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Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis Marie Horká a, * , Marie Tesarová a , Pavel Karásek a , Filip Ruži9 cka b,c , Veronika Holá b,c , b,d a Martina Sittová , Michal Roth
Institute of Analytical Chemistry of the ASCR, v.v.i., Veverí 97, 602 00 Brno, Czech Republic The Department of Microbiology, Faculty of Medicine, Masaryk University, Kamenice 53/5, 625 00 Brno, Czech Republic c The Department of Microbiology, St. Anne’s University Hospital, Brno, Pekarská 53, 602 00 Brno, Czech Republic d ská 119, Brno, 619 00 Brno, Czech Republic GeneProof a.s., Víden a
b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
We used CE for separation of MRSA and MSSA strains from clinical samples. MH agar-cultivated and blood-incubated cells of S. aureus were examined. GOTMS-modified FS etched with SCW was used for CE separation. The tested MRSA and MSSA strains were separated in CZE from each other. These methods appear to be sufficient for rapid screening of clinical samples.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2014 Received in revised form 28 January 2015 Accepted 1 February 2015 Available online xxx
Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (101–102 cells mL1) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. Recently, capillary electrophoresis (CZE) was successfully used to differentiate between the agarcultivated MRSA and MSSA strains in fused silica capillaries etched with supercritical water and modified with (3-glycidyloxypropyl)trimethoxysilane. The possible use of CZE as a fast and low-cost method for distinguishing between the blood-incubated MRSA or MSSA cells has been tested in this manuscript. Our goal was to test low amounts of bacteria (102 cell mL1) similar to those in clinical samples. The
Keywords: Capillary electrophoresis Supercritical water FS capillary Methicillin-resistant Staphylococcus aureus – MRSA Methicillin-susceptible Staphylococcus aureus – MSSA Whole human blood
* Corresponding author. Tel.: +420 5 32290221; fax: +420 5 41212113. E-mail address: [email protected] (M. Horká). http://dx.doi.org/10.1016/j.aca.2015.02.001 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Horká, et al., Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis, Anal. Chim. Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.02.001
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migration times of the purified blood-incubated cells and the agar-cultivated cells were different from each other. However, their isoelectric point was the same for all strains. ã 2015 Elsevier B.V. All rights reserved.
1. Introduction The Gram-positive bacterium Staphylococcus aureus is a human bacterial pathogen causing many nosocomial infections from skin lesions to life-threatening diseases such as bacteraemia, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, and septicaemia [1]. Treatment of these diseases has become more difficult and more expensive because of the increasing prevalence of multiresistant strains, especially methicillin-resistant S. aureus (MRSA) in recent decades. However, because MRSA increases morbidity and costs of treatment of patients compared with infections caused by methicillin-susceptible S. aureus (MSSA), many hospitals have attempted to control the spread of the former pathogen. The incidence of the majority of infections caused by S. aureus is conditioned by multiple virulence factors such as structural and secreted products. Coagulase, protein A, elastin-binding protein, collagen-binding protein, fibronectin-binding protein, and clumping factor are surface proteins involved in establishing of infection [2–6]. Enterotoxin B, a-toxins and TSST-1 belong to the secreted products needed for an outbreak of infection disease [2,6]. Most MRSA bacteria are able to form biofilm enabling them to colonize artificial prosthetic surfaces. Biofilm protects the microorganisms against host defenses and antimicrobials. And it can also be considered an important factor of virulence [2,6–9]. Currently, there are many techniques used for MRSA and MSSA strain differentiation. The techniques can be divided into three groups, phenotyping, genotyping, and mass spectrometry. Phenotyping methods include antibiogram, phage typing, serotyping, and protein electrophoresis. Plasmid analysis, analysis of chromosomal DNA, southern hybridization, pulsed-field gel electrophoresis, PCR typing, and repetitive element sequence-based PCR belong to the genotyping methods [10]. In most cases, phenotyping methods are easy to perform, cheap, and readily available in the routine laboratories. The major disadvantages are poor discriminatory ability and lack of reproducibility. Most of the abovementioned techniques are time-consuming (6–19 h). Potential disadvantages of genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. On the other hand, these methods alone are rapid and they have good reproducibility and repeatability. The major advantage of PCR and other molecular techniques is the ability to detect and differentiate between MSSA and MRSA directly from blood cultures [10]. The presence of charged groups on the outer microbial surface and the ensuing electric mobility of the cells can be utilized for differentiation and characterization of MRSA and MSSA by capillary electrophoretic techniques (CE) [11–21] such as capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE), according to their isoelectric points, pI,[14,22–30] and electrophoretic mobilities [28,31], respectively. Previously, the isoelectric points of MSSA and MRSA strains has been found to be the same at 3.4 [21,32,33]. Identification of bacteria directly from blood is difficult and time-consuming [10,34–39], and a rapid detection of low number of S. aureus cells (101–102 cells mL1) in blood is necessary to detect bloodstream infection. A fast detection and identification of MRSA and MSSA also makes it possible to select an appropriate therapy. Since the volumes of the collected blood samples are usually between 10 and 20 mL, about 100–2000 bacterial cells are usually
