Utilization of Red Nonionogenic Tenside Labeling, Isoelectric

Article Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/aidcbc

Utilization of Red Nonionogenic Tenside Labeling, Isoelectric Focusing, and Matrix-Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometry in the Identification of Uropathogens in the Presence of a High Level of Albumin Marie Horka,́ *,† Jirí̌ Š alplachta,† Filip Růzǐ cǩ a,‡ and Karel Š lais† †

Institute of Analytical Chemistry of the CAS, v. v. i., Veveří 97, 602 00 Brno, Czech Republic The Department of Microbiology, Faculty of Medicine, Masaryk University and St. Anne’s University Hospital, Pekařská 53, 602 00 Brno, Czech Republic

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‡

ABSTRACT: 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. KEYWORDS: uropathogens, high level of albumin, red nonionogenic tenside, preconcentration and preseparation, isoelectric focusing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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the separation capillary can be a limiting factor to the applicability of CE in pathogen identification. The preparation of the microbial samples must therefore include a reliable and effective method for concentrating microorganisms from large sample volumes.1 Recently, the potential of cellulose-based preparative isoelectric focusing (IEF) for preseparation and preconcentration of various microorganisms was verified.1,11,12 The analytes, proteins, or microorganisms are preseparated during preparative IEF according to their different isoelectric points (pI) from a high conductivity matrix and simultaneously preconcentrated into the zones. The final positions of the zones of analytes in the cellulose bed are usually indicated by colored pI markers.13−15 However, the number of proper pI markers which enable harvesting of the narrow zones of microorganisms is limited. Exact removal of the zone reduces the risk of its contamination by other components of the analyzed

erious infections, epidemics, and biological threats require rapid identification and monitoring of a low number of etiological agents often in complicated biological matrixes. Many different phenotyping and genotyping methods have been developed for these purposes,1−4 the polymerase chain reaction in particular. This method has great potential in the diagnostics of tens to hundreds of cells in 1 mL of a sample.5−7 Another fast and reliable technique for identifying different microorganisms, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS),5,8 generally requires at least 1000 cells9,10 for identification. The suggested methods often require various sample preparation steps to get rid of interfering components. The entire analysis time is significantly prolonged if the incubation step is used prior to pathogen identification. Capillary electrophoretic techniques (CE), namely zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), represent another efficient way of pathogen identification.1,3,4 Because a minimum of hundreds of microbial cells are required for UV detection, low sample volume (tens of nanoliters) injected into © XXXX American Chemical Society

Received: February 4, 2019 Published: June 5, 2019 A

DOI: 10.1021/acsinfecdis.9b00045 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

Article

sample. Nevertheless, a purified sample is a key prerequisite of successful analyte identification, especially in the case of MALDI-TOF MS. The use of colored microbes is particularly advantageous when using micropreparative methods such as IEF.1,11 Unfortunately, the microbial cells are generally not visible to the naked eye, and most of them are also virtually invisible under an optical microscope without cell staining. Recently, increased sensitivity of the UV/vis detection of proteins and microorganisms was reached using a chromophoric (red) nonionogenic tenside, 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester (PAPAN), which was prepared and examined as a highly effective dynamic labeling agent.7 This tenside could be useful for the presented preparative IEF. In this study, we evaluated the labeling of microorganisms by PAPAN with respect to the reliable identification of common urinary tract pathogens in urine samples using the recently developed preparative IEF in combination with CIEF and MALDI-TOF MS. The uropathogens Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Candida albicans, and Candida parapsilosis3,7,12,16−18 were chosen for these experiments. The uropathogens were identified by the recently developed preparative IEF in combination with CIEF and MALDI-TOF MS together with the control of cell viability after preparative IEF.

