New 3-Xa Vi Khuan Bang MS

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International Journal of Mass Spectrometry xxx (2013) xxxxxx
Contents lists available at SciVerse ScienceDirect
International Journal of Mass Spectrometry

journal homepage: www.elsevier.com/locate/ijms
Review
Identication of bacteria using mass spectrometry techniques

Luks Krsny a,b, , Radovan Hynek a , Igor Hochel a
a
b
Institute of Chemical Technology in Prague, Department of Biochemistry and Microbiology, Czech Republic
Institute of Microbiology of the Academy of Science CR, Czech Republic
a r t i c l e
i n f o
Article history:
Received 14 February 2013
Received in revised form 16 April 2013
Accepted 16 April 2013
Available online xxx
Keywords:
Mass spectrometry
Bacteria
Identication
BioTyper
Saramis
Vitek
a b s t r a c t
The possibility to rapidly identify bacteria is required in many different elds. Due to rapid progress
in the development of mass spectrometry devices during the last few years, identication by means
of mass spectrometry has become a very powerful and usable tool. These methods offer fast analysis
of biomarker ions, providing reliable information on bacteria characterization even at the sub-species
level. Therefore, these approaches have been successfully established as routine methods, together with
classical biochemical tests and genome sequencing. This review focuses on common biomarkers and on
different mass spectrometry techniques which have been used for bacteria identication throughout the
third millennium.
2013 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction and short historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GC/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Pyrolysis GCMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
SPME-GCMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MALDI MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
MALDI-based sequencing of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
MALDI MS protein proling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
MALDI MS identication based on lipid and lipopolysaccharide analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ESI MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
PCR/ESI-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
ESI MS identication based on the analysis of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
ESI MS identication based on lipid and LPS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: 2-AA, 2-aminoacetophenone; A, adenine; BAMS, bioaerosol mass spectrometry; BHP, bacteriohopanepolyol; C, cytosine; CE, capillary electrophoresis;
CI, chemical ionization; DART, direct analysis in real time; DESI, desorption electrospray ionization; DGDG, diglycosyldiacylglycerol; DMAN, 1,8-bis(dimethylamino)
naphthalene; DNA, deoxyribonucleic acid; DPA, dipicolinic acid; dTTP, 2-deoxythymidine-5- triphosphate; dUTP, 2-deoxyuridine-5- triphosphate; EI, electron
ionization; ESI, electrospray ionization; FAB, fast atom bombardment; FAME, fatty acid methyl ester; G , Gram-negative; G, guanin; G+ , Gram-positive; GC/MS,
gas chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; LC, liquid chromatography; LDI, laser desorption/ionization; LOS,
lipooligosaccharides; LPS, lipopolysaccharides; MAB, metastable atom bombardment; MALDI, matrix-assisted laser desorption/ionization; MALDI-RE, matrix-assisted
laser desorption/ionization re-sequencing; MRM, multiple reaction monitoring; MRSA, methicillin-resistent Staphyloccocus aureus; MS, mass spectrometry; NIH,
National Institute of Healths; PCA, principle component analysis; PCR, polymerase chain reaction; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PSD,
post-source decay; Py, pyrolysis; RNA, ribonucleic acid; SASP, small acid soluble proteins; SIFT, selected ion ow tube; SPA, Selective proteotypic-peptide analysis;
SPME, solid phase micro-extraction; T, thymine; TOF, time-of-ight; U, uracil; UV, ultra-violet; VNTR, variable-tandem repeats; VOC, volatile organic compound.
Corresponding author at: Institute of Chemical Technology in Prague, Technick
5, Prague, 16628, Czech Republic. Tel.: +420 220 443 216.
E-mail address: krasnyl@vscht.cz (L. Krsny).

1387-3806/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijms.2013.04.016
et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

Please cite this article in press as: L. Krsny,
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6.
7.
8.
Ambient techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
DESI and DART MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
SIFT MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bioaerosol mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Insights into the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction and short historical overview

The detection or identication of bacteria is required in many
different spheres of human activity. Rapid and correct pathogen
identication is crucial in medicine as well as in other elds
such as food analysis, veterinary science, ecology, agriculture, etc.
Various methods for identifying microorganisms, such as microscopic observation, biochemical testing, immunochemical assays
and molecular biological methods, have been developed. Each of
these methods has some particular advantages and disadvantages.
Biochemical testing is a non-targeted form of analysis; however, a
pure microbial culture is required. Immunochemical assays can be
performed in mixtures of microbial cultures, but the afnity and
specicity of the antibody is the limiting factor. Molecular biology
can also be performed in a mixture of microbial cultures, and even
non-cultivable microorganisms may be identied. On the other
hand, these benets are reected in the higher cost of the analysis.
Mass spectrometry (MS) is very attractive for work in microbiology because of its speed. Methods based on intact cell MALDI-TOF
experiments provide comprehensive information within 5 min; the
other advantage is that just one single colony is required for analysis. However, it was not always like this.
Despite the enormous application of mass spectrometry after
the Second World War, the rst experiments to identify individual microorganisms using this technique were performed in the
1970s in conjunction with the mission to Mars and analysis of the
Martian soil [1,2]. In these studies, pyrolysis-gasliquid chromatograph coupled with mass spectrometer was used for the analysis
of soil and Micrococcus luteus and Bacillus subtilis whole cells. The
rst bacterial Py-MS mass ngerprint was measured by Meuzelaar and Kistemaker using Curie-point pyrolysis-MS in 1973 [3],
however, the measured m/z range between 10 and 50 Da was not
sufcient. The real breakthrough was performed by Anhalt and
Fenselau [4], who measured the pyrolysis products of ubiquinone,
phospholipids, and other volatile compounds from in source heated
bacteria. Lower temperature used for pyrolysis enabled recording of the mass spectra up to 700 Da. Subsequent experiments
performed by pyrolysis-gas chromatography/mass spectrometry
allowed for example the characterization of microorganisms [5]
or Gram-type differentiation [6,7]. However, the soft ionization
techniques that emerged in the 1980s have proven to be more
effective. Fast atom bombardment (FAB) ionization was used by
Heller et al. [8] to measure lipid biomarkers from the whole bacteria
lysate. This method appeared promising; thus, correlation functions and a library-matching system for bacteria identication were
created [9]. Nevertheless, insufcient sensitivity and an insufcient
dynamic range and condence level have disqualied FAB from
practical use.
In comparison with FAB, electrospray ionization (ESI) offers high
sensitivity and is very useful for the ionization of macromolecules
such as protein biomarkers. Early experiments were carried out
with bacterial lysates [10]; however, Goodacre et al. [11] developed a method for the intact cell characterization of bacteria.
The major disadvantage of this approach is the clogging of the
electrospray needle and the high complexity of obtained spectra,
which often cannot be resolved easily. Despite this, ESI MS is used
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for the identication of bacteria, spores [12], and viral particles

