Separation of Methicillin-Resistant from Methicillin-Susceptible

Article pubs.acs.org/ac

Separation of Methicillin-Resistant from Methicillin-Susceptible Staphylococcus aureus by Electrophoretic Methods in Fused Silica Capillaries Etched with Supercritical Water Marie Horká,*,† Pavel Karásek,† Filip Růzǐ čka,‡ Milada Dvořać ǩ ová,‡ Martina Sittová,‡,§ and Michal Roth† †

Institute of Analytical Chemistry of the ASCR, 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, Brno, Pekařská 53, 602 00 Brno, Czech Republic § GeneProof a.s., Vídeňská 119, 619 00 Brno, Czech Republic ‡

ABSTRACT: Identification and prevention of Staphylococcus aureus-caused infections may benefit from a fast and dependable method to distinguish between the methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. The current methods involving polymerase chain reaction and/or other molecular tests are usually laborious and time-consuming. We describe here a fast and low-cost method employing capillary zone electrophoresis (CZE) to distinguish between MRSA and MSSA. The method makes use of a supercritical water-treated fused silica capillary, the inner surface of which has subsequently been modified with (3-glycidyloxypropyl)trimethoxysilane. With optimized proportions of suitable additives to the background electrolyte, a CZE separation of MRSA from MSSA may be completed within 12 min. The cells were baseline-resolved, and resolution was determined to be 3.61. The isoelectric points of MSSA and MRSA were found to be the same for both groups of these strains, pI = 3.4.

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of virulence factors by different isolates of S. aureus, and only a few of these seem to be produced as invariants.8−10 Rapid and accurate identification of MRSA isolates is essential to select an appropriate therapy as well as to control the infection effectively. Standardized microbiological methods for MRSA detection and identification involve cultivation followed by identification based on biochemical and morphological properties, and by methods of antimicrobial susceptibility testing (disk diffusion tests, broth microdilution methods, E-tests, etc.)2 requiring from 2 to 4 days.11 MRSA can be also detected using chromogenic and selective media,12 and various DNA-based genotyping techniques,13 pulsed-field gel electrophoresis,2,14 polymerase chain reaction (PCR),2,15 and multilocus sequence typing (MLST).2,16 Real-time PCR and other molecular tests are able to detect and differentiate MSSA and MRSA directly from blood cultures and are becoming a useful

ethicillin-resistant Staphylococcus aureus (MRSA) is a prevalent bacterial pathogen responsible for both hospital and community-associated infections of humans and of animals worldwide.1 These infections are associated with high morbidity and mortality compared with infections caused by methicillin-susceptible S. aureus (MSSA).2 Generally, it is not known whether MRSA has a greater capacity to cause invasive infection than MSSA strains.3 The ability of S. aureus strains to adhere to biotic and/or abiotic surfaces followed by the biofilm formation is considered as the virulence factor. It protects the bacterial cells against the action of antibiotic as well as the host immune system3 and allows them to rapidly colonize human body surfaces as well as medical implants and food products. Different adherences of the MRSA and the MSSA strains to the components of a host extracellular matrix are assumed.4−6 Little is known about the mechanisms.7 Despite a significant progress in understanding the pathogenesis of MRSA infection and the virulence mechanisms of MRSA strains, daunting challenges remain.3 Recent proteomics studies have revealed an enormous diversity in the production © 2014 American Chemical Society

