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RSC Advances
REVIEW
Bacterial identification: from the agar plate to the mass
spectrometer
Cite this: RSC Advances, 2013, 3, 994
Patricia Aparecida Campos Braga,a Alessandra Tata,a Vanessa Gonçalves dos Santos,a
Juliana Regina Barreiro,b Nicolas Vilczaki Schwab,a Marcos Veiga dos Santos,b
Marcos Nogueira Eberlina and Christina Ramires Ferreira*a
For more than a century, bacteria and fungi have been identified by isolation in culture followed by
enzymatic reactions and morphological analyses. The identification of environmental microorganisms,
however, remains a challenge because biochemical and staining protocols for bacteria identification are
tedious, usually stepwise, can be long (days) and are prone to errors. Molecular techniques based on DNA
amplification and/or sequencing provide more secure molecular identification of specific bacteria, but
identification based on mass spectrometry (MS), mainly on MALDI-MS, has been shown to be an
alternative accurate and fast method able to identify unknown bacteria on the genus, species and even
subspecies level based profiles of proteins and peptides derived from whole bacterial cells. Breakthroughs
such as non-culture-based identification of bacteria from biological fluids and MS detection of antibiotic
Received 6th September 2012,
Accepted 24th October 2012
resistance have recently been reported. This review provides an overview of the traditional bacterial and
fungal identification workflow and discusses the recent introduction of MS as a powerful tool for the
identification of microorganisms. Principles and applications of MS, followed by the use of high-quality
DOI: 10.1039/c2ra22063f
databases with dedicated algorithms, are discussed for routine microbial diagnostics, mainly in human
www.rsc.org/advances
clinical settings and in veterinary medicine.
Introduction
If a microbiologist working in the first decades of the 20th
century stepped into a current microbiology laboratory, it is
likely that he would not have a hard time feeling at home. In
fact, most methods for bacterial isolation and identification
have remained unchanged and are still based on the use of
specific culture media for isolation and on classical morphological, staining and biochemical enzymatic assays for microorganism identification.1
Improved assays, high-throughput microbiological analysis
with automated systems, molecular technologies based on
DNA amplification/sequencing for microorganism identification and the detection of antimicrobial resistance are
available.2,3 However, challenges such as the urgent identification of microorganisms and their antibiotic resistances in
septicemic patients and in infants, the occurrence of genetic
rearrangements in microorganisms that alter their behavior in
enzymatic assays, and the need to identify less frequent or rare
microorganisms are difficult to tackle with classical microa
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas, Campinas, 13083-970, SP, Brazil. E-mail: [email protected];
Fax: +55 19-3521 3073; Tel: +55 19-35213049
b
University of São Paulo, School of Veterinary Medicine and Animal Science,
Pirassununga, 13635-900, São Paulo, Brazil. E-mail: [email protected];
Fax: +55 19-5616215; Tel: +55 19-3565 4240
994 | RSC Adv., 2013, 3, 994–1008
biological approaches and may lead to incorrect identifications.
In response to these challenges, a paradigm break for
microbiology has been the introduction of mass spectrometry
(MS)-based microorganism identification. This strategy
emerged after the development of electrospray ionization
(ESI)4 and matrix-assisted laser desorption/ionization
(MALDI)5 at the end of the 1980s. Since the early 1990s, more
than 13 000 Pubmed indexed manuscripts on microbiology
associated with MS have been published. However, microorganism identification by MS is not only performed for
research purposes. Dedicated instruments equipped with
automated database search functions for almost real-time
microorganism identification are being installed in hospitals,
clinical institutes and commercial settings, mainly in Europe.
In the United States, the Food and Drug Administration has
still not approved any MALDI-MS system for organism
identification, which limits the widespread implementation
of this approach in this country.6
This review provides a general overview of classical
microbiological approaches and the MS ionization strategies
that have been mostly applied in microbiology. The most
recent achievements in MS-based microorganism identification, such as the identification of uncommon pathogens and
non-fermenting bacteria, non-culture identification of bacteria
in biological fluids, identification of antibiotic resistance and
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mixed culture analysis, are also described and discussed.
Finally, real-world perspectives on moving bacterial identification from the agar plate to the mass spectrometer are
presented.
Traditional bacterial and fungal identification based on
bacterial isolation by culture and enzymatic assays
There are several methods for routine microbial identification
in clinical and research microbiology laboratories, but microbiologists have continuously pursued efficient systems for
microorganism identification. These methods are mainly
based on morphological and biochemical characteristics of
bacterial colonies. For example, bacterial colony morphology
can be evaluated under defined growth conditions to show
hemolytic capacity, by Gram staining (Fig. 1a–b), and by
examining growth performance on selective media that allow
only specific bacteria to multiply, the potential to ferment
sugars, specific biochemical reactions (Fig. 1c), metabolic
characteristics, antigenic and pathogenic capacity, and antibiotic susceptibility.7
When determining the characteristics of a microorganism to
provide its identification, a pure culture population of
identical cells is needed; this means that these cells originate
from the same parental cell. However, microorganisms in
nature are typically found in mixed cultures with many
different species occupying the same environment.
Therefore, in a microbiological laboratory, the first step in
the microorganism identification workflow is the isolation of
the various species contained in a specimen. For this purpose,
there is an extensive list of available commercial tools, and the
chosen strategy depends on numerous factors. The main
considerations are the source of the sample, the species that
are expected to be present and the nutritional needs of these
microorganisms. For example, the isolation medium may
contain specific compounds that inhibit or prevent general
Fig. 1 (a) Staphylococcus aureus (left) and Staphylococcus spp. (right) on blood
agar. Note the hemolysis caused by S. aureus; (b) Bright light microscopy
analysis: Gram-positive stained cocci; (c) Biochemical tests used to identify
bacteria usually include color codes.