[13].
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry entered the eld of microorganism identication in 1996
[1416] and took the leading position in just a few years. This success is based on MALDIs short analysis time, high sensitivity, and
intact cell measurements, as well as the possibility of automation;
therefore, MALDI mass spectrometry has become the method of
choice. The other advantage of MALDI is its compatibility with different analyzers and its wide m/z range. On the other hand, the
process of MALDI ionization brings some limitations and problems,
such as suppression effects, sweet spots, and selective ionization
[17].
In the last few years, DESI-desorption electrospray ionization
[18,19], SIFT-selected ion ow tube [20], and BAMS-bioaerosol
mass spectrometry [21] have appeared and become more
widespread. Although their use is more limited than that of MALDI,
they are able to provide useful information about lipids and/or
various metabolites (e.g., volatile organic compounds, ammonia,
etc.).
In this review, we summarize both common and rare biomarkers used in bacteria identication. We summarize developments
and innovations in mass spectrometry methods applied to bacteria
identication over the last decade, and compare current commercial systems designed for clinical use. The review focuses on mass
spectrometry; thus, it will deal only with those biomarkers measurable by current mass spectrometry techniques.
2. Biomarkers
Biomarker is a word used in many different contexts in
medicine, cell biology, microbiology etc. The ofcial National Institute of Healths (NIH) denition of a biomarker is: a characteristic
that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological
responses to a therapeutic intervention [22]. Nevertheless, this
denition is inadequate for the needs of microbiology. Hence, other
denitions have arisen, such as: A biomarker is a molecule that
allows for the detection and isolation of a particular cell type or
A biomarker can be any kind of molecule indicating the existence,
past or present, of living organisms. These t better. In any case,
the specicity of a particular molecule (or molecules) is a key characteristic. From the perspective of bacteria identication, the ideal
biomarker is present in all bacteria, but it is specic for each of them
and measurable.
If we take a look at molecules present in a bacterial cell,
we nd biopolymers (nucleic acids, polysaccharides), small
organic molecules (lipids, saccharides, metabolites), and inorganic
molecules. Nucleic acid is the molecule which almost meets the
requirements of an ideal biomarker. The genome is fundamental
and encapsulates unique information about each organism in existence. But there is only one copy of DNA per cell, which makes it
difcult to measure by any technique without previous amplication. Mass spectrometry is no exception. Fortunately, PCR products
are measurable using soft ionization techniques such as MALDI or

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ESI; hence, we can consider nucleic acid as a measurable biomarker

[23].
Over 50% of the cell dry weight consists of proteins [24]. This
large abundance is very attractive, because it allows good detection
sensitivity. Proteins represent the phenotype of the organism and
in spite of their high diversity there are several thousand different
proteins in a cell they are the most often used and most successful biomarkers for bacteria identication, especially in combination
with MALDI mass spectrometry.
Lipids constitute approximately 50% of the weight of cellular
membranes and 510% of the prokaryotic cell dry weight, but
it means the highest number of molecules per cell [25]. Lipid
biomarkers were extensively investigated by pyrolysis-GC/MS and
FAB in the early days of mass spectrometry bacteria identication.
Py-GC/MS helped to introduce the MIDI system for the bacteria
identication which is still provided by MIDI Inc. [26]. This system
is based on the analysis of the fatty acid methyl esters (FAMEs) by
gas chromatography. The chromatographic proles are then compared with the library. However, there is no MS in routine analysis,
since ame ionization detector is used.
Lipids were of interest for soft ionization techniques as well;
nevertheless, their analysis is not as widespread as protein proling. The main complication is the lipids dependency on growth
conditions. In spite of this, several studies using DESI have recently
been published, which indicates the possible renaissance of lipid
proling.
Several approaches using lipopolysaccharides or Lipid A
have been considered as alternative methods of identication.
Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are components of the G bacteria outer cell membrane and are responsible
for the antigenic properties of bacterial cells. LPS consists of Lipid
A, which is considered to be the most conserved part (although
variations between or within genera may be observed), and
oligosaccharidic core and O-antigen, which constitute conversely
the most variable part and thus may be used as a specic biomarker.
Some experiments with LOS and LPS were performed using MALDI
and ESI ionization; however, the results were not entirely convincing [27,28].
The diversity of bacteria offers various metabolism pathways
that can be used for identication. This approach is more targeted than protein proling. Techniques like GC/MS [29], SIFT
(selected ion ying tube) [20] and DESI [19] are applied with varying degrees of success. The requirement of measurability is crucial
in this eld, because the concentration of the analytes could be
very low and, unlike nucleic acid, there is no possibility to amplify
them.
3. GC/MS
3.1. Pyrolysis GCMS
It was GC/MS which was the early pioneer of bacteria identication by mass spectrometry. Many studies have been performed
using pyrolysis-MS and pyrolysis-GCMS (Py-GCMS), in which
whole intact bacteria were analyzed [37]. Currently, there are
new techniques like SPME-GCMS analysis of bacterial culture
headspace, but Py-GCMS still maintains its position. Since the
rst experiments in the 1970s, Py-MS has changed slightly. The
70 eV electron ionization (EI) was changed to low energy EI or
replaced by soft ionization (chemical ionization CI, metastable
atom bombardment MAB), and thus, despite the dominance of
MALDI, Py-MS still nds applications even in the third millennium.
Classic electron ionization was used in the study by Miketova
et al. [7], where G+ and G bacteria were separated based on their
peak patterns. Six Gram positive species (Bacillus cereus, Bacillus
subtilis, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes, Bacillus anthracis) and the same number of Gram
negative species (Enterobacter aerogenes, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Brucella neotomae and
Francisella tularensis) were measured and the biological origins of
product ions were identied. Carbohydrate products were specic
for G+ bacteria, while lipid biomarkers dominated in the spectra
of G bacteria. More specic data were presented by Dworzanski et al. [30]. They found 2-pyridinecarboxamide as the dominant
component in the spectra of G+ bacteria, while G spectra contained
signals for compounds derived from LPS.
Various other biomarkers have been found useful for pyrolysis.
The ash Py-GCMS method for the detection of bacteriohopanepolyols (BHPs) producing bacteria has been developed by
Sugden et al. [31]: fragment ions were found to originate from
C18:1 and C19:1 fatty acids and protein biomarkers (fragments of
tryptophan) were identied in Brucella neotomae [32,33]; Wilkes
et al. performed the phenotypic characterization of Salmonella
enterica [34] and Vibrio isolates [35] using Py-MAB-MS; Goodacre
et al. [36] found the pyridine ketonium ion specically in pyrolysis
spectra of Bacillus spores. It is assumed that pyridine ketonium is
a thermal degradation product of dipicolinic acid (DPA), the spore
specic biomarker, as was proved by White et al. [37] two years
later (see above). Beverly et al. [38] used in situ methylation of DPA
for detection of Bacillus anthracis spores.
3.2. SPME-GCMS
Solid phase micro-extraction (SPME) in combination with
GC/MS is very popular. It enables the extraction of analytes from liquid or gas matrices and thus has a wide eld of application. From
the microbiological point of view, the analysis of volatile organic
compounds (VOCs) is the most important ability of SPME. It can
be used for the analysis of breath [29,39] or bacterial suspension
headspace [39,40], for the detection of microbial growth in food
[41], or in environmental research [42].
A set of various VOCs was analyzed in the breath of patients
infected by Helicobacter pylori [29]. Although the obtained data
indicated that isobutane, 2-butanone and ethyl acetate in breath
might serve as signs of infection, statistical analysis showed that the
results were not so unambiguous. Better results have been obtained
for 2-aminoacetophenone (2-AA) as a potential breath biomarker
of Pseudomonas aeruginosa in the lungs of patients with cystic brosis [39]. In that study, the concentration of 2-AA was increased in
the culture headspace after 24 h of growth as well as in the breath
of patients with the P. aeruginosa infection, while non-infected
patients had a signicantly lower level of 2-AA. Twenty-eight new
volatile compounds of P. aeruginosa were identied using SPME
coupled with two-dimensional GCMS in recent work by Bean
et al. [40]. Preti et al. [43] have observed several characteristic
compounds in the culture headspace for six bacteria commonly
associated with sinusitis.
The analysis of fatty acid methyl esters (FAMEs) proles of bacteria by SPME-GCMS has been conducted by Lu and Harrington
[44]. The cells of Enterococcus faecalis, P. aeruginosa, E. coli, S. enterica, Vibrio parahaemolyticus and Listeria innocua were mixed with
tetramethylammonium hydroxide methanolic solution, generated
FAMEs were extracted by SPME, and the fatty acid composition
was analyzed. Gram positive bacteria were well separated from
Gram negative ones using PCA and the results were in good agreement with previous works by Basile [6] and Dworzanski et al. [30].
Despite the fact that Lu and Harrington were even able to identify bacteria, they achieved only a classication accuracy of 87%
within these six species, which is still below the proling accuracy
for proteins.

