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Rapid detection and identification of Conventional techniques currently used for the identification
microorganisms is a challenging and important of microorganisms are based on the culture of microorganisms
aspect in a wide range of fields, from medical to and the determination of the phenotypic characteristics thereof.
industrial, affecting human lives. Unfortunately, However, these methods are labor-intensive, time-consuming
classical methods of microorganism identification (taking up to 3 days), and often inadequate for the differentiation
are based on time-consuming and labor-intensive of phenotypically similar species (8–12). These limits are
approaches. Screening techniques require the especially important from a medical diagnostics point of view
rapid and cheap grouping of bacterial isolates; because the rapid and proper identification of pathogens is an
however, modern bioanalytics demand essential factor in the implementation of the appropriate therapy
comprehensive bacterial studies at a molecular (10, 11). In addition, screening identification of microorganisms
level. Modern approaches for the rapid is also a key issue in areas such as the pharmaceutical industry, food
identification of bacteria use molecular techniques, QC, and environmental research (1, 11, 13). Therefore, much
such as 16S ribosomal RNA gene sequencing emphasis has been placed on the development of alternative
based on polymerase chain reaction or methods for the rapid and unambiguous identification of
electromigration, especially capillary zone microorganisms directly from tested material (8–11).
electrophoresis and capillary isoelectric focusing. Nowadays, in the taxonomy of microorganisms, molecular
However, there are still several challenges with the biology methods—such as 16S ribosomal RNA (rRNA) gene
analysis of microbial complexes using sequencing (8, 14), polymerase chain reaction (PCR; 15), and
electromigration technology, such as uncontrolled other related PCR-based methods (16–18)—are very popular.
aggregation and/or adhesion to the capillary These methods are characterized by high sensitivity and
surface. Thus, an approach using capillary reproducibility. 16S rRNA gene sequencing is considered the
electrophoresis of microbial aggregates with UV most accurate method and deemed the gold standard for the
and matrix-assisted laser desorption ionization identification of microorganisms at the species level (19).
time-of-flight MS detection is presented. Unfortunately, due to the high cost of this analysis, in routine
diagnostics, it is impossible to use this method. Moreover, such
studies are usually performed by external institutions, resulting
M
icroorganisms are well known for both positive and in prolonged wait times for results (14, 20).
negative properties. They are used on a wide scale in One of the strategies to reduce the time required for the
the food industry, biotechnology, and modern genetic identification of microorganisms in routine diagnostics is the
engineering. However, some microorganisms can cause food use of semiautomatic and automatic systems based on
spoilage or serious disease. Because of these two negative biochemical methods. Such methods allow the attainment
effects, the unequivocal and rapid identification of of results in maximum of 24 h (21–23). However, this time
microorganisms in real samples represents a very important frame is still too long, and the obtained results are not always
area of focus (1, 2). This is particularly important in medical satisfactory (24). An alternative could be MS methods, such as
diagnostics. Microbial infections can lead to dangerous diseases, matrix-assisted laser desorption ionization time-of-flight
such as sepsis (3), diabetic foot infections (4), or meningitis (5). mode (MALDI-TOF MS) or electromigration techniques
Rapid and unambiguous identification of the pathogen causing (8). These methods allow the performance of quick and
the infection is a necessary factor for the implementation of an clear analyses of microorganisms directly from infected
appropriate therapy to save patient lives (6, 7). liquid samples while maintaining unit costs low (25–30).
The most recent approach involves the use of multivariate
techniques, such as PCR electrospray ionization (ESI) MS
Received May 24, 2017. Accepted by RR May 24, 2017.
Guest edited as a special report on “Chromatography – Theory and (31, 32), PCR-microchip capillary electrophoresis (ME; 33),
Applications. In memoriam of Prof. Dr. hab. Edward Soczewiński” by PCR-capillary electrophoresis (PCR-CE; 34), capillary isoelectric
Tomasz Tuzimski. focusing (CIEF)-MALDI-TOF MS (34), or micropreparative
Corresponding author’s e-mail: bbusz@chem.umk.pl solution CIEF (sCIEF)-HPLC (35). Such connections allow
This work was supported by Maestro 6 No. 2014/14/A/ST4/00641
(2015–2017) and Opus 11 No. 2016/21/B/ST4/02130 (2016–2019) from
the attainment of conclusive results in a very short amount
the National Science Centre of Poland. of time and have great potential for further research
DOI: 10.5740/jaoacint.17-0207 (24, 31–35).
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2 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
Table 1. Review of the biochemical methods for the identification of different groups of microorganisms
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BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017 3
complementary to the sequence of the researched gene). The which are not allowing the recreation in laboratory (14, 20). This
product of this reaction is then subjected to gel electrophoresis method is characterized by high specificity and sensitivity.
(GE) to visualize the DNA molecule of the expected size. In a Moreover, unambiguous and reproducible results can be
next step, the amplified products are sequenced. PCR techniques obtained at the species level with this method. However, the
can be used to quantify nucleic acids [i.e., quantitative PCR technique is limited by the inability to determine more than one
(qPCR)], making disease progression monitorable. However, the species of microorganism per sample. Kommedal et al. (20)
PCR method requires expensive equipment and the need for a conducted research for the simultaneous identification of several
separate laboratory space to reduce the possible sample microorganisms using three pairs of primers—a pair for each
contamination (8, 48, 51). group of microorganisms. As a result, they analyzed samples
PCR methods have been described for almost all known containing several microorganisms from same or different
microorganisms (48, 51). Dušková et al. (8) used genus- groups and obtained several overlapping chromatograms.
specific PCR to identify Lactobacilli isolated from dairy Pendharkar et al. (14) carried out amplification of the whole
products and retail meat (Table 2). In one result from their 16S rRNA gene by PCR and then sequenced it to identify
research, the occurrence of cross-reaction between L. lactobacilli from vaginal swabs. Markiewicz et al. (57)
paracasei and L. casei was observed, indicating that the use identified the same bacteria using a combination of species-
of PCR can lead to cross-reactions in phylogenetically related specific PCR and 16S-amplified rDNA restriction analysis
species (8, 15). However, Callaway et al. (15), during their (16S-ARDRA) of the 16S-23S rDNA with two restrictases. The
identification of Lactobacilli from a human oral cavity 16S-ARDRA method was also successfully applied by Blasco
(specifically from carious dentin), did not observe this et al. (58) for the determination of Propionibacterium strains
phenomenon. Of 87 clinical isolates, Callaway et al. in cheeses. As result of the preceding body of research, species-
correctly identified 81 (93%) as lactic acid bacteria at the specific assignment was possible for all the tested strains of
species level (15). In recent years, much attention has also Propionibacterium.
