A pilot study on fingerprinting Leishmania species from the Old World using Fourier transform infrared spectroscopy
Anal Bioanal Chem
A pilot study on f ingerprinting Leishmania species from the Old World using Fourier transform infrared spectroscopy
Andrea Hornemann 0 1 2
Denise Sinning 0 1 2
Sofia Cortes 0 1 2
Lenea Campino 0 1 2
Peggy Emmer 0 1 2
Katrin Kuhls 0 1 2
Gerhard Ulm 0 1 2
Marcus Frohme 0 1 2
Burkhard Beckhoff 0 1 2
0 Global Health and Tropical Medicine Center (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa , Rua Junqueira 100, 1349-008 Lisbon , Portugal
1 Division of Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences , Hochschulring 1, 15745 Wildau , Germany
2 Department 7.2 Cryophysics and Spectrometry, Physikalisch-Technische Bundesanstalt , Abbestr. 2-12, 10587 Berlin , Germany
Leishmania species are protozoan parasites and the causative agents of leishmaniasis, a vector borne disease that imposes a large health burden on individuals living mainly in tropical and subtropical regions. Different Leishmania species are responsible for the distinct clinical patterns, such as cutaneous, mucocutaneous, and visceral leishmaniasis, with the latter being potentially fatal if left untreated. For this reason, it is important to perform correct species identification and differentiation. Fourier transform infrared spectroscopy (FTIR) is an analytical spectroscopic technique increasingly being used as a potential tool for identification of microorganisms for diagnostic purposes. By employing mid-infrared (MIR) spectral data, it is not only possible to assess the chemical structures but also to achieve differentiation supported by multivariate statistic analysis. This work comprises a pilot study on differentiation of Leishmania species of the Old World (L. major, L. tropica, L. infantum, and L. donovani) as well as hybrids of distinct species by using vibrational spectroscopic fingerprints. Films of intact Leishmania parasites and their deoxyribonucleic acid (DNA) were characterized comparatively with respect to their biochemical nature and MIR spectral patterns. The strains' hyperspectral datasets were multivariately examined by means of variance-based principal components analysis (PCA) and distance-based hierarchical cluster analysis (HCA). With the implementation of MIR spectral datasets we show that a phenotypic differentiation of Leishmania at species and intra-species level is feasible. Thus, FTIR spectroscopy can be further exploited for building up spectral databases of Leishmania parasites in view of high-throughput analysis of clinical specimens.
Fourier transform infrared spectroscopy; Hierarchical cluster analysis (HCA); Principal components analysis (PCA); Leishmania; DNA; Multivariate differentiation
Leishmaniasis is a parasitic disease caused by intracellular
protozoan parasites belonging to the genus Leishmania,
family Trypanosomatidae and are transmitted to mammals
through the bite of female phlebotomine sand flies. These
protozoa comprise approximately 53 species with an ongoing
debated taxonomical structure. Currently 31 species are
parasites of mammals and at least 21 Leishmania species are
known to cause disease in humans [
Over the past 20 years, the existence of natural hybrids is a
result of genetic recombination between different Leishmania
species such as L. panamensis/L. braziliensis, L. braziliensis/L.
peruviana, and L. major/L. infantum described in [
Depending on the parasite species and the immune system of
the host, the disease can cause different clinical forms ranging
from localized self-limiting and self-healing cutaneous lesions –
cutaneous leishmaniasis (CL) – to visceralizing infections – i.e.,
visceral leishmaniasis (VL), which is fatal if left untreated [
Depending on the Leishmania species, the disease can be
zoonotic or anthroponotic. Frequent animal hosts are dogs and
other canids, rodents, hyraxes, and marsupials and, more
recently, cats [
]. The World Health Organization (WHO)
states in its Fact Sheet [
] leishmaniasis have more than 90
different sand flies capable of transmitting Leishmania.
Leishmania infections can also be transmitted via contaminated
] and potentially from mother to child [
Leishmaniasis occurs in more than 98 predominantly
tropical and subtropical countries on four continents with an
estimated number of new cases of 0.7–1.2 million for cutaneous
leishmaniasis (CL) and 0.2–0.4 million for visceral
leishmaniasis (VL) per year, with an overall prevalence estimated to be
12 million [
Biological and genetic traits of both host species and
Leishmania strongly determine how the disease will evolve.
Thus, a correct identification of the parasite(s) is essential, as it
may have implications for diagnosis, epidemiology, treatment,
and control of the disease [
1, 14, 15
The most common diagnostic method for leishmaniasis is
the detection of Leishmania amastigotes (non-flagellated
intracellular forms) by microscopic observation in Giemsa
stained tissue biopsies of infected patients. Additional evidence
of an infection is the presence of Leishmania parasites in
cultures inoculated with suspected biological tissue samples.
However, these methods are not always successful and lack
differentiation capacity, as Leishmania species cannot be
distinguished morphologically. In the 1980s, isoenzyme analysis, also
called multilocus enzyme electrophoresis (MLEE), became the
‘gold standard’ for typing Leishmania at the species and
intraspecies levels [
]. MLEE needs cultured parasites, which is
labor-intensive, time-consuming, and can only be performed in
specialized laboratories. Moreover, it has limitations concerning
discriminatory power [
]. Currently this technique is being
complemented and will likely be replaced in the future by
molecular approaches. These approaches are based on the detection
of parasitic DNA in clinical material or from cultured parasites
amplified by polymerase chain reaction (PCR). Fragment size
or sequence analysis of the PCR amplicons enables further
characterization as well as species and strains discrimination.
Most commonly used methods are restriction fragment length
polymorphism (PCR-RFLP) analysis and sequencing of single
markers or multilocus sequence typing (MLST) as well as
multilocus microsatellite typing (MLMT) [
1, 13, 20, 21
In developing countries, where usually simple and
inexpensive techniques are required, the need for trained personnel
and well equipped laboratories still comprises a huge obstacle.
Thus, cost-effective and simple methods for the early-stage
diagnosis and parasite identification are needed [22–24]. To
overcome these obstacles, optical methods such as vibrational
spectroscopy using infrared radiation in the mid-infrared
(MIR) spectral range can be implemented. For rapid and
accurate identification as well as discrimination of
microorganisms at the genus, species, and strain level, only small sample
amounts without any complex manipulation are required [25,
26]. Fourier transform infrared spectroscopy (FTIR)
instrumentation is available in highly equipped laboratories;
however, the sample preparation can also take place in low
resource settings with an easy transfer of the substrate slides to
the specialized ones. FTIR spectroscopy allows the sensitive
and noninvasive analysis of IR light interaction with a
molecule, and hence functional groups can be determined by
absorption, emission, or reflection profiles.
