latest improvements in cief: from proteins to microorganisms

Proteomics 2012, 12, 2927–2936 2927DOI 10.1002/pmic.201200136

REVIEW

Latest improvements in CIEF: From proteins

to microorganisms

Jirı Salplachta, Anna Kubesova and Marie Horka

Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic

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

Keywords:

Bacteria / CIEF / Fungi / Microbiology / Proteins / Viruses

Received: March 30, 2012Revised: May 18, 2012

Accepted: June 6, 2012

1 Introduction

1.1 CIEF of proteins

CIEF was introduced by Hjerten and Zhu in 1985 whenthe authors adapted equipment for high-performance elec-trophoresis to IEF [1]. CIEF is a high-resolution, rapid, andautomatable separation technique requiring only a smallamount of sample. Moreover, it can be easily coupled withother separation techniques or MS [2–5]. Generally, CIEF isused for the separation of ampholytes, especially proteins.In this technique, proteins are separated according to theirpIs in a pH gradient generated by carrier ampholytes underthe influence of a direct electric field. Once the proteins arefocused, either mobilization or whole-column imaging arenecessary for their analysis. Various aspects of CIEF analy-sis of proteins are discussed in several reviews published in

Correspondence: Dr. Jirı Salplachta, Institute of Analytical Chem-istry of the ASCR, v. v. i., Veverı 97, 602 00 Brno, Czech RepublicE-mail: [email protected]: +420 541212113

Abbreviations: LIF, laser-induced fluorescence; PB-PEG, PEG 4-(1-pyrene)butanoate; PEO, poly(ethylene oxide); WCID, whole-column imaging detection

the last decade [2–4, 6–10]. CIEF was also successfully usedin some proteomic studies including biomarker discoverysummarized in recent reviews [11–15]. In addition to proteinanalysis, CIEF was found an efficient tool for investigation ofvarious microorganisms [16, 17]. Since the microorganismscarry charged or chargeable groups on their outer surface,mostly due to the presence of amino acid residues, CIEF canbe used for their separation and pI value determination.

1.2 CIEF of microorganisms

Microbial strains are spreading out of their original place ofincidence and previously sufficient morphological identifi-cation methods often must be accompanied by phenotypiccharacterizations, e.g. by PCR or MALDI-TOF MS, whichare a front line diagnostic and screening tools in human,animal, and plant health as well as in biosecurity. Recently,electrophoretic techniques have shown their big potential formicrobial characterization. These techniques can be advanta-geously used for preconcentration, separation, and detectionof whole cells taken directly from real samples. Several re-views covering different aspect of microbial analysis usingCZE and CIEF have been published [4, 16–21]. In this paper,we summarize studies dealing with CIEF of microorganisms.

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2928 J. Salplachta et al. Proteomics 2012, 12, 2927–2936

Recent developments in this field including optimization ofseparation conditions are also mentioned.

1.2.1 Microbial surface properties

Microorganisms belong to the amphoteric colloidal biopar-ticles which electrophoretic behavior is largely a function ofthe cell wall surface in contact with the surrounding environ-ment [22–25]. Microbial cells carry a surface charge depend-ing on the pH of the surrounding solution [17, 25, 26] as aresult of dissociation or protonation of carboxyl, phosphate,and amino groups in a three-dimensional surface of the ion-penetrable layer. Charged surface leads to the formation ofan electric double layer between the solid surface and thesurrounding liquid that is characterized by the electrokineticpotential, � potential (zeta potential) [25,27]. Generally, parti-cles differing in size and � potential can be characterized byelectrophoretic techniques [22,26]. Therefore, � potential, pH,and the ionic strength of the carrier electrolytes are importantparameters in optimization of microbial separation [22].

Analogous to proteins, the pH at which the electrophoreticmobility of particular microorganism is zero is referred to aspI [23,24,28,29]. The pI value of microbial cells is dependenton their size, low aggregation corresponds to high � poten-tials [20]. Generally, pI value of the most bacteria is in thepH range of 1.5–4.5 [30] with few exceptions [24, 25, 30, 31].It seems that the pI value of microbes is more appropriateparameter than the electrophoretic mobility for predictingthe steric properties of cell surface polymers and their con-sequences for cell adhesion [24, 28]. Interpretation of elec-trophoretic mobility in terms of � potential and electrokineticcharge requires information about the mobile charge insidethe bacterial wall [24, 32]. Cell surface hydrophobicity (de-pending on the cell wall composition) is a major parameterindicating affinity of cells to the inner surface of separationcapillary [33–35]. This parameter differs from one microor-ganism to another [36].

