Abstract Book of EAVLD2012 - eavld congress 2012

S4 - K - 01
MALDI-TOF AND OTHER NEW DIAGNOSTIC
Markus Kostrzewa
Bruker Daltonik GmbH, Bioanalytical development, Bremen, Germany
Microorganism identification, FT-IR spectroscopy, Raman spectroscopy, mass spectrometry, MALDI-TOF MS
Introduction – the “status quo”
Since more than one hundred years, identification of
microorganisms mainly is performed by methods investigating
their phenotypic characteristics. Gram stain together with
microscopy in combination gives a first quick hint about identity.
The screening of series of tests for biochemical capabilities is
used to generate a pattern which is more or less species specific.
Although these tests now have been automated for the detection
of the most prevalent microorganisms, the accuracy of
biochemical tests is limited, in particular for organisms with few
characteristic biochemical features like Gram negative nonfermenting
bacteria of anaerobes. Even worse is the situation for
rare bacteria, mycobacteria or fungi where mainly labourintensive
manual methods like API are applied for identification.
Therefore, there is an urgent need for modern automated
methods for identification which can be introduced in routine
laboratories to shorten time-to-result and decrease the overall
costs of the laboratories. Promising approaches appearing in the
areas of molecular biology, optical technologies and mass
spectrometry have appeared through the recent years and are on
the way to revolutionize microbial analysis.
Methods based on molecular biology have already been
introduced in many laboratories, mainly PCR based tests to
screen for dedicated microorganisms, e.g. food pathogens. While
having the advantage of high sensitivity and short analysis time
one drawback of PCR based tests is that they generally need a
pre-assumption which microorganism is searched for, i.e. a
dedicated primer design. To apply DNA analysis to broad species
identification, PCR typically is coupled with subsequent sequence
determination of the amplicon. Sequencing of the 16S rDNA
region nowadays is regarded the gold standard for species
identification. Nevertheless, 16S rDNA sequencing has never
made it to the routine but is mainly a second- or third-line method
for cases where other methods have failed. Reasons are the high
costs, elaborate sample handling, and PCR-related
contamination risk. Further, in quite a number of cases,
determination of the 16S ribosomal RNA gene sequence is not
sufficient for the discrimination of closely related species and
further gene sequences have to be analysed, increasing time to
result, costs and labour, significantly. Novel next generation
sequencing technologies might change the position of
sequencing in future by their enormous throughput capability, but
today they are still expensive and labour-intensive research tools.
The upcoming alternatives
Vibrational spectroscopy
Optical technologies have been shown to be powerful for
microorganism identification as well as for analyses on the
subspecies level. These techniques are based on a molecular
fingerprint of the entire biomass, proteins, carbohydrates, lipids,
nucleic acids. Analyses are not depending on reagent kits, labels
or drugs, and these technologies are applicable to a broad
spectrum of organisms.
Raman spectroscopy is a non-destructive optical technique,
based on scattering of light by molecules, the so called “Raman
effect”. The atoms in a molecule move in so called vibrational
modes. When light, i.e. a photon, and a molecule interact, some
of the energy of the photon can be transferred to the molecule.
Thereby, one of the vibrational modes in the molecule is excited.
The energy of the scattered photon is reduced by the exact
amount of energy received by the molecule. The energy required
to excite a molecular vibration is exactly defined and depends on
the masses of the atoms involved in the vibration, on the type of
chemical bonds between these atoms, on the structure of the
molecule and on interactions with its environment and other
molecules. The spectrum of the scattered light is used as a
pattern to identify microbes.
In contrast, Fourier-transformation infrared spectroscopy uses
absorption of an infrared light spectrum (not a single wavelength)
to create a fingerprint of the whole cell composition. Mainly
transmission of the light is used to create the characteristic
profile. The first deviation of the transmission profile spectrum is
generated and sophisticated mathematical algorithms are used to
identify strains by matching against a database or to build
relational dendrograms for classification.
Restrictions of the optical technologies is mainly that they require
a strict standardization of cultivation conditions (time, media,
temperature) as these are influencing the cell content and
thereby the optical fingerprint. The speed of the analyses is also
counteracted by the fact that a pure culture is required (i.e. often
no analysis from primary culture can be performed).
Mass spectrometry
Some technological approaches for identification and further
analysis of microorganisms based on mass spectrometry may
have the most impact on microbial characterization in the near
future. One of these approaches is based on a combination of
molecular biology and electrospray time-of-flight mass
spectrometry (ESI-TOF MS). In a first step, several target regions
of the DNA from the unknown microorganism are amplified with
generic primer pairs. This is creating a set of amplification
products with specific molecular masses. Subsequently, the PCR
products are purified with a magnetic bead based system and
then injected in liquid phase into the ESI-TOF mass
spectrometer. During the ionization process, the two DNA strands
of an amplicon are separated. The mass of both DNA strands is
measured in the ESI-TOF very accurately. From the combination
of the accurate masses of the both pairing strands the
composition of the polynucleotides (i.e. the number of A, C, G,
and T, respectively) can be determined. The projection of the
base compositions of the selected PCR products into a database
is used for the identification of an organism. The more PCR
products are analysed, the more accurate/discriminative the
identification can be. One advantage of this technology is that in
parallel to species identification also other characteristics, e.g.
resistance genes or virulence factors, can be detected. On the
other hand, this approach still relies on a relatively complex
(although in parts automated) workflow, and both the instrument
as well as consumable costs are significant.
The second, currently most successful and promising mass
spectrometry technology is matrix-assisted laserdesorption/ionization
time-of-flight mass spectrometry (MALDI-
TOF MS). In contrast to electrospray ionization the MALDI
process (generating the ions measured in the TOF analyser) is
performed from a solid sample. For this, the sample is deposited
on a target plate, overlaid with the so called matrix, generally an
organic acid related to cinnamic acid, which co-crystalizes with
the sample. This matrix supports the evaporation of the sample
and its ionization after absorption of the energy of a pulsed laser
beam. One advantage of this technology is that the sample can
be easily prepared offline before measurement and then
transported or stored until measurement.
For one molecular biology based approach, MALDI-TOF MS is
used to detect specific RNA fragments generated by PCR
followed by transcription of both DNA strands and the cleavage of
the transcripts by RNAses. The exact masses of the fragments
can be used as pattern for identification and typing of
microorganisms. Alternatively, minisequencing/primer extension
of specific regions after PCR amplification, followed by mass
determination in a MALDI-TOF MS, has been shown to have
some potential for typing or resistance detection.
While none of these DNA based mass spectrometry approaches
made it to routine yet, MALDI-TOF MS whole cell protein profiling
has entered many clinical and veterinary microbiology
laboratories, already. This MALDI-TOF fingerprinting is based on
the specific pattern of high abundant proteins of microbial cells
which can be measured quickly and accurately by MALDI-TOF
mass spectrometry. Sample preparation is very simple, fast and
cheap: A small amount of a bacterial colony is transferred to a
position on a MALDI sample target and smeared on it as a thin
film. More robust cells can be broken by a short extraction with