This methodology in combination with various
multivariate statistic analysis tools could be successfully implemented
for distinct identification and differentiation of biological
microorganisms such as bacteria [27, 28], e.g., for taxonomic
differentiation of Lactobacilli  or Streptomyces .
FTIR supported by artificial neural network (ANN) analysis
has shown its potential for accurate discrimination of Listeria
strains . FTIR is an easily accessible, label-free, and
potentially powerful tool for studies on Leishmania parasites.
The data acquisition in the MIR window reflects the overall
composition that often differs with respect to their molecular
make-up, thus providing unique fingerprint signatures for
differentiation. An initial approach to infrared (IR) data-based
differentiation of parasite sample films of three Leishmania
species has been conducted successfully by Aguiar et al.
. Further, studies on malaria parasites have been reported
. However, as there is an insufficient number of IR-based
approaches on identification of protozoan parasites, further
studies are needed in order to elucidate their biochemical
diversity under the implementation of computerized
chemometric tools. Our systematic pilot study in the MIR spectral region
from 3900 to 400 cm–1 is the first step to achieving this
ultimate goal by unraveling the molecular composition and
complexity of the selected Leishmania strains, the hyperspectral
datasets of which were analyzed by supervised and
unsupervised multivariate statistics tools, namely principal
components analysis (PCA) and hierarchical cluster analysis
(HCA). A scheme on this approach is illustrated in Fig. 1.
The aim of this work is to use several Leishmania species
and species-hybrids, such as L. infantum, L. major, L. tropica,
L. donovani, L. tarentolae, L. infantum/L. major hybrids, and
their molecular building blocks, such as their DNA, to assess
the efficiency of FTIR in terms of reliable and comparative
high-throughput analysis, especially in view of diagnosis. In
association with chemometric tools , it is shown that FTIR
methodology accurately discriminates structural patterns of
spectral datasets and elicits a specific hierarchy that may help
to achieve a better understanding of complex biological
systems diversity and the associated biochemical nature.
The applicability of FTIR spectroscopy as a routinely
implementable bioanalytical tool based on robust and
be studied, enabling discrimination by characteristic modes and their
intensity variations (c). Multivariate analysis tools enable
varianceweighted cross-correlation of MIR fingerprints (PCA) and
distancebased species differentiation (HCA) for studying the multi-dimensional
reproducible MIR spectral features of various Leishmania
samples will be presented. Our pilot study focuses on the
evaluation of MIR fingerprints in order to build up spectral
databases enabling identification of the causative species/
strains of Leishmania infections in the near future.
Materials and methods
Several strains and clones of the humanpathogenic species
Leishmania infantum, L. major, L. donovani, L. tropica, and
L. infantum/L. major hybrids were included in this study, in
addition the non-pathogenic species L. tarentolae (Table 1).
The hybrid L. infantum/L. major strains were reported for the
first time by Ravel et al. in Portugal [
Cultivation of Leishmania strains
Leishmania strains were maintained in cell culture flasks
(Sarstedt) with M199 medium (Sigma Aldrich) supplemented
with 2.2 g/L NaHCO3, 10% fetal calf serum (BioChrom AG),
1% L-glutamine, and 0.5% penicillin/streptomycin (Sigma
Aldrich). A neutral pH was ensured by the addition of 1 M
HEPES-NaOH buffer solution (pH 6.9). The cultures were
kept in an incubator at 26 °C and every 3 to 4 d fresh medium
was added by diluting the cultures 1:5 to 1:10. The density of
promastigotes (flagellated stage of the parasites) was
Leishmania species and hybrids and respective strains used in the present study
IMT 151 Cl1
IMT 151 Cl2
LV 561 Cl1
LV 561 Cl2
LV 561 Cl3
IMT 208 Cl1
IMT 211 Cl1
VL, visceral leishmaniasis; CanL, canine leishmaniasis; CL, cutaneous leishmaniasis; n.a., not available;
a Number of parasitic film preparations
b Number of DNA film preparations
c Pathology such as for the uncloned strain
d Outgroup (Sauroleishmania)
determined by microscopy using a Neubauer improved cell
counting chamber (VWR).
Isolation of Leishmania DNA
A volume of 3–12 mL parasite culture with a density of
approximately 106 parasites/mL was used for DNA extraction.
After centrifugation (3000 rpm) for 8 min, the supernatant was
discarded and the remaining pellet was washed twice with 1
mL ultrapure water (18.2 MΩ∙cm, Merck KGaA), and
centrifuged at 3000 rpm for 8 min. Then, the pellet was resuspended
in ultrapure water and centrifuged at 3000 rpm for 8 min. The
purified pellet was redissolved in 1 mL lysis buffer (50 mM
NaCl, 10 mM EDTA, and 50 mM Tris-HCl, pH 7.4), followed
by the addition of SDS to a final concentration of 0.5% and
proteinase K (20 mg/mL) to a final concentration of 100 μg/
mL and transferred to an Eppendorf tube.
The batch for cell lysis was incubated over night at 55 °C
with moderate shaking (300 rpm) in a thermo-mixer
(Thermomixer Comfort, Eppendorf). An equal volume of
phenol/ chloroform/isoamyl alcohol (25:24:1 v/v/v) was
added and the tube was gently shaked for 2 to 3 min.