Received: June 19, 2014 Accepted: September 10, 2014 Published: September 10, 2014 9701

dx.doi.org/10.1021/ac502254f | Anal. Chem. 2014, 86, 9701−9708

Analytical Chemistry

Article

tool for rapid detection of bacteremia.2 All of these techniques are time-consuming (from 6 to 19 h),17 laborious, expensive, and sometimes provide false-negative or false-positive results.11,18 Currently, a microfluidics chip coupled with surfaceenhanced Raman scattering spectroscopy was developed for rapid detection of and differentiation between pure MSSA and MRSA cultures.17 This technique shows advantages over traditional molecular typing methods, MLST and PCR, for ultrafast, automated, and reliable detection. Another powerful method for rapid and effective identification of staphylococcal infections is matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), which can reliably differentiate MRSA from MSSA.19 Capillary electrophoretic techniques (CE) can be one of the possibilities for the separation and the identification of MSSA and MRSA cells. In particular, capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE) represent effective analytical tools for differentiation and characterization of many microorganisms according to their respective isoelectric points20−31 and electrophoretic mobilities.27,32 The cells of S. aureus have a broad spectrum of cell wall-associated compounds (murein, teichoic acids, various polysaccharides, binding proteins for tissue and plasma factors).6 The measurements of their zeta potential should be very useful in distinguishing between closely related strains.33,34 Understanding the differences between S. aureus strains could help to improve our knowledge about S. aureus pathogenicity and to monitor for and respond to the emergence of more virulent strains.33 Based on the electrokinetic theory of biocolloids, the microbial surface charge strongly changes in response to environmental factors,35 which leads to the formation of an electric double layer between the solid surface and the surrounding liquid.36 The differences in mobility or isoelectric point, pI, allow for the separation and characterization of these microorganisms20 through, for example, the consequences of the differences for cell adhesion.37 In addition to other properties,38−40 different adherences of MSSA and MRSA to the compounds of host matrix4−6 can be a major parameter indicating the affinities of MSSA and MRSA cells to the fused silica (FS) capillary surface. Compared to the above-mentioned methods for identification of MRSA isolates, the CE techniques are less time-consuming, less laborious, and less expensive. These features may provide valuable advantages, particularly in demanding environments such as those of field hospitals in military missions, for example. The isoelectric point of the bacteria can be determined by measuring the electrophoretic mobility as a function of the pH41 or by potentiometric proton titration where the binding of protons and hydroxide ions to the cell walls was determined at a given pH.42 However, the isoelectric point of MSSA strain has been found to be 3.443,44 by CIEF. Although CIEF allows discrimination of microbes with close pI values, when the separation is performed in a wide pH gradient,43,45,46 the question arises whether the isoelectric points and/or electrophoretic mobilities of MSSA and MRSA will be sufficiently different and under what conditions. The aim of this study was to demonstrate the potential of rapid CE separation of MSSA and MRSA. This involves the selection of a suitable treatment of the FS capillary inner surface and application of suitable additives in the electrolyte systems.31 Within the surface treatment, the original FS will be etched with supercritical water (SCW), which is able to modify the inner surface of silica-based material.47,48 SCW does not contaminate the treated surface

with any residual impurities. The hydrophobicity of the etched FS capillary and the electroosmotic flow (EOF) will be modified with (3-glycidyloxypropyl)trimethoxysilane (GOTMS).46,47,49,50