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microbial growth yet simultaneously are appropriate for
growing the species that are determined to be present
(Fig. 1a).8 After isolation of the microorganism, phenotypic
characteristics such as enzymatic profiles, sensitivity to
antibiotics and chromatographic analysis of fatty acids can
be performed to characterize the strain.9
Microscopy is frequently used to characterize microorganisms (Fig. 1b), and there are two main microscopic techniques:
light microscopy and electron microscopy. Light microscopy is
routine in the microbiology setting and can be performed with
bright field, dark field, fluorescence, and phase control.
Constant development and refinement of the techniques used
for optical microscopy allow one to perform additional
specialized functions, such as evaluating biochemical processes occurring within living cells.10
For close to a century, bacterial identification relied on the
interpretation of a skilled microbiologist using a microscope,
specific media and antibiotics, but technological improvements over the past 50 years have allowed for both automation
and simplification of analysis.2
Automated testing for bacterial identification in laboratories
has become necessary as microbiologists attempt to answer
higher demands and the need for fast diagnoses, such as in
cases of patients with septicemia or neonatal infections.
Automation also allows increasing numbers of specimens to
be studied.11 Usually, the microorganism cell count can be
determined in real time by incubators equipped with a system
to measure the light absorption of liquid cultures because
turbidity can be related to the number of cells. Another
strategy of an automated laboratory system is based on testing
the susceptibility of bacteria to a large number of antibiotics.10
Advances in the molecular biology field in the early 1980s
resulted in novel approaches for microbial identification and
characterization based on polymerase chain reaction (PCR) to
amplify specific gene sequences of bacteria. PCR allows in vitro
amplification of specific DNA or RNA sequences, the latter
being performed following the synthesis of complementary
deoxyribonucleic acid (cDNA). PCR is a technique with high
specificity and applicability, and hundreds of methods have
been described.12 The most important feature of PCR is its
ability to exponentially amplify copies of DNA from small
amounts of material. For PCR analysis, nucleic acids must be
extracted, and several protocols using specific reagents and
different strategies have been described for this purpose.10
Though time-consuming, costly and difficult in the case of
multiplex assays, PCR-based bacterial identification is now a
common and often indispensable technique used in medical
and biological research for a variety of other applications,
including forensic DNA typing, clinical diagnosis, DNA
amplification for cloning or sequencing, paternity testing,
construction of DNA libraries and detection of mutations.12–14
For example, Campylobacter, which is the most common cause
of acute bacterial gastroenteritis in the world, may be detected
in pork samples using this approach. Sixty Campylobacter
strains isolated from porcine rectal swabs and from different
areas in a pork processing plant were shown by PCR analysis to
be mostly C. coli (86.9%) and C. jejuni.(13.1%).15
PCR is less prone to errors compared to traditional methods
of identifying microbes that rely exclusively on phenotypic
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features and some the morphological characteristics of the
organism to identify the strain, such as enzyme profiles,
antibiotic sensitivity profiles, and chromatographic analyses of
fatty acids.9 However, the expanding and impressive capabilities of MS-based microorganism identification are causing a
revolution in the field of microbiology.
Even though bacterial identification represents the largest
interest in routine microbiology, invasive fungal disease plays
an important role in the morbidity and mortality of immunocompromised patients.16 Approximately 60 years ago,
Wickerham described a broth method that is specific for
metabolic assimilation and fermentation testing of yeasts.17,18
Assimilation tests determine the ability to use various
substrates as the sole source of carbon (e.g., sucrose) or
nitrogen (e.g., KNO3). This method is still considered a
powerful tool and was used to characterize and determine
the taxonomy of yeasts. Most Wickerham media are not
commercially available and are produced in the laboratory.
This process is usually long and arduous and is employed only
by a few laboratories because basal media and various
substrates need to be prepared, sterilized, and dispensed
prior to inoculation. Since many types of yeast can ‘‘carry-over’’
nutrients from the isolation medium, one must run negative
controls for each test type and organism. Metabolic assimilation tests are read for turbidity, fermentation and gas
production for up to four weeks. Due to their time-consuming
nature, these ‘‘gold standard’’ assays have been replaced by
more practical methods that are currently available.
The first technical progress to improve the speed and
sensitivity of yeast identification from fungal blood cultures
and histological practices was the development of highly
sensitive and specific molecular techniques, including PCR. At
the molecular level, genetic sequence variation offers an
alternative to culturing for the detection and identification of
fungi. For example, ribosomal genes have conserved sequence
regions that are ideal for primer targeting as well as regions of
variability that are useful for species identification. DNA
amplification techniques, with subsequent species-specific
probing of the amplicons or PCR-enzyme immunoassay, have
also been introduced to overcome the problems of sensitivity,
specificity, and delay that are encountered with conventional
methodology. These methods have already shown great
promise in the field of diagnostics. The use of species-specific
probes, however, is not always an efficient approach in
mycology, given the large number of potentially pathogenic
fungi.19,20
In parallel with the development of molecular methods,
there has also been an emphasis on improving commercial
kits based on substrate utilization or hydrolysis. The results
are determined by increased turbidity, the generation of
colored products, or the detection of fluorescent products.
Some colored products require the addition of reagents to
reveal the color, while others are self-revealing. These kits
enable presumptive identification of the most important
etiologic agents of yeast infections. Another focus of these
rapid tests has been to screen for species that are commonly
associated with resistance to antifungal compounds.21 Some
kits are read manually, while others are read automatically.
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Many systems employ the aid of computer algorithms for rapid
and reproducible data analysis.21
Merits of mass spectrometry in microorganism identification:
time-saving and reliablity
ELECTROSPRAY-BASED BACTERIAL IDENTIFICATION (PCR-ESI-QTOF).
Scientific advances in MS, such as the development of the
‘‘soft’’ ionization techniques MALDI5 and ESI,4 have allowed
the ionization, detection and characterization of large intact
biomolecules. An improved understanding of the limitations
associated with MS analysis of nucleic acids led to the
ionization of intact PCR products by ESI.4,22 This capability
resulted in the development of MS analysis of nucleic acids for
microorganism identification, which was first described in
2005 by Hofstadler and co-workers.23–26
This approach, previously termed TIGER (Triangulation
Identification for Genetic Evaluation of Risks) or PCR-ESIQTOF-MS, uses broad-range primers for PCR analysis to
amplify products from diverse organisms, such as viruses,
bacteria, fungi and protozoa within a taxonomic group, that
are present in samples combined with PCR product calculations using MS (Fig. 2).
