit anti-fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non-specific rabbit IgG showed no effect. The 99mTc-labeled anti-fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm-bred and specific pathogen-free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm-bred rabbits were labeled, but specific pathogen-free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen-free rabbits labeled with 99mTc-labeled anti-fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.     

Fluorimetry of bovine myotendon junction by fibre-optics and microscopy of intact and sectioned tissues

Summary The autofluorescence of tendon, epimysium and endomysium at the myotendon junction of the deep digital flexor in the bovine forelimb was measured with a fluorescence microscope and with a bifurcated light guide composed of quartz optical fibres. Data were adjusted for spectral variation in the radiance of the halogen illuminator used to standardize the photometer. Samples of myotendon junction were examined intact, in slices several millimetres thick and after being frozen in liquid nitrogen and sectioned at 20 µm. Sections were examined with and without a mounting medium and with and without immersion oil objectives. Type I collagen fibres were identified by their scarcity of branching, relatively large size and yellow staining with silver. Type III collagen fibres were identified by their extensive branching, small size and black staining with silver. Purified Types I and III collagen were also examined. Type I collagen fibres had a strong fluorescence emission peak between 410 and 450 nm and a shoulder at 510 nm. For the strong peak, results obtained by fibre-optics were positively biased relative to those obtained by microscopy. Type III collagen reticular fibres lacked a strong emission peak at 410 to 450 nm. Although their overall fluorescence was weaker than that of Type I collagen fibres, Type III collagen fibres had similar or slightly stronger emissions around 510 nm. The Type I emission spectrum of collagen fibres was converted to a spectrum similar to the Type III spectrum by conditions that caused the fading of fluorescence (storage as dry or mounted sections and exposure of sections to UV light). It is suggested that, with fibre-optic fluorimetry of intact tissues, Type I collagen fibres may emit a pre-fading spectrum while Type III collagen fibres may emit a post-fading spectrum, and that the preservation of Type I and the fading of Type III collagen is a consequence of the surface to volume ratio of their fibres.     

UV-A screening in plants determined using a new portable fluorimeter

UV screening by plant surfaces can be determined by exposing plant organs to UV radiation and measuring the chlorophyll (Chl) fluorescence elicited. From this fluorescence, the UV transmittance can be derived: the more intense the screening the lower the reporter Chl fluorescence and the lower the UV transmittance. The relationships between UV screening at 375 nm, as determined in the field by a portable UV-A-PAM fluorimeter, and UV screening at 314 and 360 nm, measured in the laboratory with the non-portable XE-PAM fluorimeter, were investigated in leaves of grapevine (Vitis vinifera L. cv. Bacchus) and barley (Hordeum vulgare cv. Ricarda), as well as in white grape berries. With leaves, linear trends were observed between XE-PAM measurements at 314 nm and UV-A-PAM measurements at 375 nm but the relationship between transmittance at 360 and 375 nm in barley was curved: a simple model calculation suggests that this curvi-linearity arises from particularly weak absorbance of barley flavonoids at 375 nm relative to absorbance at 360 nm. Transmittance values at 314 nm plotted against 375 nm yielded a much smaller slope in grapevine leaves than in barley leaves, which was attributed to screening in the short-wavelength UV by hydroxycinnamic acids in the former but not in the latter species. With grape berries, a poor correlation was detected between transmittances at 314 and 375 nm which might arise from high scattering of UV radiation at the berry surface. Such artefacts appear to be confined to the UV-B region, as berry transmittance at 360 nm correlated very well with that at 375 nm. Thus, assessment of UV screening in the field at short UV wavelengths using 375 nm readings from a UV-A-PAM fluorimeter is possible provided that information is available on the relationship between the transmittance at the UV wavelength of interest and at 375 nm for the sample tissue being investigated.     