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RESULTS AND DISCUSSION CIEF of Microorganisms. This study was undertaken to demonstrate the feasibility of a combination of preparative IEF in cellulose-based separation medium with either CIEF or MALDI-TOF MS for a reliable identification of different microbial species in urine samples with high content of human serum albumin (HSA). The samples were first subjected to the preparative IEF followed by fraction collection and processing. Processed fractions were finally cultivated or analyzed by either CIEF or MALDI-TOF MS. A schematic overview of the analysis workflow is shown in Figure 1. The scheme contains approximate times of the individual steps of the developed method. The whole procedure can be done in 15 h when CIEF or MALDI-TOF MS is employed. The first step in this study was to optimize the experimental conditions for CIEF of PAPAN-labeled microorganisms. Unlike the CIEF experiments described in our previous study,7 PAPAN was used in this study to make colorless microbial zones visible in the separation medium in preparative IEF. The microorganisms labeled with PAPAN were harvested from the trough and subsequently checked for their purity and concentration by CIEF analysis. On the basis of our previous experiments, a very short incubation time of about 2 min is sufficient for the adsorption of PAPAN onto the cell surfaces before preparative IEF or CIEF. In CIEF experiments, the zones of the focused microbes were detected at 280 nm without their dynamic modification during the separation process (PAPAN was not added into the catholyte (4 × 10−2 mol L−1 sodium hydroxide) nor the anolyte (0.1 mol L−1 orthophosphoric acid)). Both the catholyte and the anolyte contained the following additives: 5% (v/v) EtOH and 0.3% (w/v) PEG 10 000. The CIEF separation of C. albicans CCM 8261 (pI 2.8), C. parapsilosis CCM 8260 (pI 3.8), E. coli CCM 3954 (pI 4.6), S. aureus CCM 3953 (pI 3.4),7 biofilm-negative S. epidermidis CCM 4418 (pI 2.3),7,19 and E. faecallis CCM 4224 (pI 2.0),20 each of them 1 × 107 cells mL−1, in physiological saline solution (PSS) labeled with PAPAN

Figure 1. Schematic overview of the analysis workflow with approximate times of the individual steps, including sample handling.

(0.001% (w/v)) at the optimized conditions is shown in Figure 2A. The pI values of the microbes were determined from the dependence of the pI values of pI markers on their migration times, Figure 2B. The pI values of labeled microorganisms remained unchanged, which is in accordance with our previous findings.7 The average migration time for each of the examined microorganisms was obtained from ten independent measurements. The relative standard deviation (RSD) of the migration times was under 1.2%. Very good qualitative and quantitative responses (coefficient of determination R2 = 0.99) were reached in the concentration range from 1 × 106 to 1 × 107 cells mL−1. Optimization of the PAPAN Concentration for Preparative IEF of Microorganisms. Preparative IEF analyses of the examined microorganisms are shown in Figures 3Aa and 3B. Microbial strains were separated into pairs (S. aureus and E. coli, E. faecalis and S. epidermidis, C. albicans and C. parapsilosis) for better clarity. For the same reason, the pH gradient created in the trough was bounded by the pI markers pI 2.0 and 10.1 so that they did not interfere with the zones of the focused microorganisms. First, 100 μL of each microbial suspension (106 cells mL−1 in PSS) labeled with PAPAN (0.006% (w/v)) was immediately injected into the trough. The boundaries for S. aureus and E. coli are depicted in Figure 3Aa. The markedly red zones of the focused microbes in the cellulose-based separation medium can be seen in the photos of the troughs after 14 hours of the IEF analysis (Figure 3Aa). The zones were harvested and treated according to the procedure described in our previous study,1 and the obtained cells were resuspended in 10 μL of PSS. Their CIEF analyses are shown in Figure 3Ab. The CIEF analysis confirmed that B



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Figure 2. CIEF separations of microorganisms in the pH gradient pH 2.0−5.3. Conditions and designations: (A) sample mixture of C. albicans CCM 8261, C. parapsilosis CCM 8260, E. coli CCM 3954, S. aureus CCM 3953, biofilm-negative S. epidermidis CCM 4418, E. faecallis CCM 4224 in PSS, each of them 107 cells mL−1, labeled with 0.001% (w/v) PAPAN; (B) sample of pI markers (pI 2.0, 2.7, 3.7, 4.0, and 5.3, each of them 25 μg mL−1), the dependence of their pI on the migration time, t (min), full circle: pI markers, empty circle: microorganisms; rinsing procedure: acetone for 5 min and then back-flushed with the catholyte for 5 min.