The Ibis T5000 Universal Biosensor is an automated platform
for pathogen identification that is based on TIGER technology.
In its commercial form, Ibis T5000 is capable of identifying and
strain typing a broad range of pathogens in a blinded panel
from human or animal samples.28–30 Because the Ibis T5000
provides digital signatures of identified microorganisms, this
technology allows the collection and dissemination of epidemiological information in real-time.31–33 A major advantage of
this methodology is the ability to characterize an organism
without prior knowledge by the instrument operator as well as
rapid sample preparation. Since rapid pathogen identification
significantly reduces rates of patient mortality, technologies for
the correct and timely diagnosis of bloodstream infections are
urgently needed. PCR-ESI-MS has been used as a new strategy
for detecting bloodstream infections and has provided high
concordance with results from standard methods, particularly
at the genus level. The results from this technique can also be
obtained in five to six hours, whereas culture and biochemical
characterization techniques typically require one to five days for
confirmation of microbial identification.34,35
Rapid detection and identification of Ehrlichia species, a
tick-borne pathogen responsible for causing Ehrlichiosis
disease, was performed by PCR-ESI-MS directly from crude
blood samples without microorganism culture. The results
from an enzyme immunoassay that was also performed
showed 100% agreement with the PCR-ESI-MS results.36 The
detection of bloodstream infections can be biased, as blood
cultures are reported to be negative in more than 50% of the
cases where bacteria are believed to exist. This approach
allows the detection and identification of both culturable and
unculturable organisms by the same method, in addition to
the identification of mixed populations of bacteria. The PCRESI-MS platform not only identifies organisms that are present
in a clinical sample but is also capable of providing
information about the strain type.37 This approach has been
applied for the genotypic characterization of S. aureus isolates
and to detect the presence or absence of genetic elements that
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Fig. 2 An overview schematic of the traditional bacterial identification workflow and the recent integration of mass spectrometry techniques. Figure adapted from
Drake et al., 201127 with permission from John Wiley and Sons.
encode potential virulence factors and antibiotic resistance
elements. PCR-ESI-MS can also distinguish S. aureus from
other coagulase-negative staphylococci (CoNS) isolates.38,39
Members of the genus Acinetobacter, which are aerobic
Gram-negative organisms that are widely distributed in soil
and water in the natural environment and are important
nosocomial (hospital-acquired) pathogens, were isolated from
infected soldiers and civilians involved in an outbreak in the
military health care system associated with the conflict in Iraq.
Through the PCR-ESI-MS technique, it was possible to
distinguish at least 16 Acinetobacter species, and the
genotyping of A. baumannii showed a genetic relationship
between endemic European isolates and many of the isolates
found in patients and in military hospitals, indicating that this
approach provides a better understanding of the origins of
these infections and will improve infection control and
prevention measures.40
The application of this technique has also been described
for the genotyping of pathogens related to food-borne illnesses, and it proved to be highly effective in differentiating C.
jejuni isolates in a panel of 50 Campylobacter isolates as well as
determining the correct classification of C. coli instead of C.
jejuni (Fig. 3).41
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The Ibis 5000 platform has also been applied in aquatic
environmental analysis to identify different Vibrio species
directly from natural aquatic samples. From 278 total water
samples that were screened, nine different Vibrio species were
detected, and 41% of samples were positive for V. cholerae, a
pathogen responsible for cholera disease. The results also
indicated that V. mimicus could be correctly identified and
distinguished from the close species V. Cholerae.42 PCR-ESIMS has been described as a high-throughput method to
simultaneously identify, based on genotype, a number of
bacterial species from complex mixtures in respiratory
samples taken from military recruits during respiratory
disease outbreaks and follow up surveillance at several
military training facilities.43 PCR coupled to ESI-MS has also
been described as a powerful tool for detecting other
microorganisms such as viruses.44–50
Use of ambient desorption/ionization techniques for direct
bacterial identification
Ambient desorption/ionization describes a new set of MS
techniques that are performed in an open atmosphere directly
on samples in their natural environments or matrices or by
using auxiliary surfaces. Ambient MS has greatly simplified
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Fig. 3 Deconvoluted ESI-TOF mass spectra of PCR amplicons of the tkt
housekeeping genes from six different C. jejuni strains. Both the forward (e) and
the reverse (#) strands of the PCR amplicons from each strain are clearly evident
in the spectra (e.g., for strain RM4197, the forward strand is A49, G22, C26 and
T45 and the reverse strand is A45, G26, C22 and T49). As can be observed in the
stacked spectra, differences due to variations in the sequence (and thus the base
composition) are readily discernible. Note that any mass differences resulting
from changes in the number of guanosines are enhanced by the use of 13C
guanosine (G*). The T5000 software automatically determines the base
composition of each strain and provides a strain association by using a set of
eight primer pairs. Reproduced from ref. 41 with permission from the American
Society for Microbiology.
and increased the speed of MS analysis, and especially after
2004, this approach has experienced a large trend towards realworld rapid chemical analysis of untreated samples in
ambient conditions.51,52 Recently, several new high-throughput ambient desorption/ionization methods have been
reported. One of the most studied ambient ionization methods
is desorption electrospray ionization (DESI), which was
introduced by Cooks and co-workers in 2004.53 DESI involves
spraying untreated samples with ionized solvent droplets from
a pneumatically-assisted electrospray. Desorption and ionization of analytes occurs through interactions of the charged
droplets with the surface from which they pick up organic
molecules, and they are delivered as desolvated ions into the
mass spectrometer (Fig. 4a).
