Determination of Pathogens by Electrophoretic and Spectrometric Techniques.

In modern medical diagnostics, where analytical chemistry plays a key role, fast and accurate identification of pathogens is becoming increasingly important. Infectious diseases pose a growing threat to public health due to population growth, international air travel, bacterial resistance to antibiotics, and other factors. For instance, the detection of SARS-CoV-2 in patient samples is a key tool to monitor the spread of the disease. While there are several techniques for identifying pathogens by their genetic code, most of these methods are too expensive or slow to effectively analyze clinical and environmental samples that may contain hundreds or even thousands of different microbes. Standard approaches (e.g., culture media and biochemical assays) are known to be very time- and labor-intensive. The purpose of this review paper is to highlight the problems associated with the analysis and identification of pathogens that cause many serious infections. Special attention was paid to the description of mechanisms and the explanation of the phenomena and processes occurring on the surface of pathogens as biocolloids (charge distribution). This review also highlights the importance of electromigration techniques and demonstrates their potential for pathogen pre-separation and fractionation and demonstrates the use of spectrometric methods, such as MALDI-TOF MS, for their detection and identification.

Capillary electrophoretic methods for classification of methicillin-resistant Staphylococcus aureus (MRSA) clones.

This study describes differentiation of methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to different genotype groups by the combination of electrophoretic techniques, transient isotachophoresis and micellar electrokinetic chromatography. MRSA isolates were separated in fused silica capillary with roughened inner surface prepared by etching with supercritical water. Separation temperature together with the rinsing procedure of the capillary turned out to be the key factors of successful analysis. The individual genotype groups were baseline-resolved in 40 min. Partial separation of the individual isolates within the groups was also observed. Relative standard deviations of the migration times of the isolate zones ranged from 0.32 to 0.79%. In addition, capability of the developed CE method to concentrate and separate MRSA isolates in clinical samples was proved by the analysis of blood sample.

Classification of clinical Cutibacterium acnes isolates at phylotype level by capillary electrophoretic methods in roughened fused silica capillary.

Despite being commensal bacterium involved in the maintenance of healthy skin, Cutibacterium acnes is also associated with inflammatory diseases. Since inflammatory and immunogenic properties vary between C. acnes phylotypes, reliable classification of clinical C. acnes isolates is important for determining their pathogenicity. Combination of optimized separation methods, polymer-enhanced transient isotachophoresis and sweeping of the charged bacterial cells in micellar electrokinetic chromatography in the roughened fused silica capillary, was used for the separation of twenty clinical C. acnes isolates. Their correct classification into the individual phylotypes was achieved in 20 min at laboratory temperature. In addition, decrease in the separation temperature to 15 °C led to the separation of the individual isolates of some phylotypes. Relative standard deviations of migration times of both intra- and inter-day analyses did not exceed 1.7%. Linearity of the proposed method in the concentration range from 5 × 105 to 1 × 107 cells mL-1 was characterized by the coefficient of determination R2 = 0.9985. Limit of detection of 5 × 105 cells mL-1 (50 cells in 100 nL of the injected sample) was determined for all the examined bacteria.

Sensitive identification of milk protein allergens using on-line combination of transient isotachophoresis/micellar electrokinetic chromatography and capillary isoelectric focusing in fused silica capillary with roughened part.

A method for on-line concentration of milk proteins from large sample volumes using combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) in fused silica capillary with an inner roughened part has been developed. The method utilizes reversible dynamic adsorption of proteins onto a thin layer of PEG 4000 on the roughened surface of the capillary. In addition, the tITP/MEKC method was combined with capillary isoelectric focusing (CIEF) for on-line concentration, separation, identification and sensitive determination of proteins in skimmed milk. The method allows analysis of up to 50 µL of sample. This study has focused on the four important whey proteins, bovine serum albumin (BSA), α-lactalbumin (α-LA), and two genetic variants of ß-lactoglobulin (ß-LG A and ß-LG B). The proteins were identified on the basis of their migration times and characteristic pI values. The pI values of BSA, α-LA, ß-LG A, and ß-LG B were determined as 4.7, 4.4, 5.1, and 5.2, respectively. Limits of detection for BSA, α-LA and both ß-LG variants were found as 1.2, 1.0 and 1.0 pg mL-1, respectively. The linearity of calibration curves was characterized by the R2 = 0.9982. The method provided highly reproducible results as the relative standard deviations of the migration times and peak areas of the examined proteins did not exceed 1.6%.

Novel chip-based isoelectric focusing device for fractionation of bacteria prior to their mass spectrometry identification.

We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry.

Bacteriophage replication on permissive host cells in fused silica capillary with nanostructured part as potential of electrophoretic methods for developing phage applications.