In the experiments shown in Figure 4, 100 μL of PSS suspension containing 1000 microbial cells (initial concentration 104 cells mL−1) labeled with PAPAN (0.001% (w/v)) was injected into the trough. Detailed photos of the preparative IEF results are depicted in Figure 4A. The zones of labeled microorganisms were clearly visible and could be precisely harvested from the trough. The collected zones together with the cellulose bed were inoculated into a suitable medium (Figure 4Ba) and cultivated according to the procedure described in the Experimental Section. The cells were viable without a significant presence of other microorganisms, see Figure 4Bb. Although preparative IEF is a suitable technique for preconcentration and preseparation of such a low number of cells, a concentration of 104 cells mL−1 is too low for CIEF or MALDI analysis. MALDI-TOF MS Identification of Uropathogens in a Urine Sample with an Increased Level of Albumin. In the case of kidney diseases, urinary tract infections may be associated with an increase in urinary albumin, which is known as albuminuria.21 However, the pI values of albumin (pI 4.8) and the most prevalent causative agent of this infection, E. coli (pI 4.6), are very close to each other. In this respect, the possibility of identification of E. coli was investigated in a urine sample containing an increased amount of albumin. First, the HSA labeled with PAPAN and dissolved in PSS in concentrations ranging from 0.005 to 5.000 mg mL−1 was analyzed by preparative IEF, and the result is shown in Figure

none of the harvested zone was contaminated significantly with the other microbe from the mixture. Cell recovery was calculated from the results of the CIEF of the individual microorganisms and the CIEF of the corresponding fractions collected after preparative IEF of the cell mixture (Table 1). The recovery was calculated for each number of cells (in the range of 5 × 106 to 1 × 107 cells mL−1) and their peak heights which correspond approximately to 97%. The concentration of 1 × 106 cells mL−1 was found to be the minimal concentration that can be reliably analyzed by CIEF without previous preparative IEF (Table 1). The goal of the labeling optimization is to accurately visualize a relatively low number of the cells in the trough. In this respect, the number of cells was decreased to 105 cells mL−1, which was related to the anticipated reduction in PAPAN concentration. Preparative IEF of a blank sample, 100 μL of 0.001% (w/v) PAPAN, is shown in Figure 3B. The pH gradient was traced with lavender pI marker pI 5.3 together with pI markers pI 2.0 and 10.1. No significant red zone of the PAPAN dye was detected in the trough. In the case of our examined microbial pairs shown in the subsequent trough photos in Figure 3B, the microorganisms were focused into red zones of different color intensities. It can therefore be assumed that the individual cells bind a different amount of dye which was not distinguishable at a higher concentration of PAPAN in the sampled suspension. C



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Figure 3. Preparative IEF of labeled microorganisms by PAPAN in the cellulose bed (Aa, B) and CIEF analysis of the collected fractions (Ab); conditions and designations, see Figure 2.

shown in Figures 5Ba, 5Bb, and 5Bc, respectively. Zones of the separated analytes were clearly visible, which significantly increased the precision of their collection from the separation medium. In the case of preparative IEF, shown in Figure 5Bb, 10 mL of sterile urine was spiked with E. coli to the concentration of 5 × 103 cells mL−1; this sample volume was reduced to 1 mL and further processed, as described in the

5A. The pH gradient was traced by the pI markers pI 2.0, 5.3, and 10.1. Zones of the focused HSA were visible when the concentration of HSA in the analyzed sample was ≥0.05 mg L−1 (Figure 5Ac). For the next experiments, sterile urine was spiked with HSA and/or E. coli. Examples of the preparative IEF of urine samples spiked with HSA, E. coli, and both HSA and E. coli are D