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EXPERIMENTAL SECTION Chemicals. High-resolution ampholyte, pH 2−4, ampholyte pH 3−4.5, 2-morpholino-ethanesulfonic acid monohydrate, 3morpholino-propanesulfonic acid, and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxy-propanesulfonic acid were purchased from Fluka Chemie GmbH (Buchs, Switzerland). The solution of synthetic carrier ampholytes (Biolyte, pH 3−10) was obtained from Bio-Rad Laboratories (Hercules, CA). N-(2acetamido)-2-aminoethanesulfonic acid and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid were obtained from Merck (Darmstadt, Germany). L-Aspartic acid was obtained from LOBA Chemie (Vienna, Austria), and other spacers including the tartaric, malic, formic, succinic, acetic, pivalic, glutamic, and nicotinic acids, as well as the nonionogenic detergent Brij 35, GOTMS, acetone, and ethanol (EtOH) were purchased from Sigma (St. Louis, MO). Polyethylene glycol (Mr 10 000, PEG 10 000) was obtained from Aldrich (Milwaukee, WI). Specifications51,52 of the simple ampholytic electrolytes used as spacers were described elsewhere.43 The low-molecular pI markers, pI = 2.7, 3.7,53 4.0,54 and 4morpholinyl acetic acid43 were synthesized at the Institute of Analytical Chemistry of the ASCR, v. v. i. All chemicals were of electrophoresis, analytical grade. Bacterial Strains and Growth Conditions. The strains of MSSA, CCM6188, CCM1484, CCM2551, CCM4223, CCM3953, and MRSA CCM4750 were obtained from the Czech Collection of Microorganisms (Brno, Czech Republic). The strains of MRSA, FS133, FB125, FB124, FB123, and FB107, were isolated from clinical material and stored in Collection of Microbiology Institute, Masaryk University and St. Anne’s University Hospital (Brno, Czech Republic). The MRSA strains were detected using Cefoxitin Disk Screen Test55 and by cultivation on selective chromogenic medium Brilliance MRSA 2 Agar (Oxoid, United Kingdom). The identification was verified by detection of mecA gene by PCR reaction. Primers for PCR detection (mecA/for 5′-ACTGCCTAATTCGAGTGCTACT-3′and mecA/rev 5′-ATGGTAARGGTTGGCAAAAAGAT-3′) were manually designed to identify mecA gene in all available SCCmec types (GenBank accession numbers e.g. AJ810120.1, AB037671.1, AJ810121.1, AB781450.1). The tested strains included in this study were stored at −70 °C in Itest cryotubes (ITEST plus, Czech Republic). Before each experiment, the strains were thawed quickly at 37 °C and cultivated on Mueller−Hinton agar (Oxoid, United Kingdom) at 37 °C for 24 h. Fabrication of Etched FS Capillaries. Constant-diameter capillaries with roughened inner surfaces in several roughness degrees were prepared from commercially available cylindrical FS capillary (100 μm I.D., 360 μm O.D., Agilent Technologies, Waldbronn, Germany, part no. 160-2634-10) by etching with supercritical water in our original apparatus48 employing a procedure similar to that described before.44 For the present application, the etching apparatus was modified to allow for an independent control of the SCW−silica surface contact time (tcon) and the mass of SCW passed through the thermostated zone (mSCW). The etching was carried out at fixed conditions of 320 °C and 400 bar. These modifications were needed to 9702



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mL−1) for monitoring the pH gradient used (2.0−4.0). The injection time, tinj., of the spacers segment, the sample segment, and the segment of carrier ampholytes and pI markers was 25, 10, and 35 s, respectively. For CZE separations, 1−2 × 10−2 mol L−1 phosphate buffers pH 2−11, with addition of 0−5% (v/v) EtOH and 0.5% (w/v) Brij 35 or 0−0.1% (w/v) PEG 10 000, were used as BGE. The other conditions were the same as in the CIEF experiments. The cell suspensions of S. aureus strains were adjusted to concentrations from 5 × 107 to 1 × 108 cells mL−1. Thiourea was used as a neutral marker of EOF at the measurements of a pH effect on the electroosmotic mobility, μEOF. The injection time ranged from 5 to 16 s. Before each CIEF or CZE run, the capillaries were rinsed with acetone for 5 min and then back-flushed with the catholyte or BGE for 5 min. For this purpose, a single-syringe infusion 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. Resolution between the MRSA and MSSA cells, R, was calculated via eq 1 from the migration times, t, of the MRSA and MSSA cells and their respective baseline peak widths, w (in temporal units)