focused on the examination of the molecular function of Molecular biology methods are also used for the rapid
microalge, especially diatoms (18). Adelfi et al. (18) identification of pathogens directly from clinical samples.
selected reference genes for qPCR using Pseudo-nitzschia Deggim-Messmer et al. (16) used PCR-based assays for the
multistriata and P. arenysensis as the model system. Their identification of Mycobacterium tuberculosis (MTB) and
results indicated that tubulin a- and b-chains and cyclin- nontuberculous mycobacteria (NTM). For this purpose they
dependent kinase A can be the most reliable reference carried out the 16S rRNA gene sequencing by using 283 and
genes for diatoms. 264 primers for PCR amplification and the Mbakt-14 primer for
A newer version of the PCR technique is PCR with real-time sequencing. Molecular-based detection gave concordant results
reading. This method was developed by Higuchi et al. (161) in for 97.7% and 97% of the specimens for MTB and NTM,
the early 1990s. Real-time PCR enables the tracking of amplicon respectively. PCR-based techniques also allowed for the
formation in real time after each cycle of the amplification explicit distinction of closely related bacteria, such as
reaction. In this method, detection of product occurs thanks to distinguishing Bacillus anthracis from other bacteria of the B.
the measurement of the fluorescence emitted by samples through cereus group, a distinction that is not possible by any other
fluorescent tracers, which is proportional to the amount of method. Wielinga et al. (59) developed a new multiplex real-time
formed PCR product (51). This solution significantly reduces PCR assay to uniquely identify B. anthracis—a life-threatening
analysis time (30–40 min) and the risk of sample contamination pathogen. Ahmod et al. (60) also developed a rapid
(51). Real-time PCR, like conventional PCR, can be used for pyrosequencing assay to distinguish B. anthracis from the B.
quantification. Spanoghe et al. (17) developed a quantitative cereus group.
SYBR green real-time PCR assay based on a new specific Technology based on PCR and DNA sequencing is
primer pair targeting the ribosomal ITS1-5.8S-ITS2 region of considered a very sensitive tool for the precise identification
Kazachstania servazzii. Their results indicated that this test of microorganisms (61). However, today, practical
allowed the detection of even small amounts K. servazzii in applicability is still scarce for the clinical diagnosis of
food samples. Quantitative real-time PCR is also an effective tool diseases mainly because of the expensive and complex
in the diagnosis of fungal infections. Walsh et al. (54) devices required for sophisticated temperature control,
developed a rapid and sensitive real-time PCR assay for rendering PCR-based systems unavailable to many resource-
the genus- and species-specific identification of Aspergillus limited environments (62). Emerging alternatives for this
infections with TaqMan technology. Using pan-Aspergillus system could be an isothermal nucleic acid amplification
primers, they were able to detect 100% of cases of confirmed method in which a DNA/RNA amplification reaction takes
or probable invasive pulmonary aspergillosos. Rahn et al. (55) place at constant temperature (63). Such methods include
developed a comprehensive set of fungal real-time PCR assays nucleic acid sequence-based amplification, loop-mediated
(fuPCR). fuPCR allowed the simultaneous detection and isothermal amplification (LAMP), strand-displacement
differentiation of fungi species, which was important from a amplification, multiple-displacement amplification, rolling-
medical perspective, directly from breath and blood samples. circle amplification, helicase-dependent amplification
Another method of molecular biology was developed by (HDA), or recombinase polymerase amplification (RPA),
Woese and Fox (56) and entails the sequencing of the most which differ depending on enzymatic mechanisms adapted
conservative sequence of the 16S rRNA gene. The 16S rRNA from natural biological processes (64–66). The use of these
gene facilitates the identification of microorganisms because it techniques obviated the need for thermocyclers and allowed for
is composed of a variable sequence that is particular to a specific the application of simplified and miniaturized instrumentation,
species. Amplifying and sequencing the 16S rRNA gene allows thereby significantly reducing both the time and cost of analysis
the identification of microorganisms, the conditions of life of (67). The most widely used non-PCR technique is LAMP, first
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4 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
described in 2000 by Notomi et al. (68). LAMP is based on both already been used as sensitive tools for pathogen
autocycling strand displacement DNA synthesis performed by detection, such as methicillin-resistant S. aureus (MRSA;
B. stearothermophylus DNA polymerase carried out at 79, 80), coronavirus-caused severe acute respiratory
60–65°C within 45–60 min in a simple water bath. This syndrome (81), or MTB (74), with an amplification time
assay requires the use of a set of four to six specially ranging between 0.5 and 2 h. Both methods are subject to
designed primers (inner and outer), which recognize four to extensive studies in their application to laboratory-on-a-chip
six distinct regions on the target DNA, ensuring high selectivity technology (64).
for target amplification (69, 70). The greatest advantages of
LAMP are high sensitivity (the ability to detect as few as six Spectrometric Techniques
copies of DNA in the sample) and higher resistance to various
inhibitory compounds, as compared with PCR, thus skipping MALDI-TOF MS is the latest next generation tool being
the extensive DNA purification step altogether (68, 71). This used for the rapid identification and classification of
method has been successfully applied for the detection of microorganisms (Figure 1). The principle of this method is
Staphylococcus aureus (72) or Salmonella enterica (73). based on the gentle ionization of intact microorganism cells
Currently, two other isothermal amplification techniques in with short laser pulses and then accelerating the particles in a
particular are rapidly gaining popularity—HDA and vacuum by way of an electric field. As a result of
RPA—mainly because they require only two primers microorganism ionization, a specific molecular fingerprint
combined with one enzyme to separate double-stranded (spectra profile) of the microorganism can be registered
DNA, simplifying the reaction scheme and the design of (82). Identification of the microorganism occurs as a result
sequence-specific probes (74). The HDA technique mimics of comparing the spectral profile of the analyzed
replication fork mechanisms and is based on the separation of microorganism against a database using an automated
DNA into single-stranded templates for further primer program (82). The most commonly used MS technique for
annealing and subsequent primer extension by a DNA the identification of microorganisms is protein analysis (83).
polymerase (64, 75). The major advantages of HDA are high Using protein profiles to differentiate bacteria was first
speed (100 base pair/s) and processivity (10 kb/binding; 76). introduced by Cain et al. (84) in 1994. Protein profiles can
On the other hand, the RPA method uses recombinase–primer not only be obtained for raw lysates, but also for the whole
complexes to scan double-stranded DNA and facilitate primer cell. The obtained spectrum allows the identification of
binding to template DNA (77). Compared with other bacteria at the species level. Research has indicated that the
techniques, RPA does not require temperatures above 37°C proteins predominantly detected by MALDI-TOF MS are
and is tolerant of fluctuations in the incubation temperature ribosomal (82–85). MALDI-TOF MS can be used in either
between 37 and 42°C (78), which is what makes this technique positive- or negative-ion mode. The most frequently used
ideal for cases in which precise temperature control is matrix for the identification of microorganisms is a-cyano-4-
technically challenging (64). HDA and RPA methods have hydroxycinnamic and 2,4-dihydroxybenzoic acids (85–87).
