Quorum sensing in <Emphasis Type="Italic">Erwinia</Emphasis> species

The term quorum sensing (QS) refers to the ability of bacteria to regulate gene expression according to the accumulation of signalling molecules that are made by every cell in the population. The erwiniae group of bacteria are often phytopathogens and the expression of a number of their important virulence determinants and secondary metabolites is under QS control. The erwiniae utilise two types of QS signalling molecules: N-acyl homoserine lactones and AI-2-type signalling molecules. Here, we review the regulatory networks involving QS in the soft rot erwiniae.     

Different iron-chelating properties of pyochelin diastereoisomers revealed by LC/MS

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. It is isolated from bacterial media as a mixture of two epimers, which readily equilibrate in most solvents. Experiments based on high-performance liquid chromatography/electrospray ionization mass spectrometry are reported here, allowing the investigation of the different Fe(III)-chelating properties of pyochelin diastereomers in solution without the need for labourious isolation. It is demonstrated in this study that only one of the two pyochelin diastereomers is able to chelate Fe(III); no Fe(III) complexes of the other diastereomer could be detected. The Fe(III)–pyochelin complex exhibited a 1:1 metal-to-siderophore ratio and no evidence for other stoichiometries was found.      

The role of quorum sensing in the pathogenicity of the cunning aggressor Pseudomonas aeruginosa

Recent decades have revealed that many bacterial species are capable of communicating with each other, and this observation has been largely responsible for a paradigm shift in microbiology. Whereas it was previously believed that bacteria lived as individual cells, it is now acknowledged that bacteria preferentially live in communities in the form of primitive organisms in which the behavior of individual cells is coordinated by cell–cell communication, known as quorum sensing (QS). Bacteria use QS for regulation of the processes involved in their interaction with each other, their environment, and, particularly, higher organisms We have focused on Pseudomonas aeruginosa, an opportunistic pathogen producing more than 30 QS-regulated virulence factors. P. aeruginosa causes several types of nosocomial infection, and lung infection in cystic fibrosis (CF) patients. We review the role of QS in the protective mechanisms of P. aeruginosa and show how disruption of the QS can be used as an approach to control this cunning aggressor.     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

Characterization and quantification of anthocyanins in selected artichoke (Cynara scolymus L.) cultivars by HPLC–DAD–ESI–MS n

The anthocyanin pattern of artichoke heads (Cynara scolymus L.) has been investigated by high-performance liquid chromatography–electrospray ionization mass spectrometry. For this purpose a suitable extraction and liquid chromatographic method was developed. Besides the main anthocyanins—cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3,5-malonyldiglucoside, cyanidin 3-(3′′-malonyl)glucoside, and cyanidin 3-(6′′-malonyl)glucoside—several minor compounds were identified. Among these, two peonidin derivatives and one delphinidin derivative were characterized on the basis of their fragmentation patterns. To the best of our knowledge this is the first report on anthocyanins in artichoke heads consisting of aglycones other than those of cyanidin. Quantification of individual compounds was performed by external calibration. Cyanidin 3-(6′′-malonyl)glucoside was found to be the major anthocyanin in all the samples analyzed. Total anthocyanin content ranged from 8.4 to 1,705.4 mg kg⁻¹ dry mass.      

Chemosensors in environmental monitoring: challenges in ruggedness and selectivity

Environmental analysis is a potential key application for chemical sensors owing to their inherent ability to detect analytes on-line and in real time in distributed systems. Operating a chemosensor in a natural environment poses substantial challenges in terms of ruggedness, long-term stability and calibration. This article highlights current trends of achieving both the necessary selectivity and ruggedness: one way is deploying sensor arrays consisting of robust broadband sensors and extracting information via chemometrics. If using only a single sensor is desired, molecularly imprinted polymers offer a straightforward way for designing artificial recognition materials. Molecularly imprinted polymers can be utilized in real-life environments, such as water and air, aiming at detecting analytes ranging from small molecules to entire cells. Figure       

Iron fractionation for corrosion products from natural gas pipelines by continuous-flow sequential extraction

The forms and quantities of iron species in corrosion product samples from natural gas pipelines were examined, using a continuous-flow sequential extraction system. Sequential extraction consists of four steps that dissolve water soluble iron (FeSO₄), acid soluble iron (FeCO₃), reducible iron (Fe-(oxyhydr)oxides) and oxidisable iron (FeS₂) fractions, respectively. Selectivity of extracting reagents for particular iron species was evaluated by determination of co-extracted anions, using ion chromatography, and evolved CO₂, using indirect flame atomic absorption spectrometer (FAAS). Iron was found predominantly in the reducible fraction (61–99%), indicating that Fe-(oxyhydr)oxides are the major constituents of the corrosion products.      

Digital bioanalysis

Digital microfluidics has recently emerged as a new paradigm in the world of lab-on-a-chip technology. A wide variety of bioanalyses have been successfully implemented in this format. This paper reviews the various techniques that have been adapted to digital microfluidic systems, and the current state of the field. Figure A multiplexed digital microfluidic device. Six analytical platforms are wired in series, allowing multiple independent analyses to be performed simultaneously from a single set of controls.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

Antioxidative effect of <Emphasis Type="Italic">Bacteroides thetaiotaomicron</Emphasis> extracts: superoxide dismutase identification

Bacteroides thetaiotaomicron, a bowel anaerobic commensal, seems to release enzymes detoxifying reactive oxygen species according to our recent work. This opportunistic pathogen would be beneficial in the case of an inflammatory process. To explore its role after an oxidative or nutritive stress, six to seven separate experiments were performed. The bacteria were grown on media restricted in growth factors or supplemented with bile. Their viability was checked after surface protein extraction. The extracts underwent 2D electrophoresis. Gel images were statistically analysed to construct “master” gels. Proteins were identified (peptide-mass fingerprinting technique). The effect of each extract on superoxide anions was evaluated (spectrophotometric method). Superoxide dismutase was identified and a major superoxide anion inhibition was shown by extracts obtained after a nutritive and oxidative stress without significant bacterial death. So, a therapeutic antioxidant potential is firmly hoped for. Figure These intestinal commensal Gram negative anaerobic bacilli are able to release a high level of functional superoxide dismutase when grown in minimal medium. Presented at the Annual French National Symposium on Mass Spectrometry, Electrophoresis and Proteomics, 17–20 September 2007 in Pau, France.     