produce significant effects on the inner surface of the capillary while preserving a constant internal diameter throughout the length of the capillary. Two roughness degrees of the etched capillaries were chosen and employed below, namely, slightly etched FS capillaries (SEFS) with roughness of 0.350 μm (etching conditions: 320 °C, 400 bar, tcon = 6 min, mSCW = 0.51 g) and long-term etched FS capillaries (LEFS) with roughness of 2 μm (etching conditions: 320 °C, 400 bar, tcon = 24 min, mSCW = 2.04 g). Modification of FS Capillaries. The FS capillaries, both original and etched, were rinsed by the solution of 5% (v/v) GOTMS in distilled water for 1 h at the flow velocity of 5 μL min−1, then washed with water for 1 h and immediately used. CIEF and CZE Equipment and Procedures. CIEF and CZE were carried out using a laboratory-made apparatus43 at a constant voltage (−20 kV on the detector side) supplied by a Spellman CZE 1000 R high-voltage unit (Plainview, NY). Each separation capillary consisted of SCW-treated and original (untreated) segments. The total lengths of the FS capillaries, 100 μm I.D. and 360 μm O.D. (Agilent Technologies, Santa Clara, CA), were 45 cm, with 20 cm long untreated part and 25 cm long SCW-treated part. The detection window was created in the untreated part of the capillary at the distance of 25 cm from the anodic end of the capillary. The capillaries employed included original FS and the FS etched with SCW and/or modified with GOTMS. The ends of the capillary and the electrodes were placed in 3 mL glass vials filled with an anolyte or a catholyte or background electrolyte (BGE). A LCD 2082 on-column UV−vis detector (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ), was operated at either 235 or 280 nm. Light absorption (optical density) of the bacterial samples was measured by use of a DU 520 UV−vis spectrophotometer (Beckmann Instruments, Palo Alto, CA) operating at 550 nm. Sample injection was performed by siphoning action as described in our previous paper.45 Height difference of the reservoirs for the sample injection, Δh, was 20 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 injection of the bacterial 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 10 times. The detector signals were acquired and processed with the Clarity Chromatography Station (ver. 2.6.3.313, DataApex, Prague, Czech Republic). For CIEF separations, 4 × 10−2 mol L−1 sodium hydroxide and 0.1 mol L−1 orthophosphoric acid, both with addition of 3% (v/v) EtOH and 0.5% (w/v) Brij 35 or 0.5% (w/v) PEG 10 000, were used as the catholyte and the anolyte solutions, respectively. Segmental injection into the capillary was employed in CIEF analysis.43,45 Solutions necessary for the CIEF run (including the sample) were injected in three consecutive segments. The first segment of spacers, a solution of selected simple ampholytic electrolytes, 15 × 10−5 mol L−1, was dissolved in the catholyte. The second segment was the cell suspension of the examined S. aureus strains. The cell concentration was adjusted to 1 × 108 cells mL−1. The third segment was a 5% (w/v) aqueous solution of the commercial carrier ampholytes Biolyte pH 3−10, ampholytes pH 3.0−4.5, and pH 2.0−4.0, mixed in the 1:2:5 ratio, respectively. This segment also contained low-molecular-mass pI markers (pI 2.7, 3.7 and 4.0; concentration of individual pI markers 25 μg

R=2

tMRSA − t MSSA wMRSA + wMSSA

(1)

Safety Considerations. The potentially pathogenic microorganisms, S. aureus, from a 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; an effective treatment and preventive measures are available, and the risk of spreading is limited. Therefore, a biosafety level 2 is necessary to be maintained.