Another well-explored ambient ionization technique is the
direct analysis in real time (DART) method, first described by
Cody and co-workers in 200554 (Fig. 4b). Due to the increasing
importance of rapid identification of bacteria for food,
biosafety and medical analysis, ambient desorption/ionization
methods are of substantial interest.55 Both the DESI and DART
techniques allow direct and rapid analysis of condensed phase
samples without any sample preparation or the need to
introduce the samples into the vacuum system of the mass
spectrometer. DESI and DART have been widely utilized in
many different applications, including bacterial analysis and
identification.51 The most important characteristic of DESI
and DART-MS approaches is the absence of sample preparation, so the real-time identification of microorganisms by
these approaches appears to be feasible.56,57
Cooks and co-workers were the first to recognize the
potential of these ambient desorption/ionization methods for
microorganism identification when they performed DESI-MS
998 | RSC Adv., 2013, 3, 994–1008
Fig. 4 Schematic of ambient MS techniques used for direct microorganism
analysis. These approaches usually make use of untreated samples, which are
desorbed and ionized from surfaces or solutions under normal atmospheric
conditions. (a) For DESI, an ionized solvent is pneumatically sprayed onto the
sample, forming a thin film in which the sample molecules are dissolved.
Secondary droplets containing ionized analytes are then delivered in the
direction of the mass spectrometer inlet. (b) DART uses an electrical potential
applied to a gas with a high ionization potential (typically nitrogen or helium) to
form a plasma of excited-state atoms and ions, which desorbs low-molecular
weight molecules from the surface of a sample. (c) In LTP-MS, there is no need
for any solvent. The ion source consists of a glass tube with an internal
grounded electrode centered axially and an outer electrode of copper tape
surrounding the outside of the glass tube. An alternating voltage is applied to
the outer electrode with the center electrode grounded to generate the
dielectric barrier discharge. The discharge AC voltage is provided by a custombuilt power supply with total power consumption below 3W. Helium is used as
the discharge gas, and it is fed through the glass tube to facilitate the discharge
to direct the plasma onto the sample surface and to transport analyte ions to
the mass spectrometer.
on freshly harvested cells from untreated E. coli and
Pseudomonas aeruginosa samples deposited on a polytetrafluoroethylene (PTFE) target. The characteristic DESI mass
spectra for each microorganism that was analyzed demon-
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strated the potential of the technique for microbiological
application.58
The similarity of the spectra for different samples of the
same culture or for E. coli different cultures was evaluated and
indicated that DESI-MS for microorganisms was reproducible.
The characteristic constituents of bacteria were observed
without chemical derivatization; in particular, acylium ions
of fatty acids were observed directly, not as the usual methyl
esters. Principal component analysis (PCA) was performed on
the DESI-MS data to differentiate the bacteria studied into two
well-separated groups that could be identified based on the
first principal component (PC1), which corresponds to the
differentiation between E. coli and S. typhimurium. Because the
mass spectra were recorded directly from freshly harvested
microorganisms, and no chemical reagent or other processing
step was used to disrupt the cells before MS analysis, any
variations in the final spectra associated with sample
pretreatment were eliminated in collaboration with the
separation. Although only two different species were evaluated, this study demonstrated the possibility of performing in
situ identification using DESI-MS, including sub-species
differentiation of microbiological agents.56
Meetani et al. (2007) also applied DESI-MS to bacterial
identification for a larger number of different samples. In this
study, seven different bacterial species were evaluated on the
basis of their spectra profiles. Incubated bacterial cells were
transferred from agar plates, washed to remove media
components, applied on glass slides and directly subjected
to DESI-MS analysis. The mass range from 50–500 was used,
and the observed ions in the mass spectra revealed the
presence of free fatty acids, such as palmitic acid (16:0) at m/z
257 for the protonated molecule and m/z 279 for the sodium
adduct. Further comparisons of the mass spectra with lowmass matrix-assisted laser/desorption ionization (MALDI)
mass spectra of bacteria did not show common ion signals,
indicating the likely complementary nature of DESI-MS and
MALDI-MS for whole-bacteria identification. The mass spectra
of negative ions were also evaluated, showing a greater
number of detected ions than their counterpart positive ion
spectra throughout the measured m/z range.57
In vivo recognition of bacteria was evaluated in another
study from the Cooks group that examined direct profiles of
intact biofilms of Bacillus subtilis by DESI-MS, noting that the
biofilms were still viable after the experiment. The authors
reported that the DESI plume primarily desorbs materials
from the bacterial cell envelopes outer layers together with
excreted metabolites. Bacteria with a rigid cell wall can
withstand the impinging sprayed droplets. This assumption
was corroborated by experimental results from Gram-negative
and Gram-positive bacteria. The outermost layer of Gramnegative bacteria is the cell outer membrane, while the
outermost layer of Gram-positive bacteria is a thicker cell
wall. For Gram-negative species, the outer membrane is
relatively easy to break, and its major phospholipids (PL) can
be readily ionized, which leads to PL dominance in the
resulting DESI mass spectra. However, the thicker cell walls of
Gram-positive species are more difficult to break, and their
major components, which correspond to 90% of glycans, are
much more easily ionized than PL. Consequently, the excreted
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metabolites are observed as the dominant species in the
resulting DESI mass spectra, especially those lipopeptides that
are produced in large quantities, are surface-active and ionize
with high efficiency, such as the surfactins.59
Fernandez and co-workers have also applied DART-MS to
two different bacterial samples.60 They describe the detection
of fatty acid methyl ester (FAME) ions from whole bacterial cell
suspensions and their identification by accurate-mass orthogonal TOF-MS. This study is interesting because the ‘‘gold
standard’’ methods routinely used in bacterial taxonomy and
classification are based on the determination of microbial
FAME composition after culturing, a process that forms the
basis of the commercial Sherlock microbial identification
system (MIDI Inc., Newark, Delaware, US). Routine FAME
analysis involves lengthy sample preparation, starting with the
hydrolysis of bacteria cells followed by fatty acid methylation.
Gas chromatography coupled to mass spectrometry (GC-MS) is
then used for separation and the detection of FAME composition. Each GC-MS run generally takes 20 to 30 min, whereas
the total DART-MS analysis takes less than 10 min.55,61 FAME
were generated from approximately 107 cell mL21 Streptococcus
pyogenes and E. coli. After incubation, cells were washed with
TRIS-sucrose buffer, suspended in water, and diluted with a
solution of tetramethylammonium hydroxide (TMAH) to
produce thermal hydrolysis and methylation of bacterial
lipids. An aliquot of the whole bacterial cell suspension mixed
with TMAH was deposited in the bottom of the capillary tube.