present in the whole blood samples. However, blood is a complex matrix containing many components such as cells (erythrocytes, leucocytes, and thrombocytes), proteins, glucose, amino acids, fatty acids, products to be disposed of (carbon dioxide, urea, and lactic acid), and other components (serum albumin, blood-clotting factors, immunoglobulins, lipoproteins particles, and electrolytes). These components cause higher background and poor identification of bacteria during separation. It is necessary to purify the bacterial blood sample and remove the blood components from it [39]. One of the procedures to purify the blood samples was used in our previous article [40], and this procedure was modified and employed in the present work. This study demonstrates the possibility of rapid CE separation of MSSA and MRSA directly from whole human blood using FS capillary etched with supercritical water (SCW) [21,40–43]. The adherence of the MSSA and MRSA cells to the inner surface of the modified FS capillary etched with SCW together with the differences in their migration times at the CZE separation were monitored before [21]. The hydrophobicity of the etched FS capillary and the electroosmotic flow (EOF) were modified with (3-glycidyloxypropyl)trimethoxysilane (GOTMS) [21]. The bacterial adhesion to different kinds of surfaces of orthopaedic implant materials was discussed before [44]. The adhesion depends on the interactions which occur between cells, on the cell structures, shapes, and surface features at different levels of roughness of the surface. These properties were used in this work. 2. Material and methods 2.1. Chemicals GOTMS, acetone and ethanol (EtOH) were purchased from Sigma (St. Louis, MO, USA). Polyethylene glycol (Mr 10,000, PEG 10,000) was obtained from Aldrich (Milwaukee, WI, USA). All chemicals were of electrophoresis or analytical grade. 2.2. Bacterial strains and growth conditions The strains of MSSA, CCM6188, CCM1484, CCM2551, CCM4223, CCM3953, and MRSA CCM4750 were obtained from Czech Collection of Microorganisms (Brno, Czech Republic). The S. aureus strains, CCM3953 and CCM4750, were always used as positive control at the presentation of the single strain separation in Section 3. The MSSA strains, FB11, FB129, FB130, FB131 and MRSA strains, FS133, FB125, FB124, FB123, FB128, FB127, FB126, 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 Test [45] 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 50 -ACTGCCTAATTCGAGTGCTACT-30 and mecA/rev 50 -ATGGTAARGGTTGGCAAAAAGAT-30 ) 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). Before each experiment, the tested strains were cultivated on Mueller-Hinton agar (MH agar, Oxoid, United Kingdom) at 37 C for 24 h and the standardized microbial suspensions were prepared as described previously [46].
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The microbial cultures were resuspended in physiological saline solution (PSS). The concentration of the resuspended microorganisms was estimated by measurement of the optical density of the suspension using a DU 520 UV–vis spectrophotometer (Beckmann Instruments, Palo Alto, CA, USA) operating at 550 nm, according to the calibration curve which was defined by reference samples. The numbers of microorganisms in the reference samples were controlled by serial dilution and plating of 100 mL of the suspension on MH agar. The colonies were counted after the cultivation at 37 C for 24 h. 2.3. Microbial sample preparation The required number of cells in 1 mL of the suspension has been achieved by serial dilution in 15 103 mol L1 PSS. The number of staphylococcal (MRSA or MSSA) cells in the initial suspension was 108 or 109 cell mL1. The whole human blood was spiked with cell suspension of MRSA and/or MSSA from 102 to 108 cell mL1, vortexed, and incubated for 1 h at 37 C in Thermomixer comfort (Eppendorf, Hamburg, Germany). These cells were isolated and purified using the procedure described below. One milliliter of distilled water was added to 1 mL of blood spiked with the examined bacteria. The obtained suspensions were vortexed for 30 min at 25 C. Subsequently, these suspensions were centrifuged at 1000 g for 10 min on the Concentrator 5301 (Eppendorf, Hamburg, Germany). Then, supernatants were removed. Cells on the bottoms of vials were covered with 1 mL of PSS, vortexed at 25 C for 5 min, centrifuged at 1000 g for 10 min, and supernatants were again removed. This procedure was repeated five times. Distilled water was used instead of PSS in the last purification run. The suspensions were again centrifuged at 10 000 g for 15 min. The resulting purified, blood-incubated MRSA cells (MRSA-B) or MSSA cells (MSSA-B) were re-suspended in 15 103 mol L1 PSS. Each sample was vortexed and then immediately injected into the capillary. 2.4. Fabrication of etched FS capillaries Constant-diameter capillaries with roughened inner surfaces were prepared from commercially available cylindrical FS capillary (100 mm I.D., 360 mm O.D., Agilent Technologies, Waldbronn, Germany, Part No. 160-2634-10) by etching with SCW in our original apparatus [42] employing a procedure similar to that described before [21,41]. In the present work, the etched FS capillaries with 2-mm inner surface roughness were employed (etching conditions: 320 C, 400 bar, tcon = 24 min, mSCW = 2.04 g). 2.5. Modification of FS capillaries The original and SCW-etched FS capillaries were rinsed by the solution of 5% (v/v) GOTMS in distilled water for 1 h, then washed with water for another 1 h, and immediately used afterwards [21]. 2.6. CZE equipment and procedures CZE was carried out using a laboratory-made apparatus [46] at a constant voltage (20 kV on the detector side) supplied by a Spellman CZE 1000R high-voltage unit (Plainview, NY, USA). The lengths of the FS capillaries, 100 mm I.D. and 360 mm O.D. (Agilent Technologies, Santa Clara, CA), were 35 cm, 20 cm to the detector. SCW-etched FS capillaries were modified with GOTMS. The ends of the capillary and the electrodes were placed in 3-mL glass vials filled with a background electrolyte (BGE). An LCD 2082 on-column UV–vis detector (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ, USA), was operated at 280 nm. Sample injection was