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The results from CIEF analyses show that the differences between the recoveries for HSA and E. coli dissolved/ resuspended in PSS and in the sterile urine were negligible. CIEF alone allows for an analysis of HSA at a concentration higher than 0.05 mg mL−1 under the given conditions (see Table 1). The recovery for HSA (from its CIEF analysis alone and CIEF of HSA from the corresponding fraction collected after preparative IEF, and calculated for each concentration of the HSA, except the concentration of 0.05 mg mL−1) was 97% as well as for E. coli (see Table 1). With respect to the MALDI-TOF MS analysis, the cultivated E. coli suspended in water was analyzed first to obtain a reference mass spectrum of the examined bacterial strain (Figure 5Ca). Because the identification of uropathogens, including E. coli, using a combination of preparative IEF with MALDI-TOF MS was described in our recent study,12 the following MS analyses were performed with the aim to find out whether PAPAN labeling has any effect on the MALDI analysis of bacterial samples and whether a pathogenic microorganism can be identified in the urine sample with a high level of urinary albumin. The next sample analyzed by MALDI-TOF MS was the zone of the E. coli collected after preparative IEF. Mass spectrum of the E. coli fraction (depicted in Figure 5Bb) is shown in Figure 4Cb. The obtained data revealed that PAPAN labeling does not affect the mass pattern of the analyzed E. coli, as can be seen in Figures 5Ca and 5Cb. Despite minor differences in the peak intensities, the same protein patterns were observed after MALDI-TOF MS analysis of the cultivated E. coli and the collected E. coli fraction. For the subsequent experiments, the urine samples, spiked with

Table 1. Comparison of the Results (Peak Heights) Obtained from Direct CIEF and CIEF Performed after Preparative IEFa E. coli (pI 4.6)

HSA (pI 4.8) peak height (mAU)

mg mL

−1

5.00 1.00 (U) 0.50 0.10 (U) 0.05

CIEF 803 157 72 15

peak height (mAU)

preparative IEF → CIEF 799** 144** 714 147 68

cells mL 1 5 1 1 1

× × × × ×

−1

107 106 106 105 (U/PSS) 104

CIEF 82 40 15

preparative IEF → CIEF 813 383 76 13/14

a

Analyzed samples: HSA and E. coli suspended in PSS or urine (U) and labeled with 0.001% (w/v) PAPAN. CIEF analysis: 100 nL of sample was injected into the capillary. Preparative IEF: 100 μL of sample was loaded into the trough; collected fractions were resuspended in 10 or 100** μL of PSS. Peak heights values are mean values of five independent measurements

Experimental Section. The amount of the bacterial cells loaded into the separation trough was therefore 5 × 103 (100 μL of 5 × 104 cells mL−1 sample). In the next experiment, sterile urine was spiked with both E. coli (104 cells mL−1) and HSA (0.1 mg mL−1) and analyzed using preparative IEF. Two distinct zones of the separated analytes, E. coli and HSA, were easily detected due to PAPAN labeling after analysis, as shown in Figure. 5Bc. Zones of the focused HSA and E. coli were collected, processed, and analyzed by CIEF and MALDI-TOF MS (the only zone of E. coli).

Figure 4. Details of the trough after preparative IEF of the examined microorganisms (A), the collected zones of microorganisms labeled with PAPAN together with the cellulose (Ba), and cultivation of the collected zones on Mueller−Hinton’s agar (Bb). E



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Figure 5. Preparative IEF of PSS (A) and sterile urine (B) samples spiked with HSA (A, Ba, Bc) and E. coli (Bb, Bc) after the focusing run. (C) MALDI-TOF mass spectra of E. coli: (Ca) water suspension of the cultivated bacterial cells, (Cb) IEF fraction of the bacterium (urine sample spiked with E. coli only), and (Cc) IEF fraction of the bacterium (urine sample spiked with both E. coli and HSA).

responses (coefficient of determination R2 = 0.99) were reached in the concentration range from 1 × 106 to 1 × 107 cells mL−1 at CIEF. The recovery of preparative IEF was determined to be 97% for the cell concentrations in the range of 5 × 106 to 1 × 107 cells mL−1. The possibility of identification of E. coli (103 cells, pI 4.6) in a urine sample containing an increased amount of albumin (0.1 mg mL−1, pI 4.8) was also investigated. Labeling by PAPAN has no effect on the MALDI-TOF MS analysis of microbial samples. It was confirmed that the E. coli cells can be reliably identified in the urine sample with a high level of urinary albumin. The viability of the cells was confirmed by their cultivation.