Phage therapy could offer a safe and effective alternative to antibiotic treatment of infections caused by Gram-positive bacterium Staphylococcus aureus that have emerged as a significant threat in hospital and community environment and is attracting growing interest among clinicians. The legislation process of approving the phage therapeutics by pharmaceutical authorities requires rapid analytical techniques for assessment of phage activity. Here, we present a three-step method for on-line monitoring the phage effect on bacterial cells dynamically adhered from microliter volumes of high conductivity matrix onto the inner surface of fused silica capillary with a part etched with supercritical water. Phage K1/420 particles of the Kayvirus genus generated by propagation on the host S. aureus cells together with the uninfected cells were concentrated, separated and detected using capillary electrophoretic methods. The phage interactions with selected S. aureus strains exhibiting differences in phage susceptibility were compared. The method allowed determination of the phage burst size and time of phage latent period in analyzed strains. Apart from enumeration of bacteriophages by the plaque assays, the proposed method is suitable for phage activity testing.

Rapid Isolation, Propagation, and Online Analysis of a Small Number of Therapeutic Staphylococcal Bacteriophages from a Complex Matrix.

A method for the fast isolation, propagation, and characterization of very low count bacteriophages active against pathogenic bacterial strains is described in this study. Bacteriophages with a count of 102 phage particles were dynamically adhered from the maximum 10 mL blood plasma sample onto the nanostructured part of the fused silica capillary. One-step propagation of phage particles of genus Kayvirus inside the etched capillary on 104Staphylococcus aureus host cells increased their number to 6 × 104 phage particles. Phage particles were concentrated online and separated by capillary electrophoretic methods. No phage replication occurred when the phage-resistant S. aureus or Escherichia coli cells were used. Two-step phage propagation in the capillary allowed an increase in the total virion count to up to 6 × 105 phage particles and subsequent off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the phage zone collected after capillary electrophoresis. Relative standard deviations of the phage peak area were at most 2.3%. We expect that the method of isolating bacteriophages from blood plasma and their simultaneous identification will facilitate clinical studies of phage preparations and contribute to pharmacokinetics studies during phage therapy. This approach is also suitable for capturing and enriching new phages from the environment when a susceptible indicator strain is available.

Identification of Aspergillus Conidia in Bronchoalveolar Lavage Using Offline Combination of Capillary Electrophoresis in Supercritical Water-Treated Fused Silica Capillary and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Diagnosis of fungal infection in lung parenchyma is relatively difficult. Bronchoscopy with bronchoalveolar lavage is very useful in its diagnosing. Therefore, a method for rapid online concentration and analysis of Aspergillus conidia in bronchoalveolar lavage fluid using the combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) with subsequent off-line identification of the separated conidia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described in this study. In the proposed procedure, conidia were first dynamically adhered onto the roughened part of the inner surface of a fused silica capillary prepared by etching with supercritical water. Then the adhered conidia were desorbed, concentrated, and separated using a combination of tITP and MEKC. Finally, the fractions containing the separated conidia were collected from the capillary and analyzed by MALDI-TOF MS. The adhesion efficiency under the optimized experimental conditions was about 80%. This rapid diagnosis will contribute to timely initiation of therapy and increase the patient's chances of survival.

Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification.

The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 µL of the prepared sample at the optimized flow rate of 6.5 µL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

Online Concentration of Bacteria from Tens of Microliter Sample Volumes in Roughened Fused Silica Capillary with Subsequent Analysis by Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

This study presents a timely, reliable, and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of a single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of microliter sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 µL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 1.8 × 102, 1.7 × 103, and 1.0 × 103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83% and it was independent of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry required at least 4 × 103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2%. The adhered bacteria, either individual or in a mixture, were online analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.

Utilization of Red Nonionogenic Tenside Labeling, Isoelectric Focusing, and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Identification of Uropathogens in the Presence of a High Level of Albumin.

Cellulose-based preparative isoelectric focusing was used for preseparation and concentration of uropathogens Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Candida albicans, and Candida parapsilosis in a urine sample containing a high concentration of human serum albumin. For the visibility of the colorless microbial zones in the separation medium, the microbial cells were labeled with red nonionogenic tenside (1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN). A very short incubation time, about 2 min, was sufficient for the adsorption of 0.001% (w/v) PAPAN onto the cell surface at the optimized conditions. As low as 103 cells of E. coli (pI 4.6) resuspended in 100 µL of urine sample and spiked with 0.1 mg mL-1 of human serum albumin (pI 4.8) were successfully preseparated and concentrated using this method. Because the pI values of the labeled microorganisms remained unchanged, the focused red zones of microbial cells were collected from the separation media and further analyzed by either capillary isoelectric focusing or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The viability of the cells extracted from the collected zones was also confirmed. The proposed method provides reliable, relatively fast, and cost-effective identification of uropathogens in urine specimens with a high level of albumin.

Capillary electrophoresis with preparative isoelectric focusing preconcentration for sensitive determination of amphotericin B in human blood serum.