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accomplished by siphoning action as described in our previous paper [47] or by a single-syringe infusion pump (Cole-Parmer, Vernon Hills, IL, USA) equipped with a 10-mL syringe (SGE Analytical Science, Victoria, Australia). The flow rate was 166 nL min1. Height difference of the reservoirs for the sample injection by siphoning action, Dh, 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, USA) 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. Detector signals were acquired and processed with the Clarity Chromatography Station (ver. 2.6.3.313, DataApex, Prague, Czech Republic). At the separations, 2 102 mol L1 phosphate buffers pH 5 with addition of 5% (v/v) EtOH and 0.1 % (w/v) PEG 10 000 were used as BGE. Before each run, the capillaries were rinsed with acetone for 5 min and then back-flushed with BGE for 5 min. For this purpose, a single-syringe infusion pump (Cole-Parmer, Vernon Hills, IL, USA) with a 100-mL syringe (SGE Analytical Science, Victoria, Australia) was used at a flow rate ranging from 3 to 20 mL min1. The cell suspensions of the discussed S. aureus strains (cultivated on MH agar or purified, blood-incubated cells) were adjusted to concentrations from 1 102 to 1 108 cells mL1.The injection time was 8 s for siphoning action which represents 100 nL of the microbial suspension injected into the capillary. The injection time for the syringe pump was 290 s. The sample segment was composed of 200 nL of BGE, 100 nL of the microbial suspension, and again 500 nL of BGE in the 10-mL syringe. 2.7. 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. 3. Results and discussion 3.1. Optimization of the CZE conditions and microbial sample pretreatment The examined bacteria, MSSA-B and/or MRSA-B cells (sonicated and vortexed), were separated on the original FS capillary modified by GOTMS in the preliminary experiments. However, only a broad peak of unresolved MSSA-B and MRSA-B cells was detected instead of a narrow peak like in Ref. [21]. This was probably caused by a change in the surface properties of the cells after their incubation in whole blood which corresponds to the earlier findings [44]. S. aureus possesses a number of adhesins, such as polysaccharide intercellular adhesin, which enables the adhesion of staphylococcal cells to each other and by this way facilitates the formation of staphylococcal aggregates [48]. Moreover, S. aureus belongs to the plasma-coagulase positive staphylococci that produce virulence factors such as coagulase [49] and clumping factor A [15,50,51] which also support the staphylococcal cells aggregation in blood environment due to conversion of fibrinogen to fibrin. This property also causes a rapid agglomeration of the purified cells of S. aureus, MSSA-B and MRSA-B. Therefore, rigorous sonication and vortexing of the sample suspension prior to the separation by CE is necessary. Fig. 1A shows the record of the CZE separation of carefully sonicated and vortexed MSSA-B and
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with certainty whether the RSD only reflects the measurement error in the discussed set of strains with very divergent surface properties, or whether at least the migration times of these strains [2–9] are different. Without doubt, the difference between the average migration times of the cultivated MSSA and MRSA cells was approximately 0.35 min and that between the MSSA-B and MRSA-B was 1.85 min at our experimental conditions. The increase in the migration times of MH agar-cultivated or purified, bloodincubated cells of MSSA and MRSA strains is associated with the changes in their surface energies [45] after incubation of the cells in blood. The surface energy changes probably influence the adherence of the cells to the inner surface of the separation capillary. We also examined whether the isoelectric point of purified cells of S. aureus, MSSA-B and MRSA-B, in comparison to the cells cultivated on MH agar was changed. The isoelectric point for MSSA and MRSA strains, agar-cultivated, was previously determined to be 3.4 [21]. The individual strains or the mixtures of the first five strains listed in Table 1 were gradually focused by CIEF. .In accordance with the previous report [21], only one narrow peak was detected in the migration time which corresponds to the isoelectric point of 3.4. Certainly, differences of the order of thousandths to hundredths of pI units between the individual strains of S. aureus can be assumed as described before [21]. Fig. 1. CZE separation of S. aureus strains cultivated on MH agar and purified, bloodincubated staphylococci on modified FS capillaries etched with SCW. Conditions: FS capillaries, 100 mm ID, 360 mm OD, 350 mm length (200 mm length to the detection window, 150 mm toward the electrode vial); FS was modified with the solution of 5% (v/v) GOTMS; applied voltage ()20 kV; UV detection 280 nm; BGE: 2 102 mol L1 phosphate buffer pH 5 with additives: 0.1% (w/v) PEG 10,000 and 5% (v/v) EtOH; sample composition: (A) MSSA-B and MRSA-B, 5 107 cell mL1 each; (B) MSSA-B and MRSA-B, 5 107 cell mL1 each, and 5 107 cell mL1 MSSA and 2 107 cell mL1 MRSA, samples were immediately sonicated and vortexed before separations; siphoning injection (Dh = 20 cm, 8 s); rinsing procedure, by acetone for 5 min. and then back-flushed with the catholyte for 5 min; t, migration time (min).