The capillary was positioned so that the bottom of the tube
came in contact with the DART He stream directly in front of
the mass spectrometer inlet orifice after sliding the sample
holder arm. The protonated FAME C9:0, C10:0, C11:0, C12:0,
C14:0, C15:0, C17:1/cycloC17:0, and C19:1/cycloC19:0 were
found to be present only in E. coli, while C11:1 was uniquely
detected in S. pyogenes. C17:1/cycloC17:0 and C19:1/cycloC19:0
were found in E. coli at relatively high abundances but were
not detected in the S. pyogenes spectrum, which is in
accordance with the membrane characteristics of Gramnegative bacteria. Some FAME ions were common to E. coli
and S. pyogenes; however, clear differences existed in the
relative abundances of these ions in the mass spectra.
Differences among samples were thus observed in the spectral
temporal and intensity domains.60
Recently, a new ambient ionization technique termed low
temperature plasma mass spectrometry (LTP-MS) was applied
for bacterial identification.60 This ambient ionization method
was introduced in 2008 by Cooks and co-workers, and the
absence of any solvent is the distinguishing feature of this
plasma-based method (Fig. 4c).55 LTP-MS was employed to
detect fatty acid ethyl esters (FAEE) from bacterial samples in a
direct way. Positive ion mode FAEE mass spectrometric
profiles for 16 different bacterial samples were obtained
without extraction or other sample preparation. Data were
examined by PCA to determine the degree of possible
differentiation among the bacterial species. Growth media
effects were observed, but in this case, they did not interfere
with species recognition based on the PCA results.55
Ambient desorption/ionization techniques can therefore be
applied to bacterial identification, but in some cases, such as
for DESI that uses a high velocity nebulizing gas, it is necessary
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to secure or fix bacterial cells onto the DESI probe surface to
prevent sample dispersion and/or aerosolization during
analysis. This procedure is required for the analysis of
pathogenic bacteria, and heating (for example, at 220 uC for
30 s) or long-term drying (.1 h at room temperature) of
bacterial cells are effective means of fixing the sample onto a
glass slide surface.57 Different groups using the same ambient
ionization technique have presented different results due to
the dependence of mass spectral profiles of intact bacteria on
the experimental design and the way in which growth media
was used and/or prepared. Future investigations in this area
appear to require detailed evaluation of the experimental
design and its influence on the data output.57 However, the
successful demonstration of identification by ion composition
from whole bacterial cells via ambient MS analysis, mainly for
segregating bacterial strains according to Gram status,
constitutes the first step that could in the future lead to the
successful development of new approaches for high-throughput microbial identification from a variety of biological, food,
and water samples in the open air with minimum sample
preparation.57
MALDI-MS based platforms: real-world breakthroughs in
microbiology
PROTEOMIC STUDIES FOR THE IDENTIFICATION OF BIOMARKERS.
Approaches that use proteomics as a tool for studying
expressed proteins are increasingly being utilized to address
diverse biomedical questions. Via the identification of
specific and conserved biomarkers, such as ribosomal
proteins, peptides and lipids, it is possible to provide cancer
diagnoses, study inflammatory and degenerative diseases and
to determinate pathogens responsible for a broad range of
diseases.62–66
Protein profiles obtained from direct MALDI-MS analysis of
intact microorganisms or protein extracts have revealed robust
biomarkers that are mostly related to conserved and specific
ribosomal proteins. Currently, bacterial species identification
by MALDI-MS is the MS approach that has the highest impact
in the field of microbiology.67,68 This approach is based on the
acquisition of ribosomal protein fingerprints directly from
protein extracts from intact organisms. Interestingly, these
protein profiles, which primarily contain ribosomal proteins,
have been found to vary considerably and allow the proper
characterization of different microorganisms. These protein
markers are rapidly being incorporated into human clinical
microbiology routines due to the availability of bioinformatics
tools for databank searches, allowing secure identification and
high laboratory reproducibility.67,68 MALDI-MS has been
proved by many reports to be easier, faster and sometimes
more reliable than classical protocols even when compared to
more sophisticated DNA analysis-based technologies.69,70
MALDI-MS-based microorganism identification has rapidly
been introduced in laboratory and clinical settings and
delivers fast and reliable diagnostic results, not only for genus
level identification but also at the species level for bacteria,63,70–81 fungi,16,82–91 algae,92 viruses93–96 and protozoa.97
An essential step in the identification of microorganisms at
the species level by MALDI-MS has been the use of dedicated
databases with rigorous data quality control and powerful
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algorithms for comparison with mass fingerprinting. These
databases can be run in parallel with MS acquisition data,
giving almost real-time bacterial identification results.33,98,99
Because much effort has focused on MALDI-TOF-MS, this
approach is already in use in clinical diagnostic laboratories.68,100–107 The observed biomarkers in the mass spectrum
enable not only the detection of pathogenic bacteria but also
the ability to distinguish them from corresponding nonpathogenic species.108
Many Campylobacter species and Helicobacter strains cause
gastrointestinal diseases and can be discriminated via protein
profiles observed by MALDI-MS.109–113 Biomarker assignment
also makes it possible to distinguish subspecies of members of
the Enterobacteriaceae family so that their fingerprints can be
used as family-specific biomarkers for accelerated bacterial
identification via database searches.114
Biomarker monitoring by MALDI-MS can also be used to
identify environmental toxin producers. MS analyses of
peptides and polyketides from intact cyanobacteria were used
to identify toxic and nontoxic water blooms115–117 and
pathogens isolated from seafood, which are associated with
food-borne diseases. This approach was also used to further
study the different protein profiles of azaspiracid toxin
biomarkers in contaminated and non-contaminated blue
mussels (Mytilus edulis).118
Burkholderia cepacia, which are important agents of chronic
pulmonary disease in cystic fibrosis patients and are problematic to accurately identify due to their complex taxonomy,
have been successfully discriminated by MALDI-MS.119–122
Mycobacterial species, which are responsible for causing
significant morbidity in humans by diseases such as tuberculosis, and Haemophilus spp., which are well known etiological
agents of pneumonia, meningitis and conjunctivitis, have
been identified by MALDI-MS via their protein profile
spectra.123–125
In the veterinary field, bacteria isolated from cows presenting subclinical mastitis, a common and easily disseminated
disease in dairy herds, were diagnosed in a few minutes
through the analysis of ribosomal protein biomarkers isolated
from microorganisms present in milk samples by MALDI-MS
with the use of the Biotyper database, a commercial software
for MALDI-MS-based microorganism identification that allows
earlier treatment with appropriate antibiotics.126
Immunoproteomic analyses of Mannheimia haemolytica, the