HSA and E. coli (to different concentrations) and labeled with PAPAN, were subjected to the preparative IEF, and the collected E. coli fractions were analyzed by MALDI-TOF MS. The obtained mass spectra were identical (if peak intensities were not taken into account) to the reference mass spectra of E. coli. No signal of HSA was detected in any of the recorded mass spectra of the E. coli fraction when the urine sample spiked with both HSA and E. coli was analyzed. As an example, the mass spectrum of the E. coli fraction marked in Figure 5Bc is shown in Figure 5Cc.

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CONCLUSION This study describes a reliable, fast (compared to conventional microbiological methods), and cost-effective method for the identification of uropathogens in urine specimens in the case of the high concentration of HSA. Uropathogens were labeled with PAPAN for their visibility in a cellulose-based separation medium in preparative IEF. The purity of the red zones of the focused microorganisms and number of cells were determined by CIEF. Microbial cells were detected at 280 nm without their further dynamic modification by PAPAN. A very short incubation time of about 2 min was sufficient for the adsorption of 0.001% (w/v) PAPAN onto the cell surfaces before preparative IEF or CIEF. The RSD of the migration times was under 1.2%. Very good qualitative and quantitative

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EXPERIMENTAL SECTION Chemicals. Amphiphilic dye, PAPAN 1000, was synthesized according to the procedure described previously.7 The solution of synthetic carrier ampholytes (Biolyte, pH 3−10) was obtained from Bio-Rad Laboratories (Hercules, CA, United States). High resolution ampholyte, pH 2−4, ampholyte pH 3−4.5, 2-morpholino-ethanesulfonic acid monohydrate, 3-morpholino-propanesulfonic acid, and N[tris(hydroxymethyl)-methyl]-3-amino-2-hydroxy-propanesulfonic acid were purchased from Fluka Chemie GmbH (Buchs, Switzerland). N-(2-Acetamido)-2-aminoethanesulfonic acid and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid F



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on a power supply and fixed by inserting the working electrodes into the trough. Then, 9 μL of the pI markers solution (pI 2.0, 5.3, and 10.1, each of them 5 μg mL−1) was loaded into the central third of the trough. The power supply was turned on, and the trough was covered with a plastic lid to retard the evaporation of water from the separation medium.1 After 2 h, 100 μL of the sample (cultivated microorganisms or HSA suspended/dissolved in PSS or sterile urine and labeled with PAPAN) was loaded into the central third of the trough. The IEF device was left running for an additional 14 h (overnight), and then the fractions, defined by the positions of the red zones of the labeled microorganisms and colored pI markers, were collected and either directly used for cultivation or processed for CIEF and MALDI-TOF MS, as described in our previous study.1 CIEF in the pH Gradient pH Range 2.0−5.3. CIEF was carried out using a laboratory-made apparatus25 at a constant voltage (−20 kV on the detector side) supplied by a Spellman CZE 1000 R high-voltage unit (Plainview, NY). The total lengths of the FS (fused silica) capillaries, 100 μm I.D. and 360 μm O.D. (Agilent Technologies, Santa Clara, CA), were 35 cm, of which 20 cm was the separation part. The detection window was created at the distance of 15 cm from the cathodic end of the capillary. The ends of the capillary and the electrodes were placed in 3 mL glass vials filled with an anolyte or a catholyte. A LCD 2082 on-column UV−vis detector (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ, United States), was operated at 280 nm. Sample injection was performed by a siphoning action as described in our previous paper.26 The height difference of the reservoirs for the sample injection, Δh, was 10 cm. Cell clusters were deagglomerated by sonication in a Sonorex ultrasonic bath (Bandelin Electronic, Berlin, Germany) and then vortexed using a Yellowline TTS 3 Digital Orbital Shaker (IKA Works, Wilmington, DE) immediately before the injection of the microbial sample into the capillary. The sonication was performed at 25 °C and 35 kHz for 1 min for each sample. Each experiment was repeated at least ten times. The detector signals were acquired and processed with a Clarity Chromatography Station (ver. 2.6.3.313, DataApex, Prague, Czech Republic). Sodium hydroxide (4 × 10−2 mol L−1) or orthophosphoric acid (0.1 mol L−1) were used as the catholyte or anolyte, respectively, both with the additives of 5% (v/v) ethanol (EtOH) and 0.3% (w/v) PEG 10 000. Solutions necessary for the CIEF run (including the sample) were injected in three consecutive segments.25,26 The first segment of spacers, a solution of selected simple ampholytic electrolytes (15 × 10−5 mol L−1) was dissolved in the catholyte. The cell suspensions of the examined cells and/or HSA in PSS or sterile urine labeled by PAPAN were included in the second segment. The third segment was a 5% (w/v) aqueous solution of a commercial carrier ampholytes Biolyte pH 3−10, ampholytes pH 3.0−4.5, and pH 2.0−4.0, mixed in a 1:2:5 ratio, respectively. This segment also contained low-molecular-mass pI markers (pI 2.0, 2.7, 3.7, 4.0, and 5.3; concentration of individual pI markers 25 μg mL−1) for monitoring the used pH gradient 2.0−5.3. The injection times, tinj, of the spacers segment, the sample segment, and the segment of carrier ampholytes and pI markers were 25, 15, and 35 s, respectively. Before each CIEF run, the capillaries were rinsed with EtOH/acetone (1/1) for 5 min and then backflushed with the catholyte for 3 min. For this purpose, a single-syringe infusion