Table 2. Review of molecular biology methods used for the identification of different microorganisms
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Currently there are three commercially available databases: Carbonnelle et al. (89) showed that Bruker MS properly
Bruker BioTyper, SARAMIS (bioMérieux), and Andromas identified 94.9% of 317 routine bacterial isolates at the
(Andromas SAS; 6, 19). The latest version of Bruker species level versus the Shimadzu MALDI-TOF MS system
BioTyper MSP contains more than 4500 species; its principle at 93.4%. Both systems mistakenly identified Shigella sonnei
of operation is based on the frequency of species-specific peaks and Streptococcus species. Martiny et al. (90) led a study of 73
occurring in the identified sample compared with standard mass isolates from anaerobic bacteria in clinical samples. They
spectra (19). The SARAMIS database, on the other hand, is obtained identification accuracies of 61.6 and 75.3% with
available in two versions: directly connected to a Shimadzu Bruker BioTyper and VITEK MS, respectively. Marko et al.
Axima mass spectrometer or a VITEK MS system. The latter (91) compared these same two systems for the identification of
allows the identification of approximately 3000 microorganisms. nonfermenting Gram-negative bacilli, identification of which
In the VITEK MS system, each peak on the reference spectrum is was very difficult both manually and through automated
weighted on a scale of 0–40 according to the specific species; the biochemical identification systems. They analyzed 200
result is expressed as the sum of the fitting m/z values (88). The isolates from patients who had cystic fibrosis. In this case,
Andromas database is the least frequently used and contains Bruker MS had a proven advantage over VITEK MS in
about 700 bacterial strains (83). combined species–complex–genus-level identification (97
Many studies have compared the effectiveness of Bruker versus 89.5%). Recently, Deak et al. (92) conducted a study
BioTyper and the VITEK MS system. Cherkaoui et al. (19) to assess preferences for the application of VITEK MS and
analyzed 720 clinical isolates using a Bruker MS and a VITEK Bruker Microflex LT with BioTyper software for the routine
MS system and compared their results to biochemical tests and identification of bacteria and yeast in a clinical microbiology
16S RNA gene sequencing. Bruker MS allowed the high- laboratory. As a result of this research, which was conducted on
confidence identification of 94.4% of isolates, whereas the 477 isolates, a nonstatistically significant difference between the
VITEK MS system identified 88.8%. Only 0.9% of isolates accuracies of both methods was recorded. Nevertheless, VITEK
were incorrectly identified by Bruker MS and only 0.5% by MS was still considered the preferred method because of several
VITEK MS. Both systems were characterized by an overall operational aspects, such as ease of sample preparation, in
accuracy of more than 99%. Moreover, both Bruker MS and comparison to Bruker MS. A similar comparative evaluation was
VITEK MS correctly identified 69 and 38% of clinical isolates, carried out by Lee et al. (93) for Gram-positive cocci identification.
which could not have been otherwise determined by Overall, they were able to correctly identify, at the species level, 97.2
conventional biochemical tests. Research conducted by and 94.7% by VITEK MS and Microflex LT, respectively.
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Currently, MALDI-TOF MS is being successfully used in first protocol for the direct analysis of blood was introduced by
clinical microbiology laboratories (94); in the veterinary field; Stevenson et al. (25). They separated bacteria from plasma
(95) in monitoring water quality (96) and food (26, 97); and in proteins and red blood cells in several centrifugation steps.
detecting and identifying fungi (98), yeasts (94), and bacteria Collected bacteria were lysed and proteins extracted with 70%
(99, 100; Table 3). Currently used protocols allow direct analysis ethanol and 70% formic acid. The prepared sample was analyzed
of infected liquid samples, such as urine, milk, or blood (25). The using the BioTyper database. Of the 212 tested isolates, only 170
Table 3. Review of MALDI-TOF MS methods used for the identification of different microorganisms
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were identified due to a insufficient number of bacteria in the associated with waste disposal (108). In addition, the
sample. Among them, 162 (95.3%) were properly identified. The preparation of each sample requires a tailored approach.
same procedure also allows for the analysis of milk samples (26). Ferroni et al. (109) conducted protein extraction using a mild
Haiko et al. (27) applied a method called “urine short incubation detergent (5% solution of saponin) and trifluoroacetic acid. van
MALDI-TOF” for the rapid identification of urinary tract Veen et al. (94) applied two-step extraction with 70% ethanol and
pathogens. They were able to correctly identify 86% of Gram- 70% formic acid.
negative bacteria; however, they failed to identify Gram-positive Another problem associated with the identification of
bacteria with this method. They found that the method was microorganisms using the MALDI technique is related to
suitable for the rapid identification of Escherichia coli—the the quality and accuracy of available databases. As a
most common cause of urinary tract infections. It reduced consequence, today, many researchers are working to
analysis time to 4–6 h versus the 24 h needed for supplement commercially available databases. A recent
conventional methods. study conducted by Sogawa et al. (110) showed that
The main advantages of modern MALDI-TOF MS systems supplementing the BioTyper database with an additional
include the speed and accuracy of identification and the spectrum of 229 routine clinical isolates has increased the
differentiation of microorganisms (8). This method identifies identification rate from 87.1 to 98%. Due to limitations of the
bacteria not only at the genus and species level, but in some database and difficulties associated with the development of
cases, even to the subspecies level (8). For further analysis, small an effective method for protein extraction, MALDI-TOF MS
amounts of biological material suffice (less than 100 ng), and the may not be widely used in clinical mycology (111). Lau et al.