MRSA-B cells (5 107 cell mL1 each) on SCW-etched FS capillary modified with GOTMS at the optimized conditions. The separation of a more complex mixture of S. aureus cells is shown in Fig. 1B. Here, the MSSA-B and MRSA-B cells (the same mixture as in Fig. 1A) were added directly into suspension of MSSA (5 107 cell mL1) and MRSA (2 107 cell mL1) cells immediately before the separation. Two zones in each group of these examined cells were detected. The first group corresponds to the cells cultivated on MH agar and the second group to the cells incubated in blood. The migration times of these strains are listed in Table 1. The average migration times for each of the strains were obtained from a minimum of eight independent measurements. The relative standard deviation (RSD) of the migration times was under 1.0%. Very good qualitative and quantitative responses (coefficient of determination R2 = 0.99) were reached. However, we cannot say
Table 1 The migration time, t, of examined strains of S. aureus cultivated on MH agar (MSSA, MRSA) and purified, blood-incubated cells (MSSA-B, MRSA-B). Strains
CCM 6188 CCM 1484 CCM2551 CCM4223 CCM3953 FB11 FB129 FB130 FB131 taverage
t (min)
Strains
MSSA
MSSA-B
9.58 9.48 9.41 9.61 9.49 9.55 9.51 9.60 9.59 9.55
10.22 10.25 10.19 10.23 10.24 10.10 10.25 10.22 10.17 10.22
CCM4750 FS133 FB125 FB124 FB123 FB107 FB128 FB127 FB126 taverage
t (min) MRSA
MRSA-B
9.89 9.93 9.90 9.95 9.80 9.92 9.91 9.87 9.86 9.90
12.06 12.16 12.09 12.18 12.01 12.08 12.02 11.99 12.07 12.07
3.2. CZE separation of clinically important levels of MSSA-B and MRSAB cells Rapid detection of low numbers of pathogens (101–102 cells mL ) in blood is necessary to detect bloodstream infection. Since the collected blood sample volumes usually range between 10 and 20 mL, around 100–2000 bacterial cells are present in a whole blood sample. In the following experiments, we intend to demonstrate the feasibility of direct injection of nL-sample volumes [52] of the microbial suspensions containing clinically significant number of S. aureus cells per 1 mL, and their subsequent CZE separation and detection. First, a representative standard sample was prepared from the first five strains of S. aureus cultivated on MH agar or purified, blood-incubated cells listed in Table 1. The sample was injected into the capillary by syringe pump and separated as described in the Experimental section. The resultant electropherogram from the sample mixture composed of the MSSA and MRSA (1 107 cell mL1 each) and MSSA-B and MRSA-B (2 107 cell mL1 each) strains is presented in Fig. 2A. Only four narrow peaks were detected, i.e., the migration velocities of MSSA, MRSA, MSSA-B, and MRSA-B cells are characteristic for each group of the examined strains. The dependence of peak areas of the examined strains on the number of injected cells is shown in Fig. 2Ai and Bi. A linear calibration was obtained for all test groups of MSSA or MRSA strains cultivated on MH agar or purified, blood-incubated staphylococci throughout the cell concentration range covered. Very good quantitative responses (coefficient of determination R2 = 0.99) were achieved. The peak areas of MSSA and MRSA (circle) or MSSA-B and MRSA-B (triangle) were not significantly different (full vs. empty circle or triangle, respectively). However, the peak areas of MSSA-B and MRSA-B were approximately twice higher than those of MSSA and MRSA, respectively. The dotted lines in Fig. 2Ai show the numbers of cells injected into the capillary by the syringe pump: 5 103 cells of MSSA and MRSA and 104 cells of MSSA-B and MRSA-B. The number of injected cells approximately corresponds to the peak areas in the electropherogram presented in Fig. 2A. We performed repetitive 100-nL injections of different microbial suspensions mentioned above by the syringe pump. RSDs of the migration time for all tested strains were below 1.8% whereas the peak area RSDs were within 5%. 1
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from MSSA-B strains and adequate for rapid selection of appropriate antibiotic therapy. The largest loss of S. aureus cells occurs during their purification procedures. The solution of this problem may include work with large volumes of blood (e.g., 10 mL) and/or improvement of the purification procedures. 4. Conclusions In this pilot study, we have demonstrated the utility of capillary zone electrophoresis as a fast and low-cost method to detect serious bloodstream infection caused by MRSA strains. The blood-incubated methicillin-resistant and methicillin-susceptible S. aureus strains were differentiated from each other by CZE on GOTMS-modified FS capillaries etched with supercritical water. The number of injected purified cells was sufficient for rapid screening of clinical samples. A minimum of two hundred MRSA cells from 2 mL of whole human blood were detected but the RSDs varied between 7 and 40% for the peak areas. This qualitative value provides sufficient information for the initiation of effective antibiotic therapy. Specifically, the purification procedure should be improved. If the purification procedure is more efficient, we will be able to analyze a lower number of cells. That will be interesting for clinical practice. The CZE also allows discrimination between the MH agar-cultivated and the blood-incubated MRSA or MSSA cells due to their different surface properties. The isoelectric point of both MH agar-cultivated and blood-incubated cell has not changed. Acknowledgements This work was supported by the Ministry of the Interior of the Czech Republic (Grant VG20102015023), by the Czech Science Foundation (Grant P106/12/0522), and by the Academy of Sciences of the Czech Republic (Institutional Support RVO:68081715). References
Fig. 2. CZE of MSSA and MRSA strains cultivated on MH agar or purified, bloodincubated staphylococci on the etched FS capillary modified with GOTMS, syringe pump injection. Conditions and designations, see Fig. 1; sample: (A, Ai) the sum of the first five strains of MSSA, MSSA-B and MRSA, MRSA-B listed in Table 1; MSSA and MRSA, 1 107 cell mL1 each; MSSA-B and MRSA-B, 2 107 cell mL1 each; (B, Bi) MSSA-B 200 cells, MRSA-B 800 cells; (Ai, Bi) the dependence of the peak area, A (mAU s) on the number of injected cells, N; circle, MSSA and MRSA; triangle, MSSA-B and MRSA-B; empty circle or triangle, methicillin-resistant strains; full circle or triangle, methicillin-susceptible strains; dotted lines, measured A and the corresponding N; tinj, 110 s BGE, 36 s microbial sample and 36 s BGE, flow rate 166 nL min1.