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4. MALDI MS
4.1. MALDI-based sequencing of DNA
One of the rst to use MALDI MS for the detection of DNA was
G.B. Hurst in 1996 [23]. In his study, 108- and 168-base PCR products from Legionella were detected in negative ion mode using
3-hydroxypicolinic acid as a matrix. The same method was used for
the detection of Methylosinus and Methylomicrobium specic genes
two years later [45].
DNA measurement by MALDI is signicantly limited. Doublestranded nucleic acid is not usually detected due to the acidic
nature of matrices. The protonation of bases disables WatsonCrick pairing; hence, double-strand nucleic acid denatures and two
single-stranded nucleic acids are detected [46]. Other problems
arise due to fragmentation [47] or salt formation of the phosphate
backbone.
For this reason, DNA is transcribed to the RNA form. The RNA
molecule is more stable during MALDI ionization [48], which is used
for the MALDI-resequencing method (MALDI-RE) [49]. This method
is based on the PCR amplication of several housekeeping genes or
variable-tandem repeats (VNTRs). Amplicons are then transcribed
in vitro and the resulting RNA is cleaved by specic RNAses. The
RNA fragments are desalted and the mass spectrum is measured in
linear mode in the mass range up to 10,000 m/z. Final spectra are
compared to the in-silico sequence digest via a pattern matching
algorithm to identify studied species. The concordance observed
between the MALDI-based resequencing method and classic Sanger
MLST sequencing exceeds 98% [49].
In 2002, von Wintzingerode et al. developed a rapid method
for 16S ribosomal RNA mass spectrometry typing [50]. The amplicon of 16S rDNA was used as a template for another PCR with
biotinylated primers and dUTP instead of dTTP. The PCR products
were then isolated by streptavidin-coated paramagnetic beads and
abasic sites were generated using uracil-DNA-glycosylase. After
denaturation, the phosphate backbone was cleaved and the nal

mix of rDNA fragments was obtained. These fragments consisted
of C, G and A nucleotides and were cleaved in the place where the
T (U) nucleotide is normally present. Different strains of Bordetella,
Alcaligenes and Achromobacter were used as a model of the closely
related microorganisms and their identication was successfully
performed. Single base pair mutation can also be detected in the
studied gene sequence. Mutation can create a new specic site for
cleavage, or, vice versa, the cleavage site may become lost because
of the shift in the reading frame. Hence, changes in the molecular
weight of the RNA fragments can be observed.
The MassARRAY system by Sequenom Inc. is currently available,
although, not for use in diagnostics. This system with iSEQ software
enables microbes, viruses and other organisms to be identied via
the transcription of targeted DNA and base-specic RNA cleavage
(Fig. 1). The main advantage of this method is that any genomic
region may be chosen for amplication.
In contrast to classic 16S rDNA sequencing, the cost is the main
benet of a MALDI-based procedure [51]. The possibility of automation and standardization, a 384-well format allowing 96 samples to
be identied at one stroke, the small amount of microbial culture
required, and even the identication of mixed samples are other
benets of this method. On the other hand, two whole working
days are required to achieve results.
4.2. MALDI MS protein proling
MALDI analysis of proteins is without doubt the most widely
used MS technique for bacteria identication. The differentiation
of bacteria using protein proling was introduced in 1994 by Cain
et al. [52], who measured the mass spectra of bacterial cell lysates.
The principle underlying this study has been conrmed by Holland
et al. [14] and Krishnamurthy et al. [15]. They have independently
proven that protein proles can be obtained not only from crude
lysates but also from whole cells, the obtained spectra allowing
Fig. 1. The SEQUENOM MassARRAY iSEQ workow.