were obtained from Merck (Darmstadt, Germany). L-Aspartic acid was obtained from LOBA Chemie (Vienna, Austria). HSA (Mr 66 500, pI 4.8) and the chemicals for preparative IEF11 were purchased from Sigma-Aldrich (Schnelldorf, Germany). 3,5-Dimethoxy-4-hydroxycinnamic acid (SA) and protein calibration mixture ProMix2 were purchased from LaserBio Laboratories (Sophia-Antipolis Cedex, France). Colored lowmolecular-mass pI markers, pI 2.0 (green), 5.3 (lavender), and 10.1 (violet) for preparative IEF, and pI 2.0, 2.7, 3.7, 4.0, and 5.3 for CIEF were developed and synthesized at the Institute of Analytical Chemistry of the CAS, v. v. i.11,13−15,22 The chemicals and buffers used in this work were of electrophoresis or MS grade. Specifications of the simple buffers used as spacers in CIEF were described elsewhere.11,23−27 Microorganisms and Cultivation Conditions. The strains included in this study, C. albicans CCM 8261, C. parapsilosis CCM 8260, E. coli CCM 3954, S. aureus CCM 3953, biofilm-negative S. epidermidis CCM 4418, and E. faecallis CCM 4224, originated from the Czech Collection of Microorganisms (Brno, Czech Republic). The examined strains were stored at −70 °C in Itest cryotubes (ITEST plus, Hradec Kralove, Czech Republic) and thawed quickly at 37 °C before analysis and were cultivated at 37 °C for 24 h. Bacterial strains were cultivated on Mueller−Hinton agar (BioRad Laboratories), and yeast strains were cultivated on Sabouraud 4% maltose agar (Merck, Germany). The microbial cultures were suspended in PSS, and the concentrations were estimated by measuring the optical density at 550 nm (DU 520 UV−vis spectrophotometer, Beckmann Instruments, Palo Alto, CA). The concentrations of microorganisms in reference samples were measured by serial dilution and by plating 100 μL of the tested suspension on either Mueller−Hinton or Sabouraud maltose agar. The colonies were counted after cultivation of the strains at 37 °C for 24 h. In addition, the viability of microorganisms labeled with PAPAN after preparative IEF was tested by the cultivation of the collected IEF fractions. The viability was confirmed on the basis of colony formation after 24 and 48 h of cultivation at 37 °C on the nutrient agar. Sample Preparation. The cultivated microorganisms and HSA were suspended or dissolved in PSS or sterile urine and then labeled with PAPAN (the final concentration of PAPAN in the labeling solution was in the range of 0.01−0.001% (w/ v)). The concentration of microorganisms and HSA was 102− 107 cells mL−1 and 0.005−5 mg mL−1, respectively. Prepared samples were analyzed within 10 min. Urine spiked with microorganisms was centrifuged at 1000g for 2 min, and the supernatant containing microorganisms was labeled with PAPAN. In the case of a larger volume of sterile urine (10 mL), the urine was first centrifuged at 1000g; the supernatant was collected and then centrifuged at 6000g, and the obtained supernatant was removed. The pellet was resuspended in 1 mL of PSS and finally labeled with PAPAN. Urine samples from healthy volunteers were obtained from the Department of Microbiology, St. Anne’s University Hospital (Brno, Czech Republic). Sterility of the urine samples was examined by diluting and plating on trypticase soy agar with 7% sheep blood (Becton Dickinson, Cockeysville (MD), United States) and incubated at 37 °C for 24 h. Preparative IEF. The preparative IEF device was described in detail in our recent studies.1,11,12,22,28 First, 0.8 mL of cellulose-based separation medium11 was loaded uniformly into an empty V-shaped plastic trough which was positioned G