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RESULTS AND DISCUSSION In the preliminary experiments, we performed the optimization of the MSSA and MRSA cells separation in an original FS capillary by CIEF and CZE. Different experimental conditions were used including the combinations of additives31 added into the background electrolytes and/or samples. In the original FS capillary, however, the discussed strains were not separated from each other. Recently, tapered FS capillaries prepared by etching with SCW were used to separate similar strains of bacteria.56,57 The results reflected the influence of chemical and mechanical modification of the capillary surface in addition to the geometry of the capillary. In our current experiments, cylindrical FS capillaries with two different structures of their inner surface were prepared by etching of the original capillaries with SCW. In one type of experiments, the original FS capillaries were just slightly etched (SEFS) by SCW. The SCW created a large number of small caves with sharp edges and spikes on the inner surface (see Figure 1A), with the depth of pits being close to 0.350 μm. In turn, a prolonged time of etching causes a total loss of any sharp surface formation in the long-term-etched FS capillaries (LEFS) where the edges are smooth and rounded. The surface structures have regular, homogeneous porosity with 2 μm roughness, see Figure 1B. The effect of etching with SCW, the modification of the inner surface with GOTMS, and the effect of additives in the BGEs on the EOF and on the possible adsorption of bacteria48,56,57 9703



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Figure 2. Effect of the pH on the electroosmotic mobility in original, etched with SCW and modified FS capillaries by GOTMS. Conditions and designations, see Figure 1; FS capillaries: (1) original FS, (2) SEFS, (3) LEFS; (4) original FS modified with GOTMS, (5 and 6) LEFS modified with GOTMS; (1−5) BGE: 1 × 10−2 mol L−1 phosphate buffer from pH 2 to 11; (6) BGE: 2 × 10−2 mol L−1 phosphate buffer with additives − 0.05% (w/v) PEG 10 000 and 5% (v/v) EtOH; siphoning injection (Δh = 20 cm, 5 s); applied voltage (−)20 kV; neutral marker of EOF, thiourea; UV detection, 235 nm; μEOF, electroosmotic mobility [cm2 V−1 s−1].

Figure 1. FS capillaries etched with SCW. FS, 100 μm I.D., 360 μm O.D., 450 mm length (250 mm separation channel to the detection window (5 mm), 200 mm nontreated part toward the cathode vial); microscopically enlarged: FS capillary, 450× , (A) SEFS (roughness 0.350 μm) and (B) LEFS (roughness 2 μm), 25 000×.

In the following experiments, the etched FS capillaries inner surfaces were modified by GOTMS to decrease the EOF, and to prevent the adsorption of examined cells onto the capillary inner surface. The additives, 0.5% (w/v) Brij 35 and 3% (v/v) EtOH, were dissolved in the catholyte, 40 × 10−2 mol L−1 NaOH and the anolyte, 0.1 mol L−1 H3PO4. The results of the optimizing procedures are shown in Figures 3A,B. The pH gradient was traced with the pI markers (pI 2.7, 3.7, and 4.0). Only one narrow peak of MSSA and MRSA cells was detected in both types of the capillaries, see Figure 3A (SEFS) and Figure 3B (LEFS). The migration velocities of the analytes were higher in electropherogram shown in Figure 3B than in Figure 3A. Simultaneously, the discussed baseline peak width, w, was approximately 50% wider on LEFS than on SEFS. A tailing peak was detected. An average coccus-shaped (spherical) bacterium S. aureus is about 0.8−1.0 μm in diameter58 compared to homogeneous porosity of LEFS with 2 μm roughness. It is possible that the larger surface pores lead to the change in the peak shape. The pI of MSSA and MRSA cells are probably the same or very similar to the order of thousandths of pI values. To confirm this assumption, the migration velocity of the cells was decreased by the addition of 3% (v/v) EtOH and of 0.3% (w/v) PEG 10000 into the solution of the catholyte and the anolyte in the CIEF separation shown in Figure 3C. For the separation the LEFS capillary was used. To maintain the reproducibility of measurements, the capillaries were thoroughly washed with acetone between each focusing run. Again, only one narrow peak was detected in the electropherogram shown in Figure 3C. Neither of the different cultivated MSSA and MRSA strains were separated under these