Afterwards, tubes were centrifuged at 16,000 × g for 10 min
and the aqueous phase was transferred into a new tube. This
extraction step was repeated two times. Finally, an equal
volume of chloroform-isoamyl alcohol (24:1, v/v) was added,
gently mixed, and centrifuged as previously. The aqueous
phase was removed carefully to a new tube and 1/10 volume
of 3 M sodium acetate and an equal volume of isopropanol
were added for DNA precipitation. After mixing gently, the
tubes were kept overnight at –20 °C. After centrifugation at
16,000 × g for 30 min, the supernatant was carefully discarded
and the DNA pellets were thoroughly washed twice by
addition of 0.5 mL 70% ice-cold ethanol and centrifugation at
16,000 × g for 15 min. The reaction tubes were kept open
for complete ethanol evaporation. Finally, DNA was dissolved
in 15–25 μL of ultrapure water for several hours in a
thermomixer at 42 °C and at 300 rpm. DNA concentrations and
quality were determined with a spectrophotometer
(NanoDropTM 1000; Thermo Scientific). Details can be
found in Fig. S1 in the Electronic Supplementary Material
Sample preparation of parasites and DNA films for FTIR analyses
Critical steps that may have impact on the yield were
harvesting and washing Leishmania parasites from cultures,
application onto the optical window, and thorough drying of the intact
parasite samples into films. Another critical issue is the
different growth kinetics of Leishmania promastigotes in culture, as
variations in growth rate may result in unequal final
concentrations of cells. Therefore, all samples were collected
between the 4th and 5th d in culture to ensure a constant
concentration of the parasites. Promastigote cultures of each
strain were adjusted to equal concentrations for application
onto the FTIR windows and to avoid large film thickness
variations during FTIR assessments. However, small
variations in film thickness could be mitigated by statistical
considerations (i.e., by applying standard deviation, arithmetic
mean, and multivariate methodologies). A volume of 0.5–1
mL of culture medium containing a defined density of 106
parasites/mL was considered to be the minimum sample
a m o u n t n e e d e d f o r r e l i a b l y c o n d u c t i n g i n f r a r e d
spectroscopical measurements. These parasite culture
suspensions were centrifuged at 1000 × g for 8 min. The supernatant
was eliminated thoroughly and the respective pellet was
washed three times with centrifugation at 1000 × g for 8 min
with a saline solution (0.9 % NaCl) and, finally the pellet was
resuspended in 20 μL of ultrapure water.
From this batch, about 2 μL droplets were pipetted (three
times in total at the same place) onto reflective MirrIR
lowemissivity (“low-e”) microscope slides (Kevley Technologies)
and air-dried, in order to guarantee a dense homogeneous
sample film for FTIR spectroscopic analysis.
In the same way, several sample droplets of the respective
isolated DNA were prepared onto low-e-slides utilizing a
hotplate for drying at 30 °C.
FTIR spectroscopy – experimental setup and data acquisition
Absorbance spectra of intact Leishmania films and their
related DNA were recorded in reflection geometry in the MIR
spectral range between 3900 cm–1 and 900 cm–1. This was
done using a Vertex 80v FTIR spectrometer (Bruker Optics
GmbH) to which a FTIR Hyperion 3000 microscope was
coupled. The spectrometer was fitted with a KBr beamsplitter
and a globar was implemented as a radiation source for
For data acquisition, a lN2-cooled multi-element mercury
cadmium telluride detector, a so-called focal plane array
(FPA) detector with 1282 pixel elements and a spectral
resolution of 4 cm–1 was used. Micro-spectroscopic experiments
on sample films were conducted with a Cassegrain objective
at a 15× magnification, enabling the study of a sample area of
3452 μm2 at approximately 2.87 μm lateral resolution; the
latter corresponds to the dimension of one single pixel.
Each spectrum was collected with the Opus software v.7.2
(Bruker Optics GmbH) and consisted of 512 averaged scans
for parasite sample films. For DNA films, 128 averaged scans
were accumulated. All interferogram scans were submitted to
a Blackman Harris 3-term window function and to a
zerofilling factor of 2 prior to Fourier transformation.
Background scans were collected prior to each sample
measurement from a region free of samples, here on a clean
low-eslide, and rationed against the sample spectrum.
For the approval of instrumental invariance the Leishmania
strains were measured in duplicate. For the DNA measurements,
one to two spectral datasets comprising 480 spectra,
respectively, were analyzed (Figs. S2 and S3, ESM). Despite a low
number of DNA replicates the datasets display a similar mutual
statistical variability being reflected by boxplots (Fig. S3,
ESM). These boxplots (highlighted in green, blue, and red)
correspond to the number of sample film preparations
(Table 1). The strains L. infantum IMT 151, L. donovani BD
09, the hybrids L. infantum/L. major IMT208 Cl1 and IMT211
Cl1, and L. tarentolae were prepared one further time and
measured again, finally resulting in three sample preparations for
Duplicate and, when possible, triplicate preparations of the
Leishmania species were conducted to verify that preparation
steps for the cultivation of the Leishmania species and
PCRbased procedures were reliable and consistent.
Univariate- and multivariate data analysis
The FTIR micro-spectroscopic datasets were subjected to the
imaging software CytoSpec v.1.4.03 and cut to the 3900–900
cm–1 spectral range. Afterwards, a baseline correction
(polynomial fit procedure of 3rd order, 6–7 correction points) of areas
that comprised 420 spectra per sample system in the case of the
parasite film data, and 480 spectra per sample system in the
case of the DNA films, were further analyzed. The different
number of scans for parasite and DNA sample systems was
taken to ensure optimal peak-to-noise ratios. We also paid
attention to recording spectra in regions with approximately the
same thickness, which can be explained by the different
number of spectra taken from the parasites and DNA sample films
datasets. Furthermore, this optimization equally included a scan
number adaptation to the sample mass depositions of the
sample areas which comprised the functional groups of interest.
The data processing software Origin 9.0G was
implemented for analysis on spectral averages and their corresponding
standard deviations. Normalization of the spectral datasets
between 0 and 1 was performed for the sake of a better
comparison in the spectral range 3900–900 cm–1. For the
determination of prominent bands, peak analysis (with a threshold
height of 5%–10%) on spectral averages of the respective
Leishmania strain was performed in Origin 9.0G, as well as
the construction of boxplots. For boxplot construction, 100
normalized spectra for the respective sample system were
considered (ESM, Figs. S2 and S3). The selection of these 100
spectra was a random choice to get a full picture of their
statistical distribution. We utilized the boxplots to display
the variation and statistical distribution of the MIR datasets,
which can be considered as a statistical population. The
boxplots are divided into five points, the median, two
quartiles, and the minimum and maximum of all the data. The
position of the median provides information about the
existence of the symmetry or skewness of a distribution.
For the multivariate analysis, the software Matlab R2012a
and Toolboxes Stats Toolbox (Mathworks) and PLS Toolbox
(Eigenvector Research Inc.) were used. Multivariate analyses
were performed in diverse spectral windows in two different
(1) Principal components analysis (PCA) was performed by
applying data pretreatments such as vector-normalization,
mean-centering, and 2nd derivatives with the help of the
Savitzky-Golay-algorithm and five smoothing points. PCA
was carried out in diverse spectral windows and in combinations
of the latter for elucidating the highest differentiation capability
among the respective parasites and DNA datasets (Table 2). The
loadings spectra and scores were calculated in Matlab R2012a.
The spectral loadings were plotted in Origin 9.0G.