1.2.2 Capillary surface modification

It is well known that various microorganisms tend to ad-here strongly on the inner surface of fused silica capil-lary [24,37–39]. Band broadening in CIEF separation is oftenobserved because of cells electrophoretic heterogeneity andtheir adsorption onto the capillary surface [22, 40–43]. Cellsare preferentially partitioned between more or less hydropho-bic phases depending on their surface polarity as influencedby the cell surface charge [27,44]. To prevent cell wall interac-tions, different type of inner capillary surface modification isused [22, 24]. Poly(ethylene oxide), PEO, coatings have beenshown to reduce the adhesion of microbial cells due to the de-crease of the Lifshitz–van der Waals attraction [37, 38]. PEO,chains are attached to a capillary surface and, consequently,

reduce cell adhesion by forming a steric barrier between thecell and the capillary surface [24, 38, 45–47].

Simultaneously, it is necessary to minimize the strongEOF in the uncoated fused silica capillary. The proper solu-tion includes dynamic modification of the inner capillary sur-face [6,48] by soluble polymers [41,43,49–55] such as hydrox-ypropylmethyl cellulose, hydroxypropyl cellulose, polyvinylalcohol, or static modification of the capillaries by silanizingreagents [49, 50, 56–58] or the sol-gel technique [59]. A num-ber of polymeric materials were used as replaceable coatingagents [24,48,58,60–63]. Lifetime of polymeric film on innersurface of the capillary is often shortened by its degrada-tion due to strong acids and bases used as background elec-trolytes [24, 48]. At the same time, adsorbed microorganismsmust be washed out from the inner wall of the capillary priorto subsequent run. The rinsing procedure between individ-ual runs was shown to have a strong effect on the separationreproducibility [24, 64]. Therefore, the application of dynam-ically modified, uncoated fused silica capillary seems to bethe simplest solution. PEG of different molecular weights be-longs to the group of hydrophylic uncharged polymers usedfor the dynamic coating of a capillary and for the modifyingof EOF [24, 65]. PEG chains attached to the capillary surfaceare reported to reduce microbial adhesion [39, 45, 46]. Appli-cation of diluted PEG solution was demonstrated to increasethe efficiency of bacterial separation [24, 42, 61, 66–70].

2 CIEF separation of microorganisms

2.1 Viruses

Viruses are small infectious agents able to replicate only in-side living cells of various organisms. All virus particles (alsoknown as virions) consist of genetic material made from ei-ther DNA or RNA and a protein coat protecting these genes.Moreover, some viruses have an envelope of lipids that sur-rounds the protein coat. Due to the presence of acidic and ba-sic amino acid residues, the protein coat carries an electricalcharge under most conditions, which enables separation andcharacterization of viruses by electrophoretic techniques [18].An empty viral capsid (a protein coat), obtained by removingthe genetic material from the virus, can be used for studiesof the virus in order to reduce the hazards of dealing withan infectious virus. Although electrophoretic mobility of theempty viral capsid is different from that of the virus, its pIvalue remains the same if the capsid surface has not beenchanged, which can be advantageously used in CIEF [71].

The first CE experiment with virus sample was publishedin 1987 by Hjerten et al. who applied CZE with UV detec-tion to analysis of tobacco mosaic virus [72]. Nevertheless,Schnabel et al. were the first who published the results onCIEF of animal virus, human rhinovirus serotype 2 (HRV2)[73]. They determined pI value of HRV2 using fused silicacapillaries dynamically coated with hydroxypropylmethyl cel-lulose, which was added to the catholyte. In order to reach the


Proteomics 2012, 12, 2927–2936 2929

Table 1. List of determined pI values of the investigated microorganisms including the used pH gradient