onto the inner surfaces of examined capillaries are discussed below. Effect of Etching and Modification of FS Capillaries on the EOF. At first, only the effect of FS etching with SCW on the EOF was compared, see Figure 2. The phosphate buffer (1 × 10−2 mol L−1) was used as BGE without additives. The results are illustrated for the original FS (curve 1), the SEFS (curve 2), and the LEFS (curve 3). Throughout the pH range, the EOF in the capillaries etched with SCW was higher than in the original FS capillary. A significant decrease in EOF occurred after the modification of the original FS capillary (curve 4) and the LEFS (curve 5) with GOTMS. The reduction in EOF is needed for successful CIEF and CZE separations of MSSA and MRSA cells on the etched FS capillaries. The electroosmotic mobility was increased in LEFS approximately to the level of original FS capillary after addition of 0.05% (w/v) PEG 10 000 and 5% (v/v) EtOH into 2 × 10−2 mol L−1 phosphate buffer, see curves 1 versus 6 in Figure 2. CIEF of MSSA and MRSA Cells on the Etched and Modified FS Capillaries in the pH Gradient, pH Range of 2.7−4.0. The conditions for the CIEF separation of the cultivated MSSA and MRSA cells were optimized on both discussed types of only etched FS capillaries, SEFS and LEFS. However, only one wide zone of the analyzed cells was again detected at these conditions. Its isoelectric point was determined to be 3.4. That corresponds to the isoelectric point of MSSA.43,44 9704



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Figure 3. CIEF of MSSA and MRSA cells on etched and modified SEFS or LEFS in the pH range of 2.7−4.0. Conditions and designations, see Figure 1 and 2; (A) SEFS, (B,C) LEFS; the catholyte, 4 × 10−2 mol L−1 NaOH, the anolyte, 0.1 mol L−1 H3PO4; additives: (A,B) 0.5% (w/v) Brij 35, 3%(v/v) EtOH, (C) 0.03% (w/v) PEG 10 000, 3% (v/v) EtOH; sample composition−segment of spacers dissolved in the catholyte, tinj, 25 s, segment of carrier ampholytes, 5% (w/v) of synthetic carrier ampholytes, Biolyte, pH 3− 10, ampholyte pH 3−4.5 and pH 2−4 (1:2:5) and pI markers pI 2.7, 3.7, and 4.0 (25 μg mL−1 of each), tinj, 35 s; sample: S. aureus CCM3953 (MSSA) a CCM4750 (MRSA), each of them 108 cells mL−1, tinj, 10 s; Δh, 20 cm; UV detection, 280 nm; rinsing procedure, acetone for 5 min, and then back-flushed with the catholyte for 5 min; t, migration time [min.].

Figure 4. CZE of MSSA and MRSA cells on the etched and modified LEFS capillary in the phosphate buffer from pH 4 to pH 7. Conditions and designations, see Figures 1−3; BGE: 1 × 10−2 mol L−1 phosphate buffer, pH 7 (A), pH 6 (B), pH 5 (C), and pH 4 (D); sample: MSSA and MRSA cells, each of them 5 × 107 cells mL−1; tinj, 10 s; Δh, 20 cm; the dependence of t MSSA (full circle) and MRSA (empty triangle) on the buffer pH (E).

conditions. It can be assumed that these strains are characterized by the same isoelectric point (= 3.4). CZE of MSSA and MRSA Cells on the Modified LEFS: Effect of pH. The samples of MSSA and/or MRSA cells, each of them 5 × 107 cells mL−1, were separated in CZE using 1 × 10−2 mol L−1 phosphate buffer. The selected pH ranges (from pH 4 to pH 7) of the BGE included linear growth region of EOF on pH, see Figure 2, curve 5. We deliberately did not use any additives in the BGE in order to learn the properties of the inner capillary surface itself. The migration times, t, of the MSSA and/or MRSA zones (Figure 4A−C,E) increased with decreasing EOF from pH 7 to pH 5 in accordance with the dependence in Figure 2, curve 5. The change in the linear relationship of pH on t occurs at pH of electrolytes less than 5, see Figure 4E, near the S. aureus pI of 3.4. With the decreasing pH, the MSSA and MRSA cells gradually became separated in two different zones, but with very low resolution, see Figure 4C−E (full circle, MSSA vs empty triangle, MRSA). Therefore, the subsequent optimized CZE experiments were carried out at