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reliable bacteria identication up to species level. Indeed, today,

MALDI-TOF MS analysis of whole intact cells is a well-established
method even in clinical microbiology laboratories [53]. Veterinary
science [54], food and water quality monitoring [5557], and ecology [58,59] are some of the other elds in which this method has
been successfully applied.
The signals detected for intact cell measurement are mostly of
ribosomal-protein origin [60]. The bacterial ribosome consists of
three species of RNA and 52 species of protein composing more than
20% of total cell protein; that is, almost half of the mass of a growing cell is composed of ribosomes [61]. Ryzhov and Fenselau [60]
focused on 42 signals obtained in spectra from the Escherichia coli
whole cell. Twenty ve of these signals were assigned as potential
ribosomal proteins by comparison of the measured m/z with the
Swissprot/TrEMBL database. Moreover, ribosomal proteins are very
basic (approx. pI = 10.5). It is well known that protonation of basic
proteins is preferred during the MALDI ionization process [62].
Other proteins found by comparison with the database were coldshock proteins, DNA-binding proteins, and major outer-membrane
lipoprotein precursor [62].
Spectra obtained from whole cells are reproducible under
dened growth conditions. Hence, creating a database and comparing unknown samples with the database via a pattern matching
algorithm is possible. The benets of this approach include its
fast analysis times, low cost requirements, ease of use, and highthroughput. A further advantage is the possibility of automation.
The cost of one sample analysis by MALDI MS is approximately
1020 times cheaper than analysis by conventional automated
platforms [63,64].
Currently, three identication databases are available: Bruker
BioTyper, SARAMIS mainly provided by bioMrieux, and Andromas
by Andromas SAS.
The most recent version of the Bruker Biotyper MSP database
contains more than 4500 unique species. Analysis is usually performed on the benchtop Bruker MicroFlex (MALDI-TOF) mass
spectrometer and is based on the frequencies of species specic peaks present in standard mass spectra compared to the
unknown sample. Calculation of the identication score requires
four values: number of matches divided by number of peaks
in unknown spectrum, number of matches divided by number
of peaks in reference spectrum, peak weight ranging from 0
to 100 representing species specicity and the correlation factor of the matching peaks intensity. Maximally seventy most
intensive peaks are taken into account in the standard identication method. The obtained score is then converted into log
score, thus the highest score 1000 is represented by log score 3
[64].
The SARAMIS database originally developed by AnagnosTec
GmbH is available in two versions. The rst version is coupled with a
Shimadzu Axima mass spectrometer and is provided by Shimadzu
as a single product. The second re-developed and larger version
is also coupled with a Shimadzu Axima; however, it is provided
by bioMrieux as the VITEK MS system. About 3000 microorganisms are included. Identication score is in linear scale. Each peak
in the reference spectra is weighted from 0 to 40 points in accordance to species specicity and score is calculated as the sum of the
matching m/z values. Score is then expressed in percentile where
99.9% represents the sum value of 999 or higher [65]. Andromas
was developed by Andromas SAS and contains approximately 700
bacteria strains.
These systems are comparable with respect to the precision
of identication and all of them have been successfully confronted with classic biochemical tests and 16S RNA sequencing
[63,64,6669]. Nevertheless, it should be noted that the size and
precision of the default database plays a signicant role in reliable
identication.
Cherkaoui et al. [64] tested 720 isolates of microorganisms originating from human infections on the Bruker and Shimadzu MS
systems and compared the results to biochemical tests and 16S
RNA sequencing. The Bruker MS system provided high-condence
identication for 680 out of the 720 isolates (94.4%), while the Shimadzu system identied 639 isolates (88.8%). The high-condence
identication mean scores were 1.7 for the Bruker MS and 70%
for the Shimadzu MS system. Only 0.9% of the isolates were identied incorrectly by the Bruker MS system and 0.5% by the Shimadzu.
In a study by Martiny et al. [69], 986 out of 1003 routine isolates
were identied to species level using the three above-mentioned
databases. These isolates originated from respiratory tract infections, skin and soft tissue infections, urinary tract infections, blood
cultures, genital tract infections, epidemiological samples, and normally sterile body uids. The accuracy of the identication was
92.7%, 93.2%, and 83.8% for the BioTyper, Vitek MS, and Shimadzu
systems, respectively. Seventy-three isolates of anaerobes were
also tested in this work, resulting in an accuracy of identication
of 61.6% for the Biotyper and 75.3% for the Vitek MS. More results
from comparative studies are shown in Table 1.
Worse results and obtained spectra of low quality were reported
for Gram-positive bacteria [64,67,70,71]. The cell wall of Grampositive bacteria is composed of thick peptidoglycan. It is assumed
that the disruption of the cell wall during the laser ablation and
ionization process is hindered by the multi-layer nature of peptidoglycan. Therefore, MALDI is unable to ionize a sufcient number
of proteins. Different protocols have been evolved to help with
cell wall disruption and protein extraction. Nevertheless, there
are no standardized guidelines for sample preparation. A protocol
designed by Smole et al. [70] proposes enzymatic disruption; van
Veen et al. [53] used two-step extraction by 70% ethanol and 70%
formic acid; Ferroni et al. [72] extracted proteins by detergent and
triuoroacetic acid; La Scola and Raoult [73] presented two different protocols, in which both acetonitrile and triuoracetic acid was
used.
Application possibilities of MALDI are even greater. Serovar
Typhi of Salmonella enterica is distinguishable from other serovars
as reported by Kuhns et al. [74]. Serovars of Salmonella could
not be distinguished using common intact cell experiments with
the Bruker BioTyper database; however, several serovar-specic
biomarker ions can be observed in spectra, which allow the discrimination of Salmonella Typhi from other serovars. The identication
of spores is also possible, although spectra are not so rich in signals and, more often, MALDI is used to identify fungal rather than
bacterial spores. In contrast to bacterial cells, the accessibility of
spore biomarkers for MALDI is considerably lower due to spore
rigidity. Analysis requires the extraction of biomarkers by an acetonitrile/water/triuoracetic acid solution; sonication or corona
plasma discharge can also increase extraction efciency [75]. The
extracted spore proteins belong to family of small acid-soluble proteins [75,76]. The spores of Bacillus sp. are frequently examined
[75,7779]. Elhanany et al. [79] found four specic signals in spectra of Bacillus anthracis spore extract; Dickinson et al. [77] identied
unique peaks in spores of Bacillus pumilus.
The standard intact cell method requires the culturing of bacteria to obtain a pure colony; however, the most recent protocols
enable direct analysis from infected liquid samples such as blood
[90,96,97] or milk [54]. In 2010, Stevenson et al. [97] introduced the
rst protocol for the direct analysis of blood, in which bacteria were
separated from red blood cells and plasma proteins by several centrifugation steps. The pelleted bacteria were lysed, and the proteins
were extracted by 70% ethanol and 70% formic acid and analyzed
by BioTyper database. Of the 212 samples, 42 (19.8%) had a spectral score of <1.7 and could not be identied, most likely because of
an insufcient amount of bacteria in the sample. Of the remaining
170, a total of 162 samples (95.3%) were identied correctly. This

