Understanding SERS of bacteria

Surface‐enhanced Raman spectroscopy (SERS) has been suggested as a powerful tool to identify bacteria, drawing from its high fingerprint (vibrational) information content, its extreme sensitivity (down to the single molecule level) and its obliviousness to the aqueous environment intrinsic to biological systems. We review here in a comparative manner the various studies that attempted to utilize SERS for this important goal in light of the work carried out by our own group over the past 10 years or so. We show that SERS has an additional major advantage, namely, it introduces a new dimension of selectivity, which, on the one hand, makes it even more suitable as an analytical tool, but on the other hand, it requires gaining control of the precise manner in which the SERS‐active metal centers are produced and brought into contact with the micro‐organism. Our emphasis in this review is on understanding the spectra in terms of the nature of the SERS‐active centers and their placement within the bacterium. On the interpretation and assignment of the spectra, we constantly keep in mind the final goal of bacteria identification. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis, structural characterization and antibacterial activity of 2,6-diacetylpyridine bis(benzenesulfonohydrazide) Schiff bases and their copper(II) complexes

2,6-Diacetylpyridine bis(benzenesulfonohydrazide) Schiff bases (L¹, L² and L³) and their Cu(II) complexes of the general formula [CuL·H₂O] were synthesized and characterized by various spectroscopic techniques. The crystal structure of [CuL³·(py)]·py was investigated by single crystal X-ray structure analysis. The Cu(II) cation has near square pyramidal, penta-coordinate geometry. The binegatively charged tetradentate Schiff base is asymmetrically coordinated to the Cu(II) ion via the pyridine N atom, the azomethine N atom, the sulfonyl O atom and the deprotonated hydrazine N atom. There is a pyridine molecule apically coordinated to the Cu(II) ion. All the Schiff bases and their copper(II) complexes were screened by the disc diffusion method against multi-drug resistant (MDR) gram-negative and gram-positive bacteria. The minimum inhibitory concentration (MIC) values were also determined. These results show that the antibacterial activity of the Schiff bases against Methicillin-resistant Staphylococcus aureus (MRSA) is enhanced when they are chelated with the copper(II) ion.     

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptoundecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)₆^3−/4−were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R_ct) increased with increasing SRB concentration. A linear relationship between R_ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 × 10⁷ cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost, and time-saving monitoring of microbial populations.     

Lactococcus lactis catalyses electricity generation at microbial fuel cell anodes via excretion of a soluble quinone

Lactococcus lactis is a gram-positive, normally homolactic fermenter that is known to produce several kinds of membrane associated quinones, which are able to mediate electron transfer to extracellular electron acceptors such as Fe³⁺, Cu²⁺ and hexacyanoferrate. Here we show that this bacterium is also capable of performing extracellular electron transfer to anodes by utilizing at least two soluble redox mediators, as suggested by the two-step catalytic current developed. One of these two mediators was herein suggested to be 2-amino-3-dicarboxy-1,4-naphthoquinone (ACNQ), via evaluation of standard redox potential, ability of the bacterium to exploit the quinone when exogenously provided, as well as by high performance liquid chromatography coupled with UV spectrum analysis. During electricity generation, L. lactis slightly deviated from its normal homolactic metabolism by excreting acetate and pyruvate in stoichiometric amounts with respect to the electrical current. In this metabolism, the anode takes on the role of electron sink for acetogenic fermentation. The finding that L. lactis self-catalyses anodic electron transfer by excretion of redox mediators is remarkable as the mechanisms of extracellular electron transfer by pure cultures of gram-positive bacteria had previously never been elucidated.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

具有全新机理的DNA旋转酶抑制剂的筛选及抑菌活性

利用具有新机制的抗耐药菌DNA旋转酶抑制剂GSK299423与DNA旋转酶的晶体复合物(PDB code:2XCS)构建基于配体-受体复合物的药效团模型, 诱骗集(Decoy set)验证结果表明该药效团模型具有较强的活性识别能力. 将药效团模型与分子对接相结合用于筛选化合物库, 通过抑菌活性测定, 获得了具有抗多药耐药菌活性的DNA旋转酶抑制剂LTH02.     

Two Novel Lyso-Ornithine Lipids Isolated from an Arctic Marine Lacinutrix sp. Bacterium

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.     

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang)

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.     

Sonodynamic inactivation of methicillin-resistant Staphylococcus aureus in planktonic condition by curcumin under ultrasound sonication

Methicillin-Resistant Staphylococcus aureus (MRSA) is an important cause of difficult-to-treat infections. The present study aims to investigate sonodynamic inactivation of MRSA in planktonic condition using curcumin under ultrasound sonication. Dark toxicity of curcumin to MRSA was investigated to choose the concentration range of curcumin used in the study. The uptake of curcumin in MRSA was observed before ultrasound sonication. After sonication colony forming units (CFUs) and bacterial viability were investigated using fluorescence assay. Additionally, chromosomal DNA fragmentation was also analyzed. Curcumin showed no dark toxicity to MRSA in the concentration range of ?500 μM. The maximum uptake of curcumin in MRSA occurred in 50 min after curcumin incubation. Counting of CFUs showed that curcumin had significantly sonodynamic killing effect on MRSA in a curcumin dose-dependent manner, and 5-log reduction in CFU was observed after curcumin treatment (40 μM) at room temperature in the dark for 50 min followed by exposure to ultrasound with intensity of 1.56 W/cm² for 5 min. The ratio of green-fluorescent intensity to red-fluorescent intensity was obviously decreased after curcumin treatment under ultrasound sonication. No significant change in chromosomal DNA was found in the cultured MRSA after the combined treatment of curcumin and ultrasound. These results demonstrated that sonodynamic action of curcumin had significant inactivation of MRSA in planktonic condition.     

飞机起动箱自动测试设备研制

起动箱是飞机发动机起动电气控制系统的核心部件,控制起动附件按照时序逻辑协调工作来起动发动机;对起动箱的组成和控制时序逻辑进行了具体分析,基于ATE硬件平台设计了起动箱的自动测试电路,采用了总线程控仪器和图形化测试软件等技术,实现了不同工作电压下起动箱的电子定时机构通断时间参数、起动程序线路时序逻辑功能的自动测试和故障诊断功能;经部队使用表明,该设备测试效率高,测试精度高,自动化程度高,人机交互界面友好,操作简单。