Microorganism pI pH gradient Reference

Human rhinovirus serotype 2 6.8 3.0–10.0 [73]Norovirus virus-like particles Kashiwa (rKAV) 5.5 3.0–10.0 [76]Norovirus virus-like particles Funabashi (rFUV) 5.9 3.0–10.0 [76]Norovirus virus-like particles Norwalk (V-Nor) 5.9 3.0–10.0 [76]Norovirus virus-like particles Seto (rSeV) 6.0 3.0–10.0 [76]Norovirus virus-like particles Hawaii (HV) 6.0 3.0–10.0 [76]Norovirus virus-like particles Narita (rNAV) 6.9 3.0–10.0 [76]MS2 virus 3.9 3.0–10.0 [71,77]Bacteriophage ФX 174 6.6 5.0–7.5 [78]Swine influenza H1N1, H1N2 6.5 6.0–7.0 [79,80]Equine influenza H3N8, H7N7 6.6 6.0–7.0 [79,80]Escherichia coli 4.3 3.0–10.0 [59]Escherichia coli 4.6 2.0–5.0 [78,91]Staphylococcus epidermidis (+) 2.6 2.0–5.0 [78,91]Staphylococcus epidermidis (−) 2.3 2.0–5.0 [78,91]Staphylococcus epidermidis (+) 2.6 2.0–10.0 [87]Staphylococcus epidermidis (−) 2.3 2.0–10.0 [87]Pseudomonas syringae pv. syringae 3.1 2.0–5.0 [88]Pseudomonas syringae pv. tomato 4.0 2.0–4.9, 1.8–5.5 [89]Pseudomonas corrugata 2.4 2.0–4.9, 1.8–5.5 [89]Klebsiela pneumoniae 2.5 2.0–4.9, 2.0–3.0 [84]Agrobacterium tumefaciens 2.2 2.0–5.0 [88]Agrobacterium tumefaciens 2.2 2.0–3.3 [95]Agrobacterium rhizogenes 4.0 3.3–4.7 [95]Agrobacterium rubi 2.15 2.0–3.3 [95]Agrobacterium vitis 2.6 2.0–3.3 [95]Clavibacter michiganensis subsp. michiganensis 4.6–4.7 2.0–4.9 [89]Xanthomonas vesicatoria 4–4.1 2.0–4.9 [89]Saccharomyces cerevisiae 6.3 3.0–10.0 [81]Candida albicans 2.9 2.0–5.0 [91]Candida albicans 2.8 2.0–3.3, 2–5 [78,90]Candida dubliniensis 2.6 2.0–3.3 [90]Candida tropicalis (+) 2.4 2.0–3.3 [90]Candida tropicalis (−) 2.5 2.0–3.3 [90]Candida parapsilosis (−) 3.8 2.0–5.0 [91,94]Candida parapsilosis (−) 3.8 3.3–3.9 [92,94]Candida parapsilosis (+) 3.6 3.3–3.9 [92,94]Candida metapsilosis (+) 3.63 3.6–4.0 [94]Candida metapsilosis (−) 3.81 3.8–4.0 [94]Candida orthopsilosis 3.83 3.8–4.0 [94]Aspergillus niger 2.0–2.1 2.0–8.0 [96]Aspergillus fumigatus 3.0–3.2 2.0–8.0 [96]Aspergillus flavus 5.9 2.0–8.0 [96]Fusarium solani 4.6 2.0–8.0 [96]Penicillium chrysogenum 4.4–4.5 2.0–8.0 [96]Monilinia laxa 3.2 2.7–5.3, 2.0–4.0 [97,98]Monilinia fructicola 3.8 2.7–5.3, 2.0–4.0 [97,98]Monilinia fructigena 3.9 2.7–5.3, 2.0–4.0 [97,98]Penicillium expansum 3.7 2.7–5.3 [97]Monilia polystroma 3.7 2.0–4.0 [98]

steady-state position of virus zone (up to 60 min in this case),a hydrostatic pressure was applied against the EOF. The fo-cused virus zone was then mobilized by the pressure appliedat the inlet and detected at 260 nm afterward. Low molecu-lar mass pI markers, the synthetic compounds introduced bySlais et al. [74, 75], were used for calibration of the pH gradi-ent. The pI value of HRV2 was determined as 6.8 (see Table 1

for the determined pI values in this and in the followingpapers).

In 2004, Goodridge et al. introduced another approach forpI value determination of norovirus virus-like particles us-ing CIEF [76]. Six recombinant virus-like particles represent-ing the two genogroups, genogroup I (Funabashi, Seto, andNorwalk) and genogroup II (Hawaii, Kashiwa, and Narita),



were subjected to CIEF with whole-column imaging detec-tion (WCID) at 280 nm. Separation was performed usingshort capillary internally coated with fluorocarbon to reduceEOF. The pI values determined via CIEF-WCID were 5.5,5.9, 5.9, 6.0, 6.0, and 6.9 for Kashiwa, Funabashi, Norwalk,Seto, Hawaii, and Narita, respectively. The authors presentedCIEF-WCID as a sensitive method able to detect norovirusstrains in several minutes.