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Table 1
Identication rates of the commercially available mass spectrometry systems. Collection of data from recent works.
Mass spectromery identication system
MALDI Biotyper
MALDI VITEK MS
MALDI Saramis
MALDI Biotyper
MALDI VITEK MS
MALDI Saramis
MALDI Biotyper
MALDI Vitek MS
MALDI Biotyper
MALDI Saramis
MALDI Biotyper
MALDI VITEK MS
MALDI Biotyper
MALDI Saramis
MALDI Biotyper
MALDI Saramis
MALDI Biotyper
MALDI Biotyper
MALDI Biotyper
MALDI Biotyper
MALDI Biotyper
MALDI Biotyper
MALDI Sepsityper
MALDI Sepsityper
MALDI Sepsityper
MALDI Sepsityper
MALDI Sepsityper
MALDI Andromas
MALDI Andromas
MALDI Andromas
MALDI Saramis
MALDI Vitek MS
MALDI-resequencing
PCR-ESI-MS
PCR-ESI-MS
Identication rate [%]a at

Species level
Genus level
92.7
93.2
83.8
61.6
75.3
9.6
100
100
67.2
49.0
72.5
80.0
83.4
65.9
51.0
61.0
62.8
46.0
82.0
84.5
79.0
94.3
93.8
83.0
46.3
79.7
68.4
91.4
93.1
90.7
98.0
86.7
n.a.
95.2
100
95.5
93.6
86.9
83.5
78.0
9.6
100
100
67.2
60.7
97.0
93.0
94.9
83.8
61.0
71.0
93.2
74.0
93.0
96.4
96.9
93.8
98.1
68.4
87.2
78.0
93.8
n.a.
99.7
n.a.
94.9
97.7
96.5
n.a.
No. of samples
Microorganisms
Ref.
986
routine isolates
[69]
anaerobes
[69]
132
streptococci
[80]
290
anaerobes
[67]
200
non-fermenting G bacteria
[66]
296
routine isolates
[81]
anaerobes
[82]
lactobacilli
streptococci
G routine isolates
routine isolates
anaerobes
blood culture isolates
G+ cocci
G+ blood culture isolates
G blood culture isolates
routine isolates
G+ bacilli
routine isolates
routine isolates
Staphylococcus aureus
Staphylococcus aureus
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[89]
[90]
[90]
[91]
[92]
[92]
[68]
[93]
[94]
[51]
[88]
[95]
73
79
148
46
440
2781
152
273
32
53
285
187
59
162
2665
659
1019
767
147
273
317
n.a. not available.

a
The ratio of correctly indentied species (genera) to the total number of bacterial species (genera) tested.
procedure was commercialized by Bruker as the SepsiTyper Kit.

According to the work of Stevenson et al. [97], an average success
rate of 80% is stated in the ofcial product information material,
which agreed with recent evaluation studies [90,91,98]. The tentimes higher cost is the serious drawback of the Sepsityper kit in
comparison with in-house method presented by Martiny et al. [91].
Besides blood, the Sepsityper kit was also used for the testing
of milk samples [54]. Juliana Barreiro et al. inoculated pasteurized
and homogenized samples of whole milk with 103 108 colonyforming units (CFU) of Escherichia coli, Enterococcus faecalis and
Staphylococcus aureus per ml. Samples were processed by Sepsityper with a protocol adjusted to the milk matrix and analyzed by
the Bruker BioTyper database. The detection limit for E. faecalis and
S. aureus was 106 CFU per ml; the detection of E. coli required a level
of 107 CFU per ml.
4.3. MALDI MS identication based on lipid and
lipopolysaccharide analysis
The analysis of lipids is the major domain of methods such
as GC/MS or pyrolysis-mass spectrometry. However, the timeconsuming sample pre-treatment (hydrolysis, derivatization) and
complicated data interpretation involved in pyrolysis-GC/MS
decrease the speed of these methods; hence, soft ionization techniques have become alternative tools for lipid characterization.
Different phospholipids can be ionized by MALDI. While phosphatidylcholines, sphingomyelins, and some sterols are detectable
in positive ion mode, signals for phosphatidylinositols, phosphatidylserines and sulfates are visible in negative ion mode, and
those for phosphatidylethanolamines are visible in both. Unfortunately, the ionization is hindered by strong suppression effects,
especially in positive ion mode, and is highly dependent on the
polar head of the molecule.
The rst experiments with bacterial lipid analysis were performed by Heller et al. [99] in 1987, who measured laser desorption
mass spectra of polar lipids in the presence of KCl. They obtained
lipid mass ngerprints of E. coli and Bacillus subtilis. In 1998, Ho
and Fenselau [100] used an infra-red laser with a wavelength of
1.06 m and a bacterial suspension mixed with a liquid matrix
consisting of a cobalt or graphite particles/glycerol mixture. Peaks
observed in the spectra of the intact cells corresponded to phosphoethanolamines (PE), phosphatidylglycerols (PG) and respective
potassiated adduct ions. Two Gram-negative (Erwinia herbicola
and Escherichia coli) and two Gram-positive bacteria (Bacillus
thuringiensis and Bacillus sphaericus) were analyzed and distinguished on the basis of lipid spectra. Ishida et al. [101] introduced
UV MALDI using spotted individual bacterial colonies on a MALDI
plate as a sample. Prior to matrix deposition, 3 l of NaI solution
was added to each spot to increase the signals from lipids. Similarly to the work of Ho and Fenselau, phosphoethanolamines and
phosphoglycerols were detected. Ishida was able to differentiate
Klebsiella, Shigella, Escherichia and Salmonella species on the basis
of the masses and relative intensities of 15 specic peaks. However,
intensity is not the ideal parameter, due to problems with MALDI
repeatability. Shu et al. [102] introduced MALDI lipid ngerprinting
into bioaerosol-MALDI mass spectrometry (BAMS). They analyzed
lipid proles from Bacillus, Escherichia and Salmonella whole cell
extracts and were also able to differentiate the measured species.