most important bacterial pathogen associated with bovine
pneumonia, have been performed by MALDI-MS to search for
and identify biomarkers from outer membrane proteins that
may hold potential as candidate vaccine antigens.127
Pigments and proteins from chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium
Chlorobium tepidum, were characterized directly from organelles and bacteriochlorophyll, and homologs were detected to
provide fingerprints for these biomarkers.128
MALDI-MS analysis was used to detect the increased
expression of cold shock proteins in bacteria collected from
the Siberian permafrost, and distinct proteins and peptide
profiles were observed as a function of temperature. The
recent capability of MALDI-MS imaging has been used to study
synergism and antagonism in microbial communities in the
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RSC Advances
nests of leaf-cutting ants through the identification of
antimicrobial and antifungal compounds that are produced
to defend a symbiotic fungus, which is a major food source for
ant species.129
These numerous examples of MALDI-MS applications are
supported by the availability of dedicated instrumentation and
software containing fingerprinting databases for bacterial
identification. Such instruments are operator-friendly, and
technicians can operate them in microbiological settings and
hospitals with diagnostic purposes with minimal specialization in MS or microbiology and with high-throughput, efficient
and trustworthy results. As a result, diverse human clinical
settings start by comparing the results of traditional methods
versus MALDI-MS-based bacterial identification, and, after
some time, they move to a solely MALDI-MS-based approach
because of the many advantages and the desire to avoid
numerous time-consuming and tedious assays.
Identification of uncommon bacterial pathogens, fungi and
yeast by MALDI-MS
Microorganisms still represent the largest reservoir of biodiversity yet to be studied. It has been estimated that only
between 1 and 10% of bacteria have been properly described.
Correct microorganism identification is essential for appropriate classification, and the criteria for the identification of
diverse microorganism species are still equivocal. Some
strains can be misidentified as closely related species. This
is the case, for example, for Cronobacter spp.130 that can easily
be misidentified as apathogenic Enterobacter turices, E.
helveticus and E. pulveris. Cronobacter spp. are Gram-negative
opportunistic food-borne pathogens and are known as rare but
important causes of neonatal infections. To overcome this
problem, MALDI-MS was successfully used for rapid genus and
species-specific identification. Moreover, multi-isotope imaging mass spectrometry is contributing to understanding the
bacterial diversity of bacterioplankton and helping to link
microbial diversity to the biogeochemistry of the pelagic zone
of the aquatic system.131
The marine environment has also been proven to be a
source of diverse arrays of bioactive metabolites with great
potential for pharmaceutical and other applications. In fact,
MALDI-MS was applied to classify environmental
Sphingomonadaceae using ribosomal subunit proteins coded
in the S10-spc-alpha operon as biomarkers.132 In particular,
sponges have a largely unexplored biosynthetic potential. Sets
of bacteria were cultured from marine sponges (Isops phlegraei,
Haliclona sp., Phakellia ventilabrum and Plakortis sp. growing
on the Norwegian coast). Intact cell MALDI-MS was used for
the rapid screening and proteometric clustering of a subset of
the strain collection comprising 456 isolates. The 11 resolved
groups were also verified by 16S rDNA analyses. The results
indicated that MALDI-MS is effective for the rapid identification of isolates, for the selection of strains representing rare
species and for their dereplication, i.e., rapid grouping of
bacterial isolates for subsequent characterization.133
Screening for microbial population complexity and diversity
in the sediment of contaminated environments has also
received increased interest, particularly due to the significance
of these microbes for environmental protection. The taxono-
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Review
mical identification of microbial isolates obtained from
sediment samples contaminated with polychlorinated biphenyls was successfully performed with MALDI-MS with minimal
time demand and reduced costs.134
The contribution of MS to worldwide biodefence has also
been substantial.135,136 In fact, some potential agents for
biological attacks are microorganisms and biotoxins. MS was
demonstrated to be a valid tool for the rapid identification of
potential bioagents. Confident identification of an organism
can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass
spectra. In bottom-up proteomics, the rapid digestion of intact
protein biomarkers is again followed by MS to provide
unambiguous bioagent identification and characterization.133,134
Accurate discrimination between species of filamentous
fungi is also essential because some species have specific
antifungal susceptibility patterns, and misidentification may
result in inappropriate therapy. Direct surface analysis of
fungal cultures90,137,138 and yeasts139 by MALDI-MS has been
evaluated for species identification. The protein profiles of
intact fungal spores83,140 such as Aspergillus, Fusarium and
Mucorales demonstrated that MALDI-MS is appropriate for the
routine identification of filamentous fungi in medical microbiology laboratories.73,138
Culture collection strains representing 55 species of
Aspergillus, Fusarium and Mucorales were used to establish
one reference database for MALDI-MS-based species identification with the MALDI BioTyper 2.0 software. To evaluate the
database, 103 blind-coded fungal isolates collected in a
routine clinical microbiology laboratory were tested, and
96.8% of the isolates were correctly identified to the species
level in agreement with reference methods. Eight technical
replicates of 15 strains were also obtained to study the
variation of mass spectra. Little variation was observed for
each spectrum, whereas enough MS variation could be
observed to separate each strain (Fig. 5).90
Yeast infections cause significant mortality in critically ill
and immunocompromised patients. In particular, Candida
spp. are the 4th most common cause of nosocomial bloodstream infections in the United States, and Cryptococcus
neoformans is the most common cause of fungal meningitis
worldwide.141,142 Candida species were reliably identified by
MALDI-MS, which had superior performance over conventional methods, and it was possible to discriminate between
different molecular types of Cryptococcus neoformans and
Cryptococcus gattii with the same technique.143,144
Non-fermenting bacteria
Non-fermenting bacteria are a taxonomically heterogeneous
group of bacteria of the Proteobacteria division, which cannot
catabolize glucose. The genera Pseudomonas, Burkholderia,
Stenotrophomonas and others belong to this large group. They
are ubiquitous environmental opportunists, and some species
can cause severe infections,145 particularly in immunocompromised or cystic fibrosis (CF) patients.146 It has been
demonstrated that classical phenotypic methods can frequently misidentify non-fermenters.147,148
RSC Adv., 2013, 3, 994–1008 | 1001
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RSC Advances
Fig. 5 Three-dimensional principal component analysis (PCA) plot of the technical replicates of selected reference strains of (a) Aspergillus (seven strains), (b) Fusarium
(four strains) and (c) Mucorales (four strains). Reproduced from ref. 90 with permission from John Wiley and Sons.