Amphotericin B (AmB) is still, despite its severe nephrotoxicity, the first-line agent in the management of serious systemic fungal infections. A sensitive and reliable method is therefore required to control AmB concentration in body fluids of a patient. This study demonstrates the potential of the off-line combination of preparative isoelectric focusing (IEF) with capillary isoelectric focusing (CIEF) or capillary zone electrophoresis (CZE) in the determination of AmB in human blood serum. The required value of the isoelectric point of AmB was determined to be 6.1 using the CIEF technique. Preparative IEF served as a pre-separation and concentration technique. The pH gradient was traced by colored low molecular pI markers. The collected fraction with AmB was easily processed and then analyzed by CIEF and CZE. Tens of picograms of AmB in human blood serum sample can be determined by a combination of preparative IEF with CZE. The method was linear in the AmB concentration range of 0.3-600 ng mL-1. The recovery ranged from 93% to 98%.

Preparative and capillary isoelectric focusing for detection and identification of Aspergillus conidia in complex sample matrices.

This study describes a new method for fast identification of highly hydrophobic conidia of Aspergillus species from both simple and complex matrices. The method is based on recently developed preparative isoelectric focusing in a cellulose-based separation medium which had to be modified with respect to the highly hydrophobic surface of the conidia. Although Aspergillus conidia are colored, their zones in the cellulose bed were indicated by colored isoelectric point markers. The isoelectric point values of Aspergillus conidia were determined by capillary isoelectric focusing. Preparative isoelectric focusing was successfully used for preconcentration of individual conidia of cultivated strains of Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus parasiticus, and also for separation of the conidia in a mixture. Subsequently, red pepper powder and peanuts spiked with Aspergillus niger and Aspergillus flavus conidia, respectively, were used as complex matrices. The detection limit for identification of the conidia in these complex matrices is 104 conidia mL-1 . The presence of conidia in the focused zones was confirmed by their subsequent analysis by capillary isoelectric focusing. Their viability was confirmed by a cultivation of the conidia extracted from the collected fractions after preparative isoelectric focusing.

Identification of bacterial uropathogens by preparative isoelectric focusing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL-1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.

Preparative isoelectric focusing of microorganisms in cellulose-based separation medium and subsequent analysis by CIEF and MALDI-TOF MS.

Pre-separation and pre-concentration of bacteria is an important step especially when they are uncultured and bacterial concentration in the matrix is low. This study describes a preparative method based on isoelectric focusing of colored microorganisms in a cellulose-based separation medium from a high conductivity matrix. The isoelectric points found for the examined cells were 1.8 for Micrococcus luteus, 3.5 for Dietzia sp., and 4.7 for Rhodotorula mucilaginosa using capillary isoelectric focusing. The final positions of the zones of colored microbial cells in the cellulose-bed are indicated by colored pI markers. Segments of the separation medium with cells were harvested by a spatula, simply purified using centrifugation and analyzed by capillary isoelectric focusing and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The determined recovery ranged from 78% to 93%. The viability of the harvested cells was verified by their cultivation.

Preparative isoelectric focusing in a cellulose-based separation medium.

An improved preparative method based on isoelectric focusing of analytes in a cellulose-based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel-like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions.

Capillary electrophoresis in a fused-silica capillary with surface roughness gradient.

The electro-osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro-osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused-silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV-visible detection. First the influence of the electro-osmotic flow on the peak shape of a marker of electro-osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical-water-treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 µg/mL albumin were reached with this method.

Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS.

Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.

CIEF separation, UV detection, and quantification of ampholytic antibiotics and bacteria from different matrices.

The effect of antibiotics on the microbial cells and concentration of antibiotics in the human body is essential for the effective use of antimicrobial therapy. The capillary isoelectric focusing is a suitable technique for the separation and the detection of bacteria, and amphoteric substances from nature. However, the determination of isoelectric points of ampholytic antibiotics by conventional techniques is time consuming. For this reason, capillary isoelectric focusing seems to be appropriate as a simple and reliable way for establishing them. The separation conditions for the capillary isoelectric focusing of selected ampholytic antibiotics with known isoelectric points and pK as, ampicillin (pI 4.9), ciprofloxacin (pI 7.4), ofloxacin (pI 7.1), tetracycline (pI 5.4), tigecycline (pI 9.7), and vancomycin (pI 8.1), were found and optimized in the suitable pH ranges pH 2.0-5.3, 2.0-9.6, and 9.0-10.4. The established values of isoelectric points correspond with those found in the literature except tigecycline. Its pI was not found in the literature. As an example of a possible procedure for direct detection of both ampholytic antibiotics and bacteria, Staphylococcus epidermidis, in the presence of culture media or whole human blood, was found. The changes of the bacterial cells after their treatment with tetracycline were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Capillary isoelectric focusing allows the fast and simple determination of isoelectric points of relevant antibiotics, their quantification from the environment, as well as studying their effectiveness on microorganisms in biological samples.

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 cow's 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, cow's 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.