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Lipids such as diglycosyldiacylglycerol (DGDG), PE, and PG were

observed in the mass spectra. More information about bioaerosolMALDI analysis is presented later.
While the above-mentioned studies were conducted in positive ion mode, Calvano et al. [103] introduced a lipid ngerprinting
method in negative ion mode for the identication of lactobacilli.
Using 1,8-bis(dimethylamino)naphthalene (DMAN), different lipid
species were detected, mainly phosphatidylglycerols and cardiolipins. Diglycosyldiacyglycerols and triglycosyldiacylglycerols were
also observed within the mass range of m/z 2002000. The presence
of specic signals enabled Lactobacillus plantarum and Lactobacillus
sanfranciscensis to be differentiated.
Several studies have been devoted to LPS and LOS analysis
in both MALDI and ESI MS [27,28,104]. Gibson et al. studied Odeacylated LOS of Salmonella typhimurium, Haemophilus inuenzae
and Haemophylus ducreyi by MALDI post-source decay (PSD) mass
spectrometry in positive and negative mode and demonstrated the
usefulness of PSD in LOS structure investigation. In-source decay
also takes place and the resulting fragments increase the complexity and unpredictability of the spectra; thus, no identication
method based on LPS or LOS mass spectrometry analysis has yet
been developed.
5. ESI MS
5.1. PCR/ESI-MS
Electrospray ionization is considered as one of the softest ionization techniques. The ESI process provides much lower sample
fragmentation; thus, accurate spectra can be obtained with the high
resolution of complementary strands up to a length of 500 bp. ESI,
however, requires a very clean sample; this means that purication
of the PCR by precipitation, microdialysis, or ion exchange resin is
needed [105].
The history of nucleic acid measurements on an ESI mass
spectrometer is similar to that of MALDI. The rst studies were published by Muddiman et al. [106] and Wunschel et al. [107] in 1996.
In both cases, PCR products of Bacillus cereus and Bacillus subtilis
rRNA fragments were detected in negative ion mode.
Recent protocols are mostly based on MLST analysis. PCR amplicons of the chosen genes are analyzed by ESI mass spectrometry,
and the base composition is determined using the exact mass of
the monoisotopic peak [95,108]. The commercial fully-automated
PLEX-ID system developed by Abbott Molecular provides results
within 46 h. This system combines broad-range PCR with multiple primers, ESI-MS, and computerized triangulation to identify
the organism in the sample. More than one organism can be
determined in complex samples such as sputum. Various human
pathogens were successfully identied, including Haemophilus
inuenzae [109], Neisseria meningitides [109], Streptococcus pyogenes [109], methicillin-resistant Staphylococcus aureus (MRSA)
[95,110], Vibrio [111], Klebsiella [112], Esherichia [113], Salmonella
[113], Shigella [113], Acinetobacter [112,114], Ehrlichia [115] etc.
In the comparative study by Kaleta et al. [88], the PCR-ESIMS method was compared with the MALDI-TOF BioTyper. Both
methods achieved a similar accuracy of microorganism identication from positive blood broths. PCR-ESI-MS achieved an accuracy
of identication of 96.7% (n = 273) at the genus level and 95.6%
at the species level, while Bruker MALDI-ToF with BioTyper 2.0
achieved respective scores of 97.1% and 94.9%. The genetic basis of
PCR makes the combination of PCR/ESI-MS advantageous, because
it additionally allows access to information about resistance to
antibiotics. This method even enables uncultivable microorganisms in the sample to be detected and identied. On the other
hand, the consumables cost per sample is a strong argument in
favor of MALDI-TOF. PCR/ESI-MS requires DNA extraction, primers,

enzymes and purication, resulting in an overall cost of between
$50 and $100 per sample, while consumables for MALDI include
only media for bacteria growth, the matrix, and the calibration
standard, with an overall cost of $3 to $7 per sample. The utilization of SepsiTyper makes MALDI even faster; however, this rapidly
increases the cost.
PCR/ESI-MS enables semi-quantitative analysis using the addition of nucleic acid of known concentration to each PCR. The
sequence similarity of the added nucleic acid and the target sample
is required. The semi-quantitative analysis is based on the comparison of the analyte peak heights and standard ones. The addition
of known nucleic acid also serves as the internal positive control
[108].
Despite PLEX-ID acquiring CE certicates (European Conformity
marking), obtained at the beginning of 2012, the distribution of the
system has ceased.
5.2. ESI MS identication based on the analysis of proteins
The identication of bacteria by ESI via protein analysis is not
as successful as by MALDI. Basic ESI workow includes sample
preparation. The measurement of intact cells usually leads to needle clogging, and the direct injection of crude extracts provides
extremely complex spectra. A typical cell lysate consists of a large
number of biomolecules and cellular debris, which signicantly
reduce sensitivity and make reasonable analysis impossible. The
analysis of intact cells by Goodacre et al. [11] and that of crude
cell lysates described by Vaidyanathan et al. [116] offered complex
spectra, in which lipid signals dominated and revealed the necessity
for sample purication. Liquid chromatography [117119] or dialysis [10,24] are commonly used for proper cell lysate processing.
Electrospray ionization offers effective tandem mass spectrometry, which is the basis of the modern approach, rst proposed
by Demirev et al. [120]. As described by Dworzanski et al. [117],
information about peptide amino acid sequences can be used for
relevant bacteria identication. This method is based on whole
bacteria proteome digestion coupled with liquid chromatography
and tandem mass spectrometry (LC-MS/MS). Obtained data are
matched afterwards with the proteome database derived from
the genomes of microorganisms. Microorganisms with a known
genome can be identied straightforwardly while multivariate statistical methods can be used to characterize bacteria with unknown
genome [121,122]. The classication is based on the similarity of
the peptide-sequence of the unknown sample and known bacteria in established taxons. The similarity is determined by principal
component analysis or cluster analysis.
Selective proteotypic-peptide analysis (SPA) is a method which
enables identication of bacteria in mixed cultures. The tryptic
digest of a sample protein extract is analyzed by LC-MS/MS [123]
or CE-MS/MS [124] and specic peptide markers are monitored
at expected elution time windows. The peptide retention time
is calculated on the basis of their sequence. Afterwards, all tandem spectra are matched against a protein database to identify
proteins and their origin. Information about accurate mass and elution time increases the condence of peptide identication and
moreover enables the components of the bacterial mixture to be
distinguished and identied.
5.3. ESI MS identication based on lipid and LPS analysis
A large number of isobaric lipid ions required tandem MS
for further characterization. Better fragmentation efciency favors
electrospray ionization over MALDI. In 1995, Smith et al. [5]
observed different phospholipid fragmentation spectra of G+ and
G bacteria during measurements of their lipid extracts. Several

