Anna Kubesová

Latest improvements in CIEF: From proteins to microorganisms

In recent years, characterization and identification of microorganisms has become very important in different fields of human activity. Conventional laboratory methods are time consuming, laborious, and they may provide both false positive or negative results, especially for closely related microorganisms. On that account, new methods for fast and reliable microbial characterization are of great interest. In particular, capillary electrophoretic techniques have a great potential for characterization of microorganisms due to their unique surface properties. Cell surface proteins play a key role in this respect. Since CIEF represents one of the most efficient techniques for protein separation, it was consequently applied to the analysis of microbial cells. This review describes, after a brief introduction to CIEF of proteins, recent developments in CIEF of diverse microorganisms (viruses, bacteria, yeasts, and fungi). Possible application schemes in human and veterinary medicine as well as in plant protection and in biosecurity are outlined.

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification.

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cows milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cows milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.

The differences in the isoelectric points of biofilm-positive and biofilm-negative Candida parapsilosis strains

The isoelectric points of 39 Candida parapsilosis strains were determined by means of capillary isoelectric focusing. The value of the isoelectric point corresponded well with cell surface hydrophobicity, as well as with the ability to form biofilm in these yeasts.

Capillary and gel electromigration techniques and MALDI-TOF MS--suitable tools for identification of filamentous fungi.

Microbial strains are now spreading out of their original geographical areas of incidence and previously adequate morphological identification methods often must be accompanied by a phenotypic characterization for the successful microbial identification. The fungal genus Monilinia represents a suitable example. Monilinia species represent important fruit pathogens responsible for major losses in fruit production. Four closely related spp. of Monilinia: Monilinia laxa, Monilinia fructigena, Monilinia fructicola and Monilia polystroma have been yet identified. However, the classical characterization methods are not sufficient for current requirements, especially for phytosanitary purposes. In this study, rapid and reproducible methods have been developed for the characterization of Monilinia spp. based on the utilization of five well-established analytical techniques: CZE, CIEF, gel IEF, SDS-PAGE and MALDI-TOF MS, respectively. The applicability of these techniques for the identification of unknown spores of Monilinia spp. collected from infected fruits was also evaluated. It was found that isoelectric points, migration velocities or the protein patterns can be used as the identification markers in the case of cultivated filamentous fungi. Moreover, the results obtained by capillary electromigration techniques are independent on the host origin of the spores. On the other hand, the host origin of the fungi can play an important role in the precise fungi identification by the other techniques.

Combination of Capillary Isoelectric Focusing in a Tapered Capillary with MALDI-TOF MS for Rapid and Reliable Identification of Dickeya Species from Plant Samples

This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.

Separation of phenotypically indistinguishable Candida species, C. orthopsilosis, C. metapsilosis and C. parapsilosis, by capillary electromigration techniques.

At the current state of laboratory diagnostics, methods for fast identification of phenotypically indistinguishable species are difficult or inaccurate. An example is represented by Candida parapsilosis, which is the second most common yeast species isolated from bloodstream infections. C. parapsilosis comprises a complex of three genetically distinct groups. Genotypes II and III have been designated as the separate species Candida orthopsilosis and Candida metapsilosis, phenotypically indistinguishable. The considerable genetic variability of these newly described yeasts species has caused difficulties in the development of molecular techniques for their precise identification. Similarly, the detection of biofilm formation, which is considered as an important yeast virulence factor, is accompanied by difficulties. In this study we optimize the first precise and reproducible method for the separation and possible identification of C. orthopsilosis, C. metapsilosis and C. parapsilosis as well as the detection of their ability to form biofilm. The method is based on capillary isoelectric focusing and capillary electrophoresis with UV detection. In capillary isoelectric focusing, very narrow pH gradients were established. With such gradients, differences in isoelectric points of biofilm-negative and biofilm-positive species calculated from the migration times of the selected pI markers were below 0.03 pI units. In the capillary zone electrophoresis narrow zones of the cells of Candida species were detected with sufficient resolution. The values of the isoelectric point and the migration velocities of the examined species were independent on the origin of the tested strains. Capillary isoelectric focusing was examined also for the separation and detection of the cultivated biofilm-negative C. parapsilosis in the blood serum.

Capillary electrophoresis of conidia from cultivated microscopic filamentous fungi.

In immunocompromised people fungal agents are able to cause serious infections with high mortality rate. An early diagnosis can increase the chances of survival of the affected patients. Simultaneously, the fungi produce toxins and they are frequent cause of allergy. Currently, various methods are used for detection and identification of these pathogens. They use microscopic examination and growth characteristic of the fungi. New methods are based on the analysis of structural elements of the target microorganisms such as proteins, polysaccharides, glycoproteins, nucleic acids, etc. for the construction of antibodies, probes, and primers for detection. The above-mentioned methods are time-consuming and elaborate. Here hydrophobic conidia from the cultures of different strains of the filamentous fungi were focused and separated by capillary zone electrophoresis and capillary isoelectric focusing. The detection was optimized by dynamic modifying of conidia by the nonionogenic tenside on the basis of pyrenebutanoate. Down to 10 labeled conidia of the fungal strains were fluorometrically detected, and isoelectric points of conidia were determined. The observed isoelectric points were compared with those obtained from the separation of the cultured clinical samples, and they were found to be not host-specific.

The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing

AbstractTrace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface. FigureThe pre-concentration and separation of spores of Monilinia species and of Penicillium expansum from the real suspensions including combination of filtration and capillary isoelectric focusing were developed and the possibility of their application to real samples was verified. The coupling of the filtration cartridge with the separation capillary can improve the detection limit of the isoelectric focusing with the UV-detection by at least four orders of magnitude.

Use of electrophoretic techniques and MALDI-TOF MS for rapid and reliable characterization of bacteria: Analysis of intact cells, cell lysates, and "washed pellets"

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.

Dynamic labeling of diagnostically significant microbial cells in cerebrospinal fluid by red chromophoric non-ionogenic surfactant for capillary electrophoresis separations.

During bacterial infections of the central nervous system the number of microorganisms in the cerebrospinal fluid is often ranging from few up to hundreds of cells per milliliter. The electrophoretic techniques with the UV-detection reach a detection limit for whole cells of approximately 10(7)cells per milliliter. The coupling of the filtration cartridge with capillary isoelectric focusing can improve the detection limit by four orders of magnitude. In order to improve the detection limit the red non-ionogenic surfactant 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN 1000, has been prepared and used for the dynamic labeling of analytes before filtration of the sample with a concentration modulation in the analysis of proteins or microorganisms. Values of isoelectric points of labeled analytes have been calculated using pI markers detectable at 515 nm and have been found comparable with pI of the native compounds. Minimum detectable amounts of proteins and microorganisms were lower than nanograms and a hundred labeled cells, respectively. The introduced method, coupling of the filtration cerebrospinal fluid spiked with microorganisms and labeled by PAPAN, facilitates their rapid CIEF separation in the pH gradient pH range of 2-5 at their clinically important level 10(1) to 10(2) cells per milliliter.