In the second part of these experiments, 2 mL of whole human blood were spiked with 200 and 800 cells of MSSA and MRSA, respectively. Subsequently, the bacteria isolated from blood were injected into the capillary and separated by CZE according to the procedure described in the Section 2.6. A representative electropherogram from six repetitive CZE runs is shown in Fig. 2B. Two narrow peaks of MSSA-B and MRSA-B strains were detected. RSDs of the migration times for the tested strains were below 1.9%. The peak areas of MSSA-B and MRSA-B correspond to the injection of 120 or 750 cells into the capillary (see the dotted lines in Fig. 2Bi). Therefore, the RSDs for peak areas were found between 7 and 40%. This value is sufficient for qualitative differentiation of the MRSA-B
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis Marie Horká a, * , Marie Tesarová a , Pavel Karásek a , Filip Ruži9 cka b,c , Veronika Holá b,c , b,d a Martina Sittová , Michal Roth
Institute of Analytical Chemistry of the ASCR, v.v.i., Veverí 97, 602 00 Brno, Czech Republic The Department of Microbiology, Faculty of Medicine, Masaryk University, Kamenice 53/5, 625 00 Brno, Czech Republic c The Department of Microbiology, St. Anne’s University Hospital, Brno, Pekarská 53, 602 00 Brno, Czech Republic d ská 119, Brno, 619 00 Brno, Czech Republic GeneProof a.s., Víden a
b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
We used CE for separation of MRSA and MSSA strains from clinical samples. MH agar-cultivated and blood-incubated cells of S. aureus were examined. GOTMS-modified FS etched with SCW was used for CE separation. The tested MRSA and MSSA strains were separated in CZE from each other. These methods appear to be sufficient for rapid screening of clinical samples.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2014 Received in revised form 28 January 2015 Accepted 1 February 2015 Available online xxx
Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (101–102 cells mL1) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. Recently, capillary electrophoresis (CZE) was successfully used to differentiate between the agarcultivated MRSA and MSSA strains in fused silica capillaries etched with supercritical water and modified with (3-glycidyloxypropyl)trimethoxysilane. The possible use of CZE as a fast and low-cost method for distinguishing between the blood-incubated MRSA or MSSA cells has been tested in this manuscript. Our goal was to test low amounts of bacteria (102 cell mL1) similar to those in clinical samples. The
Keywords: Capillary electrophoresis Supercritical water FS capillary Methicillin-resistant Staphylococcus aureus – MRSA Methicillin-susceptible Staphylococcus aureus – MSSA Whole human blood
* Corresponding author. Tel.: +420 5 32290221; fax: +420 5 41212113. E-mail address: [email protected] (M. Horká). http://dx.doi.org/10.1016/j.aca.2015.02.001 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Horká, et al., Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis, Anal. Chim. Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.02.001
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migration times of the purified blood-incubated cells and the agar-cultivated cells were different from each other. However, their isoelectric point was the same for all strains. ã 2015 Elsevier B.V. All rights reserved.
1. Introduction The Gram-positive bacterium Staphylococcus aureus is a human bacterial pathogen causing many nosocomial infections from skin lesions to life-threatening diseases such as bacteraemia, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, and septicaemia [1]. Treatment of these diseases has become more difficult and more expensive because of the increasing prevalence of multiresistant strains, especially methicillin-resistant S. aureus (MRSA) in recent decades. However, because MRSA increases morbidity and costs of treatment of patients compared with infections caused by methicillin-susceptible S. aureus (MSSA), many hospitals have attempted to control the spread of the former pathogen. The incidence of the majority of infections caused by S. aureus is conditioned by multiple virulence factors such as structural and secreted products. Coagulase, protein A, elastin-binding protein, collagen-binding protein, fibronectin-binding protein, and clumping factor are surface proteins involved in establishing of infection [2–6]. Enterotoxin B, a-toxins and TSST-1 belong to the secreted products needed for an outbreak of infection disease [2,6]. Most MRSA bacteria are able to form biofilm enabling them to colonize artificial prosthetic surfaces. Biofilm protects the microorganisms against host defenses and antimicrobials. And it can also be considered an important factor of virulence [2,6–9]. Currently, there are many techniques used for MRSA and MSSA strain differentiation. The techniques can be divided into three groups, phenotyping, genotyping, and mass spectrometry. Phenotyping methods include antibiogram, phage typing, serotyping, and protein electrophoresis. Plasmid analysis, analysis of chromosomal DNA, southern hybridization, pulsed-field gel electrophoresis, PCR typing, and repetitive element sequence-based PCR belong to the genotyping methods [10]. In most cases, phenotyping methods are easy to perform, cheap, and readily available in the routine laboratories. The major disadvantages are poor discriminatory ability and lack of reproducibility. Most of the abovementioned techniques are time-consuming (6–19 h). Potential disadvantages of genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. On the other hand, these methods alone are rapid and they have good reproducibility and repeatability. The major advantage of PCR and other molecular techniques is the ability to detect and differentiate between MSSA and MRSA directly from blood cultures [10]. The presence of charged groups on the outer microbial surface and the ensuing electric mobility of the cells can be utilized for differentiation and characterization of MRSA and MSSA by capillary electrophoretic techniques (CE) [11–21] such as capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE), according to their isoelectric points, pI,[14,22–30] and electrophoretic mobilities [28,31], respectively. Previously, the isoelectric points of MSSA and MRSA strains has been found to be the same at 3.4 [21,32,33]. Identification of bacteria directly from blood is difficult and time-consuming [10,34–39], and a rapid detection of low number of S. aureus cells (101–102 cells mL1) in blood is necessary to detect bloodstream infection. A fast detection and identification of MRSA and MSSA also makes it possible to select an appropriate therapy. Since the volumes of the collected blood samples are usually between 10 and 20 mL, about 100–2000 bacterial cells are usually