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other studies between 1996 and 2000 [125128] described the

characterization of various bacteria; however, lipid analysis has
recently been used for the detection of microbial presence, as it
is suppressed by protein prole measurements. Zink et al. used
intact polar phospholipids as microbial life markers in marine
sediments [129]; Rossel et al. [130] demonstrated that the quantitative analysis of phospholipids discriminated between archeal
and bacterial biomass.
White et al. [37] developed HPLC/ESI tandem mass spectrometry for the analysis of neutral and polar lipids, lipopolysaccharides
of Gram-negative bacteria, and 2,6-dipicolinic acid, a specic spore
biomarker. The limit of detection was 100 fg of 2,6-dipicolinic acid
per l (i.e., 2.3 103 spores of B. subtilis). The linearity of the calibration curve was over three degrees of magnitude. The detection limit
of the phosphatidylglycerol assay was as much as 90 amol l1
using multiple reaction monitoring (MRM).
Besides experiments with MALDI [27], Gibson et al. [28] have
also used electrospray ionization. In their study, the structural heterogeneity of LOS from Haemophilus and Neisseria and LPS from
Salmonella were investigated. ESI mass spectrometry in combination with capillary electrophoresis was used for the detailed
investigation of Moraxella and Haemophilus lipooligosaccharides
[131,132]. Attention was also to the characterization of Lipid A
obtained from various bacteria, including Shigella exneri [133],
Haemophilus inuenzae [134], Escherichia coli [135,136], Pseudomonas aeruginosa [137] and Agrobacterium tumefaciens [135]. It
is worth mentioning that no commercial method for the identication of bacteria based on the MS analysis of lipid A, LPS or LOS mass
spectra has yet been introduced.
6. Ambient techniques
6.1. DESI and DART MS
Desorption electrospray ionization (DESI) has been used to
study bacterial biomarkers several times since its invention in
2004 [18]. The mechanism of DESI is ideally designed for the
analysis of intact bacteria. In 2007, Song et al. [19] measured
the spectra of intact cells of Escherichia coli (3 strains) and
Salmonella typhimurium (2 strains) in the mass range up to m/z
of 1600, where lipids were chiey identied. Species and even
individual strains were successfully differentiated using principal component analysis (PCA) of the obtained results. A few
months later, an extended study of seven different bacteria
(Escherichia coli, Salmonella typhimurium, Bordetella bronchiseptica, Staphylococcus aureus, Enterococcus sp., Bacillus thuringiensis,
Bacillus subtilis) was performed by Meetani et al. [138]. Signals detected in the low mass range (m/z 50500) enabled the
ne classication of all measured bacteria by PCA. The characterization of Bacillus subtilis from a still growing biolm was
demonstrated in 2009. The characterization was based on the
signals for surfactin, the antibiotic lipopeptide excreted into the
growth medium. While previous works used biolms or cells suspended in water, Zhang et al. [139] resuspended cells in 70%
ethanol. Spectra obtained from 3 l of this suspension containing approximately 3000 cfu provided signals for various lipids and
lipopeptides, including PG, PE and lysophospholipids, in the mass
range up to m/z of 1000. Using PCA, the authors reliably identied Bacillus subtilis, Escherichia coli, Staphylococcus aureus and
Salmonella. Even four different strains of Salmonella were well separated.
A direct analysis in real time (DART) method for the rapid
identication of microorganisms has been tested in a proof-ofprinciple study by Pierce et al. [140]. This work was focused on the
fatty acid methyl ester (FAME) composition of bacteria routinely
used for the identication of clinically important pathogens. The

arrangement of the DART mass spectrometer enables in situ esterication of bacterial fatty acids in the space between the ion
source exit and the inlet of the mass analyzer. FAMEs were generated from the whole cell bacteria suspensions of 5 different
species. Signals for fatty acids from C8:1 to C24:0 were registered and several species-specic fatty acids were observed, e.g.,
C19:1 for E. coli, C11:1 for S. pyogenes and C15:1 for Coxiella
brunetii; however, the authors assumed that sophisticated statistical tools would be needed for relevant identications. Although,
the results of this study were promising, no further results have
appeared.
6.2. SIFT MS
Selected ion ow tube mass spectrometry (SIFT MS) is a technique for the measurement of concentrations of trace gases and
VOCs under ambient conditions. In contrast to GCMS, the SIFT technique enables real-time analysis of samples such as air, breath
or headspace of bacterial cultures. In 1996, Smith and Spanel
[20] introduced a novel method for diagnosing Helicobacter pylori
infection, based on the measurement of the ammonia level in
breath after an oral dose of urea. They observed a signicant
increase in ammonia 2040 min after administration to a person
infected with Helicobacter against the uninfected control. Studies of breath by Enderby et al. [141] and Shestivska et al. [142]
suggest the levels of hydrogen cyanide (HCN) and methyl thiocyanate as possible diagnostic markers of Pseudomonas aeruginosa
infection of the lungs, which is major cause of mortality in
patients with cystic brosis. Other applications have also been
introduced, e.g., lactose intolerance diagnosis; thus. it appears that
the analysis of breath is a non-invasive diagnostic method with
a bright future. Nevertheless, it is limited by the unpredictability
of the human body, because basic levels of analytes in exhaled
air are individual and may be inuenced or overlaid by other
factors.
The analysis of bacterial culture headspace is another good
opportunity for the application of SIFT. It has been proven that
volatile metabolites can be analyzed in the headspace of liquid
bacterial cultures [143,144]. Allardyce et al. took advantage of this
knowledge in identifying bacteremia from blood culture medium.
They measured a set of 14 VOC levels for ve bacterial strains cultured in blood culture bottles and they selected a list of nine VOCs
for the detection and identication of species. SIFT analysis was
able to detect bacterial growth in 24 h cultures, and, in selected
ion mode (SIM), even in 6 h cultures. It is worth noting that the
conventional blood culture method takes at least 2 days to detect
bacteria. In the following experiment by Scotter et al. [145], SIFT-MS
analysis was compared with an automated blood culture system.
Blood samples from healthy patients were inoculated with 5 or
100 cfu of bacteremia-causing organisms. Positive results by SIFT
were observed for 88.3% of samples after 8 h of growth in blood
culture bottles, and 96.6% after 24 h, while the automated system
did not give positive notication of growth in less than 12 h, usually
in 1424 h. Moreover, SIFT enabled bacteria species to be identied. A similar approach was applied for studying microbial growth
in urine [146]. Sterile urine from healthy males was infected with
one of the seven bacteria strains or with Candida albicans and incubated for 6 h at 37 C. The concentrations of twenty-one VOCs were
measured and the resulting levels conrmed some ndings in previous studies [147149], for example the signicant production of
ammonia by S. aureus [147]. Although it was not possible to fully
differentiate between eight used species, the potential of the SIFT
technique for rapid microbe identication in urine samples was
demonstrated.