For this class of bacteria, molecular tools such as 16S rDNA
gene sequencing provide reliable results, but less accurate
results have been obtained at the species level. Therefore, a
reference database for MALDI-MS based on the identification
of non-fermenters was established by Mellmann et al.149,150
and 16S rRNA gene sequencing was used for comparison.
Different cultivation conditions and mass spectrometer
instruments were used, and the methodology was evaluated
with 80 blind-coded clinical non-fermenter strains. The study
demonstrated that the MALDI-MS method provides fast and
thorough identification of non-fermenting bacteria, even more
accurately than partial 16S rRNA gene sequencing for species
identification of members of the Burkholderia cepacia complex.
A large international multicenter study has also demonstrated
the high reproducibility of the identification of non-fermenting bacteria by MALDI-MS, with 98.75% correct species
identification. This study demonstrated the suitability of the
technique as an alternative to partial 16S rRNA platforms. A
very recent study also evaluated the ability of the technique to
identify non-fermenting bacteria from among a total of 182
isolates from 70 CF patients because non-fermenters are the
main cause of mortality in this type of patient.151 MALDI- MS
was found to improve routine identification, particularly by
enlarging the Biotyper 2.0 database to include the rare and
infrequent microorganisms recovered from CF patients.152
The routine identification of microorganisms that contaminate milk is mostly based on phenotypic characteristics such
as colony morphology, hemolytic potential and several
biochemical reactions, which are time-consuming and
costly.157 Additionally, these tests may fail to correctly identify
all agents, and even though methods based on PCR are
developing rapidly, there may be no agreement among the
techniques that phenotypically and genotypically differentiate
bacterial species, resulting in the false identification of agents.
Non-culture MALDI-MS identification based on protein fingerprinting from bacteria (E. coli, S. aureus and E. faecalis)
inoculated and recovered directly from milk samples has been
successful (Fig. 6). Although relatively high bacterial loads (106
to 107 bacteria mL21 of milk) must be present, the simple
incubation of an initial load of 104 bacteria mL21 of milk can
be used to facilitate bacterial replication and successful
Non-culture-based identification of bacteria from blood and
milk
In addition to being increasingly used for the rapid identification of bacteria and fungi, MALDI-MS also holds promise for
bacterial identification from blood culture (BC) broths in
hospital laboratories153–155 and bacterial identification directly
from milk samples.156
A MALDI-MS-based approach has been shown to perform
rapid (,20 min) bacterial identification directly from positive
BCs with high accuracy. Positive predictive values for the direct
identification of both Gram-positive and Gram-negative
bacteria from monomicrobial blood culture broths were
100% to the genus level. A diagnostic algorithm for positive
blood culture broths that incorporates Gram staining and
MALDI-MS should be able to identify the majority of
pathogens, particularly at the genus level.153,154
1002 | RSC Adv., 2013, 3, 994–1008
Fig. 6 MALDI-MS ribosomal protein fingerprints for the identification of
bacteria in whole milk. Data were collected in the m/z 4000–22 000 range after
processing 900 mL of whole milk that had been experimentally contaminated
with E. coli at (a) 103, (b) 104, (c) 105, (d) 106, (e) 107, (f) 108, or (g) 109 cfu ml21.
Reproduced from Proteomics ref. 156 with permission from John Wiley and
Sons.
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RSC Advances
identification. Fast, reliable and sensitive protocols for the
analysis of relatively low concentrations of bacteria present in
milk could be of great value for the dairy industry.
Testing for antibiotic resistance with MS and polymicrobial
culture analysis
Antibiotic resistance is the ability of a microorganism to
withstand the effects of an antibiotic drug, and this ability
represents a huge health concern in the medical and
veterinary fields. Recent studies have shown that individuals
are at risk of carrying antibiotic-resistant bacteria after a series
of antibiotic treatments due to the resulting selection for
antibiotic-resistant microorganisms. The most common
mechanisms of antibiotic resistance can be divided in three
classes: alteration of the antibiotic target, restriction of
antibiotic access to the target and inactivation of the
antibiotic.158–160
Recently, the ability of MALDI MS to effectively discriminate
bacteria strains which have acquired resistance to a variety of
antibiotics has been demonstrated, indicating that this
technique has the potential to differentiate bacterial strains
with varying degrees of antibiotic resistance.161
The rapid detection of resistance type is necessary to select
the best antibiotic therapy. b-Lactam antibiotics represent a
broad class of antibiotics that contain a b-lactam nucleus in
their molecular structure. These antibiotics include penicillin
derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. b-Lactam antibiotics act by inhibiting
the synthesis of the peptidoglycan layer of bacterial cell walls,
Fig. 7 (A and B) MALDI-MS of ampicillin after incubation with a b-lactamaseproducing strain (B). (C) Inhibition of hydrolysis by a b-lactamase-producing
strain was performed in the presence of clavulanic acid. Peaks corresponding to
the non-hydrolyzed form of ampicillin are highlighted in gray. Peaks
corresponding to the hydrolyzed form of ampicillin are indicated with an arrow.