(2) Agglomerate hierarchical cluster analysis (HCA) was
performed by applying the Euclidean distance measure and
Ward’s algorithm. For HCA, 20 datasets of the score matrix
per sample were used. Second derivative IR spectra to
calculate spectral distances were found to be useful for hierarchical
Results and discussion
Univariate studies – chemical analyses on intact parasite films and DNA
The main critical step for reliable spectroscopic analysis on
Leishmania parasite films entailed the thorough elimination of
the culture medium. Therefore, several washing steps were
performed, as remaining additives may cause competitive
spectral contributions. For instance, vibrational spectroscopic
features from fetal calf serum, as one of the main components
in the culture medium, may coincide with parasitic
proteinogenic amide modes.
To evaluate the reliability and robustness of FTIR spectral
datasets of the six strains, L. infantum IMT 151, L. infantum/
L. major hybrids IMT 208 Cl1 and IMT 211 Cl1, L. donovani
BD 09, L. tropica LCR-L881, and L. tarentolae were
considered. As FTIR data must show a Gaussian distribution for
submitting them to multivariate statistics, their consistency
was verified and approved with the help of boxplots of the
respective sample film preparations (Figs. S2 and S3, ESM).
Furthermore, the resulting spectra of remeasurements (i.e.,
a second measurement of the same sample films but in another
region of interest) were compared with the spectra from the
previous acquisition using PCA and were found to be very
similar, that is, clustering closely together and indicating
proper reproducibility of the instrumental setup (data not shown).
In addition, boxplot analyses (Figs. S2 and S3, ESM) display
high similarities within the film preparations of the respective
parasites and their DNA (this was tested with all 18 strains and
2–3 sample preparations per strain), and reproducibility of
their spectral datasets could be successfully envisaged.
Leishmania parasite films
Spectral datasets with their standard deviations (represented in
gray envelopes, Fig. 2) of the parasite films of all 18 strains are
listed in Table 1. Included therein are different species as well
as different strains of species resembling the position of bands
and forms typical of IR spectral datasets of whole
microorganisms such as bacteria [35, 36].
Also for diagnostics, dried films of body fluids, serum
proteins, or other fluid specimens are convenient for routine
FTIR-based probing [37, 38]. Subsequently, the difference in
several bands related to the corresponding functional groups
will be discussed for the sample films of the respective
FTIR spectra obtained from Leishmania parasite samples
exhibited characteristic molecular fingerprints, and the
replicates indicated good reproducibility for each sample
preparation (Figs. S2 and S3, ESM).
Figure 2 illustrates the calculated arithmetic means of 420
spectra for the respective sample hyperspectral dataset, which
comprise a huge number of approximately 6,532,000 data
points in total. IR datasets display a high similarity among
all parasitic strains. Previous investigations on three very
similar IR datasets of L. amazonensis, L. chagasi (or L. infantum),
and L. major parasitic strains  have shown spectral
differences between these species allowing species discrimination/
typing in the regions of polysaccharides, fatty acids
(phospholipids), nucleic acids, and proteins (amides). Indeed, modes
from molecular constituents such as polysaccharides, nucleic
acids, amino acids, lipids, and proteins can be detected for the
Leishmania sample film datasets. Similar to what was
observed for other microorganisms such as bacteria in the MIR
spectral window [
]. We also noticed spectral differences for
L. infantum IMT 373, L. tropica LCR L881, and L. tarentolae
in comparison with the remaining strains in these depicted
wavenumber regions. For instance, we can observe the
presence/absence of a shoulder at about 1739 cm–1 originating
from the amide II mode region (Table 3) for all investigated
strains. Table 3 highlights main bands observed in the
Leishmania spectra in the respective wavenumber regions,
together with associated functional group modes, which can
be expected from microorganisms according to Helm et al.
. The fatty acid region (3000–2800 cm–1) comprises
CH3, CH2, and CH stretching vibrations of functional groups
that can be found in cellular membranes. In the 1800–1500
cm–1 spectral range, amide bonds can be identified, which
include vibrations of carboxyl, carbonyl, and ketone groups
of various proteins and peptides. In this spectral region, amide
I and amide II bands can be found at about 1650 cm–1 and
1550 cm–1, respectively [
]. Bands of high intensity occur in
the wavenumber window 1580 cm–1–1465 cm–1. The
socalled ‘mixed region’, in which vibrations of proteins, lipidic
acids, and phosphate compounds can be identified, is located
at wavenumbers 1500 cm–1–1200 cm–1. Modes in the spectral
Observed bands / cm-1
3296, 3296, 3292
3220, 3135, 3000, 2930,
2885, 2850, 2798, 2779
region between 1200 cm–1 and 900 cm–1 refer to vibrations of
polysaccharides originating from the membrane surface of
Leishmania parasites (Table 3). These band intensities differ
slightly among the species complexes (i.e., L. major with
L. tropica) and within one species (L. major LV561 Cl2 with
L. major LV561 Cl3).
The spectral fingerprints comprise vibrational modes
assigned to compounds that originate from different cell
organelles. Aside from typical eukaryotic organelles such as the
Golgi apparatus, endoplasmatic reticulum, and mitochondria,
Leishmania possess a flagellum (promastigote culture forms)
and a kinetoplast that comprise DNA in the form of maxi- and
]. The plasma membrane of Leishmania is
comprised of a glycocalyx, which entails glycoconjugates
t h a t a r e a n c h o r e d t o t h e p l a s m a m e m b r a n e v i a
glycosylphosphatidylinositol (GPI). The glycoconjugates are
glycoproteins, in particular proteophosphoglycan (PPG) and
zinc-metalloprotease GP63, also called leishmanolysin and
glycolipids, whereas lipophosphoglycan (LPG) is the most
]. The LPG of Leishmania promastigotes
play key role in the parasite’s survival in both the insect host
being responsible for the docking to the sand fly intestine or
also in mammalian hosts, by decreasing phagosome fusion
properties at the onset of infection in macrophages [
PPG, on the other hand, is known to protect Leishmania
parasites from hydrolases during the sand fly’s blood meal [
Leishmanolysin GP63 also prevents complement-mediated
lysis and plays a key role with respect to the virulence of
Leishmania parasites [
]. Glycosylation of membrane
components results in vibrations mainly occurring in the
polysaccharide region. The phosphate compounds in the ‘mixed
region’ are derived from phospholipids of the plasma
membrane and DNA and RNA molecules.