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Interior of the Czech Republic (Grant VI20172020069), the Czech Academy of Sciences (Institutional Support RVO: 68081715), and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR) under the National Sustainability Programme II, Project CEITEC 2020 (LQ1601).

pump (Cole-Parmer, Vernon Hills, IL) equipped with a 100 μL syringe (SGE Analytical Science, Victoria, Australia) was used at a flow rate ranging from 3 to 20 μL min−1. MALDI-TOF MS. Ten microliters of microbial suspension (cultivated strain suspended in distilled water) was centrifuged at 5000g for 5 min; the obtained supernatant was removed, and the pellet (microbial cells) was resuspended in 10 μL of SA solution (20 mg mL−1 in ACN/0.1% TFA, 3/2, v/v). Fractions collected from the preparative IEF were suspended in 150 μL of distilled water; the suspensions were vortexed for 3 min and then centrifuged at 1000g for 4 min. The supernatants (microbial cells) were centrifuged again at 5000g for 5 min. The resulting pellets (microbial cells) were resuspended in 10 μL of SA. All the microbial suspensions (both the suspensions prepared from the cultivated strains and suspensions prepared from the collected fractions) were briefly sonicated (2 min), vortexed (2 min), and finally centrifuged at 3000g for 3 min. The supernatants were subjected to the MALDI-TOF MS analysis that was performed on AB Sciex TOF/TOF 5800 System operating in the linear positive ion mode. Spots on a MALDI plate were first overlaid with SA solution (0.5 μL per spot), and the matrix was left to dry at room temperature. Then, 0.6 μL of each microbial sample (supernatant) was deposited on the SA layer and left to dry. All sample spots were then overlaid again with 0.5 μL of the SA solution and left to dry. The microbial samples were spotted in duplicates on the sample plate, and each sample spot was analyzed five times. Each mass spectrum was automatically acquired with delayed extraction optimized for m/z 10 000 by accumulation of 1800 laser shots; 60 laser shots were delivered at a single point, and 30 different points were randomly selected within the sample spot. External calibration of the acquired mass spectra was performed using a protein calibration mixture containing equine cytochrome c (12 360 Da), equine myoglobin (16 951 Da), and bovine trypsinogen (23 981 Da). Both singly and multiply charged ions were used for the calibration. The recorded data were processed using Data Explorer (ver. 4.8, AB Sciex). Mass accuracy for protein molecular mass determination was found to be better than 0.17% for all the microbial samples. Safety. The potentially pathogenic microorganisms from risk group 2 of infectious agents were separated in this study. These pathogens are unlikely to be seriously hazardous to laboratory personnel. Laboratory exposures rarely cause an infection leading to a serious disease; effective treatment and preventive measures were available, and the risk of spreading was limited. Therefore, it was necessary to maintain a biosafety level 2.

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ABBREVIATIONS USED CE, capillary electrophoretic techniques; CIEF, capillary isoelectric focusing; CZE, capillary zone electrophoresis; EtOH, ethanol; FS, fused silica; Δh, the height difference of the reservoirs for the sample injection; IEF, isoelectric focusing; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; PAPAN, (1-[[4(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester; pI, isoelectric point; PSS, physiological saline solution; SA, 3,5-dimethoxy-4-hydroxycinnamic acid; t, migration time (min).

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+420)-5-32290221; E-mail: [email protected]. ORCID

Marie Horká: 0000-0001-9567-0741 Jiří Š alplachta: 0000-0003-1544-3691 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Czech Republic (Grant 19-00742S), the Ministry of Health of the Czech Republic (Grant 16-29916A), the Ministry of the H



Article

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