In the next study, Liu and Pawliszyn investigated the behav-ior of MS2 bacteriophage and related antibodies with CIEF-WCID including two version of detection, UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID [71, 77].The adsorption of both virus and antibodies on the capillarywall was found to be a critical issue. This problem was effec-tively solved by the addition of sodium chloride or PBS bufferto the sample mixture in the case of CIEF-UV-WCID system.Moreover, the salt addition improved peak shape and repro-ducibility. No salt was added to the sample mixture with theCIEF-LIF-WCID system because of the strong absorption offluorescently labeled secondary antibody on the capillary wall.CIEF-WCID was used for detection of the virus and antibod-ies as well as for monitoring of immunocomplex formation.The changes in microheterogenity of the immunocomplexwas observed at different antibody-to-virus ratios. To form asingle complex, the antibodies must be in excess to saturatethe virus. CIEF-LIF-WCID was then applied to improve de-tection sensitivity and prevent precipitation at high sampleconcentrations. The MS2 bacteriophage and the antibodieswere labeled with the noncovalently binding fluorescent dye,NanoOrange.

In 2007, Horka and co-workers applied CIEF to analysisof native and inactivated microorganisms including bacte-riophage ФX 174 [78]. Since the microorganism infectivitymay represent a serious problem in their analysis, the pos-sibility of CIEF with UV detection to examine the influenceof various inactivation procedures on the microbial popu-lations was investigated. For this purpose, five inactivationprocedures were used—thermal destruction at 100�C, incu-bation with 70% (v/v) ethanol for 1 h, incubation with 4%(v/v) formaldehyde for 1 h, incubation with 0.4% (v/v) perox-oacetic acid for 30 min, or incubation with 2% (w/v) Virkon Sfor 2 h. With respect to the bacteriophage, the pI value of na-tive form was determined as 6.6. Inactivation by heat or withethanol led to damage of the viral particle integrity thus it wasnot possible to determine its pI value. However, the pI valueof bacteriophage inactivated with Virkon S was found lowercompared to the native virus. In order to enhance sensitiv-ity, the bacteriophage dynamically modified with fluorescentnonionogenic tenside PEG 4-(1-pyrene)butanoate (PB-PEG)was also analyzed. Observed pI value corresponded to that ofthe native bacteriophage.

The same group developed simple method for purificationand concentration of viruses based on PEG precipitation andsedimentation in the sucrose discontinuous density gradi-ent [79]. Purified viruses were separated by CIEF with UVdetection to examine efficiency of the purification procedure.

Figure 1. CIEF of swine influenza (A) and both swine and equineinfluenza (B) in the pH gradient 2.0–7.5.

The equine and swine influenza viruses were cultivated inallantoic fluid of specific pathogen free embryonated chickeneggs and purified by using of the sucrose cushion or thesucrose discontinuity density gradient following PEG precip-itation. The pI values of equine and swine influenza viruseswere determined as 6.6 and 6.5, respectively (see Fig. 1). CIEFwas demonstrated as a method suitable for control of viruspurity and concentration in solution. The main advantage ofthis approach is determination of concentration and purity ofviruses in one test as well as the detection of possible contam-ination by another virus or strain. The proposed procedurealso enables discrimination between complete and incom-plete virus particles.

Recently, the study dealing with characterization of differ-ent subtypes of both equine and swine influenza A virusesby CZE and CIEF techniques was published [80]. With re-spect to CIEF, two different subtypes of swine influenza,H1N1, H1N2, and two different subtypes of equine influenza,H3N8, H7N7, were separated in narrow pH gradient 6.0–7.0and detected at 280 nm. The virus particles were labeled withPB-PEG to increase detection sensitivity similar to [78]. Thesame pI values were determined for both native and labeledviruses. The pI values of the viruses were found indepen-dent of their origin and were determined as 6.5 and 6.6 forswine and equine influenza, respectively. However, the viralsubtypes could not be distinguished by the proposed CIEFprocedure.

2.2 Bacteria and yeasts

Analogous to viruses, different bacteria and yeast can be dis-tinguished based on their differences in pI values. The firstCIEF of bacterial samples was introduced by Armstrong et al.