measurement and interpretation of data is relatively quick and (111) developed an alternative method of extraction and
easy (101). Tan et al. (102) analyzed 824 bacterial and 128 yeast constructed a database containing 294 individual isolates,
isolates. They showed that application of MALDI-TOF MS representing 152 species and 79 genera. Using BioTyper
shortened analysis time to about 1.45 days compared with software, they were able to correctly identify 88.9% of the
standard biochemical methods. Smaller laboratories have isolates at the species level and 4.8% at the genus level. None
limited access to using the MALDI method because of the of the tested isolates had been mistakenly identified. Using
associated high cost of equipment. However, given low the unmodified database permitted the identification of only
operating costs and use of consumables, the unit costs of this 0.7% of the isolates at the species level and 6.2% at the genus
analysis are relatively low compared with conventional methods; level.
in other words, the investment could quickly pay off (103). In 2014, Vithanage et al. (112) compared the effectiveness of
The MALDI-TOF MS technique allows the unambiguous five systems for the identification of psychrotrophic bacteria
identification of microorganisms that are very difficult to isolated from raw bovine milk. Among the methods used were
identify by conventional culture-based methods. Farfour et al. 16S rRNS gene sequencing, MALDI-TOF MS (bioMérieux),
(104) analyzed 659 isolates of Gram-positive bacteria, including and the API system. After obtaining their results from the
Listeria, Nocardia spp., Actinomyces spp., Rhodococcus equi, experiment, Vithanage et al. were able to correctly identify
Erysipelothrix rhusiopathiae, Lactobacillus, and Bacillus spp. Gram-negative bacteria at 100, 63.2, and 60.5% at the species
Although these organisms present challenges to accurate level by 16S rRNS gene sequencing, MALDI-TOF MS, and API
identification by routine methods, application of MALDI-TOF system, respectively; whereas, Gram-positive bacteria were
MS allowed for the correct identification of 98.5% of the correctly identified at 100, 95, and 90% by the same three
organisms at the species level, with exception of the Listeria methods. The speed of each test was shaped in the following
isolates, which could only be detected at the genus level. This order: MALDI-TOF MS, 16S rRNS gene sequencing, and the
technique also permitted the differentiation of closely related API system. In contrast, reproducibility was shaped in the
species of Corynebacterium, which are difficult to distinguish following order: 16S rRNS gene sequencing, the API system,
with biochemical methods. Gruenwald et al. (105) carried out and MALDI-TOF MS. The results indicated that the most
research on the differentiation of closely related species of fungi: reliable method for the identification of this bacterium in
Stachybotrys chartarum and S. chlorohalonata. The authors dairy products was 16S rRNS gene sequencing due to the
proved that MALDI-TOF fingerprint analysis clearly accuracy and reproducibility of the elements’ identification.
discerned these two species. However, the MALDI-TOF MS However, the authors emphasized that MALDI-TOF MS
technique has some limitations in its ability to identify some and the API system represented suitable techniques for
other closely related species, particularly E. coli and Shigella, the identification of Gram-positive bacteria isolated from
whose ribosomal proteins are usually well conserved (90). raw milk.
Nonetheless, Khot and Fisher (106) recently discovered a
biomarker peak using ClinProTools software (Bruker
Daltonics); on the basis of a genetic algorithm, they were able Electromigration Techniques
to generate a classification model for the differentiation of E. coli
and Shigella species. They examined 66 Shigella species and 72 In recent years, electromigration techniques have become a
E. coli isolates and correctly identified 90% of Shigella and tool of increasing interest for the analysis of microorganisms
E. coli clinical isolates at the species level. Only 3% of the (Table 4). These techniques are based on the migration of
isolates were incorrectly identified. Another disadvantage of charged particles under the influence of a homogeneous
MALDI is the need to carry out complex procedures for electric field (Figure 2). Separation of test molecules occurs
extraction when organisms have thick cell walls, such as as a result of the difference in their electrophoretic mobility,
Gram-positive bacteria, yeast, or some filamentous fungi which is proportional to their charges and inversely
(107). The need to carry out extraction leads to an increased proportional to their functional coefficient (1). The most
consumption of dangerous substances and increases costs commonly used electromigration techniques include GE,
170207_Buszewski 7
8 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
Detection
Microorganisms Technique Supporting electrolyte Conditions for separation method Refs.
Saccharomyces cerevisiae CZE TMB (4.5 mM TRIZMA base, 4.5 mM Uncoated fused-silica capillary DAD (9)
boric acid and 0.1 mM EDTA); pH 8.4a 21 cm × 100 µm U = 10 kV
BE (4.5 mM TRIZMA base, 4.5 mM
boric acid and 0.1 mM EDTA); pH 8.4;
0.0125% PEO
Gram-positive cocci and Gram- CZE TBE (4.5 mM Tris, 4.5 mM boric acid, Fused-silica capillary DAD (10)
negative rods 0.1 mM EDTA); pH 8.53; 0.0125% PEO
Escherichia coli, Klebsiella CE 1 mM Tris, 0.33 mM citric acid, 1 mg/mL Bare silica capillaries 26 cm × 75 µm UV (118)
pneumoniae, Pseudomonas CTAB; pH 7 U = – 6 kV
aeruginosa, Enterobacter
cloacae, Enterococcus
faecalis, Staphylococcus
aureus, and Staphylococcus
epidermidis
Candida spp. CIEF CZE NaOH; H3PO4; EtOH; PEG 10000; Fused-silica capillary 350 × 0.1 mm UV (29)
PEG 4000; phosphate buffer (pH 8.4) U = –20 kV
MRSA and MSSA CZE 2 × 10–2 mol/L phosphate buffers; pH 5; Fused-silica capillaries modified UV (119)
(Staphylococcus aureus) 5% EtOH; 0.1%; PEG 10000 with (3-glycidyloxypropyl) trimethoxysilane
35 cm × 100 µm U = –20 kV
Escherichia coli CZE TBE (4.5 mM Tris, 4.5 mM, boric acid, Fused-silica capillary DAD (28)
0.1 mM EDTA); pH 8.53; PEO 0.0125%
Brevibacterium taipei, Bacillus CIEF 1 mmol/L T Tris, 0.33 mmol/L citric acid Silica capillary 30 cm × 100 µm LIF (11)
megaterium, Bacillussubtilis, (pH 7); CTAB; sulfobetaine U = –3 kV
Bacillus cereus, and Candida
albicans
Monilinia spp. CIEF 2 × 10–2 mol/L NaOH; 0.1 mol/L Fused-silica capillary 300 × 0.1 mm UV (122)
orthophosphoric; 0.1% Brij 35; 3% U = –20 kV
EtOH; 0.2% PEG 1000
CZE 2 × 10–3 mol/L phosphate buffer (pH
8.0), 5% EtOH, 0.3% PEG 1000,
0.3% Brij 35
Pseudomonas syringae and CIEF CZE 40 mmol/L NaOH; 100 mmol/L H3PO4; Silica capillary 35 cm × 50 µm UV (159)
Pseudomonas corrugate 1.5 mmol/L taurine-Tris (pH 8.4); U = –20 kV
EtOH; PEG
Rhizobiaceae spp. CIEF 4 × 10–2 mol/L NaOH; 0.1 mol/L Silica capillary 300 × 0.1 mm UV (12)
orthophosphoric acid; 1% EtOH; U = –20 kV
0.2% PEG 10000
CZE 2 × 10–3 mol/L phosphate buffer (pH
6.2); 3% EtOH; 0.8% PEG 10000
Escherichia coli, ME TBE (3.94 mM Tris, 56 mM boric acid, 14 mm U = 1000 V LIF (124)
Staphylococcus aureus, and 1.3 mM EDTA); pH 10.5; 0.025% PEO
Candida albicans
Bifidobacterium, Lactobacillus ME TBE (4.0 mmol/L Tris, 4.0 mmol/L boric Glass chip LIF (30)
casei, Lactobacillus acid, 0.09 mmol/L EDTA); pH 8.5;
acidophilus, and 0.025% PEO
Enterococccus faecalis
Lactobacillus delbrueckii subsp. ME TBE (4.5 mM Tris, 4.5 mM boric acid, Glass chip 60 mm LIF (125)
bulgaricus, Lactobacillus 0.1 mM EDTA); pH 8.5; 0.0125%
rhamnosus, and PEO; lipid-based nanoparticles
Streptococcus thermophilus
a
TMB = Tetramethylbenzidine.