present in the whole blood samples. However, blood is a complex matrix containing many components such as cells (erythrocytes, leucocytes, and thrombocytes), proteins, glucose, amino acids, fatty acids, products to be disposed of (carbon dioxide, urea, and lactic acid), and other components (serum albumin, blood-clotting factors, immunoglobulins, lipoproteins particles, and electrolytes). These components cause higher background and poor identification of bacteria during separation. It is necessary to purify the bacterial blood sample and remove the blood components from it [39]. One of the procedures to purify the blood samples was used in our previous article [40], and this procedure was modified and employed in the present work. This study demonstrates the possibility of rapid CE separation of MSSA and MRSA directly from whole human blood using FS capillary etched with supercritical water (SCW) [21,40–43]. The adherence of the MSSA and MRSA cells to the inner surface of the modified FS capillary etched with SCW together with the differences in their migration times at the CZE separation were monitored before [21]. The hydrophobicity of the etched FS capillary and the electroosmotic flow (EOF) were modified with (3-glycidyloxypropyl)trimethoxysilane (GOTMS) [21]. The bacterial adhesion to different kinds of surfaces of orthopaedic implant materials was discussed before [44]. The adhesion depends on the interactions which occur between cells, on the cell structures, shapes, and surface features at different levels of roughness of the surface. These properties were used in this work. 2. Material and methods 2.1. Chemicals GOTMS, acetone and ethanol (EtOH) were purchased from Sigma (St. Louis, MO, USA). Polyethylene glycol (Mr 10,000, PEG 10,000) was obtained from Aldrich (Milwaukee, WI, USA). All chemicals were of electrophoresis or analytical grade. 2.2. Bacterial strains and growth conditions The strains of MSSA, CCM6188, CCM1484, CCM2551, CCM4223, CCM3953, and MRSA CCM4750 were obtained from Czech Collection of Microorganisms (Brno, Czech Republic). The S. aureus strains, CCM3953 and CCM4750, were always used as positive control at the presentation of the single strain separation in Section 3. The MSSA strains, FB11, FB129, FB130, FB131 and MRSA strains, FS133, FB125, FB124, FB123, FB128, FB127, FB126, 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 Test [45] 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 50 -ACTGCCTAATTCGAGTGCTACT-30 and mecA/rev 50 -ATGGTAARGGTTGGCAAAAAGAT-30 ) 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). Before each experiment, the tested strains were cultivated on Mueller-Hinton agar (MH agar, Oxoid, United Kingdom) at 37 C for 24 h and the standardized microbial suspensions were prepared as described previously [46].
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The microbial cultures were resuspended in physiological saline solution (PSS). The concentration of the resuspended microorganisms was estimated by measurement of the optical density of the suspension using a DU 520 UV–vis spectrophotometer (Beckmann Instruments, Palo Alto, CA, USA) operating at 550 nm, according to the calibration curve which was defined by reference samples. The numbers of microorganisms in the reference samples were controlled by serial dilution and plating of 100 mL of the suspension on MH agar. The colonies were counted after the cultivation at 37 C for 24 h. 2.3. Microbial sample preparation The required number of cells in 1 mL of the suspension has been achieved by serial dilution in 15 103 mol L1 PSS. The number of staphylococcal (MRSA or MSSA) cells in the initial suspension was 108 or 109 cell mL1. The whole human blood was spiked with cell suspension of MRSA and/or MSSA from 102 to 108 cell mL1, vortexed, and incubated for 1 h at 37 C in Thermomixer comfort (Eppendorf, Hamburg, Germany). These cells were isolated and purified using the procedure described below. One milliliter of distilled water was added to 1 mL of blood spiked with the examined bacteria. The obtained suspensions were vortexed for 30 min at 25 C. Subsequently, these suspensions were centrifuged at 1000 g for 10 min on the Concentrator 5301 (Eppendorf, Hamburg, Germany). Then, supernatants were removed. Cells on the bottoms of vials were covered with 1 mL of PSS, vortexed at 25 C for 5 min, centrifuged at 1000 g for 10 min, and supernatants were again removed. This procedure was repeated five times. Distilled water was used instead of PSS in the last purification run. The suspensions were again centrifuged at 10 000 g for 15 min. The resulting purified, blood-incubated MRSA cells (MRSA-B) or MSSA cells (MSSA-B) were re-suspended in 15 103 mol L1 PSS. Each sample was vortexed and then immediately injected into the capillary. 2.4. Fabrication of etched FS capillaries Constant-diameter capillaries with roughened inner surfaces were prepared from commercially available cylindrical FS capillary (100 mm I.D., 360 mm O.D., Agilent Technologies, Waldbronn, Germany, Part No. 160-2634-10) by etching with SCW in our original apparatus [42] employing a procedure similar to that described before [21,41]. In the present work, the etched FS capillaries with 2-mm inner surface roughness were employed (etching conditions: 320 C, 400 bar, tcon = 24 min, mSCW = 2.04 g). 2.5. Modification of FS capillaries The original and SCW-etched FS capillaries were rinsed by the solution of 5% (v/v) GOTMS in distilled water for 1 h, then washed with water for another 1 h, and immediately used afterwards [21]. 2.6. CZE equipment and procedures CZE was carried out using a laboratory-made apparatus [46] at a constant voltage (20 kV on the detector side) supplied by a Spellman CZE 1000R high-voltage unit (Plainview, NY, USA). The lengths of the FS capillaries, 100 mm I.D. and 360 mm O.D. (Agilent Technologies, Santa Clara, CA), were 35 cm, 20 cm to the detector. SCW-etched FS capillaries were modified with GOTMS. The ends of the capillary and the electrodes were placed in 3-mL glass vials filled with a background electrolyte (BGE). An LCD 2082 on-column UV–vis detector (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ, USA), was operated at 280 nm. Sample injection was
