Eur J Clin Microbiol Infect Dis (2014) 33:949–955
DOI 10.1007/s10096-013-2031-5
ARTICLE
Rapid detection of antibiotic resistance based on mass
spectrometry and stable isotopes
J. S. Jung & T. Eberl & K. Sparbier & C. Lange &
M. Kostrzewa & S. Schubert & A. Wieser
Received: 31 August 2013 / Accepted: 29 November 2013 / Published online: 14 December 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract With the emergence and growing complexity of
bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies
that assist in predicting the effectiveness of empiric antibiotic
therapy are of great interest. Although the use of matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for
MALDI-TOF MS susceptibility testing are restricted to very
limited conditions. Here, we describe a technique that may
allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable
isotope labelling by amino acids in cell culture (SILAC)-like
approach. This technique was used to visualise the growth of
bacteria in the presence of an antibiotic. Pseudomonas
aeruginosa was chosen as the model organism, and strains
were incubated in normal medium, medium supplemented
with 13C6-15 N2-labelled lysine and medium supplemented
with labelled lysine and antibiotic. Peak shifts occurring due
to the incorporation of the labelled amino acids were detected
Sören Schubert and Andreas Wieser share the last authorship.
Electronic supplementary material The online version of this article
(doi:10.1007/s10096-013-2031-5) contains supplementary material,
which is available to authorized users.
J. S. Jung (*) : T. Eberl : S. Schubert : A. Wieser
Max von Pettenkofer-Institut für Hygiene und Medizinische
Mikrobiologie, Marchioninistr. 17, 81377 Munich, Germany
e-mail: [email protected]
K. Sparbier : C. Lange : M. Kostrzewa
Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin,
were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed
test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of
SILAC technology for the detection of antibiotic resistance
may contribute to accelerated and reliable susceptibility
testing.
Introduction
Over the last few years, matrix-assisted laser desorption/
ionisation time-of-flight mass spectrometry (MALDI-TOF
MS) has been successfully introduced into microbiological
laboratories worldwide. Due to its advantages in terms of
time, accuracy and cost-effectiveness, this method has largely
replaced biochemical methods for the identification of bacteria and fungi. Furthermore, the use of this technology for the
rapid detection of bacterial resistance is an attractive option,
and several studies in this field have been published to date.
These approaches rely on functional tests, such as the βlactamase assay, which detects the hydrolysis of β-lactams
after co-incubation with a β-lactamase-producing organism
[1–4]. Other tests are based on strain typing, which is only
feasible for the detection of resistance mechanisms that are
clearly related to distinct clonal groups [5–10]. Thus, MALDITOF MS can detect antibiotic resistance only to a limited
extent. In the present study, we describe a technique (MS
resist) that has the potential to overcome these limitations
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Eur J Clin Microbiol Infect Dis (2014) 33:949–955
and to address virtually any antibiotic resistance, irrespective
of the underlying mechanism or the identity of the bacterium.
The method is based on the stable isotope labelling by amino
acids in cell culture (SILAC) technique and exploits the fact
that only metabolically active microorganisms can incorporate
the labelled amino acids provided in the growth medium. This
incorporation into cellular proteins results in a peak shift that
can be detected by MALDI-TOF MS under the same conditions that are used for the identification of pathogens. If the
bacterium is no longer metabolically active due to the impact
of an inhibiting antibiotic, no peak shift will be observed in the
mass spectrum.
In this study, we tested for resistance to three antibiotics,
each representing a clinically relevant group of antimicrobials
with distinct modes of action. Meropenem was chosen as a βlactam antibiotic that interferes with cell wall synthesis in
growing bacteria. Tobramycin was used to test the applicability of the new method to aminoglycosides, which lead to the
irreversible inhibition of protein synthesis. Ciprofloxacin was
tested as a representative fluoroquinolone, which hampers the
regulation of DNA topology. Pseudomonas aeruginosa was
used as the model organism because of its high clinical relevance and the fact that it can express a great variety of
resistance mechanisms. These mechanisms can be expressed
in isolation or in combination, which makes molecular testing
rather difficult.