b
EtOH = Ethanol.
c
PEG = Polyethylene glycol.
two-dimensional GE, capillary zone electrophoresis (CZE), consequently affect the electrical double-layer formation
capillary isotachophoresis, and CIEF (1, 2). and creation of the z-potential on the surface of cells (1, 2, 9).
The cell surface contains biopolymers, such as polysaccharides, Thus, because of their size and charge, microbial cells can
phospholipids, membrane proteins, and receptors, as well as small be seen as charged biological active colloids (i.e., biocolloids;
molecules, which are products of metabolism or cellular 9). Each species of microorganism possesses a characteristic
communication molecules (1). These components contain spectroscopic fingerprint: For example, UV-spectra that are
numerous functional groups that are dissociated and recorded by popularly used DAD because of their unique
170207_Buszewski 8
BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017 9
structural and morphological qualities. Moreover, microbial each time point of growth was characterized by two distinct
cells, under specific conditions of analysis, move in the peaks. However, some differences were observed in the
electric field at different characteristic velocities. Through electropherograms for each strain, likely caused by changes in
these properties, it is possible to identify and characterize the surface charge of the tested cells at different growth stages.
microorganisms by their electromigration characteristics These results indicated that the use of CE on intact yeast cells not
(1, 9). CE for the analysis of microorganisms was first used by only allowed for the rapid identification of yeast directly from a
Hjertén et al. (113) in 1987. They analyzed tabasco moscati virus sample isolated from spontaneously fermented grapes, but also
and Lactobacillus casei. Although they observed no separation, made the assessment of their viability in a culture possible. This
their study aroused interest in the use of CE for analysis of information is very useful, especially when there is a need to
microorganisms. In 1993, Ebersole and McCormick (114) control microbial fermentation in food.
effectively separated S. agalactiae, S. pneumoniae, S. pyogenes, The main shortcomings of electrophoretic separation is that
and Enterococcus feacialis using CZE. microorganisms tend to agglomerate and interact with the
An important aspect of microorganism analysis using internal surface of the capillary (i.e., adhesion). This is due
electromigration techniques is the fact that the composition of to the fact that positively charged groups may be present on the
the surface of living organisms can change during the growth surface of microorganisms and may readily interact with the
phase of the organism or in reaction to any substitutions in their silanol groups in the capillary tube (1, 2). A dominant influence
environment (1), leading to changes in surface charge and on the aggregation of cells and, thus, on the behavior of
consequently in the value of the z-potential (1, 2, 9). microorganisms during electrophoretic separation, does
Microbial growth can be monitored by measuring the have a z-potential value (116). Research conducted by
z-potential (1). Soni et al. (115) conducted CE experiments to Kłodzińska et al. (116) showed that, in electropherograms,
measure the z-potential of bacteria in different states and growth the number and shape of peaks are dependent on the value
conditions. Their results indicated that the bacteria grown on of the z-potential. Their analysis of S. aureus and E. coli showed
nutrient-rich media were characterized by higher absolute values that, the higher the absolute value of the z-potential, the less the
of z-potential compared with dead cells cultured on starved signals are observed on the electropherograms. This is a result
media. Crispo et al. (9) used CZE for the rapid electrophoretic absolute z-potential values exceeding 30 mV during
separation of three Saccharomyces cerevisiae strains in different electrophoresis analysis because of the formation of more
growth phases. The electrophoretic profile for all strains at stable clusters. They also found that in measuring the
170207_Buszewski 9
10 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
z-potential, it is possible to differentiate the tested bacterial phenotypically difficult to distinguish. Horká et al. (29)
strains because the z-potential is different between Gram- attempted to separate phenotypically indistinguishable
positive and -negative bacteria. Candida species [the species that most frequently cause
To prevent cell adsorption into the capillary wall, numerous bloodstream infections (BSIs)]. Horká et al. developed the
procedures have been proposed. One approach suggested using first accurate and reproducible method for the separation and
polyethylene oxide (PEO) as an additive to the buffer solution to identification of Candida orthopsilosis, C. metapsilosis, and C.
dynamically modify the inner surface of the capillary. Szeliga parapsilosis by capillary isoelectric focusing and CE. In
et al. (10) used this method to rapidly identify local microbial addition, this method allowed for the determination of the
infections of the skin by CZE. They were able to identify Gram- ability of these species to form biofilm.
positive cocci and Gram-negative rods with a sensitivity of Electromigration techniques have been compared with other
88.1% and specificity of 100%. Another approach is the methods for their effectiveness in identifying microorganisms.