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accomplished by siphoning action as described in our previous paper [47] or by a single-syringe infusion pump (Cole-Parmer, Vernon Hills, IL, USA) equipped with a 10-mL syringe (SGE Analytical Science, Victoria, Australia). The flow rate was 166 nL min1. Height difference of the reservoirs for the sample injection by siphoning action, Dh, 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, USA) 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. Detector signals were acquired and processed with the Clarity Chromatography Station (ver. 2.6.3.313, DataApex, Prague, Czech Republic). At the separations, 2 102 mol L1 phosphate buffers pH 5 with addition of 5% (v/v) EtOH and 0.1 % (w/v) PEG 10 000 were used as BGE. Before each run, the capillaries were rinsed with acetone for 5 min and then back-flushed with BGE for 5 min. For this purpose, a single-syringe infusion pump (Cole-Parmer, Vernon Hills, IL, USA) with a 100-mL syringe (SGE Analytical Science, Victoria, Australia) was used at a flow rate ranging from 3 to 20 mL min1. The cell suspensions of the discussed S. aureus strains (cultivated on MH agar or purified, blood-incubated cells) were adjusted to concentrations from 1 102 to 1 108 cells mL1.The injection time was 8 s for siphoning action which represents 100 nL of the microbial suspension injected into the capillary. The injection time for the syringe pump was 290 s. The sample segment was composed of 200 nL of BGE, 100 nL of the microbial suspension, and again 500 nL of BGE in the 10-mL syringe. 2.7. 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. 3. Results and discussion 3.1. Optimization of the CZE conditions and microbial sample pretreatment The examined bacteria, MSSA-B and/or MRSA-B cells (sonicated and vortexed), were separated on the original FS capillary modified by GOTMS in the preliminary experiments. However, only a broad peak of unresolved MSSA-B and MRSA-B cells was detected instead of a narrow peak like in Ref. [21]. This was probably caused by a change in the surface properties of the cells after their incubation in whole blood which corresponds to the earlier findings [44]. S. aureus possesses a number of adhesins, such as polysaccharide intercellular adhesin, which enables the adhesion of staphylococcal cells to each other and by this way facilitates the formation of staphylococcal aggregates [48]. Moreover, S. aureus belongs to the plasma-coagulase positive staphylococci that produce virulence factors such as coagulase [49] and clumping factor A [15,50,51] which also support the staphylococcal cells aggregation in blood environment due to conversion of fibrinogen to fibrin. This property also causes a rapid agglomeration of the purified cells of S. aureus, MSSA-B and MRSA-B. Therefore, rigorous sonication and vortexing of the sample suspension prior to the separation by CE is necessary. Fig. 1A shows the record of the CZE separation of carefully sonicated and vortexed MSSA-B and
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with certainty whether the RSD only reflects the measurement error in the discussed set of strains with very divergent surface properties, or whether at least the migration times of these strains [2–9] are different. Without doubt, the difference between the average migration times of the cultivated MSSA and MRSA cells was approximately 0.35 min and that between the MSSA-B and MRSA-B was 1.85 min at our experimental conditions. The increase in the migration times of MH agar-cultivated or purified, bloodincubated cells of MSSA and MRSA strains is associated with the changes in their surface energies [45] after incubation of the cells in blood. The surface energy changes probably influence the adherence of the cells to the inner surface of the separation capillary. We also examined whether the isoelectric point of purified cells of S. aureus, MSSA-B and MRSA-B, in comparison to the cells cultivated on MH agar was changed. The isoelectric point for MSSA and MRSA strains, agar-cultivated, was previously determined to be 3.4 [21]. The individual strains or the mixtures of the first five strains listed in Table 1 were gradually focused by CIEF. .In accordance with the previous report [21], only one narrow peak was detected in the migration time which corresponds to the isoelectric point of 3.4. Certainly, differences of the order of thousandths to hundredths of pI units between the individual strains of S. aureus can be assumed as described before [21]. Fig. 1. CZE separation of S. aureus strains cultivated on MH agar and purified, bloodincubated staphylococci on modified FS capillaries etched with SCW. Conditions: FS capillaries, 100 mm ID, 360 mm OD, 350 mm length (200 mm length to the detection window, 150 mm toward the electrode vial); FS was modified with the solution of 5% (v/v) GOTMS; applied voltage ()20 kV; UV detection 280 nm; BGE: 2 102 mol L1 phosphate buffer pH 5 with additives: 0.1% (w/v) PEG 10,000 and 5% (v/v) EtOH; sample composition: (A) MSSA-B and MRSA-B, 5 107 cell mL1 each; (B) MSSA-B and MRSA-B, 5 107 cell mL1 each, and 5 107 cell mL1 MSSA and 2 107 cell mL1 MRSA, samples were immediately sonicated and vortexed before separations; siphoning injection (Dh = 20 cm, 8 s); rinsing procedure, by acetone for 5 min. and then back-flushed with the catholyte for 5 min; t, migration time (min).