Reproduced from ref. 164 with permission from the American Society for
Microbiology.
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Review
which is important for cell wall structural integrity. b-Lactam
antibiotics mainly affect Gram-positive organisms because
peptidoglycan is the outermost and primary component of
their cell wall. This antibiotic class binds in the active site of
penicillin-binding proteins (PBPs), preventing the final crosslinking (transpeptidation) of the nascent peptidoglycan layer
and disrupting cell wall synthesis.162 Since these antibiotics
are widely administered to treat infections in human and
domestic animals, many Gram-positive bacteria have already
developed resistance mechanisms.
Antimicrobial susceptibility has classically been determined
using a variety of in vitro methods such as disk diffusion and
broth microdilution as well as automated instrument-based
methods. These methods may require from a few hours, such
as for the antimicrobial susceptibility test (AST), to 24–96 h for
a pure culture of the suspected pathogen to be obtained and
subjected to disk diffusion assays.3,163 A novel MALDI-MS
method for the detection of b-lactamase resistance has
recently been reported. Resistance to b-lactam antibiotics
can be easily monitored by MS because hydrolysis of the
central b-lactam ring by b-lactamases results in the disappearance of the original ion, which is shifted 18 m/z units
higher in the spectrum of the antibiotic. In many cases,
hydrolysis is directly followed by a decarboxylation of the
hydrolyzed product, resulting in a further shift of 244 m/z
units due to detection of the hydrolyzed form. Because MS
easily monitors such m/z shifts, a MALDI-MS assay was set up
to analyze the hydrolysis reactions of different b-lactam
antibiotics (Fig. 7).164
MALDI-MS can also be applied to study bacterial resistance
to antibiotics or antimicrobial compounds secreted by other
bacterial species.68,165,166 Reportedly, antibiotic-resistant and
non-resistant strains of an important human pathogen, S.
aureus, can be differentiated by MALDI-MS by rapid and
accurate discrimination between methicillin-sensitive and
methicillin-resistant strains of this organism, which can could
lead to major improvements in the treatment strategies for
infected patients.167 The same microorganism has been
identified via biomarker analysis as one of the main pathogens
responsible for prosthetic joint infections, indicating reliable
differentiation between S. aureus and coagulase-negative
staphylococci.168
The resistance mechanism of colistin-resistant variants of
Acinetobacter baumannii was elucidated by MALDI-MS169 by
determining the phosphoethanolamine modification of lipid
A. For Bacteroides fragilis, it was recently shown that the
differentiation of cfiA gene-encoded class B metallo-b-lactamase was possible by direct MALDI-MS.170,171 The carbapenem
resistance of B. fragilis is due to a species-specific metallob-lactamase, which is encoded by the cfiA (ccrA) gene of the
organism. Almost 100% of the carbapenem-resistant bacteroid
strains were cfiA-positive B. fragilis isolates. However, such
clonal differentiation for resistant and susceptible clones
cannot be expected for the majority of bacteria. Antibiotic
resistance
in
Gram-negative
rods,
particularly
Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp.,
has been an increasing problem worldwide. Infections by
multidrug-resistant Gram-negative bacteria are usually treated
with carbapenems. This resistance is caused by an alteration
RSC Adv., 2013, 3, 994–1008 | 1003
Review
in the outer membrane of the cell wall and by the production
of carbapenemases.172 Carbapenamase activity was detected in
124 strains following comparison between well-typed bacterial
carbapenem non-susceptible isolates and clinical isolates
susceptible to carbapenems. Antibiotic resistance was demonstrated by detecting the disrupted amide bond and its
cationized forms.173
Although the microbiological methods of microorganism
culture and isolation are successful, mixed cultures or
polymicrobial cultures may occur. For example, one bacterial
isolate from subclinical bovine mastitis was identified as
Staphylococcus aureus by an enzymatic assay, and yet the same
sample, when analyzed by Biotyper software after MALDI-MS,
displayed a signature of Enterococcus faecalis mixed with S.
aureus, which was confirmed by 16S RNA sequencing.174
When bacterial identification of human clinical isolates was
performed by MALDI-MS directly on 500 blood broth cultures,
there were 27 polymicrobial cultures, and 25 (92.6%) had at
least one species correctly identified by the instrument
database. Using the same instrument and software platform,
similar results have been observed by Moussaoui et al.175 and
Christner et al.,176 who reported that 80.9% and 81.2%,
respectively, of at least one of the species present were
identified in polymicrobial cultures obtained directly from
blood culture vials.175,176
RSC Advances
around U$ 2–3 per isolate, depending on the cost of the
instrument.
Therefore, microbiology is ready to enter into a new era in
which molecular rather than morphological or biochemical
characteristics of microorganisms will be rapidly assessed
(directly from biological fluids) by MS for identification. The
use of this technique is not only limited to clinical diagnosis,
but it has been shown to have successful application for
detecting antibiotic resistance, characterizing microorganisms
that are difficult to isolate and culture and exploring the
biodiversity of microorganisms present in the environment
and in the digestive tracts of animals and humans. Mass
spectrometry-based proteomics approaches have also been
applied to gain a greater understanding of the pathophysiology and virulence of microorganisms. This approach is
providing key insights to better understand the molecular
processes involved in protein secretion, modification, synthesis and degradation.177–179
A new era has indeed emerged in which bacterial
identification seems fully prepared to make the transition
from the agar plate and visual inspection to the mass
spectrometer for characterization at the more accurate
molecular level.
References
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related to the instrument configurations. Also, most laboratories will need an internal validation period in which the
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recommended procedures and MALDI-MS using a TOF
analyzer and the Biotyper software. Besides the velocity of
the diagnosis, MALDI-MS showed substantial savings of
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