Determination of ofloxacin and moxifloxacin and their penetration in human aqueous and vitreous humor by using high-performance liquid chromatography fluorescence detection

Information on comparing the penetration of ofloxacin and moxifloxacin in the human eye is unavailable, although these two antibiotics are commonly used in ophthalmic surgery. There is a need for a rapid, reliable, and sensitive methodology for their determination in ocular fluids. We developed a robust HPLC procedure with fluorescence detection for simultaneous analysis of ofloxacin and moxifloxacin in human and rabbit aqueous and vitreous samples. The linearity of the method ranged from 10 ng/ml to 100 microg/ml with r(2) > 0.996. Most inter- and intrabatch imprecision was about 5% (range 1.6-7.6%), recoveries between 95 and 104%, and accuracies between 93 and 104% at 0.1 and 1 microg/ml. The detection limits of both compounds were 10 ng/ml (0.028 nmol/ml for ofloxacin and 0.023 nmol/ml for moxifloxacin). No sample treatment was necessary for aqueous humor and only acetonitrile precipitation was required for vitreous humor. The chromatographic time was short, 22 min. We applied this method to study penetrations of ofloxacin and moxifloxacin in aqueous and vitreous humors of human and rabbits. There was no significant difference of penetration between the two antibiotics into aqueous and vitreous but ofloxacin was found at significantly higher concentrations in aqueous than in vitreous. We also detected contralateral transfer of the antibiotics in rabbit eyes.     

Phototoxicity and photoinactivation of blebbistatin in UV and visible light

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.     

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.     

Antibiotic resistance of soil bacteria

Summary A total of 560 bacterial isolates from four rhizosphere and eight non-rhizosphere soils were examined for resistance to 7 antibiotics. There were marked differences between the overall levels of antibiotic resistance found in the different soils. The rhizosphere populations were not consistently more antibiotic resistant than their corresponding non-rhizosphere soils.     

Spectral sensitivities of photoreceptors and their role in colour discrimination in the green-backed firecrown hummingbird (<Emphasis Type="Italic">Sephanoides sephaniodes</Emphasis>)

We studied the photopic spectral sensitivity in the green-backed firecrown, Sephanoides sephaniodes, a South American hummingbird, and its possible ecological relationship with preferred flowers and body colouration. Avian colour vision is in general tetrachromatic with at least four types of cones, which vary in sensitivity from the near ultraviolet (UV) to the red wavelength range. Hummingbirds represent an important family of birds, yet little is known about their eye sensitivity, especially about the role of photoreceptors and their oil droplet complements. The photopic electroretinogram shows a main sensitivity peak at 560 nm and a secondary peak in the UV, and may be explained by the presence of four single cones (lambda (max) at ~370, 440, 508 and 560 nm), and a double cone (lambda (max) at 560 nm) screened by oil droplets. The flowers preferred by the firecrown are those in which the red-green wavelength region predominates and have higher contrast than other flowers. The crown plumage of males is highly iridescent in the red wavelength range (peak at 650 nm) and UV; when plotted in a high-dimensional tetrachromatic space, it falls in a "red + UV" purple hue line, suggesting a potential significant communication signal for sexual differentiation.     

Cell-phone-based platform for biomedical device development and education applications

In this paper we report the development of two attachments to a commercial cell phone that transform the phone's integrated lens and image sensor into a 350x microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of 150 x 50 with no image processing, and approximately 350 x 350 when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field.     

Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria

The aim of this study was to biosynthesis silver nanoparticles from the fungus Nigrospora sphaerica isolated from soil samples and to examine their activity against five human pathogenic strains of bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus using disc diffusion method. The synergistic effect of silver nanoparticles in combination with commonly used antibiotic Gentamycin against the selected bacteria was also examined. The synthesized silver nanoparticles from free-cell filtrate were characterized by using UV–Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). UV–Vis spectrophotometer analysis showed a peak at 420 nm indicating the synthesis of silver nanoparticles, FTIR analysis verified the detection of protein capping of silver nanoparticles while SEM micrographs revealed that the silver nanoparticles are dispersed and aggregated and mostly having spherical shape within the size range between 20 and 70 nm. The synthesized silver nanoparticles exhibited a varied growth inhibition activity (15–26 mm diam inhibition zones) against the tested pathogenic bacteria. A remarkable increase of bacterial growth inhibition (26–34 mm diam) was detected when a combination of silver nanoparticles and Gentamycin was used. A significant increase in fold area of antibacterial activity was observed when AgNPs in combination with Gentamycin was applied. The synthesized silver nanoparticles produced by the fungus N. sphaerica is a promising to be used as safe drug in medical therapy due to their broad spectrum against pathogenic bacteria.