In all spectra, deviations of the modes in the 3220 cm–1 and
1660 cm–1 spectral range, with respect to the presence/absence
of bands and differences in band intensities can be observed.
The mode at 1660 cm–1 displays a neighbored shoulder of the
amide I band. Basically, this mode, which is located between
1710 and 1750 cm–1, can be assigned either to C=O vibrations
from esters, which may occur in lipids or to C=O stretching
modes originating from proteins. In this case, and as it is
expected from micro-organisms usually containing more
protein than lipid-related components, we assign this shoulder to
the Amide I mode, which actually entails C=O stretching
vibrations from amino acid side-chain contributions (Table 3)
]. As bonds to atoms with a strong electronegativity of the
ester group may cause a band shift , different molecules
that can be found in Leishmania may consequently cause a
varyingly strong peculiarity of this mode. The region at 3220
cm–1 refers to stretching vibrations of adsorbed water
molecules and NH residues.
These findings may be a first indication for the feasibility
of using reflective FTIR micro-spectroscopy to study diverse
sample films of parasitic cultures with respect to their
discrimination capability, especially in the spectral region of
polysaccharides, fatty acids (phospholipids), nucleic acids, and
Leishmania DNA films
The DNA profile of each single Leishmania strain can be
considered a molecular fingerprint that reflects the parasite’s
evolution. It is characteristic to a large extent, and can be
exploited for classification of parasites by genotyping. This
is an important aspect, as some of the Leishmania species
are known to be clinically pleomorphic [
]. For this reason,
we also conducted a comparative IR spectroscopic analysis on
Figure 3 displays the calculated arithmetic means of 480
spectra per sample hyperspectral dataset, which comprises in
total about 7,464,000 data-points. The DNA spectra of the
different Leishmania strains contain typical modes at 2925
cm–1 CH and at 1043 cm–1, where the C-O-stretching
vibrations of the ribose group (Table 4) can be observed [
different type and/or amounts of bases for the respective
Leishmania strain also result in different intensities of infrared
absorption. The broad spectral region between 3500 cm–1 and
3000 cm–1 can be attributed to stretching vibrations of water
and NH molecules [
]. Furthermore, the DNA data show
modes at 1650–1610 cm–1 and at about 1500 cm–1 that can
Fig. 3 MIR spectral fingerprints including arithmetic means (each
spectral fingerprint is the arithmetic mean of 480 spectra) and standard
deviations (gray envelopes) of the Leishmania DNA films of the 18
studied strains. Analysis was performed in the 3900–900 cm–1 spectral
region at 4 cm–1 spectral resolution
be assigned to in-plane vibrations of cytosine, and stretching
vibrations of the thymine ring at 1575 cm–1 (Table 4) [
The IMT 151 and IMT 208 datasets are mutually very similar
among themselves, apart from the signatures of IMT 373, IMT
211 Cl 1, LV561, BD 17, and BD 09, the latter of which
comprise further modes in the spectral range between 1550
cm–1 and 1708 cm–1. Moreover, modes in the spectral range
between 1550 cm–1 and 1300 cm–1 can be assigned to in-plane
vibrations of residues of DNA bases and out-of-plane
vibrations (800 cm–1–760 cm–1). Modes at approximately 1225 cm–
1 and 1090 cm–1 refer to the antisymmetric and symmetric
PO2stretching vibrations, which are more pronounced in some
DNA spectra (Fig. 3) than in those of parasite films (Fig. 2).
Differentiation of intact Leishmania parasites and their
DNA by PCA
Based on the preliminary univariate results, this study
focusses now on multivariate differentiation of Leishmania
strains for which PCA was implemented. As
vectornormalization to all datasets was conducted, the intensities in
all spectral data were coherently scaled to 1. So, only spectral
differences in the respective selected wavenumber windows
(Table 2) were considered for multivariate analyses. These
comprised the highest discrimination capability, the latter of
which is reflected by the PC1 explained variances (Table S1,
ESM). Here, we tested the differentiation capability of
different Leishmania species, as well as different strains within a
species, (i.e., the inter- and intra-species variability).
For chemometric analysis of Leishmania parasite film data,
one representative strain of L. major (LV 561), L. tropica
(LCRL830), L. donovani (BD09), L. tarentolae, two strains of
L. infantum (IMT 373, IMT 151), and two L. infantum/ L.
major hybrid strains (IMT 208 cl1, IMT 211 cl1) were selected
in order to check differentiation power. This resulted in eight
principal components (PCs) for the eight sample datasets.
Under consideration of the wavenumber windows (Table 2)
W1 (3000–2700 cm–1) and W2 (1800–1500 cm–1), the
variance that can be explained for the first PC is 46.52% and the
corresponding total variance captured for this PC is 83.87%.
With respect to the output of the score diagrams, this
combination of wavenumber windows exhibits the best results for the
differentiation of Leishmania strains from PC1 to PC4 where
the variance explained per PC is about 80.35% at PC4 (Fig. 4a).
In PC1 versus PC2, a good separation of the L. infantum
strains IMT151 and IMT373 located in the second quadrant
from the remaining groups can be observed apart from a small
overlap of the datasets (Fig. 4a). The latter can be explained by
a similarity reflected in both datasets considered as two strains
of the same species originating from the same region
(Portugal). In addition, the same zymodeme MON-1 is known
to be genetically very homogeneous. On the other hand, a
separation between both datasets occurs in PC2 versus PC3
Intermol. bonded, water, carboxylic acid, NH
Weak, stretching vibration of ribose group
Stretching vibration from carboxylic acid
Shoulder, scissoring vibration in cytosine
Strong, in-plane vibration
Strong and broad, scissoring vibration of
adsorbed water molecules
Strong, bending and stretching vibration in adenine
Weak, in-plane vibration
Weak, stretching vibration
Weak, bendingand stretching vibration in adenine
Medium, in-plane vibrations
Weak, in-plane vibrations
Strong, antisymmetric stretching vibration
Strong, symmetric stretching vibration
Strong, stretching vibration of deoxyribose
Strong, stretching vibration; similar to band of
diethyl phosphate anion
Weak, out-of-plane vibrations
Medium to weak, stretching vibration
Medium to weak, out-of-plane bending
and even in higher principal components such as in PC2
versus PC4. A full separation from the remaining
noisecontaining residuals (all other datsets) can therefore be
achieved. This clustering can also be observed in the
dendrogram illustrated in Fig. 6.