Proteomics 2012, 12, 2927–2936 2931

in 1999 [42]. The authors successfully separated Escherichiacoli, Pseudomonas putida, and Serratia rubidae in a capillarycoated with methylcellulose. Focused bacteria were mobi-lized with a pressure and detected at 280 nm. In 2000, Shenet al. separated yeast cells (Saccharomyces cerevisiae) culturedto various phases of growth in fused silica capillaries coatedwith hydroxypropyl methylcellulose [81]. In this case, hydro-dynamic mobilization and UV detection at 280 nm was used.Determined pI values differed in a range from 5.2 to 6.4 independence on the growth phases of yeast cells.

Liu et al. introduced two-level CIEF method forcharacterization and identification of bacteria where thegrowth-promoting rhizobacteria were used as testing microor-ganisms [82]. At the first level, intact bacteria were character-ized based on their pI values using CIEF-WCID. Addition ofsodium chloride prevented the cellular clustering. At secondlevel, lysed bacteria were characterized by CIEF profiling ofthe intracellular proteins.

Since 2003, Horka and co-workers published sev-eral papers with CIEF of various microorganisms. Shortfused silica capillary coated with hydroxy-terminatedpoly(dimethylsiloxane) sol-gel was used for the separationof microorganisms and biopolymers by CIEF with UV andfluorescent detection [59]. Separation of the representativestrains of E. coli, Candida albicans, Staphylococcus epidermidis,and Enteroccocus faecalis, showed sensitivity and specificity ofthis approach for microbial characterization.

Studies published in 2006 were mainly focused on opti-mization of conditions for CIEF of various microbes. Newprotocol for CIEF of microorganisms in pH gradient 3–10using fused silica capillaries dynamically modified with PEG4000 was introduced [83]. In this method, a segmental in-jection of the sample pulse including segment of spacers,segment of bioanalytes, and segment of carrier ampholyteswith pI markers was applied to achieve suitable shape of pHgradient. Durability of capillaries modified with PEG 4000was at least 150 runs. Segmental injection was used in allnext studies published by Horka et al. In the following study,this CIEF method was improved and successfully applied toseparation of various microorganisms in the acidic pH gradi-ent 2–5 [84]. Proper combination of simple ampholytic elec-trolytes, commercial carrier ampholytes, and other differentadditives allows modification of linearity of the pH gradientsin the required pH ranges [83,84]. Suitable pI markers [74,75]were used for tracing of the selected pH gradients and for ver-ification of its linearity. Rinsing of separation capillary beforeeach focusing run was the basic condition of measurementreproducibility. Agglomerates and clusters of microbial cellswere disrupted by sonication and following vortexing just be-fore analysis [20, 83, 84]. Utilization of PB-PEG for CIEF ofmicroorganisms in the broad pH range 3–10 with fluorescentdetection was described in the next paper by the same work-ing group [85]. The PB-PEG was used as a buffer additivein CIEF experiments for dynamic modification and fluoro-metric detection of microorganisms. Fluorescent pI markerswere used to trace pH gradient [56]. The pI values of both

Figure 2. CIEF of C. albicans, C. parapsilosis, C. tropicalis, C.glabrata and C. krusei (A) and C. tropicalis, C. lusitaniae, Yarrowialipolytica, C. kefyr, S. cerevisiae, C. zeylanoides, Geotrichum can-didum, and Trichosporon asahii (B) modified with PB-PEG. Sepa-ration was carried out in the pH gradient 1.8–5.5 from [86], withpermission.

labeled (with PB-PEG) and native microorganisms includ-ing different bacterial and yeast strains were found the same.This method allowed detection as low as ten cells injected intothe separation capillary. In 2007, the optimized protocol forCIEF separation with fluorescent detection of yeast samplesdynamically modified with PB-PEG was published. Acidic pHgradient 2.0–5.5 was used and narrow peaks of modified an-alytes were detected (see Fig. 2) [86]. The paper describingCIEF of native and inactivated microorganisms was alreadymentioned in Section 2.1 [78]. Model bacteria and yeasts, E.coli, S. epidermidis, and C. albicans, were subjected to CIEFanalysis with both UV and fluorescent detection (using PB-PEG modification). The pI values of native forms of E. coli,biofilm-negative and biofilm-positive strains of S. epidermidisand C. albicans were determined as 4.6, 2.3, 2.6, and 2.8,respectively. No differences were found between pI valuesof native and modified form of examined microorganismsin the case of flourescent detection. Inactivation proceduresled to destruction of the cells in almost all cases. Higher pIvalue of C. albicans was observed after thermal inactivationor by using ethanol compared to native form of this yeast.In the next study, the application of CIEF to discriminatebetween biofilm-positive and biofilm-negative S. epidermidisstrains on the basis of their pI values is presented [87]. A to-tal of 73 strains isolated from blood cultures of patients withbacteremia were investigated. All strains were firstly charac-terized by standard phenotypic and genotypic methods. The