chemical modification of the fused capillary wall, in which Horká et al. (122) used five analytical techniques for the
different monomers are used, such as trimethylchlorosilane, characterization of Monilinia spp.: CZE, CIEF, gel IEF,
divinylbenzene, or acrylamide, or the application of monolith sodium dodecyl sulfate polyacrylamide GE (SDS-PAGE), and
(10). The coating can prevent cell adsorption to the capillary MALDI-TOF MS. Their results indicated that CZE and CIEF
surface. Moreover, the coating helps suppress electro-osmotic methods enabled the quick, economical, and effective
flow so cells can move through the capillary under their own differentiation of morphologically similar Monilinia species;
electrophoretic mobility. Buszewski and Kłodzińska (117) found however, they were unsuitable at distinguishing different strains
that using a variety of modifications to the inner wall of a of the same species. Gel IEF and SDS-PAGE techniques were
capillary column permitted easy and rapid identification of considerably more time consuming; however, they could
pathogens in the gastrointestinal tract. discriminate morphologically similar Monilinia spp. In
Recently, many researchers have been using electromigration addition, with the gel IEF method, the authors were able to
techniques for the screening identification of microorganisms distinguish between different strains of the same species. In
specifically in clinical samples (11, 28, 118–120). Horká et al. contrast, MALDI-TOF MS seemed to be the optimal technique
(119) set out to determine MRSA and methicillin-susceptible for differentiating morphologically similar Monilinia spp.; it was
S. aureus (MSSA) from blood using CZE. The blood-incubated fast, efficient, and accurate and allowed the identification of
MRSA and MSSA strains were differentiated by CZE on different strains of the same species. However, an important
(3-glycidyloxypropyl)trimethoxysilane-modified fused silica role in the identification of Monilinia spp. by MALDI-TOF
(FS) capillaries etched with supercritical water. In 2 mL MS plays host origin of the spores. Horká et al. suggested that
whole human blood, they detected at least 200 MRSA cells; this challenge could be resolved by applying MALDI-TOF MS in
RSD values ranged between 7 and 40%. These values provided combination with CZE or CIEF.
sufficient data for an effective antibiotic therapy to be chosen, Another interesting approach is the use of microchip
and the amount of injected purified cells was sufficient for the technology (123). ME is the miniaturized version of a
fast screening of the clinical samples. Szeliga et al. (28) conventional CE. Scientist from Ciba Geigy (123) developed
developed a rapid screening test based on CZE for the this technique in the early 1990s. Currently, ME is considered a
identification of E. coli in biological material. They simple and convenient alternative to conventional CE, offering a
examined a total of 30 E. coli-infected wounds and shorter analysis time, as well as a smaller sample size and use of
ulcerations and compared their results with those obtained less reagents. In addition, ME’s voltage requirements are lower
using conventional methods. The results indicated that due to shortened separation channels (5–15 cm); it can also
surgical wounds infected with E. coli could be determined in separate analytes from small amounts of complex matrixes (13).
just 0.5 h with 86.7% sensitivity and 85% specificity. Song et al. Nuchtavorn et al. (124) developed a new system for
(118), in 2013, used a three-plug injection method with CE for the separation and detection of fungi and Gram-positive
the rapid identification of bacteria in urine samples. Their results and -negative bacteria based on an ME coupled with a laser-
showed that this method allowed for the identification of bacteria induced fluorescence (LIF) detector with excitation and
in urine in just 10 min. In their research, one peak appeared for all emission wavelengths of 635 and 685 nm, respectively. To
bacteria species on their electropherograms. Other urine enable detection of the tested microorganisms, E. coli,
components, such as proteins and blood cells, had affected S. aureus, and C. albicans were stained with fluorescent dye
the retention time of the peak but did not have an effect on (Nile blue). The microbial charges changed as a result of the
the separation of the bacteria. Further analysis showed that the dye’s attraction for the negatively charged cell surface through
concentration limit for this method is 106 CFU/mL, and that there electrostatic interaction. Therefore, migration of microorganisms
was a good linear relationship between the height of the peak and occurred in the direction of the cathode. Tris-borate-EDTA
the concentration of bacteria. Thus, an electrophoretic technique (TBE) buffer was used for the separation because it has a pH
can also be used for the quantitative determination of of 10.5 and contains 0.025% PEO. This method allowed the
microorganisms. In fact, studies on the quantitative unambiguous analysis of the sample, which included a mixture
determination of microorganisms in natural water were carried of fungi and Gram-positive and -negative bacteria, within just
out by Oukacine et al. (121), who developed a repeatable 30–40 s. Cheng et al. (30) used a similar method to differentiate
preconcentration electrophoretic method for the analysis of probiotic bacteria. In their work, bacteria were stained with red
bacteria. They used isotachophoresis coupled with a fluorescent nucleic acid dye, and separation was carried out in
simultaneous hydrodynamic-electrokinetic injection to TBE buffer (pH 8.5) containing 0.025% PEO. This method
quantitatively determine Enterobacter cloacae with an LOD allowed for the separation of all tested bacteria within 150 s.
of 2 × 104 cells/mL. Electromigration techniques are also well This research thus confirmed that ME is an appropriate method
suited for the identification of microorganisms that are otherwise for the identification of bacteria, primarily because analysis time
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BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017 11
is short, only small quantities of sample are needed, and there is Table 5. Review of multivariate techniques used for the
less waste production (30, 124). However, one of the major identification of different microorganisms
disadvantages of the electrophoretic separation of bacteria is the
propensity of bacteria to aggregate and adhere to the inner walls of Microorganisms Method Refs.
the microchannels (1). Therefore, to minimize this effect, Wang Blood cultures PCR/ESI-MS (31, 136–138)
et al. (125) used lipid-based liquid crystalline nanoparticles as the BAL cultures PCR/ESI-MS (32, 139–141)
pseudostationary phase. By using a microchip with LIF, they could Salmonella spp. PCR/ESI-MS (133)
identify L. delbrueckii subsp. bulgaricus, L. rhamnosus, and S. Multiplex PCR-ME (33)
thermophilus. The authors suggested that surface modifications
Escherichia coli PCR/ESI-MS (133)
related to the adsorption of lipid-based liquid crystalline
Multiplex PCR-ME (33)
nanoparticles improved selectivity for microorganisms.