Furthermore, for the L. infantum strains IMT151 and
IMT373 modes at approximately 2925 cm–1 and 2850 cm–1
could be assigned to fatty acid compounds that correlate with
the PC score values. These spectral contributions can also be
observed in the PC loadings spectrum (Fig. 4b). The loadings
spectrum represents the relationship between the original
spectral data space and the new PC space and hence can be
compared with the second derivative spectra, which were the
inputs for the PC analyses.
In plot PC1 versus PC3 a differentiation of the L. tarentolae
strain compared with the other strains were observed (Fig. 4a).
These results correspond with other phylogenetic studies
based on the cytochrome b and DNA polymerase alpha gene
sequences in which L. tarentolae is found in a separate cluster
]. According to the known classification of the genus
Leishmania L. tarentolae is designated to the distinct
subgenus Sauroleishmania, whereas the remaining studied species
belong to the subgenus Leishmania. A separation of the
clusters L. donovani and L. tropica was also observed. These two
species belong to different species complexes within the genus
Leishmania and the phylogenetic relationship has been proven
among others by MLEE [
] and the sequence analysis of
many genetic loci such as the SSU (small subunit) and ITS
(Internal Transcribed Spacer) region of the ribosomal DNA
, cytochrome b [
], DNA polymerase alpha and RNA
polymerase large subunit [
], heat shock protein 70
(hsp70) , and further ones addressed in refs. [
A separation of the species L. donovani and L. tropica could
be also observed and was evidenced by Mouri et al., who
conducted cluster analysis based on mass spectrometrical
datasets originating from sample pellets of promastigote
cultures . In the PC1-PC4 plots one can see that the clusters
of L. donovani and L. tropica are mainly located in the domain
with positive scores with respect to PC1 versus PC2 (and for
L. tarentolae at PC1 versus PC3), whereas the scores of the
remaining strains lie in the negative codomain. This
arrangement of clustered datasets can also be observed in the
dendrogram (Fig. 6).
This multivariate approach also entailed the analysis of the
PCA loadings spectra, the latter of which can be considered
for elucidating what type of vibrational mode contributes to
which PC. The strain of L. infantum is separated in the scatter
plots in Fig. 4a at PC1 versus PC2, in the second quadrant
from the remaining strains where negative score values in PC1
and positive score values in PC2 can be found (Fig. 4b).
Modes with a negative intensity value in PC1 and a positive
intensity value in PC2 of the loadings spectrum are
responsible for the separation of the L. infantum strains from the
IMT208 Cl1, L. donovani BD09, L. infantum IMT151, L. infantum
IMT373, L. major/L. infantum hybrid IMT211 Cl1, L. tropica
LCRL830, L. major LV561, and L. tarentolae, respectively. (b)
Corresponding loadings spectra
The bands at 2921cm–1 and 2915 cm–1 are crucial for the
separation capability between the species L. donovani and
L. tropica. The modes at around 2915 cm–1 and 1660 cm–1
(fatty acid region) are crucial for the negative correlation of
the strains L. tarentolae, L. donovani, and L. tropica in PC1
and therefore are responsible for the separation from the
The modes that are about 2920 cm–1 and 2850 cm–1 from the
fatty acid regions could be successfully implemented as indicator
bands for the discrimination of different stages of malaria
parasites by taking loadings spectra into account [
]. The strain of
L. tarentolae shows spectral differences with respect to amide I
and amide II modes at around 1660 cm–1 and 1550 cm–1 (Fig. 2).
For the analyses on DNA films the strains L. donovani
BD09, L. infantum IMT151, L. infantum/ L. major hybrid
IMT208 Cl1, L. infantum/L. major hybrid IMT211 Cl1,
L. tropica LCR-L830, L. major LV561, and L. tarentolae were
considered (Fig. 5a). Differentiation and classification of the
datasets from DNA isolates of all strains was successful in
PC1 versus PC3.
The PCA was conducted in the wavenumber regions 1750–
1450 cm–1 (B) and 1450–1250 cm–1 (C) (Table 2). For PC1 a
variance of approximately 48.09% has been documented,
together with a total variance of 86.80% (Table S1, ESM). The
scatter plots of the scores in PC1 versus PC3 display
wellseparated clusters, where a complete differentiation can be
achieved at a variance of 75.51%. In the second quadrant of
PC1 versus PC2, a cluster originating from the hybrid strain
IMT208 Cl1 can be found. The third quadrant comprises the
clustering of the L. tarentolae scores, which is separated from
the remaining strains in PC1 versus PC3, and PC2 versus PC3
in the second quadrant. Along PC1 the clusters of
L. tarentolae and L. infantum/ L. major IMT208 Cl1 are neg
atively correlated towards the other strains (cf. PC1 versus
PC2, and PC1 versus PC3). These differences between the
L. tarentolae strain and strain IMT208 Cl1 are caused by the
modes at 1608 cm–1 (NH2 and C=N, adenine) and at about
1548 cm–1 and 1423 cm–1 (NH and CH base residues). A
differentiation of the strains of L. donovani, L. infantum,
L. major, and the hybrid IMT211 Cl1 is achieved in PC1
Similar to the above discussed loadings spectra of the
Leishmania strains and the loadings data for the DNA
(Fig. 5b), information is provided on modes that contribute
to their differentiation and separation. These bands are in
the spectral window between 1750 cm–1 and 1250 cm–1.
The hybrid strain IMT208 Cl1 is separated in PC1 versus
PC2 (second quadrant) from the remaining ones (Fig. 5a),
which can be explained by spectral differences of the mode
at 1610 cm–1 (cytosine ring). Together with the hybrid strain
IMT211 Cl1 a negative correlation can be observed along
PC3, which may be due to the spectral discrepancies at
approximately 1577 cm–1 (thymine ring), 1637 cm–1
(adsorbed H2O molecules), and at 1652 cm–1 (cytosine ring).
Similarity among hybrid strains can be observed in the fourth
quadrant (PC2 versus PC3), which is due to the mode at 1565
cm–1 (NH2 and C=N, adenine). The spectral feature at about
1580 cm–1 (cytosine ring) is responsible for the differentiation
of L. tarentolae, the scores values of which are clustered in
PC1 versus PC3 and PC2 versus PC3 (second quadrant),
respectively. The separation of the L. tropica strain occurs in the
scatter plot PC1 versus PC2 (fourth quadrant), for which the
mode at 1575 cm–1 is responsible (thymine ring).