The dosage of small volumes for chromatographic quantifications using a drop-on-demand dispenser system

A commercially available piezo-driven drop-on-demand dispenser was tested for its suitability for the preparation of analytical calibration standards and in a standard addition approach prior to quantitative ultra performance liquid chromatography (UPLC) analysis of homoserines. The reproducibility of the drop-on-demand dosing system was tested and the verification of the droplet volume was performed by preparing a series of 1.0 mg/L caffeine standard solutions from a 1,000.0 mg/L stock solution and analysis of the concentrations obtained by UPLC. The reproducibility was better than 1% relative standard deviation from measurement to measurement and the highest was 1.6% from day to day. The results were compared with the conventional way of generating standard solutions (pipetting). A gravimetric method and a photography-based method for the determination of the average single droplet volume were compared and found to be in very good agreement. The system was employed for the quantification of N-decanoyl homoserine by standard addition in bacterial culture supernatants containing this analyte. The agreement with conventional quantification techniques was high. The paper shows the feasibility of the approach with advantages in low sample and solvent volume consumption and very good reproducibility and reliability combined with easy usage. Figure Ejected droplet, 60 μs after application of the pulse     

A headspace solid-phase microextraction procedure coupled with gas chromatography–mass spectrometry for the analysis of volatile polycyclic aromatic hydrocarbons in milk samples

A sensitive and solvent-free method for the determination of ten polycyclic aromatic hydrocarbons, namely, naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene and chrysene, with up to four aromatic rings, in milk samples using headspace solid-phase microextraction and gas chromatography–mass spectrometry detection has been developed. A polydimethylsiloxane–divinylbenzene fiber was chosen and used at 75°C for 60 min. Detection limits ranging from 0.2 to 5 ng L⁻¹ were attained at a signal-to-noise ratio of 3, depending on the compound and the milk sample under analysis. The proposed method was applied to ten different milk samples and the presence of six of the analytes studied in a skimmed milk with vegetal fiber sample was confirmed. The reliability of the procedure was verified by analyzing two different certified reference materials and by recovery studies. Figure Milk is safe, healthy food     

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10–1000 in conditions where the common ES Q–TOF MS detection failed.      

Instrumental modification intended to save time,and volumes of sample and reagent solutions,in the atomic fluorescence spectrometric determination of mercury

Use of small membrane pumps, instead of peristaltic pumps, to introduce sample and reagent solutions into the spectrometer has several advantages in atomic fluorescence spectrometric determination of mercury. This simple modification results in a substantial saving in the time required for the measurements and so 90% of reagent solution volumes and 95% of sample solution volumes are saved, with a consequent decrease in the volume of waste generated. The sampling frequency is almost tripled, with no deterioration in sensitivity, which is similar to that obtained by use of peristaltic pumps. The relative standard deviation for ten consecutive measurements of a 1 μg L⁻¹ mercury solution was approximately 2%. Figure Small membrane pumps for the atomic fluorescene spectro metric determination of mercury     

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

Sensor technology and its application in environmental analysis

Environmental analysis is one of the fundamental applications of chemical sensors. In this review we describe different sensor systems for the gas and liquid phases that have been tested either with real-life samples or in the field during the last five years. Most field sensors rely either on electrochemical or optical transducers. In the gas phase, systems have been proposed for analysis of oxides of nitrogen, carbon, and sulfur in air, and volatile organic compounds. In the liquid phase, most detection systems used for real-life samples detect heavy-metal ions or organic contamination, for example pesticides, organic solvents and polycyclic aromatic hydrocarbons. Figure Chemical sensors for real-life environmental applications Dedicated to Professor Ulrich Nickel on the occasion of his 65th birthday.     

Quantitative biomarker assay with microfluidic paper-based analytical devices

This article describes the use of microfluidic paper-based analytical devices (μPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the μPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that μPADs can perform quantitative chemical analysis at very low cost.      

Analytical procedure for determination of the time profile of eprinomectin excretion in sheep faeces

An analytical procedure has been introduced to enable study of the time profile of eprinomectin excretion in sheep faeces. Eprinomectin was extracted from sheep faeces with acetonitrile, the extract was cleaned by solid-phase extraction (SPE), and, after derivatization by reaction with N-methylimidazole, trifluoroacetic anhydride, and acetic acid, eprinomectin was analysed by high-performance liquid chromatography (HPLC) with fluorescence detection. The method has a low detection limit (1.0 ng g⁻¹ of moist sheep faeces), a low quantification limit (2.5 ng g⁻¹ of moist sheep faeces), good recovery (in the range 78.8 to 87.1%), and good reproducibility (RSD<10%). The method was used to study the time-profile of excretion of eprinomectin in sheep faeces after a single topical administration of 0.5 mg kg⁻¹ b.w. of the drug. Because of its good recovery, precision, and sensitivity, the method has also proved applicable to further ecotoxicological studies of eprinomectin. Figure Autochthonous Slovenian dairy breed sheep – Istrian Pramenka     

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 10⁵ Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system