CZE-MALDI-TOF MS (128)
In summary, electromigration techniques are a useful tool for
the identification of microorganisms. These methods are Shigella spp. PCR/ESI-MS (133)
characterized by speed in result output and efficiency. In Multiplex PCR-ME (33)
addition, very small amounts of sample and reagents are Klebsiella spp. PCR/ESI-MS (132)
sufficient for analysis. Moreover, these techniques allow for CZE-MALDI-TOF MS (128)
the assessment of cell viability and are well suited for screening Acinetobacter spp. PCR/ESI-MS (132)
assays. However, there are numerous drawbacks with the Ehrlichia spp. PCR/ESI-MS (134)
selection of analytical conditions, which arise from Vibrio spp. PCR/ESI-MS (135)
microorganisms’ tendency to form aggregates and interact Multiplex PCR-ME (33)
with the inner wall of capillaries (1, 2, 9).
Cronobacter spp. Duplex PCR-CE–LIF (143)
Another technique that uses the ability of microorganisms to
PCR-DHPLC (160)
migrate in an electric field is electrical field-flow fractionation
Staphylococcus spp. tDNA-PCR-CE (24)
(EIFFF; 126). FFF represents a group of separation techniques in
which the separation of sample components occurs in an open Candida spp. FISH-CE (127)
separation channel, with separation occurring as a result of the sIEF-HPLC (35)
formation of layers of separated components at different levels in Lactobacillus spp. CIEF-MALDI-TOF MS (34)
the channel under the influence of various applied fields returned CZE-MALDI-TOF MS (128)
perpendicular to the laminar flow with a parabolic flow profile Arthrobacter psychrolactophilus CZE-MALDI-TOF MS (128)
and a counteracting the molecules diffusion. In ElFFF, the applied Bacillus spp. CZE-MALDI-TOF MS (128)
field is electrical, and electrodes comprise the two main walls of Micrococcus luteus CZE-MALDI-TOF MS (128)
the channel. The difference in potential between the two electrodes Pseudomonas putida CZE-MALDI-TOF MS (128)
causes charged particles to migrate to the opposite-charged wall
Pseudomonas aeruginosa CZE-MALDI-TOF MS (128)
(126). Saenton et al. (126) conducted an ElFFF analysis on three
morphologically different bacteria: Pseudomonas putida,
E. coli, and S. epidermidis). In parallel, separation was
performed using other techniques from the FFF group (i.e., PCR with MS and ESI is the most frequently used method for
flow and sedimentation FFF). The obtained results indicated the identification of microorganisms (129). The PCR/ESI-MS
that all three methods were effective in differentiation, method involves the amplification of various fragments of a
whether in living or dead cells. In addition, the techniques genome and the determination of the fragments’ mass (Figure 3).
allowed for the separation of different bacterial strains. Broad-range primers are used for the PCR—primers that are
specific to whole groups of microorganisms, species, or strains
Multivariate Techniques (129). Obtained products are subsequently analyzed by ESI-MS
to determine the molecular mass of the resulting DNA fragments.
In recent years, there has been an increase in the use of a Molecular mass determination results for the amplicons and the
combination of analytical techniques for the faster and more quantitative composition of the amplicon bases of several genes
effective identification of microorganisms. Recently, research arranged in tandem represent a species-specific fingerprint, which
was conducted on the use of molecular methods combined with can then be compared with other fingerprints in a database (129).
MS (31, 32) and capillary electromigration techniques (24, 33), The first studies on the analysis of PCR products by ESI-MS
as well as sCIEF-HPLC (35), fluorescence in situ hybridization were conducted in 1996 by Muddiman et al. (130) and Wunschel
(FISH)-CE (127), CIEF-MALDI-TOF MS (34), or CZE- et al. (131). Both published works relied on the detection of B.
MALDI-TOF MS (128; Table 5). Results indicated that the subtilis and B. cereus rRNA fragment PCR products in negative-
potential for these methods to correctly identify microorganisms ion mode. Recently, it was confirmed that the PCR/ESI-MS
is high (7, 24, 34, 127). technique can identify a multitude of human pathogens—such as
Klebsiella and Acinetobacter (132); Esherichia, Shigella, and
Salmonella (133); Ehrlichia (134); and Vibrio (135)—in
Molecular Methods in Combination with MS addition to BSI pathogens (31, 136–138; Table 5).
The PCR/ESI-MS method also allowed for the simultaneous
Analysis of PCR products using MS has proven to be a very identification of a variety of microorganisms in a sample without
promising tool for the multiplexed detection and direct identification requiring the isolation of pure cultures. This was confirmed inter
of microorganisms derived from specimens (24). This form of alia in the Kaleta et al. study (31). Kaleta et al. used the PCR/ESI-
diagnostic analysis technology is economical, fast, and accurate. MS approach to analyze microorganisms in blood samples. In
170207_Buszewski 11
12 BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017
their study, they used a sterile fluid bacteria and Candida assay, Fully automated systems are currently used for analyses.
which consisted of 16 pairs of primers duplicating antibiotic- PLEX-ID, which was developed by Abbott Molecular, was
resistant genes, rDNA regions, and genes specific for each the first PCR/ESI-MS system for bacterial DNA. This system
bacterial group. Of the 234 tested samples, a mixture of combines ESI-MS and broad-range PCR with multiple primers
microorganisms was detected in 29. Results from the and computerized triangulation to identify microorganisms in
biochemical test (Vitek 2) identified 98.7% of microorganisms samples. Huttner et al. (32) assessed the ability of the first version
at the genus level and 96.6% at species level in 5–6 h. Further of this system to rapidly identify infectious agents in clinical
studies conducted by Kaleta et al. (136) were designed to samples using bronchoalveolar lavage (BAL) samples. The
compare the performance of PCR/ESI-MS with MALDI-TOF obtained results were compared with the standard methods
MS (BioTyper) in identifying microorganisms from blood (SMs) used in their center for detecting bacteria and fungi in
samples. Accuracies in identification of the two methods were BAL samples. Overall agreement between the SMs and PLEX-
very similar. PCR/ESI-MS identified 96.7% microorganisms ID was 45%. PLEX-ID failed to identify 21% of the 183
from 273 samples at the genus level and 95.6% at the species microorganisms identified by the SMs; however, the SMs did
level versus 97.1 and 94.9%, respectively, for MALDI-TOF MS. not identify 28% of the 191 microorganisms identified by PLEX-
The authors emphasized the superiority of MALDI-TOF MS: ID. Based on these results, Huttner et al. found that the tested
fully automated, simple measurements, and low cost of analysis. by them (first vesion) of PLEX-ID is not a useful tool for
Moreover, MALDI-TOF MS can determine a single sample, independent microbiological diagnostics in patients who have
whereas by PCR/ESI-MS must simultaneously analyze at least suspected respiratory tract infections. This system was later
six samples. The main disadvantage of MALDI-TOF MS, modified (139). One of the changes was to increase the
however, is the need to isolate pure cultures, which prolongs amount of tested biological material from one to six
result times (136). specimens for simultaneous analysis to increase sensitivity
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BUSZEWSKI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 100, NO. 6, 2017 13
(139). This modification led to better performance in samples Koop et al. (24) used transfer RNA-intergenic space PCR
from lower respiratory tract secretions, as confirmed by Vincent in combination with CE to identify coagulase-negative
et al. (140). In their study, PCR/ESI-MS showed 84% sensitivity staphylococci (CNS) in goat’s milk. They then compared this
and 53% specificity. At the end of 2014, Abbott Molecular method with the API 20 Staph biochemical test. Using a modified
released a new PCR/ESI-MS system called IRIDICA. In 2016, transfer DNA (tDNA)-PCR protocol, they were able to initially
Strålin et al. (141) assessed the efficacy of IRIDICA’s ability to identify 103 of the 115 unique isolates (89.6%), whereas the API
identify microorganisms in BAL samples from patients who had Staph 20 could only identify 83 (73%). The overall yield of the
suspected pulmonary inflammation. The overall sensitivity of the API Staph 20 in differentiating CNS in goat’s milk was moderate
method was 77%. The obtained results indicated that this method to low, whereas the modified tDNA-PCR procedure had high
has potential in the diagnosis of patients who have suspected accuracy and speed and was low cost, and thus preferable.