MRSA-B cells (5 107 cell mL1 each) on SCW-etched FS capillary modified with GOTMS at the optimized conditions. The separation of a more complex mixture of S. aureus cells is shown in Fig. 1B. Here, the MSSA-B and MRSA-B cells (the same mixture as in Fig. 1A) were added directly into suspension of MSSA (5 107 cell mL1) and MRSA (2 107 cell mL1) cells immediately before the separation. Two zones in each group of these examined cells were detected. The first group corresponds to the cells cultivated on MH agar and the second group to the cells incubated in blood. The migration times of these strains are listed in Table 1. The average migration times for each of the strains were obtained from a minimum of eight independent measurements. The relative standard deviation (RSD) of the migration times was under 1.0%. Very good qualitative and quantitative responses (coefficient of determination R2 = 0.99) were reached. However, we cannot say
Table 1 The migration time, t, of examined strains of S. aureus cultivated on MH agar (MSSA, MRSA) and purified, blood-incubated cells (MSSA-B, MRSA-B). Strains
CCM 6188 CCM 1484 CCM2551 CCM4223 CCM3953 FB11 FB129 FB130 FB131 taverage
t (min)
Strains
MSSA
MSSA-B
9.58 9.48 9.41 9.61 9.49 9.55 9.51 9.60 9.59 9.55
10.22 10.25 10.19 10.23 10.24 10.10 10.25 10.22 10.17 10.22
CCM4750 FS133 FB125 FB124 FB123 FB107 FB128 FB127 FB126 taverage
t (min) MRSA
MRSA-B
9.89 9.93 9.90 9.95 9.80 9.92 9.91 9.87 9.86 9.90
12.06 12.16 12.09 12.18 12.01 12.08 12.02 11.99 12.07 12.07
3.2. CZE separation of clinically important levels of MSSA-B and MRSAB cells Rapid detection of low numbers of pathogens (101–102 cells mL ) in blood is necessary to detect bloodstream infection. Since the collected blood sample volumes usually range between 10 and 20 mL, around 100–2000 bacterial cells are present in a whole blood sample. In the following experiments, we intend to demonstrate the feasibility of direct injection of nL-sample volumes [52] of the microbial suspensions containing clinically significant number of S. aureus cells per 1 mL, and their subsequent CZE separation and detection. First, a representative standard sample was prepared from the first five strains of S. aureus cultivated on MH agar or purified, blood-incubated cells listed in Table 1. The sample was injected into the capillary by syringe pump and separated as described in the Experimental section. The resultant electropherogram from the sample mixture composed of the MSSA and MRSA (1 107 cell mL1 each) and MSSA-B and MRSA-B (2 107 cell mL1 each) strains is presented in Fig. 2A. Only four narrow peaks were detected, i.e., the migration velocities of MSSA, MRSA, MSSA-B, and MRSA-B cells are characteristic for each group of the examined strains. The dependence of peak areas of the examined strains on the number of injected cells is shown in Fig. 2Ai and Bi. A linear calibration was obtained for all test groups of MSSA or MRSA strains cultivated on MH agar or purified, blood-incubated staphylococci throughout the cell concentration range covered. Very good quantitative responses (coefficient of determination R2 = 0.99) were achieved. The peak areas of MSSA and MRSA (circle) or MSSA-B and MRSA-B (triangle) were not significantly different (full vs. empty circle or triangle, respectively). However, the peak areas of MSSA-B and MRSA-B were approximately twice higher than those of MSSA and MRSA, respectively. The dotted lines in Fig. 2Ai show the numbers of cells injected into the capillary by the syringe pump: 5 103 cells of MSSA and MRSA and 104 cells of MSSA-B and MRSA-B. The number of injected cells approximately corresponds to the peak areas in the electropherogram presented in Fig. 2A. We performed repetitive 100-nL injections of different microbial suspensions mentioned above by the syringe pump. RSDs of the migration time for all tested strains were below 1.8% whereas the peak area RSDs were within 5%. 1
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from MSSA-B strains and adequate for rapid selection of appropriate antibiotic therapy. The largest loss of S. aureus cells occurs during their purification procedures. The solution of this problem may include work with large volumes of blood (e.g., 10 mL) and/or improvement of the purification procedures. 4. Conclusions In this pilot study, we have demonstrated the utility of capillary zone electrophoresis as a fast and low-cost method to detect serious bloodstream infection caused by MRSA strains. The blood-incubated methicillin-resistant and methicillin-susceptible S. aureus strains were differentiated from each other by CZE on GOTMS-modified FS capillaries etched with supercritical water. The number of injected purified cells was sufficient for rapid screening of clinical samples. A minimum of two hundred MRSA cells from 2 mL of whole human blood were detected but the RSDs varied between 7 and 40% for the peak areas. This qualitative value provides sufficient information for the initiation of effective antibiotic therapy. Specifically, the purification procedure should be improved. If the purification procedure is more efficient, we will be able to analyze a lower number of cells. That will be interesting for clinical practice. The CZE also allows discrimination between the MH agar-cultivated and the blood-incubated MRSA or MSSA cells due to their different surface properties. The isoelectric point of both MH agar-cultivated and blood-incubated cell has not changed. Acknowledgements This work was supported by the Ministry of the Interior of the Czech Republic (Grant VG20102015023), by the Czech Science Foundation (Grant P106/12/0522), and by the Academy of Sciences of the Czech Republic (Institutional Support RVO:68081715). References
Fig. 2. CZE of MSSA and MRSA strains cultivated on MH agar or purified, bloodincubated staphylococci on the etched FS capillary modified with GOTMS, syringe pump injection. Conditions and designations, see Fig. 1; sample: (A, Ai) the sum of the first five strains of MSSA, MSSA-B and MRSA, MRSA-B listed in Table 1; MSSA and MRSA, 1 107 cell mL1 each; MSSA-B and MRSA-B, 2 107 cell mL1 each; (B, Bi) MSSA-B 200 cells, MRSA-B 800 cells; (Ai, Bi) the dependence of the peak area, A (mAU s) on the number of injected cells, N; circle, MSSA and MRSA; triangle, MSSA-B and MRSA-B; empty circle or triangle, methicillin-resistant strains; full circle or triangle, methicillin-susceptible strains; dotted lines, measured A and the corresponding N; tinj, 110 s BGE, 36 s microbial sample and 36 s BGE, flow rate 166 nL min1.
In the second part of these experiments, 2 mL of whole human blood were spiked with 200 and 800 cells of MSSA and MRSA, respectively. Subsequently, the bacteria isolated from blood were injected into the capillary and separated by CZE according to the procedure described in the Section 2.6. A representative electropherogram from six repetitive CZE runs is shown in Fig. 2B. Two narrow peaks of MSSA-B and MRSA-B strains were detected. RSDs of the migration times for the tested strains were below 1.9%. The peak areas of MSSA-B and MRSA-B correspond to the injection of 120 or 750 cells into the capillary (see the dotted lines in Fig. 2Bi). Therefore, the RSDs for peak areas were found between 7 and 40%. This value is sufficient for qualitative differentiation of the MRSA-B
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