the buffer pH 5. Although the relative standard deviation (RSD) of the migration times was acceptable at about 2.0%, very poor quantitative response was reached. This was probably due to the capture and/or partial adsorption of the cells onto the porous surface. For comparison, see the results in the following sections. CZE of MSSA and MRSA Cells on the Modified LEFS: Effect of Additives. The composition of BGE was based on the previous experiences in CIEF/CZE separation of the Staphylococcus strains. In the present CZE separation (Figure 5), selected additives, 0.1% (w/v) PEG 10 000 and 3% (v/v) EtOH, were dissolved in 2 × 10−2 mol L−1 phosphate buffer, pH 5. The suspensions of the MSSA or MRSA cells alone or the mixture of MSSA and MRSA cells (each of them 5 × 107 cells mL−1) were analyzed, and the electropherograms are shown in Figure 5A. In these CZE runs, the capillary was carefully washed between each separation run with acetone. The cells of MSSA were fully adsorbed on the inner surface of the capillary and therefore were not detected (dotted line). A 9705



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capillary was insufficiently coated with PEG in the last CZE run (Figure 5D). Optimized Condition for the Reproducible CZE Separation of MSSA and MRSA Cells. The lower concentration of PEG 10 000 than 0.1% (w/v) and higher than 3% (v/v) EtOH dissolved in 2 × 10−2 mol L−1 phosphate buffer (pH 5) as BGE should provide a good basis for separation of the discussed MSSA and MRSA cells on the LEFS modified by GOTMS. Qualitative and quantitative reproducibility of the measurement was reached at the concentration of 0.05% (w/v) PEG 10 000 and 5% (v/v) EtOH in BGE. At these conditions, the EOF on LEFS was comparable to the EOF on original FS capillary, see Figure 2, curve 6. The CZE separations of MSSA and MRSA alone or their mixture are shown in Figure 6A−D. Since the examined cells of S. aureus have a strong tendency to form agglomerates, a thorough sonication and vortexing of their samples were necessary before the CZE separation (see the Experimental Section). Without this preparation the

Figure 5. Monitoring of migration times of MSSA and MRSA cells at CZE in the etched and modified LEFS capillary without back-flushing with acetone after the measurements (A). Conditions and designations, see Figures 1−4; BGE: 2 × 10−2 mol L−1 phosphate buffer, pH 5; additives: (A) first measurement, 0.1% (w/v) PEG 10 000, 3% (v/v) EtOH, (B−D) second to fourth measurement, 3% (v/ v) EtOH; sample: (A) dotted line, MSSA cells only and (A−D) full line, the mixture of MSSA and MRSA strains (each of them 5 × 107 cells mL−1) were injected into the capillary.

narrow zone of MRSA cells identical to the MSSA and MRSA cells mixture was detected in the relatively same migration time (full line). We assumed that a greater cell hydrophobicity of MSSA beside MRSA in combination with the nonhomogenous dynamic PEG coating of the inner LEFS surface (relatively high porosity) were the cause of complete MSSA adsorption onto the capillary. In the subsequent CZE separations (see Figures 5B−D), the capillary from the experiments depicted in Figure 5A, full line, was not washed after and between each of the CZE runs. Only 3% (v/v) EtOH was dissolved in the phosphate buffer used in CZE experiments shown in Figure 5B−D. The zone of MSSA cells was detected, and resolution of MSSA and MRSA increased with the gradual elution of PEG from the capillary inner surface under the action of EtOH in the BGE, for comparison see Figure 5B versus Figure 5C,D. In the electropherograms shown in Figure 5C,D, certain resolution of MSSA and MRSA zones was reached but again with a low level of the quantitative response, similarly as in Figure 4 (BGE without dissolved additives). We suppose that the surface of the