pulmonary inflammation. Jordana-Lluch et al. (137) tested the
IRDICA PCR/ESI-MS system’s ability to identify BSIs in
immunocompromised patients. Their study was conducted on Electromigration Techniques in Combination with
463 blood samples and the results compared with blood cultures. Other Methods
The sensitivity of PCR/ESI-MS was 64.3%, of which 59.3%
were from hematological patients and 69% from oncological Many studies have been conducted on the use of
patients. It can be concluded that PCR/ESI-MS is a promising electromigration techniques in combination with other
tool in medical diagnostics. advanced analytical techniques for the rapid and unambiguous
identification of microorganisms. Lantz et al. (127) used CE in
combination with FISH to rapidly identify C. albicans in blood.
Molecular Methods in Combination with FISH is a hybridization method that enables the detection of a
Electromigration Techniques specific DNA sequence in a sample by using a fluorescently
labeled molecular probe. By combining FISH with CE, blood
In a traditional molecular analysis, PCR products are subjected analysis can be carried out in less than 2 h. In their results, the
to agarose GE. However, this process is time-consuming and surfaces of obtained peaks showed a strong linear correlation
requires large volumes of samples. On the other hand, real-time between concentrations of yeast up to ~107 CFU/mL, indicating
qPCR is reliable, fast, and sensitive; however, the high cost of that this method had great potential for the rapid, accurate, and
equipment and significant technical demands severely limits its quantitative identification of Candida spp. in blood. Their
application (142). For this reason, a combination of molecular research found that this method can be a useful tool in the
methods with more advanced electromigration techniques seems diagnosis of both human and veterinary infections.
to be a promising solution to increase the discriminatory power of Vykydalová et al. (35) used a combination of techniques
analytical methods. to identify yeast. They proposed combining sCIEF with HPLC
Ruan et al. (143) detected Cronobacter spp. in food products. to differentiate between biofilm-negative and -positive C.
The Gram-negative bacillus of the genus belongs to the parapsilosis from a vascular catheter. Intact yeast cannot be
Enterobacteriaceae family and causes a number of life- analyzed by HPLC because of their size (3–5 µm). Therefore,
threatening neonatal infections, such as meningitis or sepsis. yeast were boiled in distilled water to release surface substances
Currently, the procedures used to identify these bacteria are from yeast cells. The authors then used these substances to
laborious and lengthy, potentially taking more than 5 days to be differentiate individual yeast strains. The obtained supernatant
performed. Ruan et al. examined a total of 120 commercially was charged into nonwoven fabric strips and then analyzed by
available baby foods to confirm the presence of Cronobacter sCIEF. In a second step, HPLC was used to separate the solutions
spp. using duplex PCR combined with CE–LIF detection. The from the strips and the obtained fingerprints compared. This
LOD was only 1.6 × 101 CFU/mL Cronobacter spp., and the method allowed the differentiation of selected yeast species in
RSDs of the migration time for the detected DNA fragments were real clinical material. For this reason, it can be used to rapidly
2.01–2.91%. Based on these results, Ruan et al. concluded that identify microorganisms, especially in the field of clinical
CE–LIF with a duplex PCR method is suitable for the detection medicine, because early detection of pathogens is critical to
of small quantities of Cronobacter spp., as well as for the choosing the effective therapy.
identification of other pathogenic bacteria in the food Horká et al. (34) used CIEF in combination with MALDI-TOF
industry. Moreover, this method could provide valuable MS for the unique identification of probiotic bacteria in a real
information, such as the point of contamination. In fact, the sample from cow’s milk. They conducted their research on lactic
entire detection process could be cheaper, faster, more sensitive, acid bacteria, as model bioanalytes, within the genus
and friendly to the environment. Lactobacillus. They performed CIEF analysis using a tapered
Li et al. (33) also carried out a determination of pathogenic FS capillary. This approach increases the separation capacity and
bacteria in food. They developed a rapid and sensitive method efficiency of CIEF analyses and allows the detection of bacterial
based on multiplex PCR coupled with ME and optimization of cells at a level of 2 × 106 bacteria cells/mL. They performed their
the conditions in the genetic algorithm-support vector regression experiment by adding L. rhamnosus to cow’s milk and used
to simultaneously identify Vibrio parahemolyticus, Salmonella, CIEF to analyze the prepared sample. The bacterial cells were
E. coli, and Shigella in food samples. The LOD for the individual consequently harvested from the capillary and sedimented on
pathogens was very low—1.2 × 102, 2.9 × 102, 8.7 × 101, and culture medium. After completion of the culture, bacteria were
5.2 × 101 CFU/mL, respectively. The RSDs of the migration analyzed by MALDI-TOF MS. Horká et al.’s results (34)
times were 0.74–2.09%. Li et al. found that this method could showed that the proposed procedure could be an effective tool
be a useful tool for the efficient, inexpensive, and sensitive for the unique identification of probiotic bacteria in milk
assessment of food safety. samples.
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