Differentiation of intact Leishmania parasites and their
DNA by HCA
To perform differentiation on the variance-weighted datasets
the distance-based HCA was implemented for building up
dendrograms, the latter of which can be compared with the
current taxonomy of Leishmania [
1, 21, 63, 64
Figure 6 displays HCA results of the studied intact
Leishmania parasites that are hierarchically clustered in a
dendrogram structure where 20 scores values for each studied
strain were considered. Leishmania tarentolae is the most
distant species and is considered a member of distinct subgenus,
whereas L. tropica and L. donovani (as well as L. infantum and
L. major) belong to the subgenus Leishmania. The HCA
illustrates that L. tarentolae, L. donovani, and L. tropica are
clearly delimitable forming separate species-specific clusters
L. infantum/L. major hybrids IMT 208 Cl1 and IMT 211, L. tropica
LCR-L830, L. major LV 561, and L. tarentolae, respectively. (b)
Corresponding loadings spectra
(based on a single strain). This also applies to L. infantum, but
here, additionally, also strain-specific sub-clusters can be
recognized, as two strains of this species were included. The
subclusters of the two L. infantum strains are separated at H~2.25
× 10–3. There is no consistent cluster for each of the hybrid
strains (IMT211 Cl1 and IMT 208 Cl1) as well as for L. major
LV561. Of notice is that the species complexes L. major and
L. tropica are not closer related to each other than to the
L. donovani complex as should be expected from DNA
sequence-based classification of the Leishmania genus. This
is due to the different band positions in the amide I and amide
II region, the latter of which are mutally closer to each other
for L. tropica and L. donovani. Also the distant phylogenetic
relationship of L. tarentolae as member of a distinct subgenus
is not reflected in the present dendrogram.
In the present study, HCA on L. major, which is the main
representative of the L. major species complex, also shows
that the score values (branches) are scattered into two different
sub-clusters. These sub-clusters are intermingled with the two
L. infantum/L. major hybrid strains. This is due to spectral
differences in the polysaccharide-, phosphodiester bond-,
and fatty acid region.
At a heterogeneity level (H) of about 6 × 10–3 the strains are
clustered into two main groups: the first group includes
L. tarentolae, L. tropica LCR-L830, and L. donovani BD09 as
well as the minor part of the scores of the hybrid strain IMT208
Cl1. Within this main group, a sub-cluster of L. tarentolae is
further separated at H~3.3 × 10–3 from the sub-cluster that
comprises L. tropica, L. donovani, and part of the hybrid IMT
208 scores. The second main group comprises L. infantum
IMT151, L. infantum IMT373, L. major LV561, and the hybrid
strains IMT208 Cl1 (majority of scores) and IMT211 Cl1. The
HCA outputs basically delineate two different main clusters,
which do not agree with the current phylogeny based on genetic
markers, e.g., of L. infantum and L. donovani that should be
closely related as members of the same species complex and also
the positions of L. tropica and L. tarentolae. The dendrogram
rather shows the potential of FTIR for species discrimation or
typing rather than a phylogenetic classification.
Concerning other spectrometric approaches, several
MALDI-TOF mass spectrometric studies have been applied
for Leishmania species discrimination [
]. Culha et al.
found species-specific spectra for the four investigated
reference strains of L. tropica, L. major, L. infantum, and
L. donovani, which subsequently were successfully used to
identify cultured clinical isolates from patients . Mouri
et al. [
] were able to generate species-specific spectra from
cultured Leishmania isolates, including also L. donovani,
L. infantum, L. major, and L. tropica. The obtained dendrogram
was consistent with the classification based on reference
methods as MLST or sequence analysis of the hsp70 gene.
So far, there is only a single published FTIR-based
approach (Aguiar et al.) that addresses, in combination with
multivariate statistic tools, the discriminatory power and
classification capability of this method tested for three Leishmania
strains representing three species, namely L. amazonensis,
L. chagasi (synonym of L. infantum), and L. major, but only
by implementing HCA .
In PCA of intact Leishmania parasites, a small overlap
between L. infantum IMT151 and IMT373, both located in the
second quadrant, was observed (Fig. 4a). This can be explained
by a similarity that is reflected in both datasets as both strains are
of the same species also belonging to the genetically very
uniform zymodeme MON-1. A separation between both datasets
occurs in PC2 versus PC3. However, HCA on these strains
clearly shows that they are strictly separated forming two distinct
subclusters within the L. infantum cluster (Fig. 6). Very
promising in terms of species and strain typing is that both L. infantum
strains are mainly located in sister-groups within a
speciesspecific cluster expected from taxonomic considerations.
Interestingly, L. major LV561 and both hybrid strains do
not form any clearly separable strain-specific sub-clusters;
instead they show a mixed topology of two sub-clusters,
each including L. major LV561, and the two L. infantum/
L.major hybrids IMT 208 Cl1 and IMT 211 Cl1. This
suggests a higher similarity of the hybrids to the L. major
parent strain. This was also noticed in the PC scatter plots;
none of the six displayed plots showed a clear separation
of L. major LV561 and both hybrid strains. In a previous
study it was shown that polyploidy observed in
experimental L. infantum/L. major hybrid progenies displayed distinct
tropisms, in terms of clinical forms, depending on the
parental origin of extra chromosomes [
]. Only parts of
the L. infantum/L. major hybrid IMT208 are located close
to L. donovani. However, not in the L. infantum cluster,
which can be explained by emerging spectral outliers,
which concerns two datasets, as a split of the IMT208
scores values (Fig. 6). This may have been caused by the
high spectral variability in the spectral region between 3550
cm–1 and 3250 cm–1, which is nearly in the same order of
magnitude as for the L. donovani datasets (compare
standard deviations in this region for both strains datasets
in Fig. 2).
Interesting is the position and behavior of the remaining
hybrids – intermingled with L. major. The L. infantum/ L.
major hybrids display two subclusters here, together with a
split of L. major into two subclusters. This is due to the
following reasons: Within the spectra of the L. infantum/L. major
hybrid IMT208 spectral differences can be observed; for
instance, in the polysaccharide region at about 1174 cm–1, and
phosphodiester bond region at about 1085 cm–1, as well as in
the fatty acid region at about 3078 cm–1. This observation can
also be made for the datsets of the L. infantum/L. major hybrid
IMT 211, which display a spectral variability at about 1073
cm–1 and at about 3298 cm–1. The data of L. major LV561
only mutually differ at about 3298 cm–1.