Figure 6. Separation of MSSA and MRSA cells at the optimized CZE conditions. Conditions and designations, see Figures 1−4; BGE: 2 × 10−2 mol L−1 phosphate buffer, pH 5; additives: 0.05% (w/v) PEG 10 000, 5% (v/v) EtOH; sample: the suspension of the bacteria was not (A) and was (B−D) immediately sonicated and vortexed prior to the separations; the number of injected cells in 1 mL for each of the mentioned strains was 108 (A, dotted line) or 5 × 107 (B−D, full line); (A,B) mixture of the strains and (C,D) individual MSSA or MRSA cells were injected into the capillary. 9706



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Table 1. Migration Time, t, and Baseline Peak Width, w, of Examined Strains of S. aureus and Their Resolution, Ra t [min]

MSSA strains CCM6188 CCM1484 CCM2551 CCM4223 CCM3953 a

11.42 11.61 11.49 11.55 11.68 tM = 11.55 min

w [min]

MRSA strains

0.11 0.09 0.09 0.10 0.11

CCM4750 FS133 FB125 FB124 FB123

t [min] 11.81 12.06 11.78 11.92 12.05 tM = 11.92 min

w [min]

R

0.13 0.11 0.09 0.12 0.09

3.25 4.50 3.22 3.36 3.70 R = 3.61

The tM entries and R in the last line are the mean values of the migration time and resolution.

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ACKNOWLEDGMENTS This work was supported by the Ministry of the Interior of the Czech Republic (Grant VG20112015021), by the Ministry of Health (Grant NT/13242- 4), by the Czech Science Foundation (Grant P106/12/0522), and by the Academy of Sciences of the Czech Republic (Institutional Support RVO:68081715).

agglomerates were detected (Figure 6A, curves 1 and 2). The quantifications of these samples were impossible, compare the peak areas on the curves 1 (5 × 107 cells mL−1) and 2 (1 × 108cells mL−1) shown in Figure 6A. However, the enveloping curves of MSSA and MRSA agglomerates copy approximately the zones of the identical but vortexed samples, see for comparison Figure 6A versus Figure 6B−D. In these electropherograms, narrow zones of the analyzed cells were detected. Each of the five bacterial MSSA or MRSA strains was analyzed at least 10 times. RSD of the migration times was about 1.1%. The quantitative reproducibility (from 107 to 108 cells mL−1) was characterized by a value of the coefficient of determination R2 = 0.99. Resolution of a sample component, R, was calculated from the average of the migration time, tM, of the MSSA or MRSA strains and their baseline peak width (these values are listed in Table 1) and was determined to be 3.61. However, because of the enormous diversity3,8−10 of S. aureus strains, it is highly probable that they are characterized by different migration times at the same separation conditions. Nevertheless, due to the good resolution and reproducibility of the measurements, the method of standard addition59 can be used for faster and simpler identification of the discussed S. aureus strains in a standard laboratory.

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CONCLUSION A dependable distinction of methicillin-resistant (MRSA) from methicillin-susceptible (MSSA) S. aureus strains is very important for identification of both hospital and communityassociated infections of humans and of animals. It was found that the isoelectric points of MSSA and MRSA strains are the same, pI = 3.4. However, it is still possible to separate the MSSA and MRSA strains by CZE. Here, the experimental protocol has been optimized of the CZE separation of MRSA from MSSA. The electrophoretic separation makes use of supercritical water-treated and surface-modified fused silica capillary and of an optimized composition of the background electrolyte. In this sense, the electromigration-based distinction of MRSA from MSSA provides a relatively low-cost and fast complement to the costly, laborious, and time-consuming methods based on PCR and other molecular tests. Under the optimized operating conditions, the separation of MRSA from MSSA by capillary zone electrophoresis may be completed within 12 min.

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