presumption that extracellular polysaccharide substance pro-duced by biofilm-positive strains may lead to the change oftheir surface charge was confirmed by the suggested CIEFmethod. Determined pI value of biofilm-negative strains wasnear 2.3, while the pI value of the biofilm-positive strains wasnear 2.6. In the following study, CIEF in pH gradient 2–5 withUV and fluorescent detection (using PB-PEG) were appliedfor separation of various plant pathogens [88]. Influence ofcultivation time as well as host origin to pI values of examinedpathogens was investigated. It was found that pI values areindependent on both host origin and cultivation time.

Representative strains of genus Clavibacter, Xanthomonas,and Pseudomonas (tomato pathogens) as well as the bacterialstrains collected from the plant tissue suspension were sub-jected to free flow IEF as a preconcentration and purificationstep [89]. After preconcentration and purification, the result-ing fraction of bacteria was separated by CIEF in pH gradi-ent 2.0–4.9 and 1.8–5.5 with UV and fluorometric detection(using PB-PEG labeling). CIEF was also used for rapid deter-mination of pI values of the examined bacteria prior to thepreconcentration and purification step. Determined pI valueswere independent on both host origin and fluorescent label-ing. CIEF separation of selected Candida species, phenotyp-ically similar C. albicans and C. dubliniensis, biofilm-positiveand biofilm-negative C. parapsilosis and C. tropicalis, in the pHgradient 2.0–3.3 and 2.7–4.7 with UV detection was carriedout [90]. Differences between determined pI values of simi-lar Candida species were up to 0.3 units. Furthermore, fastand simple lysis procedure was developed in order to obtaincharacteristic capillary or gel IEF fingerprints of the exam-ined yeasts. The fingerprints were found species specific. Thechromophoric nonionogenic surfactant PEG 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, was prepared and usedfor dynamic modification of proteins and microorganismsin the following study [91]. Modified proteins and microor-ganisms including different bacterial and yeast strains wereseparated using CIEF in pH gradient 2–5 with UV detectionat 326 nm. Less than 100 microbial cells injected into thecapillary were detected using proposed method in the sam-ple of urine. In addition, no changes between unmodifiedand modified analytes were found. In 2010, two papers werepublished. Ruzicka et al. determined pI values of 16 biofilm-negative and 23 biofilm-positive strains of C. parapsilosis [92].The strains were separated by CIEF in pH gradient 3.3–3.9with UV detection. The ability of the proposed CIEF methodto unambiguously discriminate between biofilm-negative andbiofilm-positive strains of C. parapsilosis was confirmed. ThepI values of biofilm-negative and biofilm-positive strains weredetermined as 3.8 and 3.6, respectively. In the next paper [93],42 strains of genus Pseudomonas were analyzed by GC offatty acid methyl esters as a routine laboratory test and theresults were compared with those obtained from four ana-lytical techniques including CIEF in the pH gradient 2.0–4.7with UV detection. These analytical techniques, especiallyCIEF, provided more reliable identification of the examinedbacteria.

The possibility of CIEF with UV detection to distinguishbetween biofilm-negative C. orthopsilosis and both biofilm-negative and biofilm-positive C. metapsilosis and C. parapsilosiswas investigated [94]. For this purpose, very narrow pH gradi-ents were used and subsequently differences in pI values ofbiofilm-negative and biofilm-positive Candida species weredetermined below 0.03 pI units. The optimized separationconditions were then applied to detection of biofilm-negativeC. parapsilosis from the blood serum. Analogous to previousfindings, determined pI values were independent on the ori-gin of the examined strains. The study published by Suleet al. was focused on the characterization of Agrobacteiumand Rhizobium species [95]. CIEF in the narrow pH gradients2.0–3.3 and 3.3–4.7 with UV detection was used for separa-tion of 40 different bacterial strains. Based on determined pIvalues, three major Agrobacterium species, A. tumefaciens, A.rhizogenes, and A. vitis, and all Rhizobium species were clearlydifferentiated. However, too small differences between deter-mined pI values of A. tumefaciens and A. rubi were obtainedmaking their differentiation impossible. All strains were alsoclassified using several phenotypic determinative tests, PCR,and fatty acid methyl ester analysis, which confirmed resultsfrom CIEF.