For the HCA on DNA films in the 1750–1250 cm–1
spectral range, 20 score(s) values for each of the seven studied
strains have been selected (Fig. 7). It is striking that the
dendrogram comprises uniform strain-specific clusters and no
outliers can be found as in Fig. 6. In the dendrogram two
groups are separated at H of about 6 × 10–3. The first group
entails clustered datasets of L. tarentolae and of the hybrid
IMT208 Cl1, the latter of which branch out at an H~2.9 ×
10–3. The remaining strains can be found in the second
clustered group. L. tropica LRC-L830 with its branching off at the
second highest H value of about 3.9 × 10–3 reflects a relatively
distinct taxonomic entity.
One further branch can be found at H~2.5 × 10–3, which leads
to the sub-cluster of the hybrid IMT211 Cl1, and to the
subcluster that comprises L. major LV561; L. infantum IMT151
and L. donovani BD09. L. major LV561 is separated at H~1.9
× 10–3 from the subgroup of L. infantum IMT151 and
L. donovani BD09. The separation of L. infantum from
L. donovani was observed at H~1.8 × 10–3. The dendrogram
shows the highest similarity between L. infantum and
L. donovani at a value of H~1.8 × 10–3, which is in agreement
with the current taxonomy with these two species belonging to
the L. donovani species complex.
For both parasite films and those of the DNAs, the MIR
signatures are mutually very similar. However, there is a slight
difference of the molecular profiles of parasites and DNAs of
the strain since it includes (or considers) different amounts of
molecular constituents (compare Tables 3 and 4) that are
responsible for the respective PCA and HCA outputs.
DNA film spectra do not exhibit any large variations in
their PCA- and HCA-based clustering compared with the
intact parasite film datasets, which can be explained by a lesser
complex biochemical composition. The (spectral) variability
of DNA data within one strain is lower compared with the
Fig. 7 Dendrogram of the HCA
on parasite DNA films
investigated in the spectral
windows 1750–1250 cm–1
considering, respectively, 20
score(s) values for the seven
strains L. tarentolae, L. infantum/
L. major hybrid IMT208 Cl1,
L. tropica LCR-L830, L. major
LV561, L. donovani BD09,
L. infantum IMT151, and
L. infantum/L. major hybrid IMT
211 Cl1. The phenetic tree was
determined using Euclidean
distance and Ward’s clustering
parasites. This can be explained by a higher homogeneity
within sample preparation of DNA films, as the multiple
drop-casted amount comprised nearly the same molecules.
In contrast, the parasite films entailed a larger spectral variety
and complexity, which is due to the additional modes of
different molecules, i.e., modes that refer to fatty acid residues,
proteins, and peptides. In addition, the sample films
comprised microstructures, likely disordered by the whole
parasitic organisms, may have also caused spectral variablities.
Indeed, slight spectral differences could also be observed
and traced both univariately and multivariately, between
bacterial strains and some of their related PCR products .
The pilot study illustrates here that PCA scores were
successfully implemented for cluster analyses, PCA functioned as
a complementary multivariate analysis tool, whereas HCA
enabled the disposition of spectral datasets of parasite films
in a hierarchical order to phenetic dendrograms.
FTIR complementary tools, such as Raman spectroscopy,
may also provide further insight into the biochemical
composition of parasites and their DNAs by vibrational fingerprints. As
far as we are aware, there are no publications on Leishmania
parasites. This methodology was already applied for other
parasites, e.g., for diagnosis of Malaria and Toxoplasmosis [
This work comprises a pilot study on the molecular
composition of five Leishmania species, L. infantum/L. major hybrids
and their corresponding DNA from the Old World. Each was
successfully investigated by FTIR micro-spectroscopy.
Chemical univariate analysis has provided insights into
molecular structure and composition both for whole Leishmania
parasites and their extracted DNA. The parasites could be
discriminated by spectral differences because of the
polymorphism of polysaccharides, as well as different contributions
from fatty acids such as phospholipids, nucleic acids, and
proteins (amides region). Considering the DNA datasets,
discrimination capability was achieved by spectral differences of
base residues, i.e., contributions from thymine and cytosine,
and from the phosphate-deoxyribose backbone.
The Leishmania species differentiation has been illustrated in
two different spectral windows, addressing a systematic
approach underpinned by multivariate statistics tools such as
PCA and HCA.
PCA allowed a distinct identification and discrimination by
unique MIR spectral fingerprints of Leishmania and their DNAs
at the respective wavenumber windows, enabling successful
segregation between information-rich and -poor spectral
components. Considering the PCA and HCA results of the DNA, a
better differentiation could be achieved than for the parasites.
Hence, DNA may be a more reliable candidate for discrimination
due to the possible elimination of the environmentally sourced
changes on the complex biochemical content of the cells.
PCA results have shown that the clustered spectral datasets
in the scores diagrams strongly correlate with the clusterings
in the HCA. However, a phenetic classification was only
feasible in combination with HCA.
At this time, the present results indicate the suitability of
FTIR for typing/identification rather than for phylogenetic
classification purposes. Further Leishmania species and strains
must be investigated in order to elucidate species-specific
signatures that will allow correct identification. Consequently it is
crucial to strive for further investigations related to
intraspecies variability and species-specific clustering.
Another relevant limitation of the FTIR method is the need
of isolation of Leishmania parasites from biological samples
and culturing, making this process more time-consuming in
the steps that precede this methdology FTIR. An adaptation
for the direct use of clinical material should be further tested.
As vibrat ional s pec troscopic c har ac teri zati on of
Leishmania species and their molecular components (DNA)
with respect to their biochemical compositions by means of
FTIR comprises a relatively new approach, FTIR data of
Leishmania are scarce. This evokes the impetus for further
systematic evaluation based on a balanced and representative
sample set and consideration of other parasitic species  up
to the single-cell level [
Acknowledgments The authors thank Dr. Françoise Routier from the
Institute of Cellular Chemistry, Hannover Medical School, for providing
the L. tarentolae strain. Susanne Lobstein is gratefully acknowledged for
her assistance in DNA extraction.
Funding Information The authors acknowledge funding partially
provided by the Marie Curie EU-COFUND/BRAIN programme
(COFUNDGA-2013-609440) and Fundação para a Ciência e a Tecnologia for funds
to GHTM (UID/Multi/04413/2013). EURAMET is acknowledged for
funding. The research within this EURAMET joint research project
receives funding from the European Community's Seventh Framework
Programme, ERA-NET Plus, under Grant Agreement No. 15HLT01.
Compliance with ethical standards
Conflict of Interest The authors declare that they have no conflict of
Research involving Human Participants and/or Animals not
Informed consent not applicable.
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