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Fig. 2. Bioaerosol mass spectrometer as presented by Fergenson et al. [150].
7. Bioaerosol mass spectrometry

Recently, several groups have attempted to identify bacteria by
mass spectrometry at the level of single cells. Bioaerosol mass spectrometry (BAMS) is the result, in which particles can be drawn
directly into the mass spectrometer to be ionized, or they can be
covered by a matrix and dried prior to sampling.
The rst successful discrimination of species was performed by
Fergenson et al. [150], who was able to distinguish spores of Bacillus
atrophaeus and Bacillus thuringiensis in various background mixtures. The novel bioaerosol-LDI/TOF mass spectrometer was used.
Dried spores were introduced into the apparatus directly from the
environment through a nozzle and two scattering light detectors
were used for the determination of spore size and velocity. A laser
pulse at 266 nm was red to ionize analytes from the particles and
both positive and negative spectra were simultaneously collected
by two opposite direction TOF analyzers (Fig. 2).
BAMS analysis of Bacillus spores was also performed by Czerwieniecz et al. [151], Srivastava et al. [152] and Steele et al. [153].
They conrmed Fergensons results and the functionality of the
device. Tobias et al. [154] demonstrated BAMS capacity to follow
morphological and biochemical changes during the sporulation
process of B. atrophaeus cells. Growing signals for DPA adducts
were detected in negative mass spectra, which corresponds with
the endospore formation. Other work by Tobias et al. [155] was
targeted on Mycobacterium tuberculosis airborne particles. Specic
biomarkers were found in spectra for M. tuberculosis, M. smegmatis,
B. atrophaeus and B. cereus allowing particle identication.
BAMS without a matrix allows quick real-time measurements;
however, it is not possible to ionize larger molecules and biopolymers using this method. The above-mentioned works did not
exceed m/z of 400. Hence, particles coated by a matrix may be
measured instead. To maintain the possibility of real-time analysis, Stowers et al. [21] developed a unique method for matrix
deposition in 2004. The aerosol from particles was mixed with
matrix vapors in the ow cell, then the mixture was cooled and
the matrix condensed onto the aerosol. Afterwards, matrix coated
particles were pulled into the ion source and red by laser pulse.
A single peak at 1224 Da was detected in mass spectra of B. subtilis
spores coated by the matrix and it was identied as peptidoglycan.
Various matrices have also been tested. In Stowerss work, sinapinic acid, picolinic acid and 3-nitrobenzyl alcohol were used. In the
following study by van Wuijckhuijse et al. [156], a mass range up

to 20 kDa was scanned using ferulic acid as a matrix. Additional
peaks for Bacillus spores were found, two of them assigned as
small acid soluble proteins (SASP). Besides Bacillus spores, cells of
Erwinia herbicola [157] and E. coli [158] were analyzed; however,
there have been no recent developments in intact cell or spore
bioaerosol mass spectrometry of any signicance.
8. Insights into the future
The ability of mass spectrometry to identify bacteria is another
proof of its versatility; hence, microbiology has become another
eld in which mass spectrometry is used. Mass spectrometry is able
to target a wide range of biomarkers through a variety of ionization techniques. Currently, proteomic proling is commercially the
most successful method. It can be assumed that MS has the potential to completely displace classic biochemical methods in clinical
work in the near future. This process has already begun; however,
not only clinical microbiology is involved. The quality control of
food, air and water, environmental studies, defense against bioterrorism are all elds where mass spectrometry has already been
applied and will be applied on a larger scale in the future.
Despite the demise of the iSeq method, the improved analysis
of DNA and RNA could provide a denite method of identication,
which would also include information about antibiotic resistance.
Current progress in single cell mass spectrometry enables only
single cells to be analyzed, which can be considered a very good
starting point for the measurement of bacteria directly from blood
without cultivation. Alternatively, cell enrichment could help with
sample preparation. The branch of metabolomics, which began to
develop just a few years ago, shows great promise. Finally, SIFT and
GC/MS techniques have yielded their rst results and have demonstrated great potential in helping with the direct identication of
lung or stomach infections.
Acknowledgements
This work was supported by Czech Science Foundation
(P503/10/0664) and by specic university research (MSMT No
21/2012). Matthew Nicholls is greatly acknowledged for proofreading of the manuscript.

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MASPEC-14910; No. of Pages 13

Contents lists available at SciVerse ScienceDirect

International Journal of Mass Spectrometry

Identication of bacteria using mass spectrometry techniques

Introduction and short historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E-mail address: krasnyl@vscht.cz (L. Krsny).

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

1. Introduction and short historical overview

for the identication of bacteria, spores [12], and viral particles

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

ESI; hence, we can consider nucleic acid as a measurable biomarker

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

denaturation, the phosphate backbone was cleaved and the nal

Fig. 1. The SEQUENOM MassARRAY iSEQ workow.

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

reliable bacteria identication up to species level. Indeed, today,

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

Identication rate [%]a at

n.a. not available.

procedure was commercialized by Bruker as the SepsiTyper Kit.

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

Lipids such as diglycosyldiacylglycerol (DGDG), PE, and PG were

favor of MALDI-TOF. PCR/ESI-MS requires DNA extraction, primers,

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

other studies between 1996 and 2000 [125128] described the

used for the identication of clinically important pathogens. The

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

Fig. 2. Bioaerosol mass spectrometer as presented by Fergenson et al. [150].

7. Bioaerosol mass spectrometry

following study by van Wuijckhuijse et al. [156], a mass range up

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

ionization-time of ight mass spectrometry, Journal of Clinical Microbiology

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures,

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

ion-trap mass spectrometry, Journal of Mass Spectrometry 39 (2004)

V. Shestivska, A. Nemec, P. Drevnek, K. Sovov, K. Dryahina, P. Span

T. Wang, D. Smith, P. Span

[148] N. Hayward, T. Jeavons, A. Nicholson, A. Thornton, Development of specic

et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.04.016

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