2.3 Filamentous fungi

In recent years, several studies focused on CIEF separationsof different filamentous fungi were published. Conidia fromcultures of different strains of filamentous fungi togetherwith several strains isolated from clinical material, native orlabeled by PB-PEG, were analyzed by CIEF with UV and flu-orometric detection [96]. In accordance with previous studiesdealing with bacteria or yeasts, determined pI values werefound independent of the host origin and were not affectedby the modification with PB-PEG. Down to ten conidia in-jected into the capillary were detected using fluorescent de-tection. A new method for preconcentration and separationof microorganisms from real samples using combination offiltration microcartridge and CIEF was presented in 2011 [97].CIEF experiments were carried out in the pH gradient 2.7–5.3 with UV detection. Spores of Monilinia laxa, M. fructigena,M. fruticola, and Penicillium expansum were chosen as modelbioparticles. At first, the pI values of examined fungal sporeswere determined using CIEF and the optimized method wasthen verified on samples taken directly from infected apples.In this case, M. fructigena was identified as the causal agentof the rot on the apple. The filtration microcartridge wascoupled with CIEF in the next step where the spores of M.fructigena and M. laxa were detected on visually noninfectedapple surface (probably as a result of cross-contaminationduring handling and storage). Coupling of the filtration mi-crocartridge with the separation capillary in CIEF improveddetection limit by four orders of magnitude. Utilization of fiveindividual analytical techniques including CIEF for identifica-tion of filamentous fungi was described in the next study [98].


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Spores of Monilinia species, M. fructigena, M. fructicola, M.laxa, and different strains of M. polystroma, were selected asrepresentatives of filamentous fungi. The proposed analyti-cal methods were then applied to identification of Moniliniaspecies in the samples collected from infected fruits, appleand apricot. With respect to CIEF, separation was carriedout in pH gradient 2–4 with UV detection. This method wasapplied to efficient discrimination of Monilinia species inde-pendently on the host origin of respective fungi. However,the method failed in discrimination between different strainsof the same fungal species.

3 Concluding remarks

CIEF of microorganisms seems to be powerful tool for theseparation and characterization of microbial cells accordingto their surface properties. Outer surface of microorganismsis often very specific particularly in their hydrophobicity, ca-pability of the biofilm formation, etc. Separation of microbesas well as the sample preparation often requires very specificconditions including prevention of formation of heteroge-neous aggregates with different surface charge-to-size ratiosor prevention of adsorption of the microorganisms onto theinner surface of the separation capillary. There are several op-tions that can be applied to achieve reliable and reproducibleresults—addition of different additives into background elec-trolytes or into injected samples (e.g. PEG), application ofnonionogenic tensides, careful sonication, and shaking of themicrobial sample just prior to CIEF run. The last and one ofthe most rigorous conditions is a washing and a disinfectionof the internal and external surface of the capillaries, whichcome into contact with sterile electrolytes.

The pI values of many microorganisms are often similar(see Table 1), but they are independent on the host originand they are characteristic for either species or subspecies.In addition, the pI value can be used as an important iden-tification marker of pathogen. However, it can be difficult toachieve requested accuracy of a pI measurement in commonmicrobiological practice at the present time. Therefore, addi-tional identification marker is necessary to find. Fast separa-tion together with possibility to analyze samples taken fromreal matrices makes CIEF suitable for preseparation and pre-concentration of various microorganisms. Online or off-linecombination of CIEF with other phenotypical techniques (e.g.MALDI-TOF MS) or with methods of classical microbiologycould be very advantageous for unambiguous microbial char-acterization. Generally, the electrophoretic techniques can beused for detection and identification of etiological agents inhuman (e.g. nosocomial infections) or veterinary medicine.Another field of their utilization can be plant protection orbioterrorism.

This work was supported by the Grant of Ministry of InteriorNos. VG20112015021 and VG20102015023 and by the Institu-tional research plan RVO 68081715.

The authors have declared no conflict of interest.

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