ups contained Dulbecco’s modified Eagle medium (DMEM)
with low glucose for SILAC, without arginine, leucine and
lysine (Fisher Scientific, Schwerte, Germany), supplemented
with 4 g/L glucose, 60 mg/L Fe(II)SO4 and all proteinogenic
amino acids (300 mg/L each) except lysine. One set-up was
supplemented with normal lysine (set-up A), one set-up was
supplemented with 13C6-15 N2 L-lysine (set-up B) (Fisher
Scientific, Schwerte, Germany) and one set-up was supplemented with 13C6-15 N2 L-lysine plus the antibiotic to be tested
(set-up C), either meropenem trihydrate 290 mg/L
(AstraZeneca, Wedel, Germany), ciprofloxacin (as hydrogen
sulphate) 60 mg/L (Fresenius Kabi, Bad Homburg, Germany)
or tobramycin (C18H37N5O9) 120 mg/L (Sigma-Aldrich,
Steinheim, Germany). For the meropenem testing, labelled
and unlabelled lysine was added only after a 30-min preincubation time with the antibiotic. All the samples were
incubated at 37 °C with agitation. Different incubation times
were used, as indicated in the results section. After the incubation, the cells were washed with H2O and lysed in 10 μL of
70 % formic acid and an equal amount of 100 % acetonitrile.
The supernatants were spotted onto a polished steel target
(Bruker Daltonik GmbH, Bremen, Germany) in triplicate.
The spots were air-dried and overlaid with 1 μL of matrix
solution containing 10 mg/mL α-cyano-4-hydroxy-cinnamic
acid in 50 % acetonitrile/2.5 % trifluoroacetic acid (α-HCCA
portioned matrix, Bruker Daltonik GmbH, Bremen,
Germany).
Materials and methods
Spectrum acquisition
Bacterial strains
A Microflex LT benchtop instrument operated by the
flexControl version 3.3 software (Bruker Daltonik GmbH,
Bremen, Germany) was used for the MALDI-TOF MS analysis. The spectra were acquired in the linear positive mode at a
laser frequency of 60 Hz within a mass range of 2 to 20 kDa.
The acceleration voltage was 20 kV, the IS2 voltage was
maintained at 18.6 kV and the extraction delay time was
200 ns.
Representative P. aeruginosa strains with different minimum
inhibitory concentration (MIC) values and resistance patterns
were obtained from our clinical laboratory. The P. aeruginosa
ATCC strain 27853 was tested as a reference. The bacteria
were grown on MacConkey or Columbia blood agar (BD,
Heidelberg, Germany) overnight at 37 °C.
For the growth phase assays, starter cultures were set up by
inoculating a single colony into 200 ml of LB medium [10 g/L
tryptone (BD, Sparks, MD, USA), 5 g/L yeast extract powder
(MP Biomedicals, Solon, OH, USA) and 5 g/L NaCl (Roth,
Karlsruhe, Germany], followed by growth overnight at 37 °C
with shaking at 120 rpm. These cultures were used to inoculate 200 ml of LB at an OD600 of 0.05. The cells were
incubated with shaking at 120 rpm, and aliquots (1–2 ml)
were removed for assays at different time points, as indicated
below.
Sample preparation
For each test, three different 300-μL set-ups containing 3.5 ×
107 colony-forming units (CFU) were prepared. All three set-
Data analysis
A visual analysis of peak shifts was performed using
flexAnalysis software (Bruker Daltonik GmbH, Bremen,
Germany), and the spectra were baselined and smoothed by
applying a Savitzky–Golay filter (width 0.2 m/z, cycle number 1). The spectra derived from microorganism growth in the
presence of the antibiotic were compared to the reference
spectra with and without labelled lysine. If only minor peak
shifts were visible and the spectrum looked more similar to the
native spectrum without labelled lysine, the strain was considered to be susceptible to the tested substance. In contrast,
spectra looking similar to those obtained from the set-up with
labelled lysine but without antibiotic were considered to be
Eur J Clin Microbiol Infect Dis (2014) 33:949–955
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resistant. Based on the visual analysis, ten peak shifts were
selected to create an automated algorithm for data evaluation.
An in-house-developed software tool written in R was used
for the peak selection and calculation of the peak intensities
[11–13], which were further plotted according to the following
algorithm:
X
set − upC X
ið1−10Þ
Xið1−10Þ
set − upB X
normal peaks
ið1−10Þ
ið1−10Þ
heavy peaks
heavy peaks
normal peaks
X
− set − upA X
ið1−10Þ
Xið1−10Þ
− set − upC X
normal peaks
ið1−10Þ
ið1−10Þ
heavy peaks
heavy peaks
normal peaks
The division of the sum of the intensities of the labelled
peaks by the sum of the intensities of the unlabelled peaks led
to the highest values for set-up B and the lowest values for setup A. The values for set-up C were expected to be between
those for set-ups A and B. If they are closer to those of set-up
A than set-up B, the quotient is less than 1, and the strain is
considered to be susceptible. For resistant strains, the difference between the results from set-up C minus the results from
set-up A is greater than the difference between the results from
set-up B minus the results from set-up C, leading to a quotient
greater than 1. The figures were created using Microsoft Excel
2010.
Conventional susceptibility testing
MIC values were determined by the Etest, which was performed and interpreted according to European Committee on
Antimicrobial Susceptibility Testing (EUCAST) recommendations [EUCAST Disk Diffusion Method for Antimicrobial
Susceptibility Testing—Version 3.1 (April 2013)]. In brief,
Mueller–Hinton agar plates (bioMérieux, Nürtingen,
Germany) were inoculated with 0.5 McFarland standard bacterial suspensions. Etest strips (Liofilchem, Roseto degli
Abruzzi, Italy) were placed on the plates within 15 min after
inoculation, and the plates were incubated at 35 °C and 5 %
CO2 for 18 h. The MIC value was determined at the intersection of the elliptical growth margin with the Etest strip.
Results
Detection of mass shifts
First, to identify the mass shifts that occur due to the incorporation of the labelled lysine, ten P. aeruginosa strains (nine
clinical isolates and the P. aeruginosa ATCC strain 27853)
were incubated in DMEM containing either normal lysine or
labelled lysine for 3 h. Subsequently, the spectra were recorded and visually analysed for peak shifts resulting from the
incorporation of the labelled lysine into bacterial proteins and
peptides. Within a mass range of 2 to 10 kDa, 18 to 20 peak
shifts (depending on the isolate) occurred that were easily
assessable by a visual analysis (sufficient peak intensity, low
background noise). The difference between the native peaks
and their corresponding labelled peaks ranged from 16 to
64 Da. Because the incorporation of one labelled lysine into
a protein should result in a peak shift of approximately 8 Da,
we concluded that two to eight labelled lysine molecules were
incorporated into the detected protein biomarkers. The appearance of double or triple peaks was observed in many peak
shifts (Fig. 1), presumably because the unlabelled lysine cannot be completely removed from the cell or due to de novo
synthesis of lysine in the bacterial cell [14]. As a consequence,
most proteins contain a mixture of labelled and unlabelled
lysine molecules.
A growth curve-dependent analysis was performed to assess whether the growth phase of the bacteria used for inoculation into the labelled medium has an impact on the integration of the provided amino acids into bacterial proteins. The
P. aeruginosa ATCC 27853 strain and a clinical isolate of
P. aeruginosa were collected from a broth culture during the
lag phase (90 min), early (180 min), exponential (300 min)
and late growth (360 min) phases, and the stationary phase
(660 min), and re-inoculated into the labelled medium and
incubated for 180 min. The analysis of the peak profiles
obtained did not show any growth phase-related differences
in the incorporation of the labelled amino acid (Supplemental
Figure S1).
Establishment of a protocol for antibiotic susceptibility testing
In the next step, bacteria were incubated in three different setups: A, without labelled lysine; B, with labelled lysine; and C,
with labelled lysine and antibiotic. The peak profiles obtained
from the set-up with the antibiotic were compared to their
reference spectra, and the strains were defined as susceptible if
their peak profile was similar to that of set-up A or as resistant
if it was similar to the spectrum obtained from set-up B. To
determine the minimal incubation time needed for a clear
discrimination between susceptible and resistant strains, four
P. aeruginosa (two resistant and two susceptible) isolates
were tested per antibiotic, and spectra were recorded after an
incubation time of 90, 120 (data not shown), 150 and 180 min
(Fig. 2). A visual analysis of the spectra revealed that some
peak shifts occurred earlier than others, which might be due to
different turnover rates of the mainly ribosomal proteins detected by MALDI-TOF MS. The most rapid results were
obtained with tobramycin within 90 min; conversely,
180 min was required to distinguish between meropenemresistant and -susceptible isolates because a significant incorporation of the labelled lysine occurred even in the susceptible
isolates. To minimise this effect, we added a 30-min pre-
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Eur J Clin Microbiol Infect Dis (2014) 33:949–955
Fig. 1 A representative peak
shift in the matrix-assisted laser
desorption/ionisation time-offlight mass spectrometry
(MALDI-TOF MS) spectrum of
Pseudomonas aeruginosa: a
P. aeruginosa grown in a medium
containing native lysine; b
P. aeruginosa grown for 2.5 h in a
medium containing 13C-15 Nlabelled lysine. The incorporation
of the labelled lysine resulted in 2
new peaks at 5244.608 Da and
5252.747 Da
incubation period to allow meropenem to take effect
before adding the labelled and unlabelled lysine. Using
ciprofloxacin, sufficient discrimination between the resistant and susceptible strains was achieved after
150 min. Double and triple peaks were observed in all
the set-ups containing labelled amino acids, but this
effec t ap pe are d to be mor e pr omin en t in th e
ciprofloxacin-containing set-ups. Tests of four
P. aeruginosa strains were performed in parallel using
bacteria grown on Columbia blood and MacConkey
agar, and no differences in the test performance related
to the growth conditions were observed (Supplemental
Figure S2).
Establishment of an algorithm for software-assisted analysis
For the development of an automated algorithm that allows for
a rapid and operator-independent evaluation of the spectra,
species-specific peak shifts were identified that appeared to be
particularly suitable for discrimination between susceptible
and resistant isolates. These peak shifts were chosen based
on the following criteria: (i) good reproducibility for each
strain and all three antibiotics, (ii) sufficient peak intensity of
the labelled and unlabelled peaks, (iii) early appearance of the
mass shift and (iv) distinctive cessation of labelled lysine
incorporation if the antibiotic inhibits bacterial growth. By
applying these criteria, ten peak shifts in a mass range between
2 and 10 kDa were selected (Table 1).
Evaluation of the established method
The novel test and criteria established for the automated
interpretation of the recorded spectra were evaluated by testing a total of 90 strains of P. aeruginosa. The validation set
comprised 30 strains with different MIC values per antibiotic
(15 in the susceptible range and 15 in the resistant range,
according to EUCAST breakpoints). Following an incubation
time of 2.5 h for ciprofloxacin and tobramycin and 3 h for
meropenem (including the pre-incubation time), spectra were
recorded in triplicate, and mean values for the peak intensities
were calculated and processed according to the described
protocol (Fig. 3). The MS resist assay allowed for the correct
interpretation into susceptible and resistant strains compared
to conventional testing. This was true even for isolates showing MIC values close to the breakpoints, though no correlation
with the MIC values was obtained.
Eur J Clin Microbiol Infect Dis (2014) 33:949–955
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Fig. 2 Representative spectra
obtained from P. aeruginosa
incubated in a medium
supplemented with labelled lysine
and either ciprofloxacin,
meropenem or tobramycin. The
spectra of resistant and
susceptible isolates acquired after
90, 150 and 180 min are shown.
The mass peaks used for the
automated data analysis are
indicated by the grey lines
(unlabelled peaks) and the broken
lines (labelled peaks). The
labelled peaks occurring in the
susceptible isolates within the first
90 min of incubation are marked
with an arrow
Discussion
Thus far, susceptibility testing using MALDI-TOF MS technology has only been possible to a limited extent, e.g. the
detection of β-lactamase activity in bacterial isolates or the
determination of bacterial clones associated with the resistance to certain antibiotics. In contrast, the method described
here does not detect a distinct mechanism of resistance, yet
Table. 1 Ten peak shifts that were identified by visual analysis as being
suitable for automated evaluation
Peak no.
P. aeruginosa
biomarker mass
native [Da]
P. aeruginosa biomarker
mass with 13C-15 N-labelled
lysine [Da]
1
2
3337
4545
3353
4577
3
4
5
6
7
8
9
10
4996
5212
5737
6048
6350
6677
7205
9091
5020
5244
5793
6104
6382
6709
7245
9155
does measure metabolic activity in the presence of antibiotics.
Hence, this technique has the potential to allow for the susceptibility testing of a much wider range of antimicrobial
substances and any microorganism that can be cultured
in vitro. This technique relies on the analysis of characteristic
peak shifts resulting from the incorporation of isotopic labels
into the proteins of viable bacteria; thus, the presence of an
effective antibiotic will inhibit the appearance of these peak
shifts.
An approach based on the incubation of bacteria in a
completely 13C-labelled medium to detect antibiotic resistance
has been suggested very recently [15]. Our group demonstrated
in a first proof-of-concept study that the use of stable isotopelabelled amino acids is feasible for the detection of methicillinresistant Staphylococcus aureus (MRSA) [13]. The present
analysis focuses on Gram-negative bacteria and was intended
to further investigate the applicability of this concept to a wider
range of antibiotic substances. We hypothesised that the mechanism of action of the antibacterial agent has a significant
influence on the performance of the test. Indeed, we observed
that meropenem, which exerts its action by interfering with cell
wall synthesis, showed a slightly delayed effect in our assay.
However, the limiting factor was not the time needed for the
incorporation of the labelled amino acids into cellular proteins
but the halt of protein metabolism in isolates susceptible to the
tested substance, as a significant incorporation of labelled
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Eur J Clin Microbiol Infect Dis (2014) 33:949–955
Fig. 3 A total of 30
P. aeruginosa isolates were tested
for each antibiotic. The isolates
testing susceptible according to
the new assay exhibited values
smaller than one, whereas
resistant isolates exhibited values
greater than one. For comparison,
the MIC values obtained from a
conventional Etest are displayed
amino acids was still observable within the first 90 min of
incubation. We could minimise this effect by adding the labelled lysine after a pre-incubation time of 30 min, which
allowed the antibiotic (meropenem) to act.
Lysine was used as the isotopic labelled amino acid because
it is known to be highly prevalent in ribosomal proteins [15,
16]. Any amino acid may be applied, but different peak shifts
will be obtained. The proposed algorithm allowed for an
unbiased semi-automated evaluation after an incubation time
of 2.5 to 3 h. We selected species-specific peak shifts for this
algorithm, which resulted in a particularly good discrimination
between resistant and susceptible isolates. However, a potential
drawback of this type of data processing might be that it
requires a pre-defined set of label- and species-specific peak
shifts prior to testing. MALDI-TOF MS for species identification is a technology that has already been optimised for the
detection of peptides and proteins [16], thereby allowing for
the acquisition of the spectra in the same mode that is used for
bacterial identification. No special calibrations are needed,
which makes this technique even more attractive for routine
laboratory use.
In this study, we were able to demonstrate that the MS resist
assay is capable of distinguishing between susceptible and
resistant strains; however, as with many methods for the rapid
detection of resistance, e.g. all genotypic tests, no correlation
with MIC values was obtained [17]. Further studies should
address whether refinement of the method in terms of the
experimental conditions and data analysis can overcome this
potential drawback. Bioinformatics that take into account the
double and triple peaks occurring with the labelled amino
acids (e.g. the measurement of the area under the curve) may
be one approach. In terms of spectrum acquisition, the relative
quantification of biomolecules is an ongoing issue [18, 19];
indeed, nonlinearity in the relationship between the relative
intensity and concentration of the analyte is observed predominantly in complex samples. Therefore, it is of great relevance
to keep the samples as simple as possible. Furthermore, for a
correct quantification, it is essential to measure the analyte
within its dynamic range, which might be an important aspect
when selecting the biomarkers used for the evaluation algorithm. Measurements at different dilution steps may also be
helpful for remaining within the linear range. However, to
obtain a real correlation with MIC values, multiple measurements at different concentrations of the antibiotic in combination with a longer incubation time might be necessary. In our
assay, we focused on the detection of highly abundant ribosomal proteins, which are known to provide reproducible
spectra largely independent of the growth conditions. The
feasibility of measurements within other mass ranges for this
type of assay might be the subject of future studies.
Eur J Clin Microbiol Infect Dis (2014) 33:949–955
Potential fields of application for this novel test might
include clinical situations in which a timely diagnosis is
important and susceptibility testing of slow-growing or otherwise fastidious organisms.
Acknowledgements This study was supported by a grant from the
Bayerische Forschungsstiftung (Forschungsverbund “ForBIMed—
Biomarker in der Infektionsmedizin”) to S.S. and M.K.
Conflict of interest Katrin Sparbier, Christoph Lange and Markus
Kostrzewa are employed at the mass spectrometry company Bruker
Daltonik GmbH. The other authors declare that